USE OF AND PROCESS FOR PRODUCING A METASTASIS INHIBITOR AND FOR A CELL ADHESION INHIBITOR RESPECTIVELY CONTAINING AN ANTIBODY

Abstract: A monoclonal antibody against a peptide containing at least a peptide sequence of the N-domain of a human carcinoembryonic antigen glycoprotein and a partial or whole peptide sequence of the domain III of said glycoprotein, or a sequence homologous therewith; and a cell adhesion inhibitor containing the antibody as the active ingredient. The antibody inhibits the adhesion activity of cancer cells to thereby inhibit the metastasis of cancer cells, which has been difficult to inhibit effectively, and hence it is efficacious as cell adhesion inhibitor such as cancer metastasis inhibitor.

Description: Technical Field This invention relates to a monoclonal antibody against a peptide which impart adhesion activity to human cancer cells when expressed on cell surfaces and also to a cell adhesion inhibitor making use of the monoclonal antibody. Background Art Carcinoembryonic antigen (hereinafter abbreviated as "CEA") is a glycoprotein discovered in 1965 by Gold and Freedman as an antigen which is found in both human colon cancer and the digestive organs of a 2-6 months old human embryo and having a molecular weight of 180,000 to 200,000 [Gold, P. & Freedman, S.O., J. Exp. Med., 121 , 439 (1965)]. Its usefulness in cancer clinic has been widely recognized. It is a tumor marker which is used most often these days. Further, it became evident by subsequent research that substances similar to CEA, that is, CEA-related antigen also exists in normal human tissues. The term "CEA-related antigen" is a generic term for a group of antigens which are extremely similar to CEA in both proteinchemistry and immunochemistry. Known typical CEA-related antigens include a nonspecific cross-reacting antigen which is a glycoprotein found in the lungs and spleen of a normal human being and having a molecular weight of about 90,000 [hereinafter abbreviated as "NCA"; von Kleist, S. et al., Proc. Natl. Acad. Sci. USA, 69, 2492 (1972)], NCA-2 found in embryonic feces [Burtin, P. et al., J. Immunol., 111 , 1926 (1973)], and NFA (normal fecal antigen) found in normal adult feces [Kuroki, M. et al., Cancer Res. 41, 713 (1981)]. They are however still unknown in many aspects, including their detailed molecular structures and their differences in molecular structure. Keeping step with changes in the dietary of Japanese in recent years, colon cancer patients are increasing. About 80% of colon cancer is said to metastasize to the liver, and colon cancer is also said to metastasize to other organs such as the lungs. Recently, there has also been reported the possibility that the cell adhesion activity of CEA may take part in the metastasis of colon cancer to the liver [Jessup, J.M. et al., Cancer and Metastasis Reviews, 8 , 263 (1989)]. Using a molecular biological technique, the primary structure of peptide has recently been ascertained with respect to CEA [Oikawa, S. et al., B.B.R.C.(Biochem. Biophys. Res. Commun.), 142 , 511 (1987) and Japanese Patent Laid-Open No. 177794/1988]. NCA [Tawaragi, Y. et al., B.B.R.C., 150 , 89 (1988)], BGPI [Hinoda, Y. et al., Proc. Natl. Acad. Sci. USA., 85 , 6959 (1988)] and W272 (CGM6) [Arakawa, F. et al., B.B.R.C., 166 , 1063 (1990)]. According to the results of a cDNA analysis, CEA peptide is composed of 668 amino acids as shown, for example, in FIG. 3 of Japanese Patent Laid-Open No. 177794/1988. As is evident from the figure, CEA peptide can be divided into five domains, that is, domain N (1-108) extending from the N terminal to the 108th amino acid, domain I (109-286), domain II (287-464) and domain III (465-642) having mutually very homogeneous repetitive structures and composed individually of 178 amino acids, and domain M (643-668) composed of twenty-six, primarily hydrophobic amino acids on the side of the C terminal. Domains I, II and III may each be divided further into sub-domains (which may also be called "domains") which consist of 92 amino acid residues and 86 amino acid residues, respectively. These individual sub-domains may also be referred to as "1A", "1B", "2A", "2B", "3A" and "3B", respectively. Domain M was first considered to be anchored on a cell membrane. It is however known that, after translation, domain M is processed and PI-G (phosphatidylinositol glycan) is added instead and is anchored on a cell membrane [Hefta, S.A. et al., Proc. Natl. Acad. Sci. USA., 85 , 4648 (1988); Takami N. et al., J. Biol. Chem., 263 , 12716 (1988)]. All 12 cysteine residues are found in domains I, II and III, each domain having 4 residues at the same positions as others. Two loops are formed, one being located between the first cysteine and the second cysteine and consisting of 47 amino acids and the other between the third cysteine and the fourth cysteine and 39 amino acids, whereby CEA is considered, as a whole, to have a shape in which six loops are held. NCA, on the other hand, contains domain N consisting of 108 amino acids and domain I consisting of 178 amino acids. Portions corresponding to domains II and III of CEA are however not found and, in continuation with domain I, there is domain M composed of 24 amino acids which are primarily hydrophobic. The structure of domain I of NCA shows homology slightly lower than 90% to that of CEA in terms of amino acids and the positions of the four cysteine residues contained therein are exactly the same. BGPI is a CEA-related antigen which is found in bile [Svenberg, T. et al., Int. J. Cancer, 17 , 588 (1976)]. By a cDNA analysis, BGPI has been found to contain domain A', transmembrane domain and a cytoplasmic domain, which are characteristic to BGPI, after domain N and domain I. W272 is a CEA-related antigen isolated from human leukocytes (granulocytes) and has a domain structure similar to NCA. Biological activities of the CEA family have long remained unknown but, recently, CEA and NCA have been found to have cell adhesion activity [Oikawa, S. et al., B.B.R.C., 164, 39 (1989); Benchimol, S. et al., Cell, 57 , 327 (1989)]. Namely, it has been found that cells with both CEA and NCA expressed on their surfaces firmly adhere with each other and also that cells with CEA expressed thereon and those with NCA expressed thereon strongly adhere with each other. However, it has not been ascertained in which regions of CEA and NCA cell adhesion activity is present. This has remained as a bottleneck for the efficient production of a monoclonal anti-CEA antibody which inhibits the cell adhesion activity of CEA. Any monoclonal antibody against the whole CEA molecule unavoidably contains many antibodies which in turn recognize peptides as epitopes other than the region (domain) having the cell adhesion activity of CEA. To permit efficient production of a monoclonal antibody capable of inhibiting the cell adhesion activity of CEA, it has been necessary to identify the region having the cell adhesion activity of CEA and also to determine its minimum peptide unit. Disclosure of the Invention The present inventors found that determination of a portion as defined in claim 1 on the molecule of CEA, said portion being involved in cell adhesion, makes it possible to synthesize a peptide corresponding to the amino acid sequence of the region and hence to obtain an antibody capable of inhibiting cell adhesion or, by using Escherichia coli or animal cells in a manner known per se in the art, to produce in a large quantity an antibody capable of inhibiting cell adhesion. It was also found that an anti-domain N CEA cell adhesion monoclonal antibody produced using the above peptide can be used, for example, as an active ingredient of a cell adhesion inhibitor such as cancer metastasis inhibitor. Based on these findings, the present inventors have conducted extensive research according to a molecular biological technique with a view toward identifying the adhesion domain by establishing cell lines, which express a deletion antigen of CEA, a chimeric antigen with NCA, and BGPI and W272, both CEA-related antigens, respectively, and checking the cell adhesion activities of various combinations. As a result, it has hence been found that a peptide, which contains at least the peptide sequence of domain N and the peptide sequence of a part or the entire part of domain III in the glycoprotein of the human CEA or peptide sequences homologous to these peptide sequences, is involved in cell adhesion activity and use of the peptide as an antigen enables one to obtain an antibody with inhibitory activity of cell adhesion, leading to the completion of the present invention. The term "peptide sequence having homology" as used herein means a sequence which includes substitution, deletion, addition or the like of one or more amino acids in a part of the sequence, is not identical to the original peptide sequence but is mostly the same as the original peptide sequence and, moreover, has substantially the same function as the original peptide. Brief Description of the Drawings FIG. 1 diagrammatically illustrates adhesion activities of CEA-expressing CHO cells in the presence of monoclonal antibodies of the present invention against CEA; and FIG. 2 diagrammatically depicts percent adhesion inhibitions by monoclonal antibodies of the present invention against CEA. Best Mode for Carrying out the Invention A peptide useful as an antigen to produce an antibody with inhibitory activity of cell adhesion according to the present invention can be obtained, for example, by cutting out, from a CEA expression vector, a DNA sequence encoding the peptide sequence of domain N and a DNA sequence encoding the peptide sequence of a part of or the whole part of domain III, joining them together, inserting the thus-joined DNA sequences in an suitable vector and then expressing them in appropriate cells. Described specifically, a CEA expression vector is first digested with a suitable restriction enzyme to eliminate DNA fragments encoding domains I and II of CEA. As an alternative, the CEA expression vector is digested by a suitable restriction enzyme to divide it into DNA fragment encoding the respective domains and a DNA fragment encoding domain N and another DNA fragment encoding a part of or the entire part of domain III are joined together with ligase. By a method known per se in the art, the gene so obtained is next inserted in a plasmid which can be expressed in host cells. Host cells ( Escherichia coli , yeast or animal cells) are transformed, followed by culture. Since the peptide sequences of domains N and III (or domains 3A and 3B) of CEA are both known, it is also possible to produce a peptide according to the present invention or a peptide having homology therewith by a synthetic process instead of the above processes. To produce the monoclonal antibody of this invention by using the resulting peptide having cell adhesion activity (which may hereinafter be called an "antigen peptide"), a mammal is immunized with the antigen peptide by a method known per se in the art and the spleen is excised to obtain spleen cells. These spleen cells are then fused with subcultured cells. The cells so fused are subjected to screening in an appropriate manner to isolate fused cells in which the target antibody has been expressed. It is then necessary to culture the thus-isolated fused cells in an adequate culture medium. The above screening can be conducted, for example, by conducting selective culture on HAT culture and, with respect to the culture supernatant, confirming a reaction with the antigen by EIA. If necessary, it is possible to combine a further step in which the culture supernatant is reacted with domain III antigen. Confirmation as to whether the resulting monoclonal antibody inhibits cell adhesion or not can be achieved, for example, by such a model experiment as will be described next. Namely, a plasmid capable of expressing CEA in animal cells is produced in a manner known per se in the art. The plasmid is then introduced into CHO (Chinese hamster ovary) cells to establish a cell line which can express a CEA antigen on cell surfaces. Next, in the presence of the monoclonal antibody, the cell adhesion activity of the CEA expressing cell line is investigated. The results of the investigation are then compared with the results of an experiment conducted in the absence of the monoclonal antibody. The cell adhesion activity can be measured by any known appropriate method. For example, a cell line is allowed to grow in a 24-well plate in advance. Another cell line labeled with a fluorescent substance or radioisotope is then added to each well and, subsequent to incubation for a predetermined time, each well is washed several times with a buffer to eliminate unadhered cells. Then, the adhered cells are separated, for example, with trypsin or are dissolved with a surfactant such as 1% NP-40. By an appropriate measuring instrument, the intensity of fluorescence or radioisotope of the adhered cells is measured. The monoclonal antibody of this invention available as described above can inhibit the cell adhesion activity of CEA, said cell adhesion activity being considered to take part in the metastasis of colon cancer, and a pharmaceutical preparation containing the monoclonal antibody can be used as a cell adhesion inhibitor, for example, a cancer metastasis inhibitor. It is also possible to produce a mouse-human chimeric antibody and a humanized antibody from the mouse monoclonal antibody of this invention by known techniques [see Nature 312 , 643-646 (1984) and Nature 332 , 323-327 (1988)]. Use of these antibodies are expected to solve the problem of antigenicity due to continued administration, the problem of in vivo half-life, etc. Such mouse-human chimeric antibody and humanized antibody are obviously included in the present invention. Described specifically, production of the chimeric antibody requires joining of the DNA encoding variable region of H and L chains of a mouse, said chains having cell adhesion inhibiting activity, with the DNA encoding constant region of human immunoglobulin, followed by the insertion into an adequate expression vector. The chimeric antibody can then be mass-produced in animal cells. To produce the humanized antibody, on the other hand, only DNA encoding hypervariable regions (CDR1, CDR2 and CDR3), antigen binding sites, out of the DNA encoding variable region of H and L chains of a mouse are transplanted to DNA encoding human immunoglobulin, followed by further treatment as in the case of the chimeric antibody. Mass production therefore becomes feasible likewise. Production of the cell adhesion inhibitor according to the present invention can be conducted by formulating the monoclonal antibody in an amount as needed into a dosable preparation in a manner known per se in the art. As an administration route for the cell adhesion inhibitor, parenteral administration using an injection, drip infusion additive, suppository or the like is preferred. In some instances, however, it is also possible to adopt oral administration which makes use of tablets, powder, granules, capsules, syrup or the like. Upon formation of each dosable preparation, a known liquid or solid, diluent or carrier can be used. To formulate an injection, for example, it is only necessary to dissolve the antibody of this invention in injection-grade physiological saline and then adding a conventional salt and excipient, for example, sodium chloride, mannitol, aminoacetic acid and the like so that the resulting solution becomes isotonic with the physiological osmotic pressure. More specific dosable preparations include, for example, an injection which has been obtained by dissolving 20 mg of the antibody of this invention in 1 ml of injection-grade distilled water and then making the resulting solution isotonic with the salts described above. No particular limitation is imposed on the amount of the monoclonal antibody according to this invention to be used in the cell adhesion inhibitor. As the antibody has low toxicity and high safety, it is generally sufficient to use the monoclonal antibody in an amount of 1-1,000 mg in the case of parenteral administration. The present invention will hereinafter be described in further detail by the following Referential Examples and Examples. Referential Example 1 Preparation of N-III expression vector and development of antigen (CEA N-III) A vector expressing deleted CEA antigen N-III was prepared as will be described next. Fragments of 6122 bp and 2293 bp, which had been obtained by digesting a CEA expression vector pdKCR-dhfr-CEA [Oikawa, S. et al., B.B.R.C., 164 , 39 (1989)] with PstI (product of TAKARA SHUZO CO., LTD.), were joined together with T4DNA ligase, whereby an N-III expression vector pdKCR-dhfr-CEA N-III was prepared. Introduction of the expression vector into cells and establishment of expression cells were conducted by methods known per se in the art [Oikawa, S. et al., B.B.R.C., 164 , 39 (1989)], so that CEA N-III was obtained as an antigen. The antigen contained, as domain N, a sequence consisting of the 1st to 108th amino acids of CEA and, as domain III, a sequence consisting of the 465th to 642th amino acids of CEA. Referential Example 2 Preparation of soluble CEA N-III fraction CHO cells into which pdKCR-dhfr-CEA N-III had been introduced were subjected to PI-PLC treatment as described below, whereby CEA N-III was obtained as an antigen. (PI-PLC Treatment) 1.75 x 10 8 CHO N-III cells were treated with PBS containing 0.125% of trypsin and 0.01% of EDTA and suspended. Those cells were suspended in 5 mℓ of PBS, followed by the addition of PI-PLC (product of Funakoshi, Inc., Tokyo) to a final concentration of 0.2 unit/mℓ. They were incubated at 37°C for 2 hours. Example 1 Preparation of hybridoma Preparation of a hybridoma was conducted following the technique proposed by Milstein in Nature, 256, 495 (1975). CHO N-III cells with a CEA N-III expression vector introduced therein or a soluble N-III fraction prepared from the cells was suspended in the Freund's complete adjuvant. The suspension was injected three times at intervals of 7-10 days into the abdominal cavity of a (BALB/c) mouse. Three days after the last immunization, the spleen of the mouse was taken out. Its cells (spleen cells) and mouse myeloma cells were caused to fuse by using polyethylene glycol. Selective culture of fused cells (hybridoma) was then conducted on HAT culture medium (which had been obtained by adding hypoxanthin, aminopterin and thymidine to 10% serum-added RPMI 1640 medium) by a method known per se in the art. Primary screening was thereafter conducted with respect to the culture supernatant by ELISA in view of the reaction to purified CEA. The hybridoma which had been judged positive by the primary screening was subjected further to subcloning by the limiting dilution analysis so that a monoclone was obtained. The sub-class of an antibody (MoAb) produced by the monocloned hybridoma was determined using a commercial mouse globulin identification kit. In the manner described above, there were obtained seven hybridoma clones capable of producing an antibody having cell adhesion inhibiting activity, namely, hybridoma clones C249, E55, F287, F361, G125, G213 and G292. Incidentally, hybridomas and monoclonal antibodies produced by the hybridomas will be designated by the same symbols in the present specification. Of the seven hybridoma clones, hybridoma G125 which produces the representative antibody was deposited February 14, 1992 under FERM BP-3750 with Fermentation Research Institute, Agency of Industrial Science and Technology. Example 2 Production of monoclonal antibody The hybridomas obtained in Example 1 were inoculated to the abdominal cavity of a BALB/c mouse, and ascitic fluid was recovered. The thus-obtained ascitic fluid (20 mℓ) was centrifuged and clarified and was then caused to pass through a 35-mℓ protein A-Sepharose CL-4B column (product of Pharmacia LKB AB). After the column was washed with a 1.5 M glycine buffer (pH 8.9) which contained 3 M of sodium chloride, the monoclonal antibody adsorbed on the column was eluted with 0.1 M citric acid buffer (pH 4.0). Seven kinds of monoclonal antibodies C249, E55, F287, F361, G125, G213 and G292 obtained as described above were found to belong to immunoglobulin sub-classes shown in the following Table. Group Clone Class C249 IgGl(κ) 1 E55 IgG2a(κ) F287 IgGl(κ) 2 G125 IgGl(κ) F361 IgGl(κ) 3 G213 IgGl(κ) G292 IgGl(κ) Example 3 Preparation of Fab fraction Each monoclonal antibody obtained in Example 2 was incubated at 22°C for 8 hours together with papain-bound beads (commercial product) so that it was cut. After the supernatant was collected, it was passed through a protein A-Sepharose CL-4B column and a flow-through fraction was obtained as an Fab fraction. Example 4 Measurement of cell adhesion inhibiting activity Measurement of cell adhesion inhibiting activity was conducted by a conventional technique, that is, following the cell adhesion assay described by Oikawa, S. et al. in B.B.R.C., 164 , 39 (1989). Namely, 1 x 10 7 of CHO cells expressing CEA (CHO/CEA cells) were fluorescein-labelled with PKH2 (product of Xynaticis Company) and then suspended in 3 mℓ of a culture medium. 0.1 mℓ of the suspension was added to the non-labelled CHO/CEA cell line which had grown as a monolayer in each well of a 24-well plate, followed by incubation at 37°C for 30 minutes in a 5% CO 2 incubator. After each well was washed twice with PBS, adhered cells were separated by 0.1% trypsin and the fluorescence intensity was measured by a fluorescence intensity meter. Adhesion of the labelled CHO/CEA cells to CEA non-expressing CHO cells was used as a blank. Example 5 Inhibition of CEA-mediated cell adhesion by monoclonal antibody Using the Fab fraction prepared from each antibody, the cell adhesion assay was conducted at 37°C for 30 minutes. Namely, it was investigated in the presence of 27 µg/mℓ of the Fab fraction how much the homotypic adhesion activity of CEA would be inhibited. Incidentally, "None" indicates cell adhesion activity in the absence of an antibody. The results are shown in FIG. 1. From FIG. 1, the percent adhesion inhibition was calculated in accordance with the following formula: Adhesion inhibition, % = A 2 - A 1 A 2 - B x 100 A 1 : Homotypic adhesion activity of CEA in the presence of Fab fraction A 2 : Homotypic adhesion activity of CEA in the absence of Fab fraction (None) B: Adhesion activity of blank One example of the above results is diagrammatically illustrated in FIG. 2. As is seen from the results of FIG. 2, an epitope-dependent difference was observed with respect to the adhesion inhibiting activity of each antibody (Fab fraction). Namely, the domain N-recognizing antibody exhibited relatively high inhibiting activity compared with the domain III-recognizing antibody. In particular, G125 (Group 2) had strong activity of 100 %. Further, C249, E55 and F287 (Group 1) showed intermediate inhibition of from 51.8 % to 82.1 %. Example 6 Inhibition to liver metastasis by monoclonal antibody In the presence of 50 µg of each monoclonal antibody (Fab fraction), 2 x 10 6 of CHO/CEA cells were incubated at 4°C for 1 hour. A nude mouse was incised into the abdominal cavity under anesthesia and the cell suspension obtained as described above was introduced into the spleen. Seventeen days later, the weight of the liver as well as the presence or absence of any metastasized lesion(s) and the number of metastasized nodule(s) in the liver were determined as indices of the extent of metastasis. The results are presented in Table 2 as the liver weight, incidence of metastasized mice and the metastasized nodule(s) number, respectively. As is evident from Table 2, the metastasis of CHO/CEA cells to the liver was inhibited by the C249 or G125 pre-treatment. As a comparison, an Fab fraction prepared from an IgG fraction of a normal mouse was employed. As is shown in Table 2, no inhibition of metastasis was observed at all in this comparative group. These results indicate that a monoclonal antibody capable of inhibiting the cell adhesion activity of CEA is useful as a cancer metastasis inhibitor. Industrial applicability It has heretofore been unable to effectively prevent metastasis of a cancer to other organs. Administration of a monoclonal antibody according to the present invention, however, can inhibit the adhesion activity of cancer cells and, as a result, can prevent metastasis of cancer cells to other organs. The monoclonal antibody is therefore effective as a cell adhesion inhibitor, for example, cancer metastasis inhibitor.

Claims: Use of a monoclonal antibody against a peptide consisting of a partial sequence of human carcinoembryonic antigen (CEA), said partial sequence consisting of the peptide sequence of domain N and the peptide sequence of at least a part of domain III of said antigen, wherein the monoclonal antibody recognizes domain N of human, carcinoembryonic antigen or wherein the monoclonal antibody recognizes domain N of human carcimoembryonic antigen and domain N of human non-specific cross-reactive antigen (NCA), for the manufacture of a metastasis inhibitor against cells expressing in vivo human carcinoembryonic antigen on cell surfaces. The use of claim 1, wherein the monoclonal antibody is produced by using hybridoma G125 which is deposited under FERM BP-3750. A process for producing a metastasis inhibitor against cells expressing in vivo a glycoprotein of human carcinoembryonic antigen (CEA) on cell surfaces, comprising the generation of a monoclonal antibody against a peptide consisting of a partial sequence of human carcinoembryonic antigen, said partial sequence consisting of the peptide sequence of domain N and the peptide sequence of at least a part of domain III of said antigen, and whereby the obtained monoclonal antibody recognizes domain N of human carcinoembryonic antigen or wherein the monoclonal antibody recognizes domain N of human nonspecific cross-reactive antigen (NCA). The process according to claim 3, wherein the monoclonal antibody is produced by using hybridoma G125 which is deposited under FERM BP-3750. A process for producing a cell adhesion inhibitor against cells expressing in vivo a glycoprotein of human carcinoembryonic antigen on their surfaces, comprising the generation of a monoclonal antibody according to claims 3 or 4.

Inventors: HASHINO JYUNKO, NAKANISHI TOSHIHIRO, NAKAZATO HIROSHI, OIKAMA SHINZO, HASHINO, JYUNKO, NAKANISHI, TOSHIHIRO, NAKAZATO, HIROSHI, OIKAMA, SHINZO, HASHINO, JYUNKO, SUNTORY INST. FOR BIOMED. RES., NAKANISHI, TOSHIHIRO, SUNTORY INST.FOR BIOMED. RES, NAKAZATO, HIROSHI, SUNTORY INST. FOR BIOMED. RES., OIKAMA, SHINZO, SUNTORY INST. FOR BIOMED. RES.
Applicants: SUNTORY LTD, SUNTORY LIMITED
Application year: 1993
Main classification: C12P21
IPC codes: A61K38, A61P35, C07K16, C12P21, C07K14