A pharmaceutical composition containing an anti-human Fas antibody having apoptosis inducing activity and a compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity, as active ingredients for the prophylaxis and/or treatment of an autoimmune disease or rheumatoid arthritis. According to the present invention, the amount of the anti-Fas antibody to be used can be reduced and thereby the possibility that a patient may become tolerant to anti-Fas antibody as a result of the production of antibodies against the anti-Fas antibody in the patient""s body or the like can be decreased, and thus is provided a pharmaceutical composition which can be used for a long time.

1. Field of the Invention
The present invention relates to a novel pharmaceutical composition for prophylaxis and/or treatment of an autoimmune disease or rheumatoid arthritis.
2. Background Art
Physiological death of cells as a result of normal alternation of cells in a living organism is called apoptosis, and is distinguished from the pathological death of cells, i.e. necrosis [cf. Kerr et al., (1972), Br. J. Cancer, 26, 239]. Apoptosis is a kind of so-called programmed cell death, which is observed in certain cells that are programmed, in advance, to die in a living organism. Apoptosis is characterized by a curved cell surface, condensed nuclear chromatin and fragmented chromosomal DNA, amongst others.
Apoptosis plays a role in the differentiation of lymphocytes (T cells and B cells) by eliminating cells that recognize an autoantigen. It is believed that a cause of an autoimmune disease is the presence of auto-reactive lymphocytes generated due to failure of apoptosis in differentiation of lymphocytes [cf. Nakayama et al., (1995), Mebio, 12 (10), 79-86].
Various molecules have been identified as being involved in apoptosis, including: Fas [cf. Yonehara. S., et al., (1989), J. Exp. Med., 169, 1747-1756]; tumor necrosis factor receptor [cf. Loetscher. H., et al., (1990), Cell, 61, 351-359]; CD40 [cf. Tsubata, T., et al., (1993), Nature, 364, 645-648]; and perforin/granzyme A [cf. Jenne. D. E., et al., (1988), Immunol. Rev. 103, 53-71]. Fas is a transmembrane protein present on the cellular surface, and binding of its extracellular domain to a protein called the xe2x80x9cFas ligandxe2x80x9d induces apoptosis in the cell.
It has been reported that some anti-Fas monoclonal antibodies have a cytotoxic activity and induce apoptosis in a cell in a similar manner as the Fas ligand does, and thus they can be an agent for treating an autoimmune disease, AIDS, and neoplasm [cf. Japanese Patent Application Publication (Kokai) Hei 2-237935 and International application publication in Japan (Kohyo) Hei 5-503281].
On the other hand, rheumatism, especially rheumatoid arthritis is a disease that is accompanied by various abnormalities in immunology caused by internal and external factors, of which a basic pathological change is proliferation of synovial cells, and it is considered that it is a disorder of proliferation of the synovial cells accompanied by inflammatory cell infiltration and bone erosion. Tissue destruction around the joint suffering from rheumatoid arthritis is considered to be caused by abnormalities in the production of cytokines in inflammatory synovial cells. When the state of a joint of a rheumatic patient is investigated, there are observed an unusual proliferation of synovial cells, synovial villus proliferation, multilayered synovial cells and the like (cf. Daniel J. McCarty (1985) in xe2x80x9cArthritis and allied conditions, A textbook of rheumatologyxe2x80x9d 10th Edition, Lea and Febiger). An anti-inflammatory agent or an immunity regulatory agent, such as a steroid or the like is mainly used in pharmacotherapy that is presently carried out for rheumatism. However, if such excrescence of synovial cells can be controlled with a medicine, it is considered that such a medicine would be useful as an agent for treating rheumatism.
Incidentally, it is known that proliferation of the synovial cells in rheumatism is not out of control, but is spontaneously controlled (cf. Daniel J. McCarty (1985) in xe2x80x9cArthritis and allied conditions, A textbook of rheumatologyxe2x80x9d 10th Edition, Lea and Febiger). Furthermore, it has become clear recently that apoptosis is caused in synovial cells of a rheumatic patient and that the Fas antigen appears on a membrane of the synovial cells. Nakajima et al. (cf. Nakajima, T., et al. (1995) Arthritis Rheum. 38, 485-491) and Aono et al. (cf. 38th Japan rheumatic society summary collection (1994), 487 page and Heisei 6 Japan cancer society general meeting reports 1994, 338 page) have studied whether apoptosis is induced in a synovial cell when the anti-human Fas antibody having a cytotoxic activity is added to the abnormally proliferating synovial cells originated from a rheumatic patient, and have found that apoptosis is induced at a higher rate in the abnormally proliferating synovial cells originated from a rheumatic patient, than in synovial cells which are not originated from a rheumatic patient. Accordingly, an anti-human Fas antibody can selectively induce apoptosis, not only in a lymphocyte, but also in the abnormally proliferating synovial cells, and therefore it is considered to be useful as an agent for rheumatism.
Several kinds of anti-human Fas mouse monoclonal antibodies have already been found (cf. Yonehara, S., et al (1989) J. Exp. Med. 1, 1747-1756, (1989); SCIENCE, 245, 301-305 (1989), and the like). Furthermore, as described above, it has been reported that the antibodies induce apoptosis in synovial cells of a rheumatic patient, in-vitro (cf. 38th Japan rheumatic society summary collection (1994), p.487; and Japan cancer society general meeting reports (1994), p.338). Furthermore, some anti-Fas antibodies have been found to be effective and safe for treatment in an autoimmune disease model animal or a rheumatoid arthritis model animal (cf. European patent application publication No. 0909816).
On the other hand, it is known that the effect of treatment for a rheumatoid arthritis patient can be increased by using methotrexate and a monoclonal antibody cA2 against a tumor necrosis factor xcex1 (TNFxcex1) together (Carden, the 7th international rheumatism symposium summary (1998) p.12-13). However, a synergistic effect of an anti-Fas antibody and methotrexate was not heretofore known at all.
If there is a compound which reinforces the efficacy of an anti-Fas antibody useful as an agent for the prophylaxis and/or treatment of an autoimmune disease or rheumatoid arthritis, the amount of the anti-Fas antibody to be used can be decreased by using the compound and an anti-Fas antibody together. Thereby, the possibility that a patient becomes tolerant to an anti-Fas antibody as a result of production of antibodies against anti-Fas antibodies in the patient""s body or the like can be decreased. Accordingly, there has been a need for a prophylactic or therapeutic agent consisting of a combination of an anti-Fas antibody and a compound reinforcing the efficacy of the anti-Fas antibody, which can be used for a long time.
The xe2x80x9csynergy effectxe2x80x9d shown in this application means a coordinated or correlated action by an anti-human Fas antibody and a compound having a folate antagonistic activity or dihydrofolate reductase inhibiting activity. The coordinated or correlated action is far stronger than would be expected by a person of ordinary skill in the art.
The present invention relates to a pharmaceutical composition comprising an anti-human Fas antibody having an apoptosis inducing activity and a compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity, as active ingredients. Preferably, the anti-human Fas antibody is a monoclonal antibody CH11, anti-human Fas monoclonal antibody HFE7A produced by a mouse-mouse hybridoma HFE7A (FERM BP-5828), or humanized antibodies thereof. Preferably, the above-mentioned compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity is selected from the group consisting of: methotrexate, edatrexate, epiroprim, iometrexol, pyritrexim, trimetrexate, brodimoprim, MX-68, N-[4-[3-(2,4-diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid, N-[[5-[2-(2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]-pyrimidin-6-yl)ethyl]-2-thienyl]carbonyl]-L-glutamic acid, (R)-N-[[5-[2-(2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]pyrimidin-6-yl)ethyl]-2-thienyl]carbonyl]-L-glutamic acid, N-((2,4-diamino-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-6-yl)-ethyl)-2-thienylcarbonyl-L-glutamic acid, (S)-2-[[[4-carboxy-4-[[4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]amino]butyl]amino]carbonyl]benzoic acid, N-[4-[3-(2,4-diamino-1H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid, 2,4-diamino-6-(N-(4-(phenylsulfonyl)benzyl)methylamino)quinazoline, 2,4-diamino-5-[4-[3-(4-aminophenyl-4-sulfonylphenylamino)propoxy]-3,5-dimethoxybenzyl]-pyrimidine, N-[4-[4-(2,4-diamino-5-pyrimidinyl)butyl]benzoyl]-L-glutamic acid, N-[4-[3-(2,4-diamino-5-pyrimidinyl)propyl]benzoyl]-L-glutamic acid, N-[4-[2-(2,4-diamino-6-pteridinyl)ethyl]benzoyl]-4-methylene-DL-glutamic acid and N-(1-methylethyl)-Nxe2x80x2[3-(2,4,5-trichlorophenoxy)propoxy]imidodicarbonimidic diamide hydrochloride (PS15). Among them, methotrexate is the most preferable. The inventors of the present invention have found that the efficacy of the anti-Fas monoclonal antibody having an apoptosis inducing effect can be increased by using it together with a compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity, and thereby have completed the present invention.
As used herein, the term xe2x80x9can apoptosis inducing activityxe2x80x9d means an activity of inducing apoptosis in a cell expressing Fas on a cell membrane surface by binding to the Fas.
The anti-human Fas antibody used as the first active ingredient of the pharmaceutical composition of the present invention may be one of those that can be specifically bound to human Fas and has an apoptosis inducing effect. Preferable examples of such an anti-human Fas antibody include: anti-human Fas monoclonal antibody CH11 and anti-human Fas monoclonal antibody HFE7A produced by the mouse-mouse hybridoma HFE7A (FERM BP-5828), or humanized antibodies thereof (European patent application publication No. 0909816), but the present invention is not limited thereto. In addition, the anti-human Fas monoclonal antibody of the present invention also includes recombinants of these anti-human Fas monoclonal antibodies, which have an effect equivalent to these monoclonal antibodies. Moreover, in the present invention, there can also be used a so-called humanized antibody modified using a gene recombination technology so that the immunogenicity to humans may be reduced, without deteriorating the binding ability of the above-mentioned anti-Fas monoclonal antibody to bind to Fas and its apoptosis inducing activity.
As used herein, the term xe2x80x9cthe compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activityxe2x80x9d means a pharmaceutically acceptable compound which has an activity of antagonistically inhibiting metabolism of the folic acid that is an indispensable process in synthesis of DNA in a cell. Preferable examples of such a compound include:
Methotrexate (the following formula (I)):
Edatrexate (cf. British patent publication No. GB 2058770 B, the following formula (II)):
Epiroprim (cf. International patent application publication No. WO92/8461, the following formula (III)):
Iometrexol (cf. International patent application publication No. WO86/5181 publication, the following formula (IV)):
Pyritrexim (cf. European patent publication No. 21292, the following formula (V)):
Trimetrexate (cf. British patent publication No. GB 1345502, the following formula (VI)):
Brodimoprim (cf. British patent publication No. GB 1449387, the following formula (VII)):
MX-68 (International patent application publication No. WO97/34606, the following formula (VIII)):
N-[4-[3-(2,4-diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid (cf. J.Med.Chem. (1994) 37, 1616-1624, the following formula (IX)):
N-[[5-[2-(2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]pyrimidin-6-yl)ethyl]-2-thienyl]carbonyl]-L-glutamic acid
(cf. European patent publication No. 343801, the following formula (X)):
N-((2,4-diamino-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-6-yl)ethyl)-2-thienylcarbonyl-L-glutamic acid
(cf. Summary of U.S. cancer research society, 88th annual convention (1997) No. 660, the following formula (XI)):
(S)-2-[[[4-carboxy-4-[[4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]amino]-butyl]amino]carbonyl]benzoic acid
(cf. European patent publication No. 345308, the following formula (XII)):
N-[4-[3-(2,4-diamino-1H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid
(cf. European patent publication No. 334636, the following formula (XIII)):
2,4-diamino-6-(N-(4-(phenylsulfonyl)benzyl)methylamino)quinazoline
(cf. Summary of U.S. cancer research society, annual convention (1992) No. 2458, the following formula (XIV)):
2,4-diamino-5-[4-[3-(4-aminophenyl-4-sulfonylphenylamino)propoxy]-3,5-dimethoxybenzyl]pyrimidine
(cf. European patent publication No. 231888, the following formula (XV)):
N-[4-[4-(2,4-diamino-5-pyrimidinyl)butyl]benzoyl]-L-glutamic acid (cf. International Patent Application No. WO95/9845, the following formula (XVI)):
N-[4-[3-(2,4-diamino-5-pyrimidinyl)propyl]benzoyl]-L-glutamic acid (cf. International Patent Application No. WO95/9845, the following formula (XVII)):
N-[4-[2-(2,4-diamino-6-pteridinyl)ethyl]benzoyl]-4-methylene-DL-glutamic acid (cf. International Patent Application No. WO91/10666, the following formula (XVIII)):
N-(1-methylethyl)-N-[3-(2,4,5-trichlorophenoxy)propoxy]imidodicarbonimidic diamide hydrochloride (PS15) (cf. International Patent Application No. WO93/16037, the following formula (XIX)):
It is known that all of these compounds have a folate antagonist activity or a dihydrofolate reductase inhibiting activity. Among the above-mentioned compounds, methotrexate is the most preferable as the compound that is to be contained in the pharmaceutical composition of the present invention.
The pharmaceutical composition of the present invention can be obtained by formulating an appropriate mixture of an anti-Fas monoclonal antibody having an activity of inducing apoptosis in cells expressing Fas and a compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity.
The anti-Fas monoclonal antibody can be produced by methods known in the art using, for example, a molecule containing an extracellular domain of human Fas as an antigen. For example, the monoclonal antibody HFE7A that is one of the preferable anti-Fas monoclonal antibodies to be contained in the pharmaceutical composition of the present invention can be obtained by immunizing a Fas knock-out mouse with human Fas, subsequently fusing the spleen cells from the mouse with mouse myeloma cells, and culturing the resultant hybridoma. Specifically, it can be obtained according to the following method.
Preparation of a monoclonal antibody involves at least the following steps:
(a) purification of a biomacromolecule for use as the antigen;
(b) preparation of antibody producing cells, after immunizing an animal using injections of the antigen, bleeding the animal and assaying the antibody titer, in order to determine when to remove the spleen;
(c) preparation of myeloma cells;
(d) fusing the antibody producing cells and myeloma cells;
(e) selecting a hybridoma producing an antibody of interest;
(f) preparing a single cell clone (cloning);
(g) optionally, culturing the hybridoma cells, or growing animals into which the hybridoma cells have been transplanted, for large scale preparation of the monoclonal antibody; and
(h) testing the biological activities and the specificity, or assaying marker agent properties, of the monoclonal antibody thus prepared.
The method for the preparation of an anti-Fas monoclonal antibody is described below more in detail, in line with the above described steps. However, the method for preparing the antibody is not limited thereto. Other antibody producing cells than spleen cells and myeloma can also be used.
(a) Purification of Antigen
A recombinant protein (hereinafter referred to as xe2x80x9crecombinant human Fasxe2x80x9d), effective as the antigen, can be obtained by transfecting the monkey cell line COS-1 with the expression vector phFAS-AIC2, which encodes a fusion protein comprising the extracellular domain of human Fas and the extracellular domain of the mouse interleukin-3 receptor (hereinafter referred to as IL3R), [cf. Nishimura, Y., et al., (1995), J. Immunol., 154, 4395-4403] to express it, and collecting and partially purifying the expression product. The plasmid phFas-AIC2 was constructed by inserting DNA encoding a human Fas and mouse IL3R fusion protein into pME18S, which is an expression vector for animal cells. As noted above, the materials used, such as the DNA encoding Fas, the vector and the host, are not restricted to those mentioned.
For example, the human Fas and mounse IL3R fusion protein produced in the culture supernatant of the transformed COS-1 cells transfected with the plasmid phFas-AIC2 may be partially purified by ion-exchange chromatography using a Resource Q column (tradename; manufactured by Pharmacia).
Purified Fas obtained from the cell membranes of human cell lines can also be used as the antigen. Furthermore, since the primary structure of Fas is known [cf. Itoh, N., et al., (1991), Cell, 66, 233-243], a peptide comprising the known amino acid sequence may be chemically synthesized by a method well known in the art, and used as the antigen.
(b) Preparation of Antibody Producing Cells
The immunogen produced in step (a) is mixed with an adjuvant, such as Freund""s complete or incomplete adjuvant and alum, and an experimental animal is immunized therewith. A suitable experimental animal may be a Fas knock-out mouse, which may be produced by the method of Senju et al. [Senju, S., et al., (1996), International Immunology, 8, 423].
Suitable administration routes to immunize the mouse include the subcutaneous, intraperitoneal, intravenous, intradermal and intramuscular injection routes, with subcutaneous and intraperitoneal injections being preferred.
Immunization can be by a single dose or, by several repeated doses at appropriate intervals (preferably 1 to 5 weeks). Immunized animals are monitored for antibody titer in their sera, and an animal with a sufficiently high antibody titer is selected as the source of antibody producing cells. Selecting an animal with a high titer makes the subsequent process more efficient. Cells for the subsequent fusion are generally harvested from the animal 3 to 5 days after the final immunization.
Methods for assaying antibody titer include various well known techniques such as radioimmunoassay (hereinafter, referred to as RIA), solid-phase enzyme immunoassay (hereinafter, referred to as ELISA), fluorescent antibody assay and passive hemagglutination assay, with RIA and ELISA preferred for reasons of detection sensitivity, rapidity, accuracy and potential for automation.
Determination of antibody titer may be performed, for example, by ELISA, as follows. First, purified or partially purified Fas is adsorbed onto the surface of a solid phase, such as a 96-well ELISA plate, followed by blocking any remaining surface, to which Fas has not been bound, with a protein unrelated to the antigen, such as bovine serum albumin (hereinafter referred to as BSA). After washing, the well surfaces are contacted with serially diluted samples of the first antibody (for example, mouse serum) to enable binding of the anti-Fas antibody in the samples to the antigen. An enzyme-labeled, anti-mouse antibody, as the secondary antibody, is added to be bound to the mouse antibody. After washing, the substrate for the enzyme is added, and antibody titer can then be estimated by determining absorbance change due to color development caused by the decomposed substrate or the like.
(c) Preparation of Myeloma Cells
In general, cells from established mouse cell lines serve as the source of myeloma cells, for example, 8-azaguanine resistant mouse (derived from BALB/c) myeloma strains P3X63Ag8U.1 (P3-U1) [Yelton, D. E., et al., Current Topics in Microbiology and Immunology, 81, 1-7, (1978)], P3/NSI/1-Ag4-1(NS-1) [Kohler, G., et al., European J. Immunology, 6, 511-519 (1976)], Sp2/0-Ag14 (SP-2) [Shulman, M., et al., Nature, 276, 269-270 (1978)], P3X63Ag8.653 (653) [Kearney, J. F., et al., J. Immunology, 123, 1548-1550 (1979)] and P3X63Ag8 (X63) [Horibata, K. and Harris, A. W., Nature, 256, 495-497 (1975)]. The cell line selected is subcultured in an appropriate medium, such as 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, fetal calf serum (hereinafter referred to as FCS), and 8-azaguanine], Iscove""s Modified Dulbecco""s Medium (hereinafter referred to as IMDM) or Dulbecco""s Modified Eagle Medium (hereinafter referred to as DMEM). The cells are then subcultured in a normal medium, such as ASF104 medium (Ajinomoto, K. K.) containing 10% FCS, 3 to 4 days prior to fusion, in order to ensure that at least 2xc3x97107 cells are available on the day of fusion.
(d) Cell Fusion
The antibody producing cells to be used are plasma cells and lymphocytes which are their precursor cells, which may be obtained from any suitable part of the animal. Typical areas are spleen, lymph nodes, peripheral blood, or any appropriate combination thereof, spleen cells most commonly being used.
After the last booster injection, tissue in which antibody producing cells are present, such as the spleen, is enucleated from a mouse having the predetermined antibody titer to prepare antibody producing cells such as spleen cells. The currently favored technique for fusion of the spleen cells with the myeloma cells prepared in step (c), employs polyethylene glycol, which has relatively low cytotoxicity and the fusion procedure using it is simple. An example of this technique is as follows.
The spleen and myeloma cells are washed well with serum-free medium (such as RPMI 1640) or phosphate buffered saline (hereinafter referred to as PBS) and then mixed, so that the number ratio of spleen cells to myeloma cells is approximately between 5:1 and 10:1, and then centrifuged. After the supernatant has been discarded and the pelleted cells sufficiently loosened, 1 ml of serum-free medium containing 50% (w/v) polyethylene glycol (m.w. 1,000 to 4,000) is added dropwise with stirring. Subsequently, 10 ml of serum-free medium is slowly added and then the mixture centrifuged. The supernatant is discarded again, and the pelleted cells are suspended in an appropriate amount of HAT medium containing a solution of hypoxanthin, aminopterin and thymidine (hereinafter referred to as xe2x80x9cHATxe2x80x9d) and mouse interleukin-2 (hereinafter referred to as IL-2). The suspension is then dispensed into the wells of culture plates (hereinafter referred to as xe2x80x9cplatesxe2x80x9d) and incubated in the presence of 5% v/v CO2 at 37xc2x0 C. for about 2 weeks, with the supplementary addition of HAT medium as appropriate.
(e) Selection of Hybridomas
When the myeloma strain used is resistant to 8-azaguanine, i.e., it is deficient in the hypoxanthin guanine phosphoribosyl transferase (HGPRT) enzyme, any unfused myeloma cells and any myelomaxe2x80x94myeloma fusions are unable to survive in HAT medium. On the other hand, fusions of antibody producing cells with each other, as well as hybridomas of antibody producing cells with myeloma cells can survive, the former only having a limited life. Accordingly, continued incubation in HAT medium results in selection of only the desired hybridomas.
The resulting hybridomas grown up into colonies are then transferred into HAT medium without aminopterin (hereinafter referred to as xe2x80x9cHT mediumxe2x80x9d). Thereafter, aliquots of the culture supernatant are collected to determine anti-Fas antibody titer by, for example, ELISA. When the above-mentioned fusion protein is used as the ELISA antigen, it is also necessary to eliminate clones producing an antibody which specifically binds to the extracellular domain of the mouse IL3 receptor. The presence or absence of such a clone may be verified, for example, by ELISA using the mouse IL3 receptor, or its extracellular domain, as the antigen.
Although the above selection procedure is exemplified using an 8-azaguanine resistant cell line, it will be appreciated that other cell lines may be used with appropriate modifications to the media used.
(f) Cloning
Hybridomas which have been shown to produce specific antibodies, using a method similar to that described in the step (b) to determine antibody titer, are then transferred to another plate for cloning. Suitable cloning methods include: the limiting dilution method, in which hybridomas are diluted to contain one cell per well of a plate and then cultured; the soft agar method in which colonies are recovered after culturing in soft agar medium; a method of using a micromanipulator to separate a single cell for culture; and xe2x80x9csort-a-clonexe2x80x9d, in which single cells are separated by a cell sorter. Limiting dilution is generally the most simple and is commonly used.
The cloning procedure according to, for example, the limiting dilution method is repeated 2 to 4 times for each well demonstrating an antibody titer, and clones having stable antibody titers are selected as anti-Fas monoclonal antibody producing hybridomas. Hybridomas producing an anti mouse Fas antibody are selected by a similar method to obtain an anti-Fas monoclonal antibody producing cell line. A mouse Fas useful for this purpose, for example, is the fusion protein expressed by culturing animal cells transfected with the expression vector pME18S-mFas-AIC. This plasmid has DNA encoding a fusion protein comprising the extracellular domain of mouse Fas and the extracellular domain of the mouse IL3 receptor [cf. Nishimura. Y., et al., (1995). J. Immunol., 154, 4395-4403]. Other sources of murine Fas include purified mouse Fas and cells which express mouse Fas on their surface.
The mouse-mouse hybridoma HFE7A that produces the anti-Fas monoclonal antibody preferable as an active ingredient in the pharmaceutical composition of the present invention was deposited with the National Institute of Bioscience and Human-Technology at 1-3, Higashi 1-chome, Tsukuba, Ibaraki, Japan on Feb. 19, 1997, in accordance with the Budapest Treaty on the Deposition of Microorganisms, and was accorded the accession number FERM BP-5828. Accordingly, when preparing an antibody using the mouse-mouse hybridoma HFE7A, the preparation may be performed by following a procedure starting from the step (g) below, with the steps (a) to (f) omitted.
(g) Culture of Hybridoma to Prepare Monoclonal Antibody
The hybridoma obtained by the cloning is then cultured in normal medium, not in HT medium. Large-scale culture can be performed by roller bottle culture, using large culture bottles, or by spinner culture. The supernatant from the large-scale culture is purified by a suitable method, such as gel filtration, which is well known to those skilled in the art, to obtain an anti-Fas monoclonal antibody which the prophylactic or therapeutic agent of the present invention contains. The hybridoma may also be grown intraperitoneally in a syngeneic mouse, such as a BALB/c mouse or a Nu/Nu mouse, to obtain a large quantity of ascites containing an anti-Fas monoclonal antibody which the prophylactic or therapeutic agent of the present invention contains. Purification can also be conducted through use of commercially available monoclonal antibody purification kits (for example, MAbTrap GII Kit; Pharmacia).
Monoclonal antibodies prepared as above have a high specificity to human and mouse Fas.
(h) Assay of Monoclonal Antibody
Determination of the isotype and the subclass of the monoclonal antibody thus obtained may be performed as follows. Suitable identification methods include the Ouchterlony method, ELISA and RIA. The Ouchterlony method is simple, but requires concentration of the solution when the concentration of the monoclonal antibody is low. When ELISA or RIA is used, the culture supernatant can be reacted directly with an antigen adsorbed on a solid phase and with secondary antibodies having specificities for the various immunoglobulin isotypes and subclasses to identify the isotype and subclass of the monoclonal antibody. A method of using a commercial kit for identification, such as a Mouse Typer Kit (manufactured by Bio-Rad Laboratories, Inc.) is more simple.
Quantification of protein may be performed by the Folin-Lowry method, or by calculation based on the absorbance at 280 nm [1.4 (OD 280)=Immunoglobulin 1 mg/ml].
DNA encoding the heavy and light chains of the anti-Fas monoclonal antibody that is preferable as an active ingredient in the pharmaceutical composition of the present invention, may be obtained by preparing mRNA from hybridoma cells producing the anti-Fas monoclonal antibody, converting the mRNA into cDNA with reverse transcriptase, and then isolating the DNA encoding the heavy and/or light chains of the antibody, respectively.
Extraction of mRNA can be performed by the guanidinium thiocyanate-hot phenol method, the guanidinium thiocyanate-guanidinium HCl method, or the like, but the guanidinium thiocyanate-cesium chloride method is preferred. Preparation of mRNA from cells is generally performed by first preparing total RNA and then purifying mRNA from the total RNA by using a poly(A)+ RNA purification matrix, such as oligo(dT) cellulose and oligo (dT) latex beads. Alternatively, mRNA may be prepared directly from a cell lysate using such a matrix. Methods for preparing total RNA include: alkaline sucrose density gradient centrifugation [cf. Dougherty, W. G. and Hiebert, E. (1980), Viology, 101, 466-474]; the guanidinium thiocyanate-phenol method; the guanidinium thiocyanate-trifluorocesium method; and the phenol-SDS method. The method using guanidinium thiocyanate and cesium chloride [cf. Chirgwin, J. M., et al., (1979), Biochemistry, 18, 5294-5299] is preferable.
The thus obtained poly(A)+ RNA can be used as the template in a reverse transcriptase reaction to prepare single-strand cDNA which can then be converted to double stranded cDNA. Suitable methods therefor include the S1 nuclease method [cf. Efstratiadis. A., et al., (1976), Cell, 7, 279-288], the Gubler-Hoffman method [cf. Gubler. U. and Hoffman, B. J., (1983), Gene, 25, 263-269] and the Okayama-Berg method [cf. Okayama. H. and Berg, P., (1982). Mol. Cell. Biol., 2, 161-170]. However, the preferred method involves the polymerase chain reaction [hereinafter referred to as PCR, cf. Saiki, R. K., et al., (1988), Science, 239, 487-49] using single-strand cDNA as the template, namely xe2x80x9cRT PCRxe2x80x9d.
The double-strand cDNA obtained above may then be integrated into a cloning vector and the resulting recombinant vector can then be used to transform a micro-organism, such as E. coli. The transformant can be selected using tetracycline resistance or ampicillin resistance. If E. coli is used, then transformation may be effected by the Hanahan method [cf. Hanahan, D., (1983), J. Mol. Biol., 166, 557-580]. Namely, the recombinant vector may be introduced into competent cells prepared by co-exposure to calcium chloride, magnesium chloride or rubidium chloride. If a plasmid is used as a vector, it is necessary that the plasmid harbors a drug-resistant gene, such as mentioned above. It is also possible to use other cloning vehicles, such as lambda phages.
In order to select transformants for those which carry cDNA encoding a subunit of an anti-Fas antibody of interest, various methods, such as those described below, can be used. When the cDNA of interest is specifically amplified by the above-mentioned RT-PCR, these steps may be omitted.
(1) Method by Polymerase Chain Reaction
If all or part of the amino acid sequence of the desired protein has been elucidated, then sense and antisense oligonucleotide primers corresponding to parts of the amino acid sequence can be synthesized, and used in the polymerase chain reaction technique [cf. Saiki, R. K., et al. (1988), Science, 239, 487-49] to amplify the desired DNA fragment encoding the anti-human Fas monoclonal antibody light chain subunit and heavy chain subunit. The template DNA may be, for example, cDNA synthesized by reverse transcription from mRNA of the hybridoma producing the anti-human Fas monoclonal antibody HFE7A (FERM BP-5828).
The DNA fragment thus synthesized may either be directly integrated into a plasmid vector by using a commercially available kit or the like, or may be labelled with, for example, 32P, 35S or biotin, and then used as a probe for colony hybridization or plaque hybridization to obtain the desired clone.
Anti-Fas monoclonal antibody HFE7A that is preferable as an active ingredient in the pharmaceutical composition of the present invention is immunoglobulin GI (hereinafter referred to as xe2x80x9cIgGIxe2x80x9d) comprising a heavy chain (xcex31 chain) subunit and a light chain (K chain) subunit. The partial amino acid sequence of each of subunits mentioned above can be determined preferably by isolating each subunit by a well known method such as electrophoresis and column chromatography, and sequencing the N-terminal amino acid sequence of each subunit with an auto protein sequencer (for example, PPSQ-10 by Shimadzu Seisakusho, Corp.).
Harvesting of cDNA encoding each subunit of anti-human Fas monoclonal antibody from the appropriate transformants obtained above may be performed by well known techniques [cf. Maniatis, T., et al., in xe2x80x9cMolecular Cloning A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, NY, (1982)]. For example, the region of DNA encoding the desired subunit may be excised from plasmid DNA after separating the fraction corresponding to the vector DNA from cells.
(2) Screening Using a Synthetic Oligonucleotide Probe
If all or part of the amino acid sequence of the desired protein has been elucidated (the sequence can be that in any region of the protein, provided that is specific and contains continuous amino acids), oligonucleotides corresponding thereto may be synthesized, and used as a probe (after labelling with 32P, 35S, biotin or the like), namely, hybridized with DNA from the transformant which has been immobilized on a nitrocellulose filter to select positive strains. As the probe, there can be used one oligonucleotide which is designed considering the frequency of codons in a host, or a mixture of possible oligonucleotides. In the latter case, the number of oligonucleotides to be used can be reduced by using inosine.
DNA thus obtained may be sequenced by, for example, the Maxam-Gilbert chemical modification technique [cf. Maxam, A. M. and Gilbert. W. (1980) in xe2x80x9cMethods in Enzymologyxe2x80x9d 65, 499-576], the dideoxy chain termination method [cf. Messing J. and Vieira J. (1982) Gene, 19, 269-276] or the like.
In recent years, there has been widely used automated DNA sequencers using a fluorogenic dye, for example Sequence robot xe2x80x9cCATALYST 800xe2x80x9d and the model 373A DNA Sequencer, manufactured by Perkin-Elmer Japan, Inc.
By using systems such as those described above, determination of the DNA sequence can be performed efficiently and safely. Based on the data of the nucleotide sequences of the DNA of the present invention thus determined and the data of the N-terminal amino acid sequences of the heavy chain and the light chain thereof, the entire amino acid sequences of the heavy chain and the light chain of a monoclonal antibody of the present invention can be determined.
Construction of a mutant wherein one or more amino acids in an amino acid sequence is deleted may be performed, for example, by cassette mutagenesis [cf. Toshimitsu Kishimoto, xe2x80x9cShin-Seikagaku Jikken Kouza 2: Kakusan III Kumikae DNA Gijutsuxe2x80x9d, 242-251].
Such DNA sequences may be prepared by chemical synthesis using a conventional method, such as the phosphite triester method [cf. Hunkapiller, M., et al., (1984), Nature, 310, 105-111]. Codons for each amino acid themselves are known, and a specific codon for a desired amino acid may be selected arbitrarily, or by taking a frequency of a given codon in a host into account. Partial modification of the nucleotide sequence can be accomplished by conventional techniques, such as site-specific mutagenesis utilizing synthetic oligonucleotide primers encoding the desired modifications [cf. Mark, D. F., et al., (1984), Proc. Natl. Acad. Sci. USA, 81, 5662-5666].
Whether DNA can be hybridized with DNA encoding the light chain or the heavy chain of the anti-Fas monoclonal antibody that is preferable as an active ingredient in the pharmaceutical composition of the present invention can be determined, for example, by using a DNA probe labelled with (xcex1-32P)dCTP or the like, for example, by the random primer method [cf. Feinberg, A. P. and Vogelstein, B. (1983), Anal. Biochem., 132, 6-13], by the nick translation method [cf. Maniatis, T., et al., (1982), in xe2x80x9cMolecular Cloning A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, NY] or the like. A suitable technique is as follows.
First, the DNA to be determined is adsorbed onto a nitrocellulose membrane or a nylon membrane, for example, being subjected to alkaline treatment if necessary, and then being fixed on the membrane by heating or UV irradiation. The membrane is next immersed in prehybridisation solution containing 6xc3x97SSC (1xc3x97SSC is a solution of 0.15 M NaCl and 0.015 M tri-sodium citrate), 5% v/v Denhardt solution and 0.1% v/v sodium dodecyl sulfate (SDS), and incubated at 55xc2x0 C. for 4 hours or more. Then, the probe previously prepared is added in similar prehybridisation solution to a final specific activity of 1xc3x97106 cpm/ml, followed by incubation at 60xc2x0 C. overnight. Subsequently, the membrane is washed at room temperature by repeated washing with 6xc3x97SSC for 5 minutes and further with 2xc3x97SSC for 20 minutes, and is then subjected to autoradiography.
By using the above method, DNA hybridizable with the DNA encoding the heavy or light chain of the anti-Fas monoclonal antibody that is preferable as an active ingredient in the pharmaceutical composition of the present invention can be isolated from any cDNA library or genomic library [cf. Maniatis, T., et al., (1982), xe2x80x9cMolecular Cloning A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, NY].
Integration of DNA thus obtained into an expression vector allows transformation of prokaryotic or eukaryotic host cells, thereby allowing the DNA to be expressed in the host cell.
Suitable prokaryotic host cells include, for example, Escherichia coli, Bacillus subtilis, and the like. In order to express the gene of interest in such host cells, these host cells may be transformed with a plasmid vector containing a replicon derived from a species compatible with the host, namely an origin of replication and a promoter sequence, such as lac UV5. These vectors preferably have sequences capable of conferring a selection phenotype on the transformed cell. A suitable strain of E. coli is strain JM109 derived from E. coli K12. Suitable vectors include pBR322 and the pUC series plasmids, without being limited thereto. Other known strains and vectors can also be utilized. Suitable promoters include the tryptophan (trp) promoter, the lactose promoter (lac), the tryptophan lactose promoter (tac), the lipoprotein promoter (lpp), the lambda (xcex) PL promoter derived from bacteriophage, and the polypeptide chain elongation factor Tu (tufb) promoter, without being limited thereto.
A preferred strain of Bacillus subtilis is strain 207-25, and a preferred vector is pTUB228 [cf. Ohmura, K., et al., (1984), J. Biochem., 95, 87-93], without being limited thereto. A suitable promoter is the regulatory sequence of the Bacillus subtilis xcex1-amylase gene. If desired, the DNA sequence encoding the signal peptide sequence of xcex1-amylase may be linked to the DNA of the present invention to enable extracellular secretion.
Eukaryotic hosts include cells from vertebrate and yeast species. An example of vertebrate cells used is the monkey COS-1 cell line [cf. Gluzman, Y., (1981), Cell, 23, 175-182]. Suitable yeast cell hosts include baker""s yeast (Saccharomyces cerevisiae), methylotrophic yeast (Pichia pastoris) and fission yeast (Schizosaccharomyces pombe). The cells mentioned above are generally used as the host cell, but the host cell to be used is not limited thereto.
In general, the requirements for suitable expression vectors for vertebrate cells are that they comprise: a promoter, usually located upstream of the gene to be expressed; an RNA splicing site; a polyadenylation site; and a transcription termination sequence; and an origin of replication if necessary. A suitable plasmid is, for example, pSV2dhfr containing the SV40 early promoter [cf. Subramani, S., et. al. (1981), Mol. Cell. Bio., 1, 854-864], without being limited thereto.
Suitable expression vectors for yeasts contain, for example, the promoter of the alcohol dehydrogenase gene [cf. Bennetzen, J. L. and Hall, B. D., (1982), J. Biol. Chem., 257, 3018-3025) or the promoter of a galactose metabolic enzyme (for example, gal 10) [cf. Ichikawa, K., et. al. (1993), Biosci. Biotech. Biochem., 57, 1686-1690], without being limited thereto. If desired, the DNA sequence encoding the signal peptide sequence of a yeast gene may be linked to the DNA to be expressed in order to enable extracellular secretion.
When COS-1 cells are used as a host cell, expression vectors suitably comprise the SV40 replication origin, enabling autonomous replication, a transcription promoter, a transcription termination signal and an RNA splicing site. The said expression vectors can be used to transform the COS-1 cells by any suitable method, such as the DEAE-dextran method [cf. Luthman. H, and Magnusson. G. (1983), Nucleic Acids Res., 11, 1295-1308], the phosphate calcium-DNA co-precipitation method [cf. Graham, F. L. and Van der Eb, A. J., (1973), Virology, 52, 456-457] and the electric pulse electroporation method [cf. Neumann, E., et. al., (1982), EMBO J, 1, 841-845]. A desired transformant can be obtained by these methods.
Preferably, COS-1 cells are co-transfected with two expression vectors: one containing DNA encoding the heavy chain and the other containing DNA encoding the light chain, to provide a transformant producing the recombinant anti-Fas antibody. However, the method of producing the recombinant anti-Fas antibody is not limited thereto. For example, it is also possible to construct only one expression vector containing both the DNA encoding the heavy chain and the DNA encoding the light chain, which is expressed simultaneously, and to transfect COS-1 cells therewith.
Desired transformants obtained by the above methods may be cultured using conventional methods, the recombinant anti-Fas antibody being expressed either intra- or extra-cellularly. Suitable culture media include various commonly used media, depending on the host chosen. For example, suitable media for COS-1 cells include RPMI-1640 and Dulbecco""s Modified Eagle Medium (DMEM), which can be supplemented with, as desired, fetal calf serum (FCS).
The culture temperature for culturing the transformant may be any suitable temperature which does not markedly depress the protein synthesis capability of the cell, and is preferably in the range of 32 to 42xc2x0 C., most preferably 37xc2x0 C. If desired, the culture may be effected in an atmosphere containing 1 to 10% (v/v) carbon dioxide.
The fraction containing the anti-Fas antibody protein produced intra- or extra-cellularly by the transformants as described above may be isolated and purified by various well known methods of separation according to the physical and chemical properties of the protein. Suitable specific methods of separation include: treatment with commonly used precipitating agents for protein; various methods of chromatography such as ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography (HPLC), dialysis and combinations thereof.
According to the method described above, the highly pure recombinant, anti-Fas antibody can be readily produced in high yields.
In order to confirm that a recombinant anti-Fas antibody prepared by the above method specifically binds to Fas, an ELISA may be preferably performed in a manner similar to that described above for the evaluation of antibody titers in immunized mice.
On the other hand, a compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity, for example the compounds represented by the above formulae (I) to (XIX) can be produced according to the literature description referred to in the earlier explanation for each of the compounds. It is possible to determine whether other compounds have a folate antagonist activity or a dihydrofolate reductase inhibiting activity according to the methods for confirming a folate antagonist activity or a dihydrofolate reductase inhibiting activity, utilized in the literatures respectively.
The pharmaceutical composition of the present invention containing the above compounds as active ingredients can be used as an agent for prophylaxis and/or treatment of an autoimmune disease or rheumatoid arthritis in a mammal, for example, a human. Such a prophylactic or therapeutic agent may be administered in various forms. Examples of such forms include oral administration, with tablets, capsules, granules, powders, syrups or the like, or parenteral administration, with injection, dropping injection, suppositories or the like.
The amount of the anti-human Fas antibody and a dose of the compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity in the pharmaceutical composition of the present invention can vary depending on activity inherent in each antibody or each compound to be used. For example, when CH11 or HFE7A is used as the anti-human Fas antibody, and methotrexate is used as the compound having a folate antagonist activity or a dihydrofolate reductase inhibiting activity, it is preferable that the pharmaceutical composition of the present invention is prepared as a solution containing 0.1 to 100 mg/ml of CH11 or HFE7A and 0.05 to 5 nM of methotrexate, but the present invention is not limited thereto.
The dosage of the anti-human Fas antibody will be 0.001 to 10 mg/kg for a one time daily administration. The dosage of the compound having a folate antagonistic activity or a dihydrofolate reductase inhibiting activity will be 0.15 xcexcg/kg to 0.15 mg/kg for a one time daily administration.
The dose will vary, depending on factors such as the condition, age and body weight of the patient, but usually it may be administered in an amount of from 0.1 mg to 1,000 mg at one time as the amount of anti-human Fas antibody by a subcutaneous, intramuscular or intravenous injection.
The efficacy for treatment of the autoimmune diseases or rheumatoid arthritis by using the pharmaceutical composition of the present invention prepared as described above can be checked by culturing cells (for example, human lymphocyte cell stock HPB-ALL (cf. Morikawa, S., et al. (1978) Int. J. Cancer 21, 166-170 to reference), Jurkat (American Type Culture No. TIB-152), synovial cells originating from the rheumatoid arthritis patient or the like) in a medium to which the pharmaceutical composition of the present invention is added, and measuring viability by methods such as MTT assay (cf. Green, L. M., et al. (1984) J. Immunological Methods 70, 257-268) or XTT assay indicated in the following examples. The pharmaceutical composition of the present invention can induce apoptosis in self-reactive lymphocytes which is one of the main causes of an autoimmune disease and in synovial cells proliferating abnormally in the rheumatoid arthritis affected part at a lower dose of anti Fas antibody, compared with the case where only an anti-Fas antibody is administered, and thus it is effective in treatment of these autoimmune diseases or rheumatoid arthritis. It has been reported also in European patent Application Laid-open publication No. 0909816 that the composition containing the anti-Fas antibody accepted to be effective in the experiments using the above-mentioned cultured cells is actually effective for treatment of autoimmune disease and rheumatoid arthritis with results of the experiment using the animal experiment model.

Pharmaceutical compositions containing anti-Fas antibody


The present invention develops a manufacture method, via conventional liquid metallurgy, of finished and semi-finished metallic parts as casting, ingot, blooms and slabs in alloys base Fe, base Ni and base Co, microstructurally reinforced with complex molybdenum and titanium carbide particles, by means of their previous elaboration and latter addition to the molten alloy in the melting furnace. Then, when the alloy solidifies, they are inserted and distributed within the grains of the base metallic matrix, enhancing their mechanical properties and behavior at room as well as at high temperatures.

The composite components ceramic-metal, obtained by powder metallurgy and conformed by means of the introduction of ceramics in the metal matrices bases softer are meriting an ample acceptance by its properties of cutting and wear resistance. The ceramic powders that more usually are utilized for these applications are refractory metal carbides like those of titanium, tungsten, zirconium, etc.
On the other hand, the improvements that could be obtained, through the reinforcing with hard particles, in the properties of structural alloys and surface layers, as an increase of the mechanical resistance, to the wearing, thermal stability, etc., also are accepted and recognized.
Overall ceramic materials of reinforcement, one of most attractive is the titanium carbide, by its high hardness, thermal stability and low density, but the particle incorporation of this carbide as layering material, cermets and cast metallic products (finished or semi-finished) has great technical difficulties, due to their propensity to the oxidation, poor wettability and segregation tendency.
With the aim to resolve some of these difficulties, patent WO9303192 develops powdered products based in TiC and with additions of W, Fe, Ni, Co, etc., in order to reinforce cast iron alloys, welding material and surface layers.
Nevertheless, it has been verified that these products are inadequate for their incorporation, conventional metallurgy process, to molten alloys base Fe (steel or iron), base Ni or base Co, due, mainly, to the poor wettability of the TiC into these liquid baths.
In the other hand, patent WO9411541 develops a process for the Fe-base alloys reinforcement by means of the addition of carbides of the liquid metal. Some of the disadvantages and problems of industrial application of this patent are the following ones:                To improve the carbide wettability, it is necessary to cover it with metallic elements as iron, nickel, copper, titanium and carbon. It is not indicated how one obtains carbide nor the form to cover it but, in any case, must be expensive.        The particles have to have similar density to the one of the base material. For this reason, it is necessary the availability of complex carbides of titanium and tungsten. It is not indicated how to obtain them but, in any case, the process must be more complicated and expensive than the one from the present invention.        It is not contemplated its application to materials base Ni and base Co.
In the ferrous alloys field, its application does not include the steels with ≦0.3% C.                In the better case, the addition yield (carbides inserted in the matrix/added carbides) is not higher than 50%.
Therefore, the improvements obtained by this method is negatively affected by the mentioned aspects, as well as from the technical-operative point of view (problematic particle availability, half of added particles are coalesced together, being agglomerated in the lining of the melting furnace, delaying the heat, dirtying the metal, etc.), as economic aspect (the very high cost of particles must be multiplied by two in the end product). In addition, it does not include the steel family more indicated for its reinforcing, those of low carbon, that by their high toughness and low wear resistance, needs to increase this one without damage that one.
For that the reinforcement of the metallic materials by means of the liquid metallurgy process become technical and economically viable at industrial level it requires of the development and use of a new type of reinforcing material, technically adapted for its incorporation to the molten metal (with a similar yield to the one of the rest of the alloying materials) and with the lower fabrication cost possible, as well as of the optimization of the metallurgical process of addition of the reinforcing material, at solid state, to metallic alloys in liquid state. In addition, the properties improvement of the reinforced alloy are a direct function of the nature of the particles of reinforcement, their homogenous and uniform intragranular distribution, without coalescence among them, of the size and regular morphology of the same ones and their metallurgical coherence with the base alloy.
Then, the present invention is based, first of all, in the design and making method of the reinforcing material that fulfils the previous technical-economic requirements. In this sense, it has been verified that the complex carbides of X(MoTi)C type, where X can be Fe, Ni or Co, and with the chemical composition that will be defined below, they maintain the inherent advantages to titanium carbide and allow to overcome the technical and economical limitations above indicated in its applications as structural metallic alloy reinforcement.
Effectively, in order that any particle type could be used as metallic alloy reinforcement, by its incorporation, in solid state, to the metallic alloy in liquid state and the later metallurgical insertion once it has solidified, must be increased, to the higher level possible, the wettability of the particle into the mentioned alloy. For it, the contact angle between the solid particle and the liquid must be the smallest possible and this value almost totally depends on the chemical nature and compositions of both materials.
In this sense, during the development of the present invention it has been verified that, unlike particles of TiC and (WTi)C, the solid particles formed by carbides of (MoTi)C type are easily wet by molten alloys base Fe, base Ni and base Co.
In addition, due to the very negative formation energy of the titanium carbide (to any temperature) and of molybdenum carbine (above 1500° C.), the proposed procedure for it making is simple, economic and perfectly applicable to industrial level.
Effectively, this procedure is based on the extremely exothermic nature of both formation reactions:Ti+C→TiC+35000 cal./atom.gr.C2 Mo+C→Mo2C+30000 cal./atom.gr.C (over 1500° C.)
This means that the formation of the TiC from its constituent elements is very advantageous from the thermodynamic point of view, just like the one of the Mo2C when is reached (or exceeded) the temperature of 1500° C. On the other hand, once the first reaction begins, the temperature rises quickly (due to the liberated heat), facilitating the second reaction, unlike the case in that the complex carbide is of the type (WTi)C, where the wolfram carbide has to add previously made. The mentioned facility of reaction is advantageous, in addition, in order that the reactive elements could be diluted by means of the addition of other metallic elements, as Fe, Ni or Co, that melt to the temperatures that are being reached during the reaction and act as binders of produced carbides. During the later process of its addition to the molten alloy, the binder melts on contact with this one and increases the wettability and metallurgical insertion of the reinforcing carbide.
On condition that this reinforcement material can be obtained to a low cost, as it happens with the process that will be below described, the design of the same one also includes the proportional quantification of each chemical element (in function of the TiC), having been contrasted that the optimal results are reached with the following percentage values.    TiC: 50-90%    Mo: ≧5%    X (Fe, or Ni, or Co): ≧5%    Ti/C ratio in carbide: Approximately equiatomic (one atom of Ti by one of C).
Besides the high yield of the addition of these carbides to the alloy to be reinforced and the degree of reinforcement that is from this one, the cost of the end product is lowered by the use of appropriate techniques for the previous manufacture of such carbides. These techniques are based on the self-propagating high temperature synthesis reactions of chemical compounds strongly exothermic, as it is the case of the present carbides. To achieve the technical goal of the invention, in case that the necessary amount of carbides is small, the previous manufacture of such carbides it can be made by means of a discontinuous process, in which, once prepared and mixed a certain amount of raw materials, they are introduced in a reactor where the reaction of synthesis to high temperature takes place, and when reaction product cools, it is extracted of the reactor, repeating, in this way, the production cycle until the attainment of the required amount. Nevertheless, during the development of the present invention it has been stated that the productivity of this process is sensibly increased and the production cost decreased using a process in continuous, developed previously by FUNDACION INASMET for the obtaining of other ceramic materials. This process, along with necessary productive equipment for its application, is gathered in patent EP1060789: "Continous procedure for manufactures of powdered materials by combustion and reactor for the realisation thereof".
Due to the high exothermic character of the (MoTi)C carbide reaction formation, as well as to the facility whereupon the required thermodynamic conditions after their beginning and accomplishment can be reached, the synthesis, from the raw materials that contain the adapted proportions of C, Ti, Mo and Fe or Ni or Co, can be made also in continuous, according to the process described in the mentioned patent, being increased by this way the productivity, which allows, on the one hand, the possibility of having great amounts of reinforcement material and, on the other hand, of reducing its production costs. In addition, the necessary reactive elements can be incorporated in bi or trimetallic alloy form available in the market to smaller cost than individual metals, like Fe—Ti, Fe—Mo, Ni—Ti, Co—Ni—Mo, etc.
In any of both ways the necessary reinforcing product for each alloy family is obtained: Fe(MoTi)C for the reinforcement of steels and cast irons; Ni(MoTi)C for alloys base nickel; and Co(MoTi)C for those of base cobalt. The titanium and molybdenum carbide particles, 1-4 microns size and rounded shape, are agglomerated by the metal Fe in the first case, by the metal Ni in the second case and by the metal Co in the third case.
The second aspect of the invention lies in the process of addition of these new reinforcement materials to structural metallic alloys base Fe, base Ni or base Co.
As it has been mentioned previously, the presence of Mo in the complex carbide, sensibly improves its wettability within of Fe, Ni and Co alloys, wettability that is increased when these carbides are agglomerated by the same metal that the basic constituent of the alloy, which they are tried to add into. In addition, due to its very elevated negative formation energy, they are very stable as much in low temperatures as in the temperatures in which the mentioned alloys are in molten state (of the order of 1.500° C.). Therefore, the manipulation and processing of this material are simple and do not need special measures to avoid their deterioration in contact with the environmental atmosphere, either by oxidation or other causes, nor their decomposition in the liquid bath. Nevertheless, the yield of their addition to this one and the distribution of small carbide particles in the matrix of the metallic alloy play an important role in the final result. Thus, whichever greater it is the yield of the addition (carbides present in the final product/carbides added to the molten alloy), lower is the cost, on equal characteristics, of this final product. Also, in such case, the cleaning, deslagging and adjustment of the liquid alloy in the melting furnace are less complicated and onerous. On the other hand, the improvement of mechanical characteristics of the reinforced alloy is direct function of the homogenous one and uniforms intragranular distribution of carbides, without coalescence among them, of the rectangular morphology of them and its metallurgical coherence with the base material.
In the case of the present invention, the optimization of such parameters has been obtained, on the one hand, by means of the appropriate design of carbides and their obtaining process, as it has been described previously, and, on the other and, by means of the following sequential procedure of addition in the melting furnace:                a) Adjust of the material to be added. The X(MoTi)C reinforcement material, in a not higher than 10% proportion in weight of the total charge of the bath, is mixed with commercial Si—Ca normally used as desgasifier element of mentioned alloys, in proportion of around 10% of the reinforcement material.        b) Melt of the metallic alloy, base Fe (steel or cast iron), base Ni or base Co, according to the conventional process and in furnaces, usually used for the melting of these materials.        c) Addition of the reinforcing material mixed with Si—Ca. This begins when the base charge is in molten state and at a temperature of 150° C. higher than the "sólidus" temperature of the alloy. The operative way of the addition is similar to the one of any other material provided in the conventional metallurgical process (ferroalloys, more or less pure metals, etc.), for the adjustment of the chemical composition of alloys. One of the novel aspects and optimizers of the invention, is indeed in the positive and determining effect of Si—Ca, since, as it has been verified, if the X(MoTi)C is only added (in relatively fine and powder way), tends to coarse together and to deposit in the walls of the furnace, whereas when the Si—Ca is incorporated to the molten bath, takes place a reaction of exothermic oxidation of the Ca with the oxygen of the same one, giving rise to a reduction of the liquid bath-carbide particles surface tension and favoring the insertion and dispersion of these in its sine.        d) Once the addition has been completed, the resulting liquid material, metallic alloy+reinforcing carbides, is processed in the conventional way, being able to be poured in molds (sand, ceramic, metallic, etc.) to obtain castings, or in ingot-molds, to obtain semi-processed products.
The yield of the addition is over 80%, reason why the losses can be considered as normal as in any other metallurgical operation of adjustment of chemical composition of an alloy.
As a result, castings or semi-processed products are obtained, with a microstructure composed by the corresponding metallic matrix (base Fe, base Ni or base Co) plus titanium and molybdenum carbide particles inserted within it, of high hardness and thermal stability, 2-5 microns of size and regular shape, coherently bonded, dispersed and homogenously distributed inside the grains of the matrix. This product can later be put under any type of heat treatment, thermo-chemical or thermo-mechanical, without the carbide particles undergo any deterioration.
FIG. 1 demonstrates the microstructure of (MoTi)C carbides made using the process above, and FIG. 2 demonstrates the microstructure of the reinforced material.
Consequently, the product thus obtained has got the own characteristics of the metallic matrix base, according to the alloy and of its later processing, improved and increased by the reinforcing and hardening effect of inserted carbides. These enhanced effects are related, mainly, to the fatigue and wear resistance at room temperature, and to the creep and abrasion resistance at high temperatures. Taking as reference the values of the characteristics corresponding to the metallic alloy without reinforcement, the increase obtained on these values, with new products, is higher than 50%.

Product manufacture in structural metallic materials reinforced with carbides


The invention concerns a home kit and a method for detection of food allergies by determining the presence of antibodies against specific antigens in a stool sample.

The present invention is generally in the field of kits, and more specifically concerns kits for domestic use for the self-detection of various physiological conditions.
The xe2x80x9cfood allergyxe2x80x9d refers to adverse immunologic reactions to food. Food allergy is typically mediated by IgE antibodies directed to a specific food protein, but other immunologic mechanisms can also play a role in this phenomena. The primary target organs for food allergy reactions are the skin, the gastrointestinal tract, and the respiratory system.
Both acute reactions (hives and anaphylaxis) and chronic diseases (asthma, atopic dermatitis and gastrointestinal disorders) may be cause or exacerbated by food allergy.
Foods commonly causing these reactions in children are: milk, eggs, peanuts, soy, wheat, tree-nuts, fish and shellfish. In adults, the most common foods are: peanuts, tree-nuts, shellfish and fruit.
Several specific tests have been developed for the evaluation of IgE-mediated food allergies, in order to identify or exclude responsible foods.
One method of determining the presence of specific IgE antibodies to various allergens, among them food allergens is the xe2x80x9cprick-puncture skin testingxe2x80x9d. While the patient is not taking antihistamines, a device such as bifurcated needle or a lancet is used to puncture the skin through glycinerated extract of a food and also through appropriate positive (histamine) and negative (saline-glycerine) control substances. A local xe2x80x9cwheat-and-flarexe2x80x9d response indicates the presence of food-specific IgE antibodies, wherein a wheal having a diameter of more than 3 mm is typically considered as indicating a positive response.
Prick skin tests are most valuable when they are negative since the negative predicted value of this test is relatively high (estimated to be above 95% for IgE mediated reactions). However, the positive predicted value of these tests for such IgE mediated reactions is only on an order of 50%. Thus a positive skin test in isolation cannot be considered as a proof of clinically relevant hypersensitivity. Another disadvantage of the prick skin test is that many patients are reluctant to be repeatedly pricked for identifying the responsible food. Furthermore, the test is quite time consuming, typically requiring several test sessions at the practitioner""s office, each one for testing several suspected foods.
In vitro blood testing for specific IgE (Radioallergosorbent tests (RAST)) is another option for diagnosing food allergies. As with skin tests a negative result in RAST testing is relatively reliable in ruling out an IgE-mediated reaction to a particular food, however a positive result has low positive predicted value. Another disadvantage of RAST is that it is an invasive test requiring obtaining a blood specimen from the patient and sending it to a laboratory to be analyzed, which analysis is quite expensive. Furthermore, since the test cannot be carried out in the practitioner""s office the results are very often delayed.
Double-blind placebo-controlled food challengers are another option for diagnosis of food allergies. According to this option, patients avoid the suspected food for at least two weeks, while discontinuing antihistamine therapy. Other allergy medication, such as anti-asthma medication are reduced as much as possible. Then graded doses of either challenge food or placebo food are administered to the patient. The food is hidden under another food or is taken as capsules as it is consumed without the patient knowing its nature. Medical supervision and immediate access to emergency medications including antihistamines, steroids and inhaled beta antagonists, and equipment for cardiopulmonary resuscitation are required since the allergic reactions can be severe and may even include shock. It is clear that, while such labor intensive and potentially dangerous procedures can detect acute reaction to the food allergies, they can not detect chronic reaction and in no way can be considered as standard procedure for determining allergies to foods.
There have been a number of studies indicating the problematic high false negative results of various food allergies when blood and skin tests are performed (Thompson et al., The Lancet, Jan. 22, (1983)).
Further research involved testing the presence of antibodies in mucosal secretions (such as feces) in connection with food allergy testing. It was found that a higher percentage of children with allergy feature IgE in feces compared to the rare occurrence of IgE immunoglobulin in extracts of feces from healthy children (Kolmannskog, Int. Archs Allergy Applic. Immunol. 76:133-137, (1985)). The hypothesis was that the IgE may at least in part have been produced locally in the gut mucosa. These findings were supported by the findings that increase Copro IgE (in faecal extracts) are a specific consequence of the local immune response to food allergy simulation in gut mucosa (Sasai et at in Allergy, 47:594-598 (1992)).
Self et al. (JAMA, March 31, 707:13, 2393 (1969)) demonstrated stool precipitants against dietary antigens being in children suspected of having various food allergies. These studies indicate the initiation of antibodies to specific food antigens, which could be detected in the stool of the allergic children.
The studies by Self et al. further suggested that the precipitated antibodies are contained in the immunoglobulin A (IgA) fraction of the intestinal secretion, which finding is also in accordance with the fact that most plasma cells of the intestines contain IgA and, unlike serum, intestinal secretions contain large amounts of IgA and lesser amounts of the of other immunoglobulin.
The traditional model of clinical laboratory tests has been that a physician orders a test, a biological specimen such as blood, urine, throat culture, etc. is obtained and sent to a clinical laboratory, and days or weeks later the laboratory result is sent to the physician. The patient then returns to the physician""s office to learn result and received advice concerning his or her medical condition. While this model is appropriate for many complicated physiological conditions, there are many other physiological situations where the patient is, or can easily be, well equipped to understand the results of a lab test, has an interest or need to know the result immediately, and is capable of performing the test by himself, and understanding the test results.
Thus, over the past decades home tests for pregnancy have become very popular. Home tests for glucose for diabetic patients have also become very popular. Other tests, such as tests for cholesterol levels, are beginning to gain wide popularity. Still other tests, such as those for day of ovulation are not yet widely known, but are gaining market acceptance.
Thus, a substantial market exists for home diagnostic tests and there is constant need to develop additional home tests for further physiological conditions.
The present invention is based on the realization that there is a need for a non-invasive, fast, accurate, and user friendly method for diagnosing food allergies, both in adults and in children. The present invention is further based on the realization that detection of food allergy is of the type of detections which may be carried out at home, or at a doctor""s clinic, without involving an analyzing laboratory, since the patient (or doctor) can easily understand positive result of a food allergy test, and in many cases can even self treat himself/herself, simply by avoiding the food which caused the food allergy.
The present invention is further based on the realization it is possible to develop a kit for such a non-invasive, reliable and home (and practitioner""s office) testing.
The present invention is further based on the realization that such a home kit for determining food allergies, can be constructed for detecting antibodies against food antigens present in stool. Utilization of stool instead of skin or blood as the specimen of the diagnosis of antibodies has the advance that the test is non-invasive, and furthermore features the advantage of greater reliability of antibody testing, as most of the allergic reaction to food takes place in the mucosa of the gastrointestinal tract.
Thus the present invention by its first aspect concerns a home kit for detection of a food allergy in an individual, the kit comprising:
(a) a vessel for mixing a stool specimen with a diluting liquid to produce a diluted stool specimen;
(b) a housing holding within a substrate, the substrate comprising at least one zone containing food antigens, the housing further comprising reagents for producing a visually detected reaction when an antibody-food antigen complex is formed, the housing further comprising at least one conveying means for receiving diluted stool specimen and transferring kit to the food-antigen containing zone of the substrate;
(c) the housing further comprises an indicator for showing the presence of the visually detected reaction.
The term xe2x80x9chome kitxe2x80x9d in the contents of the present invention refers to the fact that the kit of the invention, and the detection reaction produced therein, does not necessitate any complicated machinery for collecting the specimen and preparing it, for positioning the specimen in the kit, and for reading and interpreting the resultsxe2x80x94and typically, the results can be viewed by the naked eye, or by simple optical reactor. The term does not necessarily mean that the kit is only operable at home, since due to its non-invasiveness and it is easy, user friendly manner of operation, it can be also used in a practitioner""s office or even in hospitals without involving an analytical laboratory.
The kit of the present invention comprises a vessel in which a small amount of stool specimen can be placed and diluted by a suitable diluting liquid. The vessel can then be sealed and the stool and diluting liquid shaken to produce a diluted stool specimen. The diluting liquid may be plain tap water but according to a preferred embodiment of the invention the diluting liquid is, saline, distilled water, 10% formalin solution, sodium acetate solution with or without detergent and the like, and this diluting liquid is also provided as part of the kit""s present invention, either a priori present inside the vessel or in a separate container.
The vessel may be for example in the form of a regular capped tube, having graduations, which indicate the volume of the raw stool specimen which is to be placed inside the tube, as well as the amount of the diluting liquid to be added.
The kit may also comprise a construction for collecting the stool, such as a disposable sheet to be placed inside a toilet bowl, a disposable vessel for stool collection, etc., as well as a scooping device, for example in the shape of a small spoon to pick a determined amount of stool. The scooping device (scoop) may be an integral part of the vessel""s cap.
The kit""s main component is a housing which holds within a substrate, for example, the housing may be a plastic container, having a substrate layer sandwiched between two plastic layers of the container. On a predefined zone of the substrate the food antigens are present, and by a preferred embodiment they are immobilized on the zone. Examples of the substrates are absorbent materials such as nitrocellulose sheets, gel-films, cellulose acetate, fiberglass sheet, paper, agarose gels, and in general any media featuring capillary force or absorbent forces of fluid. Typically the housing has at least one conveying means which can receive the diluted stool specimen and transfer it to said zone. The conveying of the liquid may be by capillary or absorbing flow, which are due to the inherent properties of substrate or inherent properties of a specially designed layer or by the construction of specific flow channels which bring the fluid to the antigen-containing zone.
By one option the conveying means are a combination of openings in the container associated with a construction which can transfer to diluted stool specimen to the zone on the substrate which holds the food antigens and where the antibody-antigen interaction takes place. For example, the conveying means are in the shape of an opening in the housing through which a small amount of the diluted stool can be poured. The stool is then transferred to the antigen-containing zone of the substrate, for example, by capillary forces either of the substrate itself (which is made of absorbent material) or by capillary or absorbed forces of a specially designed layer which sole purpose is to transfer the diluted stool to the antigen containing zone, or by flow in specially designed channels.
By another option the conveying means is an absorbent material or material composed of capillaries which protrudes out of the housing, for example, an absorbent stick protruding out of an opening in the housing. In such a case the protruding substrate material is dipped in the diluted stool and due to capillary forces the liquid is transferred to the antigen-containing zone which is present inside the housing.
The antigens may be immobilized on the substrate by an interaction such as covalent bonds, hydrogen bonds, electrostatic forces contained within voids of beads, etc.
Typically, large particles have to be filtered out of the diluted stool before the diluted stool is conveyed to the antigen-containing zone of the substrate. Said filtering may take place in the vessel itself, for example by constructing a two part cap: the more distal part serving as a seal, which hermetically closes the vessel and allows the user to vigorously mix its contents. However, this cap may be opened fully or partially to expose below a filter sieve which can ensure that only relatively small particles are poured from the vessel into the convening means.
Alternatively, the housing itself may comprise said filtering sieve, which for example may be present either at the mouth of the opening of the convening means, or may be present as a continuous filter sieve layer above the substrate zone on which the food antigens are present.
By a preferred option where the substrate is an absorbent material, the stool particles may be sieved on its upper layers of the substrate so that a filtered specimen reaches the layer of immobilized antigen.
The food antigens should be chosen in connection with the type of food allergy which is to be detected by the kit of the invention.
By one embodiment, the kit may comprise a single food antigen and in such a case it can give a binary (yes/no) indication, whether the individual is allergic to that specific antigen. Alternatively, the indication may be semi-quantitative, for example, by giving three shades of the same colorxe2x80x94a darker shade indicating xe2x80x9chighxe2x80x9d (level of antibody and allergy), than medium shade xe2x80x9cmediumxe2x80x9d, and light shade xe2x80x9clowxe2x80x9d.
By another embodiment, the kit of the present invention includes a plurality of food antigens, and in such a case it is possible to determine, in a single assay, whether the individual is allergic to a variety of foods.
Kits in accordance with a preferred embodiment are generally divided into two groups: kits for children and kits for adults and these are chosen with the most common foods which cause allergy in any of these two groups.
The kits for children typically include the most common in food antigens to which children are allergic and include the following:
(a) antigens from cow""s milk (which may include BLG, ovalbumin, casein) or whole extracts of milk;
(b) antigens from soy protein (which may include whole extracts of soy);
(c) antigens for peanuts which include whole extracts of peanuts;
(d) antigens from wheat which include whole extracts, or gluten from milk;
(e) antigens to eggs which include whole extracts of egg.
Kits for adults typically include antigens from various vegetables and fruits, grains and nuts.
Antigens for the above are commercially available from a plurality of sources and may include protein as well as non-protein. Typically, regions of the substrate surrounding the zone on which the antigen is immobilized are saturated by non-specific hydrophilic polymers such as bovine serum albumin, other proteins, or polyethylene glycol to block unspecific binding of the antibody to the substrate.
Interaction between the food antigens, a priori present in the kit, and antibodies which are present in the stool specimen, yields an antigen-antibody complex. In the kit of the invention the presence of such a complex should produce a visually detected reactionxe2x80x94i.e. a reaction which produces a visible indication, which may be viewed either by the naked eye, or by an optical reader. Examples of such a reaction is a color reaction (achieved by ELISA method) or a precipitation reaction which can easily be detected. A plurality of methods for producing visually detected reaction for antigen-antibody complexes are well known in the art, for example, as specified in xe2x80x9cImmunoassay Handbookxe2x80x9d by David Wild 2nd Edition, Nature Publishing Gropu, pp. 150-175 and 271-277. An example is indirect ELISA, a procedure which is used to identify the presence of antibodies utilizing, for example, anti-human antibodies conjugated to a visually detectable moiety (such as gold particles) or conjugated to an enzyme producing a color reactor). Another possibility is by detection of the presence of antibody-antigen aggregates, for example, as specified in the publication of Self (Supra).
The indicator is typically an opening in the housing (xe2x80x9ca windowxe2x80x9d), which allows direct viewing of the visually detected reaction. Typically the opening is immediately above the region on which the antigens are immobilized. The view may be by the naked eye, for example, by the detection of colored bar, or a colored dot, or alternatively may be viewed by an optical reader, to increase sensitivity.
Where a single food antigen is to be detected, a single indicator can be used which when showing the indicator (color bar, dot, etc.) indicates that there exists antibodies to the specific food antigen.
Where a plurality of food allergies are to be detected a plurality of indicators (windows) can be used so that each indicator is associated with a single food allergy. Alternatively, a single indicator giving different readings can be used wherein each reading is in accordance with the specific antigen-antibody complex formed and thus each reading is indicative of a different food antigen.
For example, where five different food allergies are to be tested, it is possible to construct a housing with five different and separate antigen containing substrate zones, so that in each zone a different food antigen will be immobilized, and for each zone there will be associated a separate indicator. This will ensure that the visually detected reaction in each zone is specific to the food antigen present on the substrate of said zone, and the indicator (xe2x80x9cwindowxe2x80x9d) will simply be specifically associated with each zone. In the above case the housing may have a single means convening for example in the form of an absorbent layer.
The liquid stool specimen is poured into an opening, and due to the fact that the substrate is absorbent, capillary forces present in the absorbent material, cause transfer of the diluted stool specimen through all the separate zones on which are immobilized different food antigens. Then, the specific visually detected reaction is formed in each separate zone which reaction can be viewed by the specific indicator associated with that zone (for example by a xe2x80x9cwindowxe2x80x9d the container through which a colored bead can be viewed).
In accordance with a preferred embodiment of the invention the kit also contains internal control. The internal control is composed of antibodies against the antigens of the kit present, a priori, in the zone of the antigens. The purpose of these, a priori, present food antibodies, is to form aggregates with the food antigens, to produce a visually detected reaction, in order to determine that the reagents used for producing the reaction are functioning properly.
The antibodies detected by the kit of the invention may be any antibody, monoclonal or polyclonal against a food antigen but typically the kit detects total immunoglobulins, or alternatively, IgA, and also IgE antibodies.
The present invention also concerns a method for detecting the presence of a food allergy in an individual, suspected of having such an allergy, the method comprising:
(a) obtaining a stool specimen from the individual;
(b) diluting the stool specimen with a diluting liquid to produce a diluted stool specimen;
(c) introducing the stool specimen into the kit of the invention; and
(d) viewing the indicator, the presence of a visually detected reaction in the indicator, indicating the presence of food allergy in the individual.

Kit and method for detecting food allergies