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WO1999031139A1 - Nouveaux anticorps monoclonaux et leur utilisation - Google Patents

Nouveaux anticorps monoclonaux et leur utilisation Download PDF

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Publication number
WO1999031139A1
WO1999031139A1 PCT/JP1998/005634 JP9805634W WO9931139A1 WO 1999031139 A1 WO1999031139 A1 WO 1999031139A1 JP 9805634 W JP9805634 W JP 9805634W WO 9931139 A1 WO9931139 A1 WO 9931139A1
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Prior art keywords
ssi
monoclonal antibody
protein
hybridoma
amino acid
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PCT/JP1998/005634
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English (en)
Japanese (ja)
Inventor
Kazuyuki Yoshizaki
Tadahiro Kajita
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International Reagents Corporation
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Publication of WO1999031139A1 publication Critical patent/WO1999031139A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to a protein involved in the regulation and control of intracellular signal transduction activated by cytokines such as interleukin-6 and interleukin-14, the expression of which is induced by STAT.
  • the present invention relates to a series of highly homologous proteins having an action of inhibiting a signal, particularly to a monoclonal antibody having a specific affinity for SSI-1, a method for producing the same, and uses thereof.
  • Inuichi Leukin-6 (hereinafter abbreviated as IL-16), Inuyu Leukin 11 (hereinafter abbreviated as IL-11), leukemia inhibitory factor (hereinafter abbreviated as LIF), ciliary neurotrophic Factor (hereinafter abbreviated as CNTF) and Oncos
  • IL-16 Inuichi Leukin-6
  • IL-11 Inuyu Leukin 11
  • LIF leukemia inhibitory factor
  • CNTF ciliary neurotrophic Factor
  • SM cytokines
  • Activated JAK2 and TYK2 further phosphorylate the C-terminal tyrosine residue of STAT3, a member of the STAT (signal transducers and activators of transcription) family. It is known that phosphorylated STAT3 forms a homo- or heterodimer, activates transcription by translocating from the cytoplasm to the nucleus and binding to a specific DNA sequence. However, the complete mechanism of STAT activation has not been elucidated.
  • SSI-1 STAT-induced STAT inhibitor-1
  • IL-4 inuichi leukin-4
  • IL-6 IL-6
  • LIF granulocyte colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • SSI-1 protein mouse-derived protein whose expression is induced by cytokines (hereinafter referred to as SSI-1 protein), which is induced by STAT3 to inhibit tyrosine phosphorylation of gp130 and STAT3.
  • mouse-derived SSI-1, JAB and mmS0CS-1 and rat-derived rrSOCS-1 mouse-derived SSI-1, JAB and mmS0CS-1 and rat-derived rrSOCS-1 (each consisting of 212 amino acid sequences), and human-derived hs SOCS-1 (211 Has a very high homology of 95-99% at the amino acid sequence level (JAB has been reported with the same amino acid sequence as SSI-1).
  • JAB has been reported with the same amino acid sequence as SSI-1).
  • the conservation of amino acid sequence is characteristic.
  • SSI-11, JAB and SOCS-1 are considered together according to the above reports and various research results, that is, they are also called SSI-1 / S0CS-1 / JAB (Narazaki ⁇ Natl. Acad. Sci. USA, Vol. 95, 13130-13134, 1998).
  • the protein is referred to as SSI-11 o (here, SOCS-1 means Suppressor of cytokines signal in g 1, and JAB means Janus kinase-binding protein JA binding protein).
  • SOCS-1 means Suppressor of cytokines signal in g 1
  • JAB means Janus kinase-binding protein JA binding protein.
  • the above series of proteins, including SS I-1, are based on similarities in the mode of expression induction and structural similarities deduced from the protein amino acid sequence level.
  • the abundance of the SSI family, including SSI-1, in living organisms is extremely small, and it is necessary to use genetic engineering techniques to obtain a large amount of SSI family.
  • An object of the present invention is to provide various types of monoclonal antibodies having specific affinity for the SSI family, particularly SSI-1, and to provide uses of the monoclonal antibodies. It is another object of the present invention to provide a hybridoma cell line that produces the monoclonal antibody.
  • the present inventors have conducted intensive studies and found that the amino acid sequence of SSI-1, whose amino acid sequence is known to be well conserved across species, has a low amino acid homology with the previously reported protein. A region having a sequence specific to the SSI-1 is selected, and a region having high hydrophilicity is selected from the above regions by the structural analysis of the protein deduced from the amino acid sequence.
  • anti-SSI-1 monoclonal antibody a monoclonal antibody that specifically recognizes SSI-1 (hereinafter simply referred to as anti-SSI-1 monoclonal antibody) and established a hybridoma cell line that produces the monoclonal antibody. The use of the monoclonal antibody was found.
  • the present invention is as follows.
  • Immune cells of a mammal immunized with an oligo (poly) peptide consisting of all or a part of at least one protein of the SSI family one protein are fused with a Myeloma cell line,
  • a method for producing a hybridoma comprising cloning, from a fused cell, a strain that produces a monoclonal antibody having a specific affinity for at least one protein among SSI family proteins.
  • Mammalian immune cells immunized with an oligo (poly) peptide comprising all or a part of the SSI-1 protein are fused with a Myeoma cell line, and the fused cells
  • a method for producing a hybridoma which comprises cloning a strain that produces a monoclonal antibody having specific affinity.
  • At least one SSI family protein comprising culturing the hybridoma produced by the production method according to (9) above and obtaining a monoclonal antibody from the culture.
  • a method for producing a monoclonal antibody having specific affinity for a protein comprising culturing the hybridoma produced by the production method according to (9) above and obtaining a monoclonal antibody from the culture.
  • a method for measuring at least one kind of SSI family protein in a sample comprising a step of reacting the monoclonal antibody according to (1) above with a sample.
  • a method for measuring SSI-1 protein in a sample comprising a step of reacting the monoclonal antibody according to any of (2) to (6) with a sample.
  • a protein having the whole or a part of the amino acid sequence of SEQ ID NO: 1 including a step of reacting the monoclonal antibody according to any of (2) to (6) with a sample; Measurement method.
  • a reagent comprising the monoclonal antibody according to any one of (1) to (6).
  • Fig. 1 is a photograph of electrophoresis showing the expression status and analysis of SSI-1 protein in COS7 cells in which the expression of SSI-1 protein and Jak2 protein was induced alone or simultaneously by genetic engineering. (Western plot image)
  • Extracts of COS 7 cells in which the SSI-11 protein and Jak2 protein were induced alone or simultaneously by genetic engineering were prepared.
  • c DNA Type of gene carried by the expression vector to be introduced
  • Lane 1 COS 7 cells expressing SSI-1 protein
  • Lane 2 COS 7 cells expressing Jak 2 protein
  • Lane 3 COS 7 cells expressing SSI-1 and Jak 2 simultaneously
  • Lysate Western blot image using cell-solubilized extract as a sample Figure 2 Expression of SSI-1 protein in COS 7 cells in which SSI-1 protein and Tyk2 protein were induced alone or simultaneously by genetic engineering It is an electrophoresis photograph showing the situation and analysis. (Western plot image)
  • Extracts of CO S7 cells in which the SSI-11 protein and the Tyk2 protein were induced alone or simultaneously by genetic engineering were prepared.
  • cDNA the type of gene to be introduced that is carried by the expression vector
  • Lane 1 CO S7 cells expressing SSI-1 protein
  • Lane 2 COS 7 cells expressing Tyk 2 protein
  • Lane 3 CO S7 cells expressing SSI-1 and Tyk2 simultaneously
  • Lysate Western blot image using cell-lysed extract as sample Figure 3.
  • SSI-1 protein in COS 7 cells in which SSI-11 protein and Jak2 protein were induced to be expressed alone or simultaneously by genetic engineering 1 is a photograph of electrophoresis showing the expression status and analysis of. (Western blot image) Extracts of COS 7 cells in which SSI-11 protein and Jak 2 protein were induced alone or simultaneously by genetic engineering were prepared.
  • anti-SSII-1 monoclonal antibody of the present invention is useful not only for Western blotting but also for immunoprecipitation.
  • cDNA gene to be introduced, which is carried by the expression vector
  • Lane 1 CO S7 cells expressing SSI-1 protein
  • Lane 2 COS 7 cells expressing Jak 2 protein
  • Lane 3 COS 7 cells expressing SSI-1 and Jak2 simultaneously
  • Lysate Western blot image using cell solubilized extract
  • the monoclonal antibody of the present invention (the anti-SSI-1 monoclonal antibody of the present invention), which has a specific affinity for SSI-1, is used regardless of the origin of animals such as humans, mice, and rats. It is characterized by recognizing a region whose amino acid sequence is highly conserved.
  • SSI-1 recognized by the monoclonal antibody means one or both of a protein expressed by genetic engineering and a naturally occurring SSI-11 whose expression is induced in the cytoplasm by cytokine or the like.
  • the epitope recognized by the anti-SSI-1 monoclonal antibody of the present invention is not particularly limited as long as the monoclonal antibody can specifically recognize SSI-11, but it is preferable. Or it exists in the region from amino acid 43 to amino acid 69 of SSI-1 (SEQ ID NO: 1). This region has low amino acid homology when the amino acid sequence of SSI-1 is compared with the amino acid of a previously reported protein, and is assumed to be highly hydrophilic by structural analysis.
  • Another embodiment of the monoclonal antibody of the present invention includes a monoclonal antibody having a specific affinity for a protein having all or a part of the amino acid sequence shown in SEQ ID NO: 1 in Sequence Listing.
  • Specific examples of the protein include mouse-derived J AB and mmSOCS-1, rat-derived rrSOC S-1 and human-derived hs SOC-1, as described above. Protein with high homology (SS I-1 / SOCS-1 / JAB, see above o
  • SSI family 1 refers to a series of proteins whose expression is induced by STAT, 2) STAT signal is suppressed, and 3) src homologous (SH2) region is contained. I do.
  • Specific examples of the proteins belonging to the SSI family include those described above. For example, SSI-1, SSI-2, SSI-3, SSI-4, SSI-5, SSI-6, and SSI-6 — 7 and so on.
  • the antibody include those having specific affinity for all of the proteins belonging to the SSI family, those which recognize each of the proteins belonging to the family 1 or some of them.
  • the monoclonal antibody of the present invention is prepared by a method commonly used in the art. That is, various antibody-producing cells having specific affinity for SSI-1 and proteins belonging to the SSI family 1 and the SSI family, respectively, are fused with myeloma cells to form a hybridoma, and the hybridoma is formed. By cloning and selecting clones that produce antibodies specific to each protein. Antigens vary depending on the specificity of interest, but for example, SSI-1 protein To obtain an antibody having specific affinity for SSI-1, all or part of SSI-1, a polypeptide having a region from amino acid 43 to amino acid 69 of SSI-1, etc. Is used.
  • a polypeptide in the region from the 43rd amino acid to the 69th amino acid of SSI-1, which is a sequence more specific to SSI-1.
  • the antigen may be altered in its amino acid sequence such as deletion, substitution, or addition of one or several amino acids.
  • an amino acid sequence having extremely high homology between proteins belonging to the family is used as an antigen.
  • the SH2 region present in the central part of each protein molecule and the SoC CS box in the C-terminal region are used.
  • a region having a sequence characteristic of the protein molecule of interest is obtained as described above. It can be selected and used as an antigen.
  • the antigen can be prepared by genetic engineering or by synthesis based on a selected partial amino acid sequence.
  • the obtained protein molecule such as SSI-11 or an oligo (poly) peptide based on the selected partial amino acid sequence is placed in an appropriate buffer such as phosphate buffer (PBS). Dissolved or suspended ones are used.
  • PBS phosphate buffer
  • the antigen solution may be usually prepared at a concentration of about 50 to 50 Ojug / ml as an antigen substance.
  • an appropriate carrier protein such as albumin or keyhole rind mosinin.
  • Animals immunized with the antigen include mice, rats, pomas, goats, and egrets.
  • a mouse Preferably a BALB / c mouse.
  • the antigen solution can be administered as a mixture with an adjuvant in order to enhance the responsiveness of the immunized animal to the antigen.
  • the adjuvants used in the present invention include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), R ib i (MPL), Rib i (TDM), Rib i (MP L + TDM), pertussis vaccine (Bordetella pertussis vaccine), muramyl dipeptide (MDP), aluminum adjuvant (ALUM), and combinations thereof
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • MPL MPL
  • Rib i TDM
  • Rib i MDP
  • ALUM aluminum adjuvant
  • the injection site, schedule, and the like can be appropriately changed depending on the type of antigen to be used and the presence or absence of adjuvant admixture.
  • the adjuvant mixed antigen solution .05 to lml antigen substance 10 to 20 injected intraperitoneally, subcutaneously, intramuscularly or intravenously (tail), boosted 1 to 4 times every 4 to 14 days from the first immunization, and After 1 to 4 weeks, final immunization is performed
  • the antigen solution is administered without using an adjuvant, the antigen amount may be increased and intraperitoneal injection may be performed.
  • Blood is collected after 6 days for examination, and the antibody titer can be measured by a usual method according to the antibody assay described later About 3 to 0.5 days after the final immunization, spleen cells are separated from the immunized animal. To obtain antibody-producing cells.
  • myeloma cells those derived from mice, rats, humans and the like are used.
  • Examples include antibody-producing cells and myeloma cells. Is preferably derived from an animal of the same species, particularly an animal of the same strain.
  • Myeloma cells can be cryopreserved or passaged and maintained in common media supplemented with poma, heron or fetal calf serum. It is preferable to use cells in the logarithmic propagation phase for cell fusion.
  • Examples of a method for forming a hybridoma by fusing antibody-producing cells with myeloma cells include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device.
  • PEG polyethylene glycol
  • spleen cells and myeloma cells are placed in a suitable medium or buffer containing about 30 to 60% PEG (average molecular weight: 1000 to 6000) in a ratio of 1: 10: 1, preferably 5-10%. : Suspended at a mixing ratio of 1: 1 at a temperature of about 25 to 37 ° (: pH 6 to 8 for about 30 seconds to 3 minutes
  • the reaction may be performed for a while. After completion of the reaction, remove the PEG solution, resuspend in the medium, inoculate the cell plate, and continue culturing.
  • the cells after the fusion operation are cultured in a selection medium to select hybridomas.
  • the selection medium is a medium in which the parent cell line can be killed and only the fused cells can grow.
  • a hypoxanthin-aminopterin-thymidine (HAT) medium is used.
  • Selection of hybridomas is usually started 1 to 7 days after the fusion procedure by exchanging part of the medium, preferably about half, with the selection medium, and culturing while repeating the same medium exchange every few days. It is done by doing. Confirm the colony is growing by microscopic observation.
  • the culture supernatant may be collected and subjected to antibody assay.
  • the antibody titer is determined by, for example, adding the supernatant to immobilized SSI-1 protein (the protein to be immobilized can be changed according to the specificity of interest), and reacting with the fluorescent substance, enzyme, It can be measured by reacting a secondary antibody labeled with HI (antiglobulin, anti-IgG, anti-IgM serum, etc.). In this way, a gel producing an appropriate antibody is obtained. Separate single clones by limiting dilution, soft agar, or a method using a fluorescence-excited cell saw.
  • a hybrid is produced by serially diluting a hybrid-macroni in a medium so as to be about 1 cell / gel, followed by culturing to isolate a clone producing the desired monoclonal antibody. be able to.
  • the obtained antibody-producing hybridoma is frozen at about 170% to about 196 ° C. in the presence of a cryoprotectant such as about 10% (v / v) dimethyl sulfoxide (DMS 0) or glycerin.
  • a cryoprotectant such as about 10% (v / v) dimethyl sulfoxide (DMS 0) or glycerin.
  • the monoclonal antibody of the present invention obtained by the above method is, for example, a mouse-derived and IgG class monoclonal antibody, which is designated as SI-122B.
  • SI-122B a mouse-derived and IgG class monoclonal antibody
  • the hybridoma is cultured under general conditions, and the class of the antibody secreted in the culture supernatant is analyzed using a commercially available kit for determining antibody class and subclass. It can be known by analyzing using
  • the hybridoma S1-126262B producing the monoclonal antibody S1-126262B was newly isolated by the present inventors.
  • the International Depositary Agency based on the Budapest Treaty, the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry of the Ministry of International Trade and Industry ( ⁇ 1-3-5 Tsukuba East, Ibaraki Prefecture 1-3-3) FERMBP-648 as a deposit number (transfer date of international deposit: September 10, 1998; original domestic deposit date: September 25, 1997, yuan FERMP-1 644 5)
  • FERMBP-648 as a deposit number (transfer date of international deposit: September 10, 1998; original domestic deposit date: September 25, 1997, yuan FERMP-1 644 5)
  • Monoclonal antibodies can be suitably obtained by culturing a hybridoma producing the antibody and collecting the culture.
  • the cultivation of the hybridoma may be performed in an animal (in vivo) or in vitro.
  • the hybridoma may be obtained from mouse ascites, cell culture, or the like.
  • other known methods can be appropriately selected.
  • a hybridoma that can grow in the mouse intraperitoneal cavity can be obtained from ascites at a high concentration of several mg / ml.
  • Hybridomas that cannot grow in vivo can be obtained from culture supernatants of cell cultures. According to cell culture, antibody production is lower than in vivo, but there is an advantage that immunoglobulins and other contaminants contained in the intraperitoneal cavity are small and purification is easy.
  • the hybridoma When the mouse is obtained from the intraperitoneal cavity of the mouse, for example, the hybridoma is injected into the abdominal cavity of a BALB / c mouse previously administered with a substance having an immunosuppressive effect such as pristane (2,6,10,14-tetramethylpentyldecane) (Approximately 10 s or more) and transplant the collected ascites about 1 to 3 weeks later.
  • a substance having an immunosuppressive effect such as pristane (2,6,10,14-tetramethylpentyldecane) (Approximately 10 s or more) and transplant the collected ascites about 1 to 3 weeks later.
  • pristane 2,6,10,14-tetramethylpentyldecane
  • the hybridoma is cultured using a culture method such as a high-density culture method or a spinner flask culture method in addition to the stationary culture method to obtain a culture supernatant containing the antibody.
  • Serum contains contaminants such as other antibodies and albumin, and has many inconveniences in antibody purification. Therefore, it is desirable to reduce the amount added to the culture solution.
  • Purification of monoclonal antibodies from ascites and culture supernatant is performed by fractionation by salting out using conventionally known ammonium sulfate sodium sulfate, polyethylene glycol fractionation, ethanol fractionation, and DEAE ion. It can be easily achieved by applying exchange chromatography, gel filtration, etc.
  • the monoclonal antibody of the present invention when the monoclonal antibody of the present invention is a mouse IgG antibody, it can be purified by affinity chromatography using a protein A-bound carrier or an anti-mouse immunoglobulin-bound carrier, which is convenient. It is. Using the novel monoclonal antibody of the present invention, it is possible to rapidly measure the SSI family including the SSI-1 protein in a sample. As a measuring method, various kinds of Imnoassays usually performed in this field can be used.
  • the method is not particularly limited as long as it includes a step of reacting a sample (analyte) with the antibody and measuring the formed immune complex, and an immunological ratio for optically detecting a precipitation reaction or an agglutination reaction.
  • a turbidity method and a labeled immunoassay using an antibody labeled with a substance that can be easily separated and detected include radioimmunoassay using RI as a label for the detection of immune complexes, enzymatic immunoassay using an enzyme such as alkaline phosphatase peroxidase, and fluorescent immunoassay using a fluorescent substance.
  • the labeling target there are a direct method of directly labeling the antibody to be detected, and an indirect method of labeling the antibody of the antibody to be detected, that is, a secondary antibody.
  • the indirect method for example, when the anti-SSI-11 monoclonal antibody of the present invention is a mouse IgG monoclonal antibody, for example, an anti-mouse IgG polyclonal antibody or the like may be used as the secondary antibody .
  • the method for preparing the secondary antibody, and the labeling of the antibody with a fluorescent substance, RI, an enzyme, and the like can be performed using a method commonly used in the art.
  • a method using the reaction of piotin-avidin (or streptavidin) in the measurement method is also available.
  • Examples of the method include a method using a combination of an anti-SSI-1 monoclonal antibody labeled with biotin and streptavidin labeled with a fluorescent substance or the like. Labeling of the anti-SSI-1 monoclonal antibody with biotin and streptavidin with a fluorescent substance or the like can be carried out using a method usually used in the art, for example, streptovavidin labeled with a fluorescent substance or the like. Avidin is also commercially available.
  • the sample to be measured by the measurement method of the present invention is not particularly limited, but cells in which expression of SSI-1 or SSI family is induced by a cytokine such as LIF or cells in which the protein is forcibly expressed by genetic engineering. Etc. are exemplified.
  • the measurement can be performed at the cell or tissue level, and can also be performed using an extract from the cell or tissue.
  • the measurement at the cell or tissue level is performed by using immunohistochemistry or immunocytochemistry observed with an optical or electron microscope.
  • the measurement using extracts from cells and tissues is performed by using the immunoblot method (Western blot method) using immunoprecipitation, electrophoresis, or the like. For these measurement methods, methods generally used in the art can be applied.
  • the novel monoclonal antibody of the present invention can be included in the reagent.
  • the reagents referred to herein include, for example, reagents for detecting SSI families such as SSI-1 in a sample, reagents for detecting molecules closely related to the protein, and reagents for purifying the protein. .
  • the reagent may be, for example, an appropriate buffer of the monoclonal antibody labeled with RI, an enzyme, a fluorescent substance, or the like. It consists of a solution for label detection, a blocking solution, a washing solution, etc., in addition to a solution dissolved in the solution.
  • a washing solution a buffer solution such as a PBS containing anionic or nonionic surfactant such as sodium dodecyl sulfate (SDS) or polyoxyethylene sorbin monolaurate, or gelatin (further containing sodium azide.
  • the blocking solution may be serum albumin (BSA) or a nonionic surfactant (eg, polyoxyethylene). And a buffer solution such as PBS containing sodium azide (which may further contain sodium azide).
  • BSA serum albumin
  • PBS polyoxyethylene
  • the blocking solution preferably further contains serum derived from animals such as goats.
  • the reagent is, for example, an unlabeled monoclonal antibody and a secondary antibody labeled with RI, an enzyme, a fluorescent substance, etc., which has specificity for the monoclonal antibody, a blocking solution, and a washing solution. And so on.
  • the reagents and the like used in the electrophoresis method can be packed together.
  • a reagent for performing fluorescence measurement by a fluorescence microscope can also be packaged with an encapsulant for the sample (cells, tissues, etc.), and such an encapsulant is preferably glycerol-containing PBS or polyvinyl alcohol-containing PBS. Is exemplified.
  • Reagents for purification of SSI families such as SSI-1 may include, for example, the monoclonal antibody, as well as necessary reagents according to affinity purification methods generally used in this field. it can. Specifically, in addition to the anti-SSI-1 (anti-SSI family 1) monoclonal antibody of the present invention, carriers and reagents for immobilizing the monoclonal antibody, buffers for washing and antigen elution Liquid and the like.
  • the detection reagents for molecules closely related to SSI families such as SSI-1 vary depending on the properties of the target molecule.For example, reagents used for detection methods using immunoprecipitation are listed. Can be Specifically, in addition to the anti-SSI-1 (anti-SSI family 1) monoclonal antibody of the present invention, a carrier for immobilizing the monoclonal antibody, a buffer for washing, and the like can be mentioned. s
  • SI-1 SSI family
  • the same reagents as those used in ordinary electrophoresis reagents and reagents used in the Western blotting method described above are exemplified.
  • Example 1 Preparation of hybridoma and anti-SSI-1 monoclonal antibody Preparation of hybridoma
  • STAT induces its expression, suppresses the STAT signal, and uses the amino acid sequence of SSI-1 protein, which is already known to have the src homology (SH2) region, to determine the amino acid sequence of the SSI-1 protein.
  • a 27-mer SSI-1-1-1 polypeptide was synthesized by the Fmoc method using a peptide synthesizer 421A (manufactured by Applied Biosystems).
  • the prepared polypeptide was purified by reverse-phase HPLC using an oligo T-120ODS column (manufactured by TO SO) and freeze-dried.
  • the purified polypeptide is weighed in a dry weight of 2 mg, dissolved in a binding buffer (Pierce), and combined with 2 mg of maleimidized keyhole limpet to Mosyanin (Pierce, hereinafter abbreviated as KLH). I let it.
  • mice with high titers are further treated with PBS after 2 weeks.
  • the 1 ⁇ 1 ⁇ 11-bound 3S-I-I prepared in 1111 was injected through the tail vein of mice to obtain final immunization.
  • the antibody titer was measured using the serum of a mouse immunized with the antigen according to the screening method described later.
  • splenectomy of BALB / c mice was performed, and spleen cells were suspended in an EMEM culture solution to prepare a suspension of spleen cells.
  • the spleen cells were washed four times with the EMEM medium, and the cell number was calculated to obtain 5.9 ⁇ 10 8 spleen cells.
  • Cell fusion was carried out using a 2-LB-6-hydroxy-8-azapurine (8-azaguanine [2-amino-6-oxy-8-azaprine]) resistant BALB / c mouse-derived myeloma cell line (P3-X63- Ag8 ⁇ 653 (hereinafter also referred to as X63 cells) was used as the parent cell line.
  • X63 cells were subcultured in RPMI-1640 culture medium (20 g / ml, containing 8-azaguanine) containing 10% of inactivated fetal calf serum (FCS), and 8 days before cell fusion. The cells were further cultured in RPMI-1640 culture solution containing 10% FCS without azaguanine, and cells in logarithmic growth phase were used. X6 3 cells RPMI - After washing 3 times with 1640 culture medium, and calculating the number of cells to obtain a 7 x 1 0 7 viable cells.
  • FCS inactivated fetal calf serum
  • polyethylene glycol 4000 is dissolved to a concentration of 50 (w / v)%, and mixed so that the ratio of the above spleen cells and X63 cells becomes 10: 1, and the well-known method is used. (Kshler and Milstein, Nature, vol 256, pp 495-497, 1975, Eur. J. Immunol, vol 6, pp 511-519, 1976) Was done.
  • the RPMI-1640 medium supplemented with 10% FCS, 1 x 10- 4 M hypoxanthine, 4 X 10- 7 aminopterin M and 1. HAT containing thymidine for 6 x 10- 5 M
  • the spleen cells were suspended in a selection medium at 2.0 ⁇ 10 s cells / ml.
  • 50 1 of the cell suspension, 96 after dispensed into each Ueru of Ueru micro evening Ita first plate of, C0 2 sterile incubator at a temperature 37 ° C, humidity 95%, C0 2 atmosphere of 8% Was performed.
  • various synthetic peptide ELISA methods were used. That is, selection was carried out by reaction between the culture supernatant of the hybridoma cell line and ovalbumin (hereinafter abbreviated as 0 VA) -bound SSI-11-I antigen immobilized riser plate. In addition, non-specific reactive clones that react with the non-specific polypeptide-immobilized riser plate were removed, and clones that specifically reacted only with the SSI-1-1-1 antigen were selected.
  • ovalbumin hereinafter abbreviated as 0 VA
  • SSI—; I—I synthetic and nonspecific synthetic polypeptides were combined with maleimide OVA. I let it.
  • OVA-bound SSI-1-1 synthetic peptide and OVA-bound non-specific synthetic peptide were prepared as antigen solutions to a concentration of 2 g / ml, respectively, and 50 ⁇ l per well were added to each microplate. After adding and adsorbing, wash with phosphate buffer containing 0.05% Tween-20 (hereinafter abbreviated as washing solution) three times, and block with phosphate buffer containing 1% BSA. Each OVA-bound synthetic peptide antigen-immobilized plate was prepared.
  • the culture supernatant of the hybridoma cell line obtained above is added to the immobilized plate, reacted at room temperature for 1 hour, washed three times with a washing solution, and further subjected to horseradish superoxidase (HRP). This was reacted with a labeled anti-mouse immunoglobulin antibody (derived from goats) at room temperature for 30 minutes.
  • HRP horseradish superoxidase
  • SSI-1-A 11 residues of amino acids 43 to 53 of the amino acid sequence of SSI-1 within the amino acid sequence of SSI-11 (hereinafter referred to as SSI-1-A), 15 residues of amino acids (hereinafter referred to as SSI-1—B) and 23 residues of amino acids 48 to 70 of the amino acid sequence of SSI-1 (hereinafter referred to as No. 10-11 AP) )
  • SSI-1-A 15 residues of amino acids
  • SSI-1—B 15 residues of amino acids
  • No. 10-11 AP 23 residues of amino acids 48 to 70 of the amino acid sequence of SSI-1
  • each OVA-bound monopeptide prepared at a concentration of 2 ⁇ g / ml was placed in a microtiter plate every 5 ⁇ l per well, allowed to adsorb overnight, and then washed three times with the washing solution. Blocked with PBS containing 1% BSA and prepared various OVA binding polypeptide (SS I-1-1, SSI-1-As SSI-1 -B and No. 10-1-1 AP) antigen-immobilized plates .
  • the culture supernatant of the anti-SSI-1 monoclonal antibody-producing hybridoma cell line screened above was reacted with each OVA-bound polypeptide solid phase for 1 hour at room temperature, and then washed three times with a washing solution, followed by HRP A labeled anti-mouse immunoglobulin antibody (from goat) was allowed to react at room temperature for 30 minutes. After this reaction, the plate is washed four times with a washing solution and reacted with o-phenylenediamine substrate solution. Then, the reaction is stopped with 2 N sulfuric acid, and a plate reader for an ELISA at a main wavelength of 492 nm and a sub wavelength of 690 nm is used. Absorbance was measured in Step 1.
  • Hypridoma cell line obtained as a single clone by cloning
  • the mouse immunoglobulin subclass of the monoclonal antibody produced was determined.
  • the mouse immunoglobulin subclass was identified using the culture supernatant of each hybridoma cell line, using a MONO Ab tp ing kit manufactured by Zymed. Using. Table 1 shows the results.
  • SS 1-1 protein which is the target of the monoclonal antibody, is a protein whose expression is induced by STAT, suppresses the STAT signal, and has a src homology (SH2) region, and belongs to the SSI family.
  • the anti-SSI-1 monoclonal antibody (SI-1262B) used in the following examples is a representative example of the monoclonal antibody obtained in the present invention, and the present invention is not limited to this clone. Not something.
  • the SSI-11 protein was co-precipitated with the simultaneously expressed Jak 2 or Tyk 2 protein, and the anti-SSI-1 of the present invention against the expressed protein was used.
  • An experiment was conducted to confirm the binding activity of the monoclonal antibody by the Destamp method.
  • 1 x 10 6 COS 7 cells 1 10 ⁇ g of SS I—1 cDNA / PEF — BOS vector or (S. Mizushima and S. Nagata, pEF-BOS, a poerfu 1 mammalian expression vector, Nucleic Acid Research, p5322, vol. 18, No.
  • This membrane was blocked with a blocking buffer (5% (w / v) skim milk / PBS) for 30 minutes at room temperature. Thereafter, the membrane was incubated for 1 hour at room temperature with an anti-SSI-1 monoclonal antibody (SI-1262B) diluted to 1 gZml. Further, the membrane was washed once for 15 minutes and twice for 5 minutes, and incubated with a secondary antibody (horseradish peroxidase (HRP) -labeled anti-mouse immunoglobulin G antibody) at room temperature for 30 minutes. The membrane was further washed with a washing solution once for 15 minutes and twice for 5 minutes, and detected with an Enhanced Chemiluminescence (ECL) detection kit (Amersham).
  • Figure 1 shows the results when coprecipitated with Jak2
  • Figure 2 shows the results when coprecipitated with Tyk2.
  • each of the expression vectors (1) to (3) was transfected into 1 ⁇ 10 6 COS7 cells by the calcium phosphate method, and then cultured for 72 hours. After culturing, collect the cells, remove the supernatant as much as possible, add 501 cell lysis buffer to solubilize the cells, add 501 SDS-treated solution, and stir at 96 ° C. Boil for 3 minutes. The mixture after the boiling treatment was subjected to electrophoresis (SDS-PAGE) on SDS-polyacrylamide gel and fractionated on a nitrocellulose membrane in the same manner as above. After transferring the resulting protein, the protein was blocked with a blocking buffer at room temperature for 30 minutes.
  • SDS-PAGE electrophoresis
  • the membrane was incubated with an anti-SSI-1 monoclonal antibody (SI-1262B) for 1 hour at room temperature.
  • This membrane was further washed once for 15 minutes and twice for 5 minutes, and incubated with a secondary antibody (HRP-labeled anti-mouse immunoglobulin G antibody) at room temperature for 30 minutes.
  • the membrane was further washed with a washing solution once for 15 minutes and twice for 5 minutes, and detected with the Enhanced Chemiluminescence (ECL) detection kit (Amersham) (lower row in Figs. 1 and 2). ).
  • ECL Enhanced Chemiluminescence
  • Example 3 Detection of SSI-11 protein by immunoprecipitation of SSI-1 protein with anti-SSI-1 monoclonal antibody
  • 1 ⁇ 10 s COS 7 cells were prepared by (1) only 10 / g SSI—1c DNA / PEF-B 0 S vector, and (2) 20 g Jak 2 cDNA. / PEF—BOS vector only, and 3 10 g of SS I—1c DNA / PEF-B0 S vector and 20 ⁇ g of Jak 2 cDNA / PEF—BOS vector, or 10 g of SSI—1 c DNA / PEF—BOS vector and 20 / g Tyk2c DNA / PEF-one BOS vector were simultaneously transfected by the calcium phosphate method.
  • the cells were collected, the supernatant was removed as much as possible, the cells were suspended well in 1 ml of cell lysis buffer, left on ice for 30 minutes, and centrifuged at 15,000 rpm for 20 minutes to precipitate. Thereafter, the supernatant was transferred to another tube, 1 g of anti-SSI-1 monoclonal antibody (SI-1262B) was added, and the mixture was incubated at 4 ° C for 24 hours. Further, 50% (v / v) Protein in G Sepharose was added to the cells, and incubated for 4 hours.
  • SI-1262B anti-SSI-1 monoclonal antibody
  • the membrane was washed once for 15 minutes and twice for 5 minutes, and incubated with a secondary antibody (HRP-labeled anti-mouse immunoglobulin G antibody) at room temperature for 30 minutes.
  • the membrane was further washed with a washing solution once for 15 minutes and twice for 5 minutes, and detected with an Enhanced Chemiluminescence (ECL) detection kit (Amersham).
  • ECL Enhanced Chemiluminescence
  • the anti-SSI-11 monoclonal antibody of the present invention is a monoclonal antibody that specifically recognizes an SSI-1 protein antigen molecule. By using the monoclonal antibody, it becomes possible to analyze various properties of the SSI-1 protein which is produced in an extremely small amount in a natural state.
  • the monoclonal antibody can be applied to a method for purifying a highly pure SSI-1 protein molecule by affinity purification from a cell extract in which expression is induced by genetic engineering. It has been suggested that it has the potential to provide useful materials for use in crystallographic analysis of SSI-1 protein, and its application range is wide.
  • monoclonal antibodies against sSI family are useful for research such as analysis of the molecular and physiological functions of SSI-1 protein and various SSI family proteins. It can be applied as a useful probe.
  • the monoclonal antibody is used not only for immunologically and histologically analyzing the control mechanism of the intracellular signal transduction system, but also for a wide range of applications not only in basic medicine and clinical medicine but also in pharmaceuticals and diagnostics. It is a novel monoclonal antibody that provides a useful tool for research.
  • This application is based on Japanese Patent Application No. 3653494 filed in Japan, the contents of which are incorporated in full herein. Sequence listing free text
  • SEQ ID NO: 1 A peptide designed to have ebitove of a monoclonal antibody having specific affinity for SSI-1 protein

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Abstract

L'invention concerne un anticorps monoclonal possédant une affinité spécifique avec la protéine SSI-1, un hybridome produisant cet anticorps monoclonal; procédé pour la production de cet hybridome; un procédé pour la production de l'anticorps monoclonal comprenant la culture de l'hybridome ou bien in vivo ou in vitro; une méthode de dosage de la protéine SSI-1 dans des échantillons caractérisée par l'utilisation de l'anticorps monoclonal; un réactif contenant l'anticorps monoclonal. L'invention concerne également un autre anticorps monoclonal possédant une affinité spécifiquement vis-à-vis de la famille des SSI; un hybridome produisant cet anticorps monoclonal; et l'utilisation de l'anticorps monoclonal. Ces anticorps monoclonaux rendent possible l'analyse des propriétés diverses de la protéine SSI-1 et de la famille des SSI. De plus, on peut les utiliser comme sondes dans l'étude des fonctions moléculaires et physiologiques de ces protéines. On peut aussi les utiliser pour l'analyse du mécanisme régulateur de transduction du signal intracellulaire, dans les domaines médicaux de base et clinique, et ils sont utiles dans les recherches visant à développer des médicaments et des diagnostics, etc.
PCT/JP1998/005634 1997-12-15 1998-12-11 Nouveaux anticorps monoclonaux et leur utilisation WO1999031139A1 (fr)

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JP9363494A JPH11169171A (ja) 1997-12-15 1997-12-15 新規モノクローナル抗体およびその用途
JP9/363494 1997-12-15

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030688A1 (fr) * 1997-01-10 1998-07-16 Tadamitsu Kishimoto Nouvelle proteine de regulation de la fonction stat

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998030688A1 (fr) * 1997-01-10 1998-07-16 Tadamitsu Kishimoto Nouvelle proteine de regulation de la fonction stat

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 387, 26 June 1997, A. TAKAHO et al., "A New Protein Containing an SH2 Domain that Inhibits JAK Kinases", p. 921-924. *
NATURE, Vol. 387, 26 June 1997, T. NAKA et al., "Structure and Function of a New STAT-Induced STAT Inhibitor", p. 924-929. *

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