EP0507948A1 - Monoklonaler antikörper, hybridome, ihre herstellung und verwendung - Google Patents

Monoklonaler antikörper, hybridome, ihre herstellung und verwendung

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Publication number
EP0507948A1
EP0507948A1 EP91901637A EP91901637A EP0507948A1 EP 0507948 A1 EP0507948 A1 EP 0507948A1 EP 91901637 A EP91901637 A EP 91901637A EP 91901637 A EP91901637 A EP 91901637A EP 0507948 A1 EP0507948 A1 EP 0507948A1
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EP
European Patent Office
Prior art keywords
bfgf
arg
asp
antibody
cell
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EP91901637A
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English (en)
French (fr)
Inventor
Yoshio Kozai
Akira Hori
Yuzo Ichimori
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Publication of EP0507948A1 publication Critical patent/EP0507948A1/de
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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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to monoclonal antibodies which im uno-neutralize the biological activity of basic fibroblast growth factor (also briefly referred to as bFGF in the present specification) proteins and have high binding sensitivity with bFGF proteins, hybridomas secreting the same, their production and use thereof.
  • basic fibroblast growth factor also briefly referred to as bFGF in the present specification
  • bFGF is a basic polypeptide hormone having an affinity for heparin and a molecular weight of about 17,000 [D. Gospodarowicz, Nature 249, 123 (1974)]. It is now known that bFGF exhibits growth promoting action on almost all cells derived from mesoblast, and acts as a differentiating factor to a mesoblast system.
  • bFGF induces the proliferation of nerve cells, the proliferation of vascular endothelial cells to cause angiogenesis, and the proliferation of cancer cells. Coupled with these functions, therefore, bFGF is involved in diseases such as tumors. Accordingly, a monoclonal antibody which inhibits the activity of bFGF would be useful as a therapeutic drugs for such diseases. However, this possibility has not been recognized till now. Further, as a means for diagnosing these diseases, the determination of blood bFGF concentrations is considered. It is however impossible to detect bFGF with great sensitivity by previously known methods. As discussed above bFGF is involved in diseases such as cancer, so that the inhibition of the growth promoting activity of bFGF to the cancer cells presents the possibility of use as a cancer therapy.
  • bFGF itself may have application as a therapeutic drug for traumas and burns.
  • the determination of fundamental information regarding bFGF is essential for the development of bFGF as a medicine.
  • the diagnosis of the disease becomes possible by tracing the blood bFGF concentrations.
  • bFGF is adsorbed by heparan sulfate existing among vascular endothelial cells, bFGF exists in blood only in trace amounts. Accordingly, there is a need in the art to develop a highly sensitive quantitative assay for measuring bFGF in the blood.
  • the present invention provides: (1) a monoclonal antibody which has the following characteristics, it immuno-neutralizes the activity of a bFGF protein and has high binding sensitivity with the bFGF protein:
  • rhbFGF mutein CS23 a mutein in which the cysteine residues at positions 70 and 88 of hbFGF are substituted for serine residues
  • HUVE human unbilical vein endothelial
  • a method for producing a cloned hybridoma which comprises fusing a spleen cell from a mammal and a homogenic or heterogenic lymphoid cell, said mammal being immunized with a mutein in which at least one cysteine residue of bFGF is substituted for a serine residue, and selecting the desired hybridoma, followed by cloning;
  • (6) a method for detecting or measuring a bFGF protein, which comprises using the monoclonal antibody described in the above item (1) .
  • FIG. 1 shows a cDNA sequence of human aFGF used in Reference Example 1
  • Fig. 2 is a schematic representation showing the construction of plasmid pTB975 obtained in Reference Example 1;
  • Figs. 3 to 5 show elution patterns obtained in Reference Example 1;
  • Fig. 6 is a graph showing the affinity of monoclonal antibody 3H3 of the present invention obtained in Example 4 for rhbFGF mutein CS23;
  • Fig. 7 is a graph showing the human bFGF-neutralizing activity of monoclonal antibody 3H3 of the present invention obtained in Example 6;
  • Figs. 8(a) and 8(b) are graphs showing the proliferation inhibition effect of monoclonal antibody 3H3 of the present invention obtained in Example 6 for HUVE cells, in which Fig. 8(a) shows the results of cultivation for 3 days and Fig. 8(b) shows the results of cultivation for 5 days;
  • Fig. 9 is a graph showing the relationship between the concentration and the absorbance of hbFGF, obtained in Example 9;
  • Fig. 10(1) and 10(2) are graphs showing the influence of heparin in a hbFGF determination system, obtained in Example 10, in which Fig. 10(1) shows the results when MAbl2 is fixed, and Fig. 10(2) shows the results when a 50-50 mixture of MAb52 and MAb98 is fixed;
  • Fig. 11(1) and 11(2) are graphs showing the reaction in a hbFGF determination system, obtained in Example 11, in which Fig. 11(1) shows the results when MAbl2 is solidified, and Fig. 11(2) shows the results when a 50-50 mixture of MAb52 and MAb98 is fixed;
  • Fig. 12 is a graph showing the anti-tumor effect of an antibody of the present invention, obtained in Example 13;
  • Fig. 13 is a schematic representation showing the construction of plasmid pTBlOOO obtained in Reference Example 2.
  • the bFGF proteins of the present invention include bFGFs and muteins in which at least one cysteine residue of bFGF is substituted for a serine residue and which have bFGF activity.
  • polypeptides containing the amino acid sequence represented by formula (I) there are preferably used polypeptides containing the amino acid sequence represented by formula (I) :
  • the bFGFs of the present invention include bFGFs derived from mammals.
  • the mammals include humans, monkeys, pigs, bovines, sheep and horses.
  • the bFGFs also include bFGFs extracted from various organs existence of which is already clarified, such as brains and pituitary glands.
  • the bFGF proteins may be produced by recombinant DNA techniques.
  • amino acid sequences of the bFGFs include the amino acid sequence represented by formula (II):
  • human bFGF is preferable.
  • the human bFGF has the amino acid sequence in which X is Thr and Y is Ser in the above formula (II).
  • the muteins in which at least one cysteine residue of the above bFGF is substituted for a serine residue include, for example, the muteins described in Seno et al., Biophys. Res. Commun. 151, 701 (1988) and European Patent Publication No. 281,822.
  • rhbFGF mutein CS23 recombinant human bFGF mutein CS23 in which each of the cysteine residues at positions 70 and 88 of the human bFGF is substituted for a serine residue.
  • Met is numbered as the first.
  • examples of the human bFGF include bFGF produced by the methods described, for example, in FEBS Letters 213, 189 (1987), Biophys. Res. Commun. 146, 470 (1987) and European Patent Publication No. 237,966.
  • mammals are immunized with the bFGF proteins or protein conjugates
  • experimental animals such as sheep, goat, rabbits, guinea pigs, rats and mice, as the mammals to be immunized.
  • rats or mice it is preferred to use rats or mice for immunization.
  • mice When mice are immunized, for example, they can be immunized by any of the subcutaneous, intraperitoneal, intravenous, intramuscular and intracutaneous routes. However, subcutaneous, intraperitoneal and intravenous injections are preferably used. In particular, subcutaneous injection is preferable.
  • the immunizing interval and the immunizing dose are widely variable, and various methods are available. For example, methods in which immunization is carried out about 2 to 6 times at intervals of 2 weeks and spleen cells are removed after about 1 to 5 days, preferably about 2 to 4 days from the final immunization are frequently used.
  • the immunizing dose it is preferred to use about 0.1 ⁇ g or more, preferably about 10 to 300 ⁇ g of the peptide per one immunization of a mouse. Further, it is desirable to carry out the fusion process using the spleen cells after confirmation of an increase in antibody titer in blood by collecting a portion of blood and measuring the antibody titer before removal of the spleens.
  • lymphoid cells are then fused with lymphoid cells.
  • lymphoid cell strains such as suitable myeloma cells [for example, P3-X-63-Ag"8UI (Ichimori et al., J. Immun. Method 80, 55 (1985))] of the same kind or a different kind (preferably the same kind) having markers such as hypoxanthine-guanine-phosphoribosyl-transferase deficient (HGPRT-) and thymidine kinase deficient (TK ⁇ ).
  • suitable myeloma cells for example, P3-X-63-Ag"8UI (Ichimori et al., J. Immun. Method 80, 55 (1985)
  • suitable myeloma cells for example, P3-X-63-Ag"8UI (Ichimori et al., J. Immun. Method 80, 55 (1985)
  • HGPRT- hypoxanthine-guanine-phosphoribosyl
  • the fused cells are produced in accordance with the method of Kohler and Milstein [Nature 256, 495 (1975)].
  • myeloma cells and spleen cells in a ratio of about 1:5 are suspended in a medium prepared by mixing Iskov medium and Ham F-12 medium in a 1:1 ratio (hereinafter referred to as IH medium), and a fusion accelerator such as Sendai virus or polyethylene glycol (PEG) is added thereto.
  • a fusion accelerator such as Sendai virus or polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • DMSO dimethyl sulfoxide
  • the polymerization degree of PEG is usually about 1,000 to 6,000, the fusion time is about 0.5 to 30 minutes, and the concentration of the suspension is about 10 to 80%.
  • the fusion is carried out efficiently by using PEG 6,000 in a concentration of about 35 to 55% for about 4 to 10 minutes.
  • the fused cells can be selectively proliferated using hypoxanthine-aminopterin-thy idine medium (HAT medium) [Nature, 256, 495 (1975)].
  • HAT medium hypoxanthine-aminopterin-thy idine medium
  • the culture supernatant of the proliferated cells is then screened for the production of the desired antibody. Screening of the antibody titer can be carried out in the following manner. First, the presence or absence of the antibody production by peptide immunization is examined by radio im unoassays (RIAs) or enzyme immunoassays (EIAs). For these methods, various modified methods are also available. As a preferred example of the assays, a method using the EIA is described below.
  • a rabbit anti-mouse immunoglobulin antibody is coupled with a carrier such as cellulose beads according to conventional methods, and then a culture supernatant or mouse serum to be assayed is added thereto, followed by reaction at a constant temperature (about 4 to 40 C, the same applied hereinafter) for a definite time.
  • a constant temperature about 4 to 40 C, the same applied hereinafter
  • an enzyme-labeled peptide a peptide is coupled with an enzyme according to conventional methods, followed by purification
  • reaction at a constant temperature for a specified time After the reaction product is thoroughly washed, an enzyme substrate is added thereto, followed by reaction at a constant temperature for a specified time. Then, the absorbance or fluorescence of the color-produced product is measured.
  • the screening of the neutralizing activity to the bFGF proteins can be carried out in the following manner.
  • the neutralizing activity can be examined using cells whose proliferation is induced by the bFGF proteins.
  • cells there can be used vascular endothelial cells, fibroblasts, nerve cells and the like. It is however desirable to know whether or not the additional effect of the bFGF proteins is inhibited by measuring the number of cells, using the vascular endothelial cells begin a new paragraph.
  • Methods for measuring the number of cells include a method for measuring directly the number of cells, a method for determining radioactivity by using tritium thymidine and a method for measuring colorimetrically using (4,5-dimethyl- 2-thiazoly)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (MTT method).
  • MTT method 4,5-dimethyl- 2-thiazoly-2,5-diphenyl-2H-tetrazolium bromide
  • the bFGF protein and the culture supernatant to be assayed are added thereto, followed by cultivation at a constant temperature (37 C) at a low concentration of oxygen (about 7% is preferable) for a definite time. Then, the culture solution are replaced by a medium containing MTT, and cultivation is further continued, whereby MTT is reduced to a formazan, a colorimetric material, in intracellular endogenous mitochondria.
  • the concentration of the formazan is made even, the absorbance at 590 nm is measured.
  • the proliferative property of cells correlates with the absorbance. Hence, if the solution containing the hybridoma supernatant is decreased in absorbance compared with a solution not containing the hybridoma supernatant, the antibody contained in the supernatant can be said to have bFGF neutralizing activity.
  • the cells in wells which proliferate in a selective medium and which produce antibodies with immuno-neutralizing activity to the peptide used for immunization be cloned by a limiting dilution analysis.
  • the supernatant of the cloned cells is similarly screened, and the selected cells which show a high antibody titer are proliferated, whereby monoclonal antibody- producing hybridoma clones showing the reactivity with the immunized peptide can be obtained.
  • the hybridoma cells thus cloned are proliferated in a liquid medium.
  • the hybridoma cells are cultivated in the liquid medium such as a medium prepared by adding about 0.1-40% bovine serum to RPMI-1640 [G. E. Moore et al., J. Am. Med. Assoc. 199, 549 (1967)], for about 2 to 10 days, preferably for 3 to 5 days, whereby the monoclonal antibody can be obtained from the culture solution.
  • the antibody can further be obtained by intraperitoneally inoculating mammals with the hybridoma cells, thereby proliferating the cells and then collecting the ascites.
  • a mouse for example, about 1 X 10 4 to 1 X 10 7 , preferably 5 X 10 5 to 2 X 10 6 of the hybridoma cells are intraperitoneally inoculated into a mouse such as BALB/c preliminarily inoculated with mineral oil and the like, and the ascites is collected after about 7 to 20 days, preferably after about 10 to 14 days.
  • the monoclonal antibody formed and accumulated in the ascites can be easily isolated as pure immunoglobulin by ammonium sulfate fractionation, DEAE-cellulose column chromatography or the like.
  • the monoclonal antibodies of the present invention have high binding sensitivity not only with the immunogen peptides, but also with the bFGF proteins, and further exhibit the neutralizing activity to the bFGF proteins. As a result of this high binding sensitivity, the monoclonal antibodies of the present invention are very useful as reagents for assaying the bFGF proteins and for purifying the bFGF proteins.
  • the bFGF proteins can be assayed to 20 pg/ml by the assays using the monoclonal antibodies of the present invention.
  • the ability to assay such an extremely small amount of bFGF ii vivo is an important development in the art.
  • diseases induced by overproduction of bFGF tumors are mentioned.
  • cancer cells proliferate directly by bFGF and the case that vascular endothelial cells react with bFGF produced from the tumors to proliferate and induce new formed blood vessels which supply nutritive substances to the tumors, which results in enlarged tumor masses.
  • these diseases can be anticipated by measuring the amount of the bFGF overproduced. Since bFGF easily adheres to the inner walls of blood vessels, it is therefore important to have a detecting or determining assay method as sensitive as possible.
  • bFGF is considered to improve the symptoms thereof effectively.
  • the monoclonal antibodies of the present invention can be used as means for tracing the amount of the mutein m. vivo, because the monoclonal antibodies also have high binding sensitivity with the mutein in which at least one cysteine residue is substituted for a serine residue.
  • the monoclonal antibodies of the present invention inactivate the bFGF in vivo and exhibit antitumor activity, because of their strong immuno-neutralizing activity.
  • the monoclonal antibodies are administered in an amount of about 100 ⁇ g/kg to 10 mg/kg. These monoclonal antibodies are also effective for treatment of enlarged tumors following angiogenesis.
  • Examples of the methods for detecting or assaying the bFGF proteins include an immunoassay for assaying the bFGF proteins by using an anti-bFGF antibody supported on a carrier and a conjugate obtained by combining an anti-bFGF antibody directly with a labeling agent, the anti-bFGF antibody differing from the antibody held on the carrier in an antigen determinant.
  • the carriers on which the antibody is held in the above-mentioned assay include, for example, gel particles such as agarose gels [for example, Sepharose 4B and Sepharose 6B (Pharmacia Fine Chemical, Sweden)], dextran gels [for example, Sephadex G-75, Sephadex G-100 and Sephadex G-200 (Pharmacia Fine Chemical, Sweden) ] and polyacrylamide gels [for example, Biogel P-30, Biogel P-60 and Biogel P-100 (Bio RAD Laboratories, U.S.A.)]; cellulose particles such as Avicel (Asahi Chemical Industry, Japan) and ion exchange cellulose (for example, diethylaminoethyl cellulose and carboxymethyl cellulose); physical adsorbents such as glass (for example, glass balls, glass rods, aminoalkyl glass balls and aminoalkyl glass rods), silicone pieces, styrenic resins (for example, polystyrene balls and polystyrene
  • the antibody In order to couple the antibody onto the carrier. methods known in the art are applied. Examples of such methods include the cyanogen bromide method and the glutaraldehyde method which are described in Metabolism, j3, 696 (1971). As a simpler method, the antibody may be physically adsorbed on the surface of the carrier.
  • the labeling agents with which the antibodies are combined include radioisotopes, enzymes, fluorescent substances and luminous substances. However, it is preferred to use the enzymes.
  • the enzymes which are preferably stable and high in specific activity, there can be used peroxidases, alkaline phosphatases, 3-D- galactosidases, glucose oxidases and the like.
  • peroxidases is preferably used.
  • Peroxidases of various origins can be used. Examples of such peroxidases include peroxidases obtained from horseradishes, pineapples, figs, sweet potatoes, broad beans and cone.
  • horseradish peroxidase (HRP) extracted from horseradishes is preferable.
  • the thiol group of Fab 1 as the antibody molecule is utilized, and peroxidase into which a maleimide group is preliminarily introduced is conveniently used.
  • a maleimide group When a maleimide group is introduced into peroxidase, it can be introduced through an amino group of peroxidase.
  • N-succinimidyl-maleimide-carboxylate derivatives can be used.
  • N-( ⁇ -maleimidobutyloxy)succinimide (hereinafter also briefly referred to as GMBS) is preferably used. A certain group may therefore intervene between the maleimide group and perxidase.
  • GMBS is reacted with peroxidase in a buffer solution having a pH of 6 to 8 at about 10 to 50°C for about 10 minutes to about 24 hours.
  • the buffer solutions include, for example, 0.1 M phosphate buffer (pH 7.0).
  • the maleimidated peroxidase thus obtained can be purified, for example, by gel chromatography. Examples of carriers used in the gel chromatography include Sephadex G-25 (Pharmacia Fine Chemical, Sweden) and Biogel P-2 (Bio RAD Laboratories, U.S.A. ) .
  • the maleimidated peroxidase can be reacted with the antibody molecule in a buffer solution at about 0 to 40 C for about 1 to 48 hours.
  • the buffer solutions include, for example, 0.1 M phosphate buffer (pH 6.0) containing 5 mM sodium ethylenediaminetetraacetate.
  • the peroxidase-labeled antibody thus obtained can be purified, for example, by gel chromatography. Examples of carriers used in the gel chromatography include Sephadex G-25 (Pharmacia Fine Chemical, Sweden) and Biogel P-2 (Bio RAD Laboratories, U.S.A. ) .
  • a thiol group may be introduced into peroxidase to allow it to react with the maleimidated antibody molecule.
  • Enzymes other than peroxidases can be directly combined with the antibodies similarly to the methods of combining peroxidases, and known methods which achieve such combining include body fluids or, for example, the glutaraldehyde method, the periodic acid method and the water-soluble carbodiimide method.
  • Test samples used in the assay system of the present invention include humors such as urine, serum, plasma and cerebrospinal fluid, extracts of animal cells, and culture supernatants thereof.
  • test sample containing the bFGF protein to be assayed is added to the antibody held on a carrier to conduct antigen-antibody reaction, and then the conjugate of the peroxidase with the anti-bFGF protein antibody obtained above is added thereto, followed by reaction.
  • the purified antibody of the present invention is coupled with a suitable carrier such as activated agarose gel beads according to conventional methods, and packed in a column. Then, a sample containing the crude bFGF protein, such as a culture supernatant or disrupted cells, is loaded onto the antibody affinity column to allow the sample to be adsorbed thereby, followed by washing. Then, elution is carried out with a chaotropic reagent such as potassium thiocyanate (KSCN) or under such acescent conditions that the bFGF is not inactivated.
  • KSCN potassium thiocyanate
  • the antibody column can be prepared by coupling the monoclonal antibody of the present invention, which is, for example, purified from ascites or other humors inoculated with the hybridoma cells, with an appropriate carrier.
  • any carrier may be used as long as the bFGF protein is specifically efficiently adsorbed thereby after coupling and suitable elution is thereafter possible.
  • carriers include agarose gels, cellulose and acrylamide polymers.
  • polyacrylamide gel beads in which primary amines of the proteins are activated so as to be easily combinable such as Affi-Gel 10 (Bio RAD), are conveniently used according to the following method.
  • the antibody is reacted with Affi-Gel 10 in a buffer solution such as a bicarbonate solution having a concentration of about 0.001 to 1 M, preferably about 0.1 M.
  • the reaction is conducted at about 0 to 20°C at a broad pH range for about 10 minutes to about 24 hours, preferably at about 4 C at a pH of about 3 to 10 for about 4 hours.
  • the larger amount of antibody is mixed with Affi-Gel 10
  • the larger amount of antibody becomes combined therewith, within the range up to about 50 mg of antibody per 1 ml of Affi-Gel 10.
  • the antibody may therefore be mixed with Affi-Gel 10 in any ratio within this range.
  • about 10 to 30 mg of the antibody is conveniently used, considering the combining efficiency and the purification efficiency in affinity chromatography.
  • the antibody-carrier combined material thus formed is thoroughly washed with the buffer solution used for the reaction. Then, residual unreacted active groups are blocked by allowing the washed material to stand for several days, by adding a compound containing a primary amine such as ethanolamine-hydrochloric acid or glycine thereto to a final concentration of about 0.05 to 0.10 M, followed by reaction at about 4°C for about 1 to 4 hours, or by reacting a protein such as 1 to 5% bovine serum albumin (BSA) therewith at 4°C overnight.
  • BSA bovine serum albumin
  • the bFGF protein-containing sample is dissolved in a buffer solution having a pH around neutrality such as phosphate buffer or Tris-hydrochloric acid buffer, followed by adsorption by the antibody column. Then, the column is washed with the same buffer, and then the bFGF protein is 5 eluted.
  • a buffer solution having a pH around neutrality such as phosphate buffer or Tris-hydrochloric acid buffer
  • the column is washed with the same buffer, and then the bFGF protein is 5 eluted.
  • the following solutions are commonly used: weakly acidic solutions such as acetic acid solutions, solutions containing polyethylene glycol, solutions containing peptides more easily combinable with the antibody than the sample, high concentration salt solutions and their
  • the substantially pure bFGF protein substantially free from pyrogens and endotoxins can be obtained.
  • the substantially pure bFGF protein of the present invention contains the bFGF protein in a concentration of 90% (w/w) or more, and preferably in a concentration of 95% (w/w) or 0 more.
  • the monoclonal antibodies of the present invention immuno-neutralize the biological activity of the bFGF proteins at low concentrations and have high binding sensitivity with the bFGF proteins, so that they can be used 5 as therapeutic drugs for treatment of diseases such as cancer, and as reagents for assaying the bFGF proteins.
  • Mouse 3H3 cells obtained in Example 2-(4) described below was deposited with the Institute for Fermentation, Osaka, Japan (IFO) under the accession number IFO 50216 on November 10, 1989. The above cells were also deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan (FRI) under the accession number FERM BP- 2658 on November 14, 1989.
  • Anti-bFGF monoclonal antibodies MAbl2, MAb52 and MAb98 described in the following Examples can be produced by the methods described in Hybridoma, 8, 209-221 (1989) and
  • the recognition site of MAbl2 is included in the amino acid sequence of from position 1 (the N-terminus) to position 9 of bFGF, and the recognition sites of MAb52 and MAb98 are included in the amino acid sequence of from position 14 to position 40 of bFGF.
  • Recombinant human aFGF which was produced by the methods described in Reference Example 1 was used.
  • rhbFGF Recombinant human bFGF
  • Escherichia coli MM294(DE3)/LysS,pTB762 which is produced in Reference Example 1 was deposited with IFO under the accession number IFO 14936 on September 12, 1989. This transformant was also deposited with FRI under the accession number FERM BP-2599 on September 20, 1989.
  • pET3c As a vector DNA, there was used pET3c [F. W. Studier et al., J. Mol. Biol., 189, 113-130 (1986)] carrying a Z$10 promoter for a T7 phage.
  • pET3c was cleaved with Ndel and the termini thereof were made flush by treatment with large fragment. Then, an Ncol linker, 5*-CCATGG-3' , was ligated thereto with T4 DNA ligase. The resulting plasmid was cleaved with Ncol, and the cleaved ites were made flush with DNA polymerase large fragments, followed by cleavage with BamHI to remove the sequence of S10.
  • This strain was cultivated in a medium containing 35 ⁇ g/ml of ampicillin and 10 ⁇ g/ml of chloramphenicol. When the turbidity reached 170 Kletts, isopropyl-6-D- thiogalactoside (IPTG) was added thereto to a final concentration of 0.5 mM, and cultivation was further continued for 3 hours. The cells were collected by centrifugation and washed with ice-cooled PBS. Then, the cells were collected again and stored at -20°C until their use.
  • IPTG isopropyl-6-D- thiogalactoside
  • This supernatant was mixed with 200 ml of an ice-cooled solution [20 mM Tris-HCl (pH 7.4), 1 mM EDTA], and the mixture was loaded onto a heparin Sepharose column (2.5 cm diameter X 4 cm) equilibrated with a buffer [20 mM Tris-HCl (pH 7.4), 1 mM EDTA].
  • the column was washed with 150 ml of a solution [20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1.5 M NaCl], and then the protein was eluted with an eluting solution [20 mM Tris-HCl (pH7.4), 1 mM EDTA, 1.5 M NaCl].
  • the eluate was fractionated into 6 ml portions and 0D fln was monitored to collect the second peak fraction (8th to 11th, the total amount: 24 ml) (Fig. 3). Twenty-two ml of the eluate was mixed with an equal volume of solution 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 M (NH. )_S0.), and the mixture was loaded onto a phenyl
  • Sepharose column (2.5 cm diameter X 8 cm) equilibrated with a buffer [20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 M (NH 4 ) 2 S0 4 ] (flow rate: 0.5 ml/min).
  • the column was washed with 20 ml of the same buffer, and eluted with a linear gradient of 0 to 1 M ammonium sulfate (flow rate: 0.5 ml/min, gradient time: 200 minutes).
  • the eluted fractions 40 to 55 (Fig. 4) were collected as purified human aFGF.
  • a 1.2 mg/ml solution of the purified human aFGF was mixed with 0.25 ml of 0.1% trifluoroacetic acid (TFA), and the mixture was applied to a reverse-phase C4 column (VYDAC) .
  • the column was eluted with a linear gradient of 0 to 90% acetonitrile in 0.1% TFA to examine an elution pattern.
  • the flow rate was 1 ml/min and the gradient time was 60 minutes (Fig. 5) .
  • the activity of the human aFGF was determined by
  • Plasmid pTB762 obtainable from E. coli MM294(DE3)/
  • Lys,pTB672 (IFO 14936, FERM BP-2599), and described in
  • Mouse BALB/c3T3 clone A31 cells [T. akunaga et al. Science, 209, 505-507 (1980)] were seeded on a 6 cm diameter dish for tissue culture having DMEM medium
  • Each of these transformed cells showed the form of a malignant cell gland and formed colonies on a soft agar plate.
  • mice female, 8 weeks old were intraperitoneally injected with 50 ⁇ g of antigen rhbFGF mutein CS23 (a mutein in which each of Cys residues at positions 70 and 88 of human bFGF were substituted for a Ser residue) which was dissolved in Freund's complete adjuvant (Difco) . Two weeks later, the mice were intraperitoneally given again 50 ⁇ g of antigen rhbFGF mutein CS23 dissolved in 0.4 ml of Freund's complete adjuvant.
  • antigen rhbFGF mutein CS23 a mutein in which each of Cys residues at positions 70 and 88 of human bFGF were substituted for a Ser residue
  • mice were additionally immunized with 50 ⁇ g of antigen rhbFGF mutein CS23 dissolved in 0.4 ml of Freund's incomplete adjuvant.
  • 50 ⁇ g of rhbFGF mutein CS23 dissolved in physiological saline was inoculated into the caudal veins of the mice.
  • Mouse myeloma cells SP2/0-AG14 (ATCC No. CRL 1581) were subcultured in DMEM medium containing 10% fetal calf serum under an atmosphere of 5% carbon dioxide and 95% air.
  • PEG 7 (2 X 10 cells) were mixed with the immunized lymphocytes g (1.5 X 10 cells) obtained by the above method, and the mixture was centrifuged. Then, 1 ml of a 45% solution of polyethylene glycol 6000 (hereinafter referred to as PEG
  • IH medium containing 20% calf serum
  • HAT 1 X 10 -4 M hepoxanthine, 4 X 10-7 M am opterin, 1.6 X 10 —5 M thymidine
  • HAT medium 1 X 10 -4 M hepoxanthine, 4 X 10-7 M am opterin, 1.6 X 10 —5 M thymidine
  • HAT medium 1 X 10 -4 M hepoxanthine, 4 X 10-7 M am opterin, 1.6 X 10 —5 M thymidine
  • a fixing buffer [0.1 M sodium hydrogencarbonate (pH 9.6), 0.02% sodium azide] containing 200 ng/ml of rhbFGF mutein CS23 was added in an amount of 100 ⁇ l/well to a 96- well polystyrene microtiter plate (Nunc) .
  • the microtiter plate was washed with a rinsing liquid (0.05% Tween 20, physiological phosphate buffer), and then 100 ⁇ l of the combined solution of 50 ⁇ l of the culture supernatant and 50 ⁇ l of a buffer for dilution (0.05 M Tris-HCl buffer pH 8.01, 1 mM magnesium chloride, 0.15 M sodium chloride, 0.05% Tween 20, 0.02% sodium azide, 0.3% gelatin) was added to the microtiter plate. After 2 hours, the culture supernatant was washed with a rinsing liquid, followed by addition of the alkaline phosphatase-labeled anti-mouse IgG goat antibody (Bio RAD) as the second antibody.
  • a rinsing liquid 0.05% Tween 20, physiological phosphate buffer
  • a buffer for dilution 0.05 M Tris-HCl buffer pH 8.01, 1 mM magnesium chloride, 0.15 M sodium chloride, 0.05% Tween 20,
  • the second antibody was washed with a rinsing liquid, and then coloring reaction was conducted by adding a reaction substrate (ELISA method) .
  • ELISA method reaction substrate
  • GIT culture solution containing various concentrations of hybridoma culture supernatants, 4 ng/ml rhbFGF and 2.5% fetal calf serum was added in an amount of 100 ⁇ l/well, and cultivation was conducted at 37°C under an atmosphere of 5% C0 and 7% 0 choir for 3 days. After 3 days, the culture solution was removed, and then GIT culture solution containing 1 mg/ml MTT (4,5-dimethyl-2-thiazolyl- 2,5-diphenyl-2H-tetrazolium bromide) and 2.5% fetal calf serum was added in an amount of 100 ⁇ l/well.
  • MTT 4,5-dimethyl-2-thiazolyl- 2,5-diphenyl-2H-tetrazolium bromide
  • Hybrid Cells The cells in this well were spread to 0.5 cell per well on a 96-well microtiter plate on which mouse thymocytes had preliminarily been spread as vegetative cells, and cloning was carried out. As a result, hybridoma mouse 3H3 cells (IFO 50216, FERM BP-2658) were obtained. The cloned cells were stored in liquid nitrogen, adding dimethyl sulfoxide (DMSO) to IH medium containing 20% calf serum to a concentration of 10%. - 33 -
  • DMSO dimethyl sulfoxide
  • Example 2-(3) The culture supernatant of 3H3 cells obtained in Example 2-(3) were reacted with various immunoglobulin samples by a subclass detecting kit (Bio RAD) . The results are shown in Table 1.
  • the IgG amount of the samples was determined according to the method described in Example 2-(2) in the following manner. Various dilutions of mouse IgG whose concentration was known and the 3H3 antibody were fixed on a 96-well polystyrene microtiter plate with a fixing buffer. After 2 hours, the alkaline phosphatase- labeled anti-mouse IgG goat antibody (Bio RAD) was added thereto. After 2 hours, coloring reaction was conducted by adding a reaction substrate (ELISA method). With respect to mouse IgG, a determination curve was drawn, and the IgG amount of the samples was determined based on this curve, whereby a 60 ⁇ g/ml solution of the 3H3 antibody was prepared.
  • Fig. 6 shows the results of the antibody titer to rhbFGF mutein CS23 measured for the monoclonal antibody thus purified, according to the method described in Example 2- (2).
  • One ⁇ g/ml of the rabbit anti-bFGF polyclonal antibody was fixed and rhbFGF mutein CS23 was added thereto.
  • 1 ⁇ g/ml of the 3H3 antibody was added and 1 ⁇ g/ml of the alkaline phosphatase-labeled anti-mouse IgG goat antibody was further added thereto.
  • rhbFGF mutein CS23 could be detected up to 3 ng/ml.
  • the antibody was purified from ascites.
  • the mouse 3H3 cell strain was injected into the mice (Balb/c) .
  • IgG was purified from the ascites according to conventional methods. Namely, 5 ml of the ascites was subjected to salt precipitation using a 45% saturated solution of ammonium sulfate, and the precipitate was dissolved in borate buffer (BBS, pH 8.5) containing 0.15 M NaCl, followed by dialysis against BBS at 4°C for 20 hours.
  • BBS borate buffer
  • the antigen recognition site of the 3H3 antibody whose antibody titer was measured in Example 4 was examined by competitive binding inhibition experiments.
  • competitive substances there were used human aFGF, rhbFGF, rhbFGF mutein CS23, synthetic peptides pep 1: Pro-Ala-Leu-Pro-Glu- Asp-Gly-Gly-Ser-Tyr [the peptide in which Tyr was added to the C-terminus of N-terminal amino acids 2 to 10 of human bFGF, Regulatory Peptides, 10, 309-317 (1985)] and pep 2: Leu-Pro-Met-Ser-Ala- Lys-Ser (corresponding to amino acids 141 to 147, refer to European Patent Publication No.
  • the synthetic peptides, heparin sodium, human aFGF, rhbFGF and rhbFGF mutein CS23 were diluted with the buffer for dilution used in Example 2-(2) to a concentration of 100 ⁇ g/ml.
  • the synthetic peptides, heparin sodium, human aFGF, rhbFGF and rhbFGF mutein CS23 were used as the competitive substances, 100 ng/ml of the 3H3 antibody and the competitive substance were suspended, and the suspension was maintained at 37°C for 60 minutes.
  • the recognition site of the 3H3 antibody is considered to be the peptide of 10th to 141st amino acids of human bFGF molecule and binding site of human bFGF to human bFGF receptor or its adjacent site, other than the heparin combining site, or a region adjacent thereto.
  • Example 6 (Examination of Immuno-Neutralizing Action)
  • the human bFGF-immunoneutralizing activity against rhbFGF was studied by the MTT method of Example 2-(3) (Fig. 7). Namely, the activity was assayed by 3-day proliferation in the presence of 2 ng/ml of bFGF of the human umbilical vein endothelial (HUVE) cell.
  • the proliferation inhibitory effect is shown by the absorbance at OD-590 nm in case of adding the monoclonal antibodies where the value is estimated as 100% both in the presence of the bFGF and in the absence of the monoclonal antibody. Referring to Fig.
  • the assay was further changed to study the proliferation inhibition effect of the HUVE cells.
  • the HUVE cells were seeded on a 24-well Linbro plate in an amount of 1 X 10 cells/well (the culture conditions were37he same as with Example 2-(3)).
  • 2 ng/ml of the bFGF and the 3H3 antibody were added thereto. followed by cultivation for 3 days and for 5 days.
  • the number of cells was measured by using a Coulter counter (Coulter Electronics, Inc.) for each case.
  • Figs. 8(A) shows the results when cultivation was carried out for 3 days
  • Fig. 8(B) shows the results when cultivation was carried out for 5 days.
  • the number of cells when the 3H3 antibody was added was expressed in percentage, taking the number of cells when the bFGF was added and the 3H3 antibody was not added as 100%.
  • - • - and - jp - indicate the effect of the 3H3 antibody in the presence of 2 ng/ml of the bFGF and in the absence of the bFGF, respectively.
  • the IC50 value of the proliferation hindrance shows 13.2 and 7.6 ng/ml in Fig. 8(A) and Fig. 8(B), respectively.
  • the 3H3 antibody did not affect the number of cells in the absence of the bFGF. It is clear from the above data that the 3H3 antibody has strong immuno-neutralizing action to the biological activity of the bFGF.
  • Example 7 (Preparation of Horseradish Peroxidase-Labeled 3H3 Antibody)
  • the purified 3H3 antibody (7 mg/ml) was dialyzed against 0.1 M acetate buffer (pH 4.5) containing 0.1 M NaCl at 4 C for 20 hours, followed by addition of pepsin (0.1 mg) (Sigma, U.S.A.). Then, digestion was carried out at 37°C for 8 hours. The solution was adjusted to pH 8 with 1 M Tris to terminate the reaction. The resulting solution was placed on a column of Ultrogel AcA44 (IBF, France) and eluted with 0.02 M borate buffer (pH 8.0) containing 0.15 M NaCl to obtain F(ab') 2 .
  • the solution containing F(ab') 2 was concentrated to 1 ml. Then, the concentrated solution was dialyzed against 0.1 M phosphate buffer (pH 6.0) at 4 C for 20 hours, and 0.1 ml of a solution [0.2 M mercaptoethyl- amine, 5 mM EDTA, 0.1 M phosphate buffer (pH 6.0)] was added, followed by reduction at 37°C for 90 minutes. The reaction solution was placed on a Sephadex G-25 fine column (1 cm diameter X 60 cm, Pharmacia Fine Chemical, Sweden) and eluted with an eluent [5 mM EDTA, 0.1 M phosphate buffer (pH 6.0)] to obtain an Fab' fraction.
  • HRP horseradish peroxidase
  • GMBS N-( ⁇ -maleimidobutyloxy)succinimide
  • DMF N,N-dimethylformamide
  • the solution of GMBS in DMF was added to the HRP solution, followed by stirring at 30 C for 60 minutes. Then, the resulting solution was placed on the Sephadex G-25 fine column (1.2 cm diameter X 60 cm) and eluted with 0.1 M phosphate buffer (pH 7.0) to obtain maleimide group- introduced HRP (maleimidated HRP).
  • MAbl2 or a 50-50 mixture of MAb52 and MAb98 was diluted with 0.1 M carbonate buffer (pH 9.6) to a concentration of 10 ⁇ g/ml. The resulting solution was poured in an amount of
  • Buffer B 0.02 M phosphate buffer (pH 7.0) containing 25% Blockace (blocking agent prepared from milk protein) (Dainippon Pharmaceutical), 0.15 M NaCl],
  • Fig. 9 shows the relationship between the concentration of the hbFGF and the absorbance. Referring to Fig. 9, - o - and - • - indicate the relationship between
  • the amount of the bFGF in the test samples was assayed - 42 -
  • rhbFGF Recombinant human bFGF
  • Buffer B 0.02 M phosphate buffer (pH 7.0) containing 25% Blockace, 0.15 M NaCl
  • Buffer D used for dissolution of o-phenylenediamine [0.1 M citrate buffer (pH 5.5) containing 0.02% hydrogenperoxide and 0.005% thimerosal],
  • Example 10 Influence of Heparin in hbFGF Assay System
  • heparin was added to buffer B to a concentration of 0, 1, 10 or 100 ⁇ g/ml, when the standard hbFGF was diluted.
  • the concentration of the hbFGF was assayed in the manner of Example 4-(l).
  • Figs. 10(1) and 10(2) there were observed no considerable changes in the standard curves with the presence of heparin, as shown in Figs. 10(1) and 10(2).
  • Fig. 10(1) indicates the results when MAbl2 was fixed
  • Fig. 10(2) indicates the results when the 50-50 mixture of MAb52 and MAb98 was fixed.
  • - • -, - o -, - ⁇ - and - ⁇ - indicate the results when heparin concentrations are 0, 1, 10 and 100 ⁇ g/ml, respectively.
  • Example 11 Reactivity of Acid-Modified hbFGF in hbFGF Assay
  • Example 11(2) indicates the results when the 50-50 mixture of MAb52 and MAb98 was solidified. Further in each figure, - o -, - • -, - ⁇ - and - - indicate the results when the hbFGF was incubated at pH 4 for 0, 1, 3 and 10 minutes, respectively. Figs. 11(1) and 11(2) show that the hbFGF modified at pH 4 is reduced in reactivity to about 1% by incubation for 10 minutes in each assay system.
  • the assay systems of Example 9 are assay systems by which only the native hbFGF can be assayed.
  • Example 12 (Assay of bFGF in Various Cells)
  • A375 human melanoma
  • A431 human squamous cell carcinoma
  • A549 human lung cancer
  • SK-Hepl human hepatic cancer
  • Table 3 A shows the results when MAbl2 was fixed, and B shows the results when the 50-50 mixture of MAb52 and MAb98 was fixed. Table 3 Amount of bFGF in Cell
  • the K1000-F1 cells obtained in Reference Example 2 were - 46 -
  • Fig. 12 (major axis) X (minor axis) 2] was calculated. The results are shown in Fig. 12. Referring to Fig. 12, - • -, - Q - and - J - indicate the results for an untreated control group, a nonimmune mouse IgG-given group and a 3H3 antibody-given group, respectively.
  • the 3H3 antibody exhibited antitumor effect against the KlOOO tumor (14 days after implantation, the 3H3 antibody-given group is decreased in tumor volume to 34% of that of the untreated control group) . The antitumor effect of nonimmune mouse IgG was not observed.

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