WO1991009875A1

WO1991009875A1 - Monoclonal antibody, hybridomas, their production and use

Info

Publication number: WO1991009875A1
Application number: PCT/JP1990/001726
Authority: WO
Inventors: Yoshio Kozai; Akira Hori; Yuzo Ichimori
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 1989-12-28
Filing date: 1990-12-27
Publication date: 1991-07-11
Also published as: EP0507948A1; JPH05503842A; CA2069872A1

Abstract

Disclosed are (1) a monoclonal antibody which immuno-neutralizes the activity of a bFGF protein and highly sensitively combines with the bFGF protein; (2) a cloned hybridoma for (1); (3) a method for producing the cloned hybridoma (2); (4) a method for producing the monoclonal (1); (5) a method for purifying the bFGF protein, which comprises using the monoclonal antibody (1); and (6) a method for detecting or determining the bFGF protein, which comprises using the monoclonal antibody (1).

Description

DESCRIPTION MONOCLONAL ANTIBODY, HYBRIDOMAS, THEIR PRODUCTION AND USE

Technical Field 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. Background Art 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. For example, 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. In addition, there is the potential that bFGF itself may have application as a therapeutic drug for traumas and burns. In this case, the determination of fundamental information regarding bFGF is essential for the development of bFGF as a medicine. Also in the diseases in which bFGF is invollved, such as cancer, the diagnosis of the disease becomes possible by tracing the blood bFGF concentrations. However, since 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.

Summary of the Invention In view of the above-mentioned circumstances, the present inventors prepared monoclonal antibodies which highly sensitive determination of bFGF proteins, as well as immuno-neutralize the activity of the bFGF. The present invention conducted further researches based thereon, thus completing the present invention.

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:

(a) it has a molecular weight of about 140,000 to 160,000,

(b) it does not cross-react with acidic fibroblast growth factor,

(c) it belongs to immunoglobulin class IgG,,

(d) it binds with rhbFGF mutein CS23 (a mutein in which the cysteine residues at positions 70 and 88 of hbFGF are substituted for serine residues), (e) it completely inhibits proliferation of a human unbilical vein endothelial (HUVE) cell by addition of 50 ng/ml thereof in the presence of 2 ng/ml of bFGF, and

(f) it can detect 20 pg/ml of the bFGF protein by a sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody MoAbl2 (solid phase) and a peroxidase- labeled antibody;

(2) a cloned hybridoma derived from a mammalian spleen cell 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;

(3) 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; (4) a method for producing the monoclonal antibody described in the above item (1), which comprises culturing a cloned hybridoma comprising a spleen cell from a mammal and a homogenic or heterogenic lymphoid cell in a liquid culture medium or in a peritoneal cavity of the mammal to produce the monoclonal antibody, said mammal being immunized with a mutein in which at least one cysteine residue of bFGF is substituted for a serine residue, and recovering the monoclonal antibody; (5) a method for purifying a bFGF protein, which comprises contacting the monoclonal antibody described in the above item (1) with a sample containing bFGF protein; and

(6) a method for detecting or measuring a bFGF protein, which comprises using the monoclonal antibody described in the above item (1) .

Brief Description of Drawings 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; and

Fig. 13 is a schematic representation showing the construction of plasmid pTBlOOO obtained in Reference Example 2. Detailed Description of the Invention 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.

As the bFGF proteins, there are preferably used polypeptides containing the amino acid sequence represented by formula (I) :

Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val- Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro (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.

Examples of amino acid sequences of the bFGFs include the amino acid sequence represented by formula (II):

Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser- Gly-Ala-Phe-Pro-Pro-Gly-His-Phe-Lys-Asp- Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly- Phe-Phe-lTeu-Arg-Ile-His-Pro-Asp-Gly-Arg- Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-

His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu- Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Cys- Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp- Gly-Arg-Leu-Leu-Ala-Ser-Lys-Cys-Val-Thr- Asp-Glu-Cys-Phe-Phe-Phe-Glu-Arg-Leu-Glu- Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg- Lys-Tyr-X -Ser-Trp-Tyr-Val-Ala-Leu-Lys- Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Y -Lys- Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe- Leu-Pro-Met-Ser-Ala-Lys-Ser (II) wherein X represents Thr or Ser, and Y represents Ser when X is Thr, and Y represents Pro when X is Ser.

As the bFGF, 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.

In particular, there is preferably used recombinant human bFGF mutein CS23 (hereinafter also briefly referred to as rhbFGF mutein CS23) in which each of the cysteine residues at positions 70 and 88 of the human bFGF is substituted for a serine residue. With respect to the number of the positions of the above amino acids, in the amino acid sequence in which Met is added to the N-terminus of the peptide in which X is Thr and Y is Ser in the above formula (II), the Met is numbered as the first. In the bFGFs produced by the above-mentioned genetic engineering techniques, 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.

When mammals are immunized with the bFGF proteins or protein conjugates, there are used experimental animals such as sheep, goat, rabbits, guinea pigs, rats and mice, as the mammals to be immunized. In order to obtain monoclonal antibodies, however, it is preferred to use rats or mice for immunization.

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. As the immunizing dose, it is preferred to use about 0.1 μg or more, preferably about 10 to 300 μg 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.

The above spleen cells are then fused with lymphoid cells. For example, the spleen cells removed from the mice are fused with 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~). For example, begin a new paragraph the fused cells are produced in accordance with the method of Kohler and Milstein [Nature 256, 495 (1975)]. For example, 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. It is of course possible to add other fusion accelerators such as dimethyl sulfoxide (DMSO). 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%. As a preferred condition, 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)].

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. After the reaction product is thoroughly washed, an enzyme-labeled peptide (a peptide is coupled with an enzyme according to conventional methods, followed by purification) is added thereto, followed by 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. As to the culture supernatant of the proliferated cells, the screening of the neutralizing activity to the bFGF proteins can be carried out in the following manner. In this case, the neutralizing activity can be examined using cells whose proliferation is induced by the bFGF proteins. As such 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). As one example of the preferred assays, there is hereinafter described a method for assaying cell proliferation by the MTT method using human umbilical vein- derived endothelial cells. After the cells are seeded, 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.

After a specified time has elapsed, SDS is added thereto to dissolve the cells, and 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.

It is desirable that 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. Specifically, for example, 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. In the case of a mouse, for example, about 1 X 10⁴ to 1 X 10⁷, preferably 5 X 10⁵ to 2 X 10⁶ 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. As diseases induced by overproduction of bFGF, tumors are mentioned. For the tumors, there are the case that 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. In these cases, 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.

Also in treating traumas and burns, bFGF is considered to improve the symptoms thereof effectively. In this case, it is more effective to use a mutein in which at least one cysteine residue of the bFGF is substituted for a serine residue and which is higher in stability than the bFGF. When the mutein is used as a therapeutic drug, 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.

As described above, many tumors are considered to be proliferated by bFGF in bodies. The monoclonal antibodies of the present invention inactivate the bFGF in vivo and exhibit antitumor activity, because of their strong immuno-neutralizing activity. In this case, the monoclonal antibodies are administered in an amount of about 100 μg/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 particles) and plates for immunoassay (for example, Nunc, Denmark); and ion exchange resins such as weakly acidic cation exchange resins [for example, Amberlite IRC- 50 (Rohm & Haas, U.S.A.) and Zeocurve 226 (Permutit, west Germany)], and weakly basic anion exchange resins [for example, Amberlite IR-4B and Dowex (Dow Chemical, U.S.A.)]. 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. As 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. In particular, 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. In particular, horseradish peroxidase (HRP) extracted from horseradishes is preferable.

In combining peroxidase with the antibody, the thiol group of Fab¹ as the antibody molecule is utilized, and peroxidase into which a maleimide group is preliminarily introduced is conveniently used.

When a maleimide group is introduced into peroxidase, it can be introduced through an amino group of peroxidase. For this purpose, N-succinimidyl-maleimide-carboxylate derivatives can be used. N-(γ-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.

As an example of the assays of the present invention, a case is hereinafter described in detail in which peroxidase is used as the labeling agent, but the present invention is not limited to peroxidase.

(1) First, a 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.

(2) The substrate of the peroxidase is added to the reaction product obtained in (1), and then the absorbance or the fluorescent intensity of the resulting substance is measured, thereby knowing the enzyme activity of the above reaction product.

(3) The procedures described in (1) and (2) are preliminarily carried out for the standard solution of the bFGF protein of a known amount to prepare a standard curve showing the relation between the amount of the bFGF protein and the absorbance or the fluorescent intensity thereof.

(4) The absorbance or the fluorescent intensity obtained for the test sample containing the bFGF protein of an unknown amount is applied to the standard curve to determine the amount of the bFGF protein in the test sample.

In order to purify the bFGF protein, 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. Thus, the bFGF protein can be efficiently purified. 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. Examples of such carriers include agarose gels, cellulose and acrylamide polymers. By way of example, 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. With respect to the mixing ratio of the antibody to Affi-Gel 10, 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. However, 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. The combined material thus treated is packed in an appropriate column to form the antibody column.

In purifying a sample with the above antibody column, 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. As eluents, 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

10. combined solutions. Solutions which do not so promote the degradation of the bFGF protein are preferred.

Eluents are neutralized with buffer solutions by conventional methods. The above purification procedure can be repeated again as needed. 5 Thus, 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

European Patent Publication No. 288,687 the disclosures of which are herein incorporated by reference. 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.

Recombinant human bFGF (rhbFGF) which was produced by the methods described in Iwane et al., Biophys. Biochem.

Res. Commun. 146, 470 (1987) and European Patent Publication No. 237,966 was used. rhbFGF mutein CS23 which was produced using transformant Escherichia coli MM294/pTB762 (IFO 14613, FERM BP-1645) by the methods described in Sano et al., Biophys. Biochem. Res. Commun. 151, 701 (1988) and European Patent Publication No. 281,822 was used. The above E. coli MM294/pTB762 was deposited with IFO under the accession number IFO 14613 on May 27, 1987. This transformant was also deposited with FRI under the accession number FERM P-9409 on June 11, 1987. This deposit was converted to the deposit under the Budapest Treaty and the transformant is stored at the FRI under the accession number FERM BP-1645.

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.

The following examples are given to illustrate embodiments of the present invention as it is presently preferred to practice. It will be understood that the examples are illustrative, and that the invention is not to be considered as restricted except as indicated in the appended claims.

Examples Reference Example 1 (Preparation of Recombinant Human aFGF) Human aFGF was produced by the following method, with reference to the methods described in Biotechnology, 5_, 960 (1987) and ICUS Short Report, vol. 8, Advances in Gene Technology; Protein Engineering and Production, Proceedings of the 1988 Miami Bio/Technology Winter Symposium, page 110, IR Press.

(a) Construction of Expression Plasmid

Plasmid pTB917 obtained by incorporating chemically synthesized human aFGF DNA (Fig. 1) into pUC18 [Methods in Enzymology 101, 20-78 (1983)] was digested with BspMI, and the cleaved sites were changed to flush ends by reaction with DNA polymerase large fragment, followed by digestion with BamHI to produce a 0.45-kb DNA fragment.

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. Then, the above 0.45-kb blunt BspMI-BamHI fragment was incorporated thereinto with T4 DNA ligase to obtain pTB975 (Fig. 2). (b) Expression of Human aFGF cDNA in E. coli IPhage DE3 [F. W. Studier et al., J. Mol. Biol. 189, 113-130 (1986)] in which an RNA polymerase gene of the T7 phage was incorporated into E_^ coli strain MM294 was lysogenized, and plasmid pLysS [F. W. Studier et al., J. Mol. Biol. 189, 113-130 (1986)] having a lysozyme gene of the T7 phage was further introduced thereinto to prepare E. coli MM294(DE3)/pLysS. This strain was transformed with _PTB975 to give E. coli MM294(DE3)/pLysS, pTB975(IFO 14936, FERM BP-2599) .

This strain was cultivated in a medium containing 35 μg/ml of ampicillin and 10 μg/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.

(c) Purification of Human aFGF The cells collected from a one liter culture were suspended in 100 ml of an ice-cooled solution [10 mM Tris-HCl (pH 7.4), 10 mM EDTA, 0.6 M NaCl, 10% sucrose, 0.25 mM PMSF], and egg white lysozyme was added thereto to a concentration of 0.5 mg/ml. The mixture was allowed to stand in ice for 1 hour, followed by incubation at 37 C for 5 minutes. Then, the product was subjected to ultrasonication twice under ice cooling for 20 seconds, and then centrifuged at 18 Krpm at 4°C for 30 minutes (Sorvall) to obtain a supernatant. 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 (NH₄)₂S0₄] (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.

(d) Reverse-Phase C4 HPLC

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) .

(e) Biological Activity

The activity of the human aFGF was determined by

3 assaying the incorporation of [ H] thymidme as an indication of the-DNA synthesis induction of mouse BALB/c3T3 cells (ATCC No. CRL 6587) according to the method of Sasada et al. [Sasada et al., Mol. Cell. Biol. 8, 588-594 (1988)].

When the test sample was added, a heparin (Sigma, Grade I) - 27 -

solution was incorporated in the medium and the test sample as required.

Reference Example 2

(a) Construction of Plasmid pTBlOOO for Expression of Human bFGF Mutein

Plasmid pTB762, obtainable from E. coli MM294(DE3)/

Lys,pTB672 (IFO 14936, FERM BP-2599), and described in

Japanese Patent Unexamined Publication (Laid-Open) No.

2-193/1990 (corresponding to European Patent Publication No.

281,822) was cleaved with restriction enzymes EcoRI and

BamHI to obtain a 0.38-kb DNA fragment containing a region coding for human bFGF mutein CS23. Additionally, plasmid pTB503 described in Japanese Patent Unexamined Publication

(Laid-Open) No. 62- 175182/1987 (corresponding to EP-225,701) was cleaved with Clal and EcoRI to obtain a 1.7- kb DNA fragment containing an LTR of a mouse leukemia virus

(MuLV) region, an SV40-derived promoter and a splicing region and a leader sequence of human interleukin 2. Further, plasmid pTB675 described in Japanese Patent Unexamined Publication (Laid-Open) No. 2-193/1990

(corresponding to EP-281,822) was cleaved with Clal and BamHI to obtain a 3.4-kb DNA fragment containing a region coding for the C-terminal side of the human bFGF, a 3'-untranslated region thereof, a plasmid pBR322-derived ampicillin-resistant gene and a replicator in EL_ coli.

These three DNA fragments were ligated using T4 DNA ligase to obtain plasmid pTBlOOO (Fig. 13). - -

(b) Transformation of Mouse BALB/c3T3 Cell and Establishment of Transformed Cell

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

[Dulbecco et al. Virology, , 396 (1959)] containing 10% calf serum. The next day, the medium was exchanged for the same medium. After 4 hours, 10 μg or 1 μg of plasmid pTBlOOO obtained in the above (1) was transfected by the calcium phosphate method [Graham et al.. Virology, 52, 456 (1973)]. After transfection, cultivation was continued in DMEM medium containing 5% calf serum. After about 3 weeks, the formed focuses (focus forming frequency: 10 to 20/μg DNA) were transferred into a new dish to cultivate them, and cloning was further conducted by the limiting dilution method. For the three transformed cell clone strains K1000-F1, K1000-F2 and K1000-3 thus obtained, the FGF activity of the culture solution and a cell extract was assayed as DNA synthesis-promoting action to the A31 cells in a still state. The results are shown in the following table.

FGF activity (ng FGF equivalent/dish) Cell Culture solution Cell extract

K1000-F1 θ7θ §76

K1000-F2 0.16 11.3 K1000-F3 0.09 4.5

A31 < 0.05 0.2

Each of these transformed cells showed the form of a malignant cell gland and formed colonies on a soft agar plate.

Example 1 (Immunization)

BALB/c mice (female, 8 weeks old) were intraperitoneally injected with 50 μg 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 μg of antigen rhbFGF mutein CS23 dissolved in 0.4 ml of Freund's complete adjuvant. Further 2 weeks later, the mice were additionally immunized with 50 μg of antigen rhbFGF mutein CS23 dissolved in 0.4 ml of Freund's incomplete adjuvant. Two weeks after the additional immunization, 50 μg of rhbFGF mutein CS23 dissolved in physiological saline was inoculated into the caudal veins of the mice.

Example 2

(1) Cell Fusion Three days after the final inoculation, the spleens were removed from the mice immunized in Example 1 to obtain cells to be used for cell fusion. These cells were suspended in IH medium.

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.

Cell fusion was carried out in accordance with the method established by Kohler and Milstein [G. Kohler and C.

Milstein, Nature, 256, 495 (1975)]. The above myeloma cells

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

6000) in IH medium was dropwise added thereto. The PEG 6000 solution was preheated to 37°C and slowly added for 1 minute. Then, 1 ml of IH medium was added for 1 minute, 1 ml for 1 minute and 8 ml for 3 minutes. The solution was thereafter centrifuged at room temperature at 1,000 rpm for 5 minutes to remove a supernatant. The resulting cell precipitate was suspended in 30 ml of IH medium containing 20% calf serum. The suspension was seeded on a 96-well microtiter plate (Nunc) in an amount of 100 μl/well. One day later, IH medium (containing 20% calf serum) supplemented with HAT (1 X 10 -4 M hepoxanthine, 4 X 10-7 M am opterin, 1.6 X 10 —5 M thymidine) was added to the microtiter plate in an amount of 100 μl/well. The IH medium supplemented with HAT is hereinafter referred to as HAT medium. Further every 3 days, one-half the amount of the medium was exchanged for HAT medium. The cells which thus grew were hybrid cells. (2) Screening of Antibody-Producing Cells

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 μl/well to a 96- well polystyrene microtiter plate (Nunc) . After 2 hours, the microtiter plate was washed with a rinsing liquid (0.05% Tween 20, physiological phosphate buffer), and then 100 μl of the combined solution of 50 μl of the culture supernatant and 50 μl 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. After 2 hours, the second antibody was washed with a rinsing liquid, and then coloring reaction was conducted by adding a reaction substrate (ELISA method) . By this method, the rhbFGF mutein CS23 combining activity was observed in 4 wells.

(3) Screening of anti-bFGF Immuno-Neutralizing Antibody-Producing Cells Human umbilical vein-derived vascular endothelial cells was suspended in GIT medium (commercially available from Wako Pure Chemical Industries, Ltd. Japan; a mammalian serum-derived composition for animal cell cultivation, produced by subjecting mammalian serum to purifying treatment including an inactivation process for contaminant microorganisms and a salting-out/desalting process; cf. U.S. Patent No. 4,654,304) containing 2.5% fetal calf serum. and the suspension was seeded in an amount of 100 μl/well (2,000 cells/well) to a 96-well microtiter plate. The next day, 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 μl/well, and cultivation was conducted at 37°C under an atmosphere of 5% C0 and 7% 0„ 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 μl/well. After cultivation was carried out at 37 C under an atmosphere of 5% C0 and 7% 0 for 4 hours, 10% sodium dodecyl sulfate (SDS) was added in an amount of 100 μl/ml. After 4 hours, the absorbance at 590 n was measured with a spectro- photometer for 96 wells (Titertek) (MTT method) . By this method, the strong neutralizing activity was observed in one well.

(4) Cloning of 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 -

Example 3 (Immunoglobulin Class of Monoclonal Antibody)

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.

Table 1

Immunoglobulin sample 3H3 antibody

IgG₁

IgM

IgA

In Table 1, "+" indicates that the reaction is positive, and "-" indicates that the reaction is negative. Table 1 shows that the antibody in the culture supernatant of 3H3 cells belongs to IgG, in the immunoglobulin class. Example 4 (Purification of Monoclonal Antibody from

Culture Supernatant and Ascites) (1) Purification from Culture Supernatant The combined solution of the culture supernatant of mouse 3H3 cells and a combining buffer [3 M sodium chloride, 1.5 H glycine (pH 8.7)] in a 1:1 ratio was loaded onto a Protein A column. After washing with a combining buffer, elution was carried out with an elution buffer [0.1 M citric acid (pH 5)]. To the eluate was added 1 M Tris (pH 8.0) to neutralize it, followed by dialysis against physiological phosphate buffer.

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 μg/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 μg/ml of the rabbit anti-bFGF polyclonal antibody was fixed and rhbFGF mutein CS23 was added thereto. Then, 1 μg/ml of the 3H3 antibody was added and 1 μg/ml of the alkaline phosphatase-labeled anti-mouse IgG goat antibody was further added thereto. By this method, rhbFGF mutein CS23 could be detected up to 3 ng/ml. (2) Purification from Ascites

Further, 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. The dialyzed solution was applied to a DE-50 column (1 cm diameter X 60 cm, Whatman, Great Britain), and the column was eluted with a linear gradient of 0.1 to 0.35 M NaCl in 0.1 M phosphate buffer (pH 8.0), whereby 7 mg of monoclonal antibody 3H3 was obtained from 5 ml of the ascites. Example 5 (Determination of Antigen Recognition Site)

The antigen recognition site of the 3H3 antibody whose antibody titer was measured in Example 4 was examined by competitive binding inhibition experiments. As 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. 288,687) and heparin sodium. 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 μg/ml. When 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 amount of antibody not combined in this solution was assayed by the EIA shown in Example 2-(2). The results are shown in Table 2.

Table 2

3H3 antibody

Human bFGF +

Human aFGF

CS23 pepl pep2

Heparin sodium

In Table 2, "+" indicates that the competitive inhibition occurred, and "-" indicates that the competitive inhibition did not occur. From these results, 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) As to the 3H3 antibody purified by the method of Example 4(1), 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. 7, - • - indicates the results of the 3H3 antibody, and - J - indicates the results of normal mouse IgG. The line of 44% indicates the number of cells when the bFGF was not added. When the 3H3 antibody was added, 50 ng/ml thereof hindered the proliferation of the HUVE cells to the number of cells in the absence of the bFGF. The normal mouse IgG did not exhibit the proliferation inhibitory effect.

The assay was further changed to study the proliferation inhibition effect of the HUVE cells. Namely, 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)). The next day, 2 ng/ml of the bFGF and the 3H3 antibody were added thereto. followed by cultivation for 3 days and for 5 days. Then, the number of cells was measured by using a Coulter counter (Coulter Electronics, Inc.) for each case. Referring to Figs. 8(A) and 8(B), Fig. 8(A) shows the results when cultivation was carried out for 3 days, and 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%. In both of Fig. 8(A) and Fig. 8(B), - • - 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 (the amount of the antibody which hinders 50% of cell proliferation when the antibody is not added) shows 13.2 and 7.6 ng/ml in Fig. 8(A) and Fig. 8(B), respectively. Further, 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.

Additionally, 10 mg of horseradish peroxidase (HRP, Behringer Manheim, West Germany) was dissolved in 1.5 ml of 0.1 M phosphate buffer (pH 7.0), and 3.5 mg of N-(γ-maleimidobutyloxy)succinimide (GMBS) was dissolved in 100 μl of N,N-dimethylformamide (DMF). 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). Fab' and maleimidated HRP were mixed with each other to a mol ratio of 1:1, followed by reaction at 4°C for 20 hours. The reaction solution was applied to an Ultrogel AcA44 column and eluted with 0.1 M phosphate buffer (pH 7.0) to - 40 -

obtain an enzyme- labeled antibody (3H3-HRP) .

Example 8 (preparation of Antibody-Sensitized Plate)

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 μg/ml. The resulting solution was poured in an amount of

100 μl/well into an immunoplate for EIA (Maxisoap: Nunc,

Denmark) , followed by standing at 4 C overnight to sensitize or fix the antibody to the plate. After washing the plate with 0.01 M phosphate buffer (pH 7.0) containing 0.15 M NaCl, 0.01 M phosphate buffer (pH 7.0) containing 0.1% BSA was poured into each well. Then, the plate was stored in a chilled place until its use.

Example 9 (Assay of Human bFGF) 1. Preparation of Calibration Curve A. Reagents

(1) Enzyme-labeled antibody obtained in Example 7,

(2) Antibody-sensitized microtiter plate obtained in Example 8, (3) Recombinant human bFGF (rhbFGF): 0-50 ng/ml,

(4) Buffer A[0.02 M phosphate buffer (pH 7.0) containing 0.15 M NaCl],

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],

(5) Peroxidase substrate solution [sodium citrate buffer (pH 5.5) containing 0.02% hydrogen peroxide and 0.15% - 41 -

o-phenylenediamine] ,

Enzyme reaction-terminating solution(2N sulfuric acid). B. Assay

Into each well of the antibody-sensitized plate 5 obtained in Example 8, 100 μl of a solution of the bFGF (0.1 to lOOOpg/ml) in buffer B was added, followed by reaction at 4°C for 24 hours. After washing each well with buffer A, 100 μl of an enzyme- labeled antibody solution diluted 200 times with buffer B was added to each well, followed by

10 further reaction at 25°C for 2 hours. Each well was washed with buffer A, and 100 μl of the peroxidase substrate solution was added thereto, followed by reaction at 25 C for 30 minutes. Then, 100 μl of the enzyme reaction-terminating solution was added thereto to terminate reaction. The

15 absorbance at 492 nm was thereafter measured by using an automatic colorimeter for microtiter plates (MTP-32. Corona). Fig. 9 shows the relationship between the concentration of the hbFGF and the absorbance. Referring to Fig. 9, - o - and - • - indicate the relationship between

20 the concentration of the hbFGF and the absorbance when the microtiter plate sensitized with MAbl2 was used and when the microtiter plate sensitized with the 50-50 mixture of MAb52 and MAb98 was used, respectively. It was revealed that 20 pg/ml of the bFGF protein could be detected, when the plate

25 was sensitized with MAbl2.

2. bFGF Immunoassay Kit and Assay of bFGF

The amount of the bFGF in the test samples was assayed - 42 -

by using the following bFGF immunoassay kit according to the following procedure: A. Reagents

(1) Enzyme-labeled antibody (3H3 antibody-HRP) obtained in Example 7,

(2) Antibody-sensitized microtiter plate obtained in Example 8,

(3) Recombinant human bFGF (rhbFGF): 0-50 ng/ml,

(4) Buffer A [0.02 M phosphate buffer (pH 7.0) containing 0.15 M NaCl],

Buffer B[0.02 M phosphate buffer (pH 7.0) containing 25% Blockace, 0.15 M NaCl],

(5) o-Phenylenediamine,

(6) Buffer D used for dissolution of o-phenylenediamine [0.1 M citrate buffer (pH 5.5) containing 0.02% hydrogenperoxide and 0.005% thimerosal],

(7) Enzyme reaction-terminating solution (2N sulfuric acid) ,

B. Assay Each well of the antibody-sensitized plate obtained in Example 8 was washed with buffer A, and 100 μl of a standard solution of the bFGF in buffer B or a solution of the test sample diluted with buffer B was poured thereinto, followed by reaction at 4 C for 24 hours. After washing each well with buffer A, 100 μl of an enzyme-labeled antibody solution diluted 200 times with buffer B was added to each well, followed by further reaction at 25°C for 2 hours. After - 43 -

washing each well with buffer A, 100 μl of a 0.15% solution of o-phenylenediamine in buffer D was added thereto, followed by reaction at 25°C for 30 minutes. Then, 100 μl of the enzyme reaction-terminating solution was added thereto to terminate reaction. The absorbance at 492 nm was thereafter measured by using an automatic colorimeter for microtiter plate (MTP-32. Corona). The calibration curve of the standard bFGF was prepared, and the bFGF concentration was determined from the absorbance obtained for the test samples.

Example 10 (Influence of Heparin in hbFGF Assay System) In Example 9-1, heparin was added to buffer B to a concentration of 0, 1, 10 or 100 μg/ml, when the standard hbFGF was diluted. Then, the concentration of the hbFGF was assayed in the manner of Example 4-(l). As a result, there were observed no considerable changes in the standard curves with the presence of heparin, as shown in Figs. 10(1) and 10(2).

Referring to Figs. 10(1) and 10(2), Fig. 10(1) indicates the results when MAbl2 was fixed, and Fig. 10(2) indicates the results when the 50-50 mixture of MAb52 and MAb98 was fixed. Further in each figure, - • -, - o -, - ^ - and - Δ - indicate the results when heparin concentrations are 0, 1, 10 and 100 μg/ml, respectively. Example 11 (Reactivity of Acid-Modified hbFGF in hbFGF Assay

Systems) A 20 μl solution of the hbFGF (in 20 mM Tris-HCl buffer - -

(pH 7.4) containing hbFGF at a concentration of 200μg/ml and containing 1 M NaCl was added in an amount of 20 μl to 180 μl of 1 M acetate buffer, followed by incubation at 25 C for 0, 1, 3 or 10 minutes. 400 μl of 1 M Tris was added thereto to neutralize it, and then the resulting solution was diluted with buffer B used in Example 9-1. Then, ^■ calibration curves were prepared for respective incubation times according to the method of Example 9-1, as shown in Figs. 11(1) and 11(2). Referring to Figs. 11(1) and 11(2), Fig. 11(1) indicates the results when MAbl2 was solidified, and Fig. 11(2) indicates the results when the 50-50 mixture of MAb52 and MAb98 was solidified. Further in each figure, - o -, - • -, - Λ - 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) (all of them were supplied by ATCC, U.S.A.) and human umbilical vein endothelial cells were each scraped in

•η an amount of 2 to 6 X 10 cells by a cell scraper. The cells were washed twice with a phosphate buffered saline (PBS, pH 7.2) containing 0.15 M NaCl, and then 0.6 ml of PBS - 45 -

was added thereto, followed by ultrasonication under ice cooling for 25 seconds. After centrifugation at 15,000 rpm, the concentration of the bFGF in the supernatant was determined according to the method of Example 9-2.

The results are shown in Table 3. In 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

Amount of bFGF

A B

Name of Cell (pg/10⁶ cells) (ng/10⁶ cells)

A375 1.4 4.0 A431 1.5 0.5 A549 8.4 8.5 SK-Hepl 4.0 11.7 HUVE 11.7 0.84

From Table 3, it is observed that the difference in measured amount of bFGF between the A assay system and the B assay system is great. From this fact, the possibility is considered that the N-terminus of the bFGF is cleaved during extraction operation, or that the N-terminus of the actually produced bFGF is cleaved or masked.

Example 13 (Antitumor Effect to K1000 Tumor)

The K1000-F1 cells obtained in Reference Example 2 were - 46 -

subcutaneously implanted in an amount of 3 X 10 cells/mouse into nude BALB/c mice. Three days after implantation, the 3H3 antibody obtained in Example 4 and nonimmune mouse IgG were given to the caudal veins in a dose of 200 μg/mouse/day continuously for 5 days. After implantation, the diameter of the tumors was measured, and the volume thereof [=0.5 X

(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.

Incorporated by reference

Nature 249, 123 (1974)

Biophys. Res. Commun. 151, 701 (1988)

European Patent Publication No. 281,822

FEBS Letters ^ _., 189 (1987)

Biophys. Res. Commun. 146, 470 (1987)

European Patent Publication No. 237,966

J. Immun. Method 8fJ, 55 (1985)

Nature 2!56_, 495 (1975)

J. Am. Med. Assoc. 192, 549 (1967)

Metabolism, , 696 (1971)

Hybridoma, £, 209-221 (1989)

European Patent Publication No. 288,687

Biotechnology, 5_, 960 (1987)

ICUS Short Report, vol. 8, Advances in Gene Technology;

Protein Engineering and Production, Proceedings of the

1988 Miami Bio/Technology Winter Symposium, page 110, IRL

Press J. Mol. Biol., 189., 113-130 (1986) Mol. Cell. Biol. Q , 588-594 (1988) Methods in Enzymology 101, 20-78 (1983) Japanese Patent Unexamined Publication (Laid-Open) No.

2-193/1990 (European Patent Publication No. 281,822) Japanese Patent Unexamined Publication (Laid-Open) No. 62-

175182/1987 (EP-225,701) Science, ^OjJ. 505-507 (1980) Virology, .8, 396 (1959) Virology, .52, 456 (1973) U.S.P. No. 4,654,304 Regulatory Peptides_, lfj, 309-317 (1985)

Claims

- 48 -CLAIMS WHAT IS CLAIMED IS:

1. A monoclonal antibody which has the following characteristics, immuno-neutralizes the activity of a basic fibroblast growth factor (bFGF) protein and highly sensitively combines with the bFGF protein:

(a) a molecular weight of about 140,000 to 160,000:

(b) does not cross-react with acidic fibroblast growth factor,

(c) belongs to immunoglobulin class IgG, ,

(d) binds with rhbFGF mutein CS23 (a mutein in which the cysteine residues at positions 70 and 88 of hbFGF are substituted for serine residues),

(e) it completely inhibits proliferation of a human umbilical vein endothelial (HUVE) cell by addition of 50 ng/ml thereof in the presence of 2 ng/ml of bFGF, and

(f) it can determine 20 pg/ml of the bFGF protein by a sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody MoAbl2 (solid phase) and a peroxidase- labeled antibody.

2. A monoclonal antibody in accordance with claim 1, in which said bFGF protein is a polypeptide containing the following amino acid sequence:

Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly- Val-Arg-Glu-Lys-Ser-Asp-Pro.

3. A cloned hybridoma derived from fusing a spleen cell of 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.

4. A hybridoma in accordance with claim 3, in which said mammal is a mouse.

5. A hybridoma in accordance with claim 3, in which said lymphoid cell is a myeloma cell.

6. A hybridoma in accordance with claim 3, in which said mutein is a polypeptide containing the following amino acid sequence:

7. A hybridoma according to claim 6 which has the characteristics of mouse 3H3 cell.

8. A method for producing a cloned hybridoma comprising 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, which comprises subjecting said spleen cell and said lymphoid cell to cell fusion, followed by cloning. - 50 -

9. A method in accordance with claim 8, in which said mammal is a mouse.

10. A method in accordance with claim 8, in which said lymphoid cell is a myeloma cell.

11. A method in accordance with claim 8, in which said mutein is a polypeptide containing the following amino acid sequence:

12. A method for producing the monoclonal antibody claimed in claim 1, which comprises culturing a cloned hybridoma comprising a spleen cell from a mammal said mammal being immunized with a mutein in which at least one cysteine residue of bFGF is substituted for a serine residue, and a homogenic or heterogenic lymphoid cell in a liquid culture medium or in a peritoneal cavity of the mammal under conditions suitable for antibody production, and recovering the antibody produced.

13. A method in accordance with claim 12, in which said mammal is a mouse.

14. A method in accordance with claim 12, in which said lymphoid cell is a myeloma cell.

15. A method in accordance with claim 12, in which said mutein is a polypeptide containing the following amino acid sequence:

16. A method for purifying a bFGF protein, which comprises using the monoclonal antibody claimed in claim 1.

17. A method for detecting or measuring a bFGF protein, which comprises using the monoclonal antibody claimed in claim 1.

18. A method in accordance with claim 17, in which said bFGF is mutein containing the following amino acid sequence:

19. A method in accordance with claim 17, in which the bFGF protein is detected or measured by an enzyme immunoassay.

20. A kit of reagents for detecting or measuring a bFGF protein which comprises the monoclonal antibody according to claim 1.

21. The kit of reagents according to claim 20, wherein the monoclonal antibody is produced by the cloned hybridoma according to any of claims 3-5.

22. The kit of reagents according to claim 21, wherein the bFGF protein is a mutein in which at least one cysteine residue of bFGF is substituted for a serine residue.

23. The kit. of reagents according to claim 21, which is used in an enzyme-linked immunosorbent assay.