Monoclonal antibody capable of recognizing the antigen, causing apoptosis of myeloid cells in the bone marrow and have the ability to cause apoptosis of myeloma cells, its fragment and hybridoma

 

The invention relates to medical biotechnology and is concerned with the obtaining of monoclonal antibodies (MAB) and its fragments using hybridoma FERM BR-4382, which produces the above-mentioned antibody. To obtain hybridoma as antigens using recombinant granulocyte colony-stimulating factor rG-CSF. Produced MAB belongs to the IgG class. The fragment of MAB is a F(ab)2the fragment. MAB can recognize the antigen, causing apoptosis of myeloid cells in the bone marrow, but also has the ability to cause apoptosis of myeloma cells, which allows its use as a drug, is effective for use in case malacitana leukemia. 3 S. and 4 C.p. f-crystals, 21 ill.

The technical field to which the invention relates the Invention relates to novel monoclonal antibodies that have the ability to cause apoptosis of myeloid cells and can therefore be considered as a useful drug for use in case malacitana leukemia, as well as fragments of such antibodies, in addition, the invention relates to hybridoma producing monoclonal antibodies.

Because monolithically antigens, which induce apoptosis in myeloid cells, but also, themselves have the ability to cause apoptosis of myeloid cells, based on these properties, they can be considered as medicines, is effective for use in case malacitana leukemia.

Background of invention Granulocyte colony-stimulating factors, such as, for example, recombinant granulocyte colony-stimulating factors [RG-CSF (rG-CSF)], were first known as humoral factors that stimulate differentiation and proliferation of granulocyte cells, while in experiments on mice in vivo was shown that the introduction of RG-CSF stimulates haematopoiesis in the bone marrow and, in addition, causes a marked extramedullary haematopoiesis implemented in the spleen with the proliferation of hematopoietic stem cells and progenitor cells present in the process of hematopoiesis in the spleen. It is considered that the mechanism of haematopoiesis in the spleen, as extramedullary by its nature, is due to modifications in the microenvironment in the process of hematopoiesis in the spleen, which arise in connection with stimulating effect of RG-CSF enhances hematopoietic potential is Eden RG-CSF, to determine the level of hematopoietic potential in the spleen, then installed the hematopoietic stroma cell line (CF-1 cells from spleen of mice, which was introduced WG-CSF, to study the existence of the fact of increasing hematopoietic potential under the influence of stromal cells containing the introduced WG-CSF demonstrated with the use of hematopoietic stromal cells, the potential effect on haematopoiesis and finally determined the colony-stimulating activity in vitro, and the ability to support hematopoietic stem cells in vitro (Blood, 80, 1914, 1992).

However, despite the fact that on the basis of a number of stromal cells in the spleen was able to get cell line (CF-1 cells) and to determine their cytological characteristics, still not received specific antibodies that recognize cell surface antigens, as not known up to the present time and the characteristics of such antibodies.

In this regard, the authors of the present invention conducted a study with the aim of obtaining, on the basis of the above information concerning the stromal cells of the spleen, as well as the results of the research, specific antibodies capable resposnes cell lines spleen as antigens for immunization, in the result, there have been new, not previously known monoclonal antibodies.

In the study of the properties of the obtained monoclonal antibodies, the authors found that these antibodies have the ability to cause apoptosis of myeloid cells, and this fact put an end to the research that formed the subject of the present invention.

Disclosure of the invention the Purpose and object of the present invention is the creation of new monoclonal antibodies, which are able to induce apoptosis in myeloid cells and in this connection it is interesting from the point of view of their use for the treatment malacitana leukemia, besides getting their fragments, as well as hybridoma capable of producing monoclonal antibodies.

Monoclonal antibody of the present invention is important from the point of view of its application as antibodies that recognize antigens that cause apoptosis [he is also known as the process of self-destruction of cells, a phenomenon in which DNA nuclear chromatin is cleaved in the nucleosome (with the formation of the so-called "ladder-sequence"] , which leads to cell death) myeloid cells, and using his characteristic functions Eden is nom case myeloid cells include cells, non-lymphoid cells, such as neutrophils, megakaryocytes, myeloblast, plasmic order has been revealed, mast cells, macrophages, monocytes and urethroplasty, so in the context of the present invention, the term "myeloid cells" has the above meanings. Still have not been known monoclonal antibodies having the ability to cause apoptosis of myeloid cells, so the monoclonal antibodies of the present invention include all monoclonal antibodies capable of inducing apoptosis in myeloid cells.

In General, monoclonal antibodies of the present invention can be obtained using the following method.

Namely, the monoclonal antibody of the present invention can be obtained, for example, by using stromal spleen cells taken from animals entered as antigen RG-CSF, immunization them, which is carried by the traditionally accepted method of merging immunized cells using standard procedures mergers and cloning the fused cells using a known method of cloning.

As the preferred method of obtaining monoclonal antibodies of the present invention may be a method, include the o which is administered RG-CSF, and which are established by the authors of the present invention as culture cell lines (Blood, 80, 1914, 1992), fusion plasma cells (immune cells) from a mammal immunized with the antigen with myeloma cells of a mammal, in particular a mouse, clone obtained fused cells (hybridomas), selection of clones producing antibody of the present invention, which is able to recognize the specified cell line among others, and cultivating them with the yield of the target antibodies.

However, this method is shown only as one possible example, so in this case for obtaining, based on the same method as in the case of CF-1 cells, antibodies, communicating with interesting myeloid cells as antigens can be used not only the above-mentioned CF-1 cells, but also cell line obtained in a manner analogous applied in the case of CF-1 cells, stromal cells in the spleen of man.

In accordance with the method of obtaining such monoclonal antibodies using those or other mammals can be immunized by the above-mentioned antigen is not bound by a particular restriction; however, the procedure merge cells while this method is preferable to work with mice, rats and hamsters.

Immunization is carried out in accordance with standard procedure, in particular through the introduction of by injection of stromal cells of the spleen, such as the aforementioned CF-1 cells in the peritoneal cavity of a mammal. More specific, it is preferable to introduce their animal when thinning or suspendirovanie in an appropriate amount of a phosphate buffer solution (FBI) or isotonic sodium chloride several times monthly. Preferably, in addition, be used as immune cells spleen cells selected after the last injection of the above-mentioned cells.

The second component necessary to merge cells, which represents the myeloma cells, preferably the use of a number of known cell lines, which include RH (RSH Hell 8.653) (y Immunol., 123, 1548, 1978), P3-V1 (Current topics in Microbiology and Immunology, 81, 1-7, 1978), NS-1 (Eur. J. Immunol., 6, 511-519, 1976), MPC-11 (Cell, 8, 405-415, 1976), SP 2/G-Ag 14 (Nature, 276, 269-270, 1978), FO (J. Immunol, Meth., 35, 1-21, 1980), S 194 (J. Exp. Med., 148, 313-323, 1978) and R 210 (Nature, 277, 131-133, 1979).

Merge cells using the above immunocytes and myeloma of kitchena et al (Methods Enzymol., 73, 3-46, 1981).

More specific, the above-mentioned cell fusion can be carried out, for example, in a simple nutrient medium in the presence of substances that accelerate the merger. As such accelerating cell fusion agent may be used polyethylene glycol (PEG) and Sandan virus (HVY), and, in addition, if necessary, increase the effectiveness of the merger practiced adding adjuvants such as dimethylsulfoxide.

As for the ratios of immune cells and myeloma cells, preferably using the first 1 to 10-fold amount relative to the second component of the mixture to merge cells. As examples of the environment, applicable for carrying out the above procedure merge, you can bring the environment R1-1640 and G environment (minimum supporting environment), which are well suited for the implementation of the mentioned proliferation of myeloma cells, as well as a number of other media conventionally used for culturing such cells which, in addition, may contain additional serum, in particular fetal calf serum (FCS).

Cell fusion is conducted by mixing the previously described quantities of the above immunocytes and myeloma cells in Viseu is heated to a temperature of about 37oWith, for example, such a PEG, which has an average molecular weight in the range from 1000 to 6000, followed by stirring. Then, by repeating the procedures of adding the appropriate media for each other, the centrifugation of the reaction mixture and removing supernatant can be obtained of the target hybridoma.

Mentioned hybridoma subjected to selection during cultivation on conventional selective medium, in particular on GAT medium (a medium containing gipoksantin, aminopterin and thymidine). The culture is grown at GAT environment for a time period sufficient for the cells, non-hybrid (naslite cells), were killed, usually this process takes from several days to several weeks. Then using the usual method of limited breeding conduct screening and cloning of hybridomas.

Received hybridoma capable of producing the monoclonal antibodies of the present invention may be further subjected to subculturing, after which they can be stored in liquid nitrogen for a long time.

For the purposes of obtaining monoclonal antibodies of the present invention from a hybrid method can be applied, including the cultivation of hybrid of piperazine in the body of a suitable mammal with getting them out of ascites. The first method is used for obtaining the antibodies of high purity, while the second is for the purpose of mass production of antibodies.

In addition, antibodies produced by the above methods can be purified to high purity by conventional purification methods, such as vysalivaniya, gel filtration and affinity chromatography.

Monoclonal antibody of the present invention may be any such antibody having a specific property, described in more detail later in the section of Examples, namely the ability to cause apoptosis of myeloid cells, all bearing such property antibodies included in the scope of the present invention, regardless of the type of antigens; monoclonal antibody of the present invention can be applied based on the realization that its properties as a medicinal product for the treatment of malacitana leukemia.

No need to say that creation on the basis of the monoclonal antibodies of the present invention are specific systems for the identification and recognition of antigens, causing apoptosis of myeloid cells, or for use as a drug for the treatment malacitana lei is istemi included in the scope of the present invention, to the extent that they are applicable in practice, using a technique that is obvious to every specialist, with an intermediate level of knowledge in this field.

A brief description of the drawings Fig.1 shows the results of immunofluorescence analysis (control in the absence of antibody, CF-1 cell).

In Fig. 2 shows the results of the study of binding capacity GSPST-1 antibodies with CF-1 cells on the basis of immunofluorescence analysis.

In Fig.3 shows the results of the study of binding capacity WMAR-1 antibodies with CF-1 cells on the basis of immunofluorescence analysis.

In Fig.4 shows the results of immunofluorescence analysis (control in the absence of antibodies, the cell of the bone marrow).

In Fig.5 shows the results of a study's ability to bind G-SPST-1 antibodies with bone marrow cells on the basis of immunofluorescence analysis.

In Fig.6 shows the results of the study of binding capacity WMAR-1 antibodies with bone marrow cells on the basis of immunofluorescence analysis.

In Fig.7 shows the results of immunofluorescence analysis (control in the absence of antibodies, NFS-60).

In Fig. 8 shows the results of a study's ability to kvazivariatsionnogo analysis (relative to control lgG1, NFS-60).

In Fig. 10 shows the results of the study of binding capacity WMAR-1 antibodies with NFS-60 cell-based immunofluorescence analysis.

In Fig. 11 illustrates a method of quantitative determination of monoclonal antibodies (VMAR-1), inhibits the proliferation of NFS-60 cells.

In Fig. 12 illustrates a method of quantitative determination of monoclonal antibodies (GS PST-1), inhibits bone marrow transplantation.

In Fig. 13 illustrates a method of quantitative determination of monoclonal antibodies (VMAR-1), inhibits bone marrow transplantation.

In Fig.14 shows [micrograph (painting hematoxylineosin) samples of bone marrow (400)] bone marrow cells (2), died on the 6th day after the introduction of monoclonal antibodies WMAR-1 of the present invention, and control (1) in the absence of antibodies.

In Fig.15 shows (picture motion electrophoresis) education ladder-sequence DNA in bone marrow cells, which may occur when administered monoclonal antibody WMAR-1 of the present invention.

In Fig. 16 illustrates a quantitative method for the determination of cytotoxicity using L-929 glue the determination of cytotoxicity using monoclonal antibodies (VMAR-1).

In Fig. 18 shows the results of immunofluorescence analysis (relative to control rat lgG2a, BWV1).

In Fig. 19 shows the results of a study's ability to bind antibodies to murine main complex pure compatibility MHC class 1 BWV1 cell-based immunofluorescence analysis.

In Fig. 20 shows the results of immunofluorescence analysis (lgG1 control for rats, BWV1).

In Fig. 21 shows the results of the study of binding capacity WMAR-1 antibodies with BWV 1 cell-based immunofluorescence analysis.

Explanation of symbols: DNA from mouse thymus, which was introduced WMAR-1 (24 hours) b: DNA from the bone marrow of the mouse, which was introduced WMAR-1 (24 hours) in: DNA from the bone marrow of the mouse, which was introduced WMAR-1 (8 hours) g: DNA from the bone marrow of the mouse, which was introduced WMAR-1 (4 hours) l: DNA from the bone marrow of untreated mouse (bone marrow cells)
e: the Marker for the determination of molecular weight
Next is a detailed description of the present invention in accordance with procedural and reference examples, not limiting the scope of the present invention.

Reference Procedure
Creating a line of stromal cells of the spleen and its characteristics

The procedure was as follows: after the introduction of RG-CSF in aseptic conditions from the animal to remove the spleen, cultivate it in the incubator at a temperature of 37oC in 5% CO2for 6 weeks in a plastic flask with a footprint of 25 cm3[Corning Co. (Corning Co.)], using the environment Dulbecco modification Iscove (IMDM) [(Boehringer-annheim Co. )] which made inactivated by heating 10% fetal calf serum[FCS (FBS [Sanko Junyaku, Tokyo (Sanko Junyaku, Tokyo)] 100 IU/ml penicillin and 100 μg/ml streptomycin, with the specified medium replaced with fresh medium to cultivate twice a week.

In confluentes culture selected populations of fused cells (stromal cells) from the flask using the FBI, not containing CA and mg, which are made of 0.95% trypsin and 0.02% EDTA (Sigma chemical Co.), and transferred into a new flask. Such action is repeated once or twice a week. First (the first ten times) the ratio of splitting cells ranges from 1/4 to 1/8, subsequently it decreases to values in the range of 1/16 to 1/32. After about the 10th transfer stromal cells become grout and sent for cell cloning using the technique of limited breeding; cell cloning was repeated twice with the establishment of lines of stromal cells (cell line CF-1).

Further, these cells support in the flask with a footprint of 25 cm3(Corning Co.) in 5 ml of medium 1 D with the addition of 10% V / V heat inactivated FCS and conduct subcultivation every five days when the splitting ratio of 1/32. Line stromal cells of the spleen can be created not only based on a mouse, but also from other animals; for example, using the above-described method can be derived stromal cell lines through cell transformation by the adenovirus vector SV-40 (J. Cell hysiol., 148, 245, 1991).

2) Characteristic of CF-1 cells
CF-1 cells installed in the manner described in the form of a cell line, was investigated using standard cytochemical methods for the analysis of alkaline phosphatase, acid phosphatase,-glucuronidase,-naphthyl-acetate esterase. In the study of CF-1 cells by the methods of enzyme histochemistry used the following monoclonal and polyclonal antibodies: macI [Cepo Tek. (Sero Tec. )] , antigen associated with factor VIII [Dakopatts (Dacopatts)], collagen type I, collagen type III and fibronectin of 1.09 μm. Sigma), and the ability of the CF-1 cells to become adipocytes was determined when exposed to phosphate hydrocortisone (Sigma) at a dose of 10-6mol/l within 4 weeks confluent culture flask with a footprint of 25 cm2.

As a result of the studies have shown that CF-1 cells do not contain Alp, antigen associated with factor VIII, mac 1, in addition, in the study of phagocytosis were obtained negative results, while positive results were shown when testing for the presence of collagen type I, collagen type III and fibronectin. CF-1 cells did not show transformation in adipocytes within 4 weeks in confluentes culture in the presence of hydrocortisone in a dose of 10 mol/l, although the CF-1 cells contain, as has been shown, the traces of lipid. Based on these results it was concluded that the CF-1 cells do not possess the properties of preadipocytes, macrophages and endothelial cells, and therefore their origin from other cells, and stromal cells.

3) the Maintenance of hematopoietic stem cells cell CF-1
In order to determine whether the CF-1 cells to support the growth of hematopoietic stem cells, using technick spleen (CFU-S) - test for colony-forming ability of splenic cells. During testing, a group of 10 mice was irradiated with a dose of 900 cGy (MBK-1520R; Hitachi, Tokyo), after which animals were injected with intravenous menagerie bone marrow cells (BM cells) (1,0105/the body, 5,0104/the body or 2.5104/the body) and CF 1 cells (1,0105/the body), and on the 12th day in the spleen were counting the number of formed colonies in SOME of the clones (splenic colony).

It was shown that when transplanted into the body irradiated mice managernew bone marrow cells (BM cells) and CF-1 cells the number of colonies of each group VM cells is greatly increased (from 1.4 to 1.8 times) as compared with mice that were not injected CF-1 cells with reduced mortality, because on the 12th day after transplantation, the survival rate of mice transplanted with BM cells and CF-1 cells was higher than in the case of mice bearing as transplanted only VM cells; these results demonstrate the ability of the CF-1 cells to support the growth of hematopoietic stem cells.

Optionmenu.

EXAMPLE
Obtaining monoclonal antibodies
1) Antigens and immunization
Immunization is carried out with the use as antigens CF-1 cells obtained by the above standard procedure. Cells cultivated in an incubator at a temperature of 37oAnd the content of 5% CO2in the environment of Dulbecco modification Iscove (1D environment) () containing 10% fetal calf serum (FCS; Sanko Junyaku).

Cells treated with 1 mm EDTA/FBI and using the pipette from the culture flask. The cells are then suspended in 1 mm EDTA/FBI until a cell concentration of about 1107/ml, after which the resulting suspension is administered to Wistar rats Imamah (Wistar Imamich) [rats, 7 weeks of age, females obtained from the research laboratory breeding and animal Breeding Research Laboratory)] . One ml of cell suspension containing about 1107cells/ml, is injected into the abdominal cavity of the rat during the primary immunization, and a month later introduce 1 ml of cell suspension containing approximately 1107cells/ml Then with an interval of one month enter a few more times 1 ml of cell suspension, the content is the body of immunized rats and CF-1 cells, administered the final dose immunization in 1 ml of cell suspension containing about 1108cells/ml After three days after administration of the final dose, rats clog and remove from them the spleen.

2) Merge cells
Spleen after removal from the body of the rat is crushed, isolated splenic cells centrifuged, suspended in 1D environment () and thoroughly washed. On the other hand, cells obtained by cultivation of the myeloma cell line of mouse Sp2/0-Agl4 (Nature, 276, 269-270, 1978) and in the environment 1D () supplemented with 10% fetal calf serum (FCS; Sanko Junyaku), washed in the above 1D environment, where they are taken 1108cells, as described previously splenic cells are selected respectively 2108cells and placed both aliquots in a centrifuge tube for implementation according to traditional methods, the process of merging cells under the influence of polyethylene glycol 4000 Naharai the Www.enl.ee (Nagarai Kadaku)] (lin. Exp. Immunol., 42, 458-462, 1980).

Next, the resulting fused cells was dispensed into 96-alopecia tablet in 1MDM medium containing 20% FCS, and cultured GAT Wednesday, continuing on her cultivation.

Since the beginning of cultivation twice a week replace supernatant on fresh GAT environment in order to continue the growth of the culture to maintain cell proliferation.

After that obtained after fusion hybrid cell clone according to traditional methods using the method of restricted breeding. Namely, in accordance with the above conventional method, using the method of restricted breeding cloned only those clones that have strong abilities to bind, keep track of their affinity to the antigens, which are connected with the antibodies, present in supernatant such hybrid cells.

3) Screening
Screening of fused cells (hybridomas) are based on the indirect fluorescence antibody using flow cytometry.

Screening of clones producing the target antibody is performed using CF-1 cells as target cells. The cells are suspended in reaction buffer (FBI with the addition of 2% FCS and 0 02% NaN3), centrifuged, and the residue is again suspended in 100 μl of culture supernatants from growing hybridomas (about 11 shall try again the above buffer, add labeled fluorescent goat antibody to rat lgG (FC) (Chemicon) and are incubated for 1 hour.

At the end of one stage of washing the cells analyzed by flow cytometry [Facscan, Becton Dickinson (FACScan, Becton Dickinson)] .

4) Purification of antibodies
After screening the 3) the method of merged cells cultured using conventional techniques, thus formed antibodies are separated from supernatant using traditional procedures and further purified.

In accordance with this procedure hybrid with high titers of antibodies to antigens selected from cells, distributed in tissue culture on a plastic Petri dish (Corning Co.), cultivated for the purpose of proliferation at a temperature of 37oAnd the content of 5% CO2and then purified by the usual methods for obtaining monoclonal antibodies GSPST-1 and WMAR-1.

To highlight GSPST-1 derived cells injected into the abdominal cavity deprived hairline mice (nude) BAl.B/ cAJc1 - nu [mouse males, 8 weeks of age, Nippon, Korea (Nippon Kurea)]. The resulting ascites reveal 10-14 days, vymalivayut using 33% ammonium sulfate and cialiswhat against the FBI. As for WMAR-1 antibodies carry out large-scale culties supernatant concentrate, vymalivayut using 33% of ammonium sulfate, dialist against the FBI, again clean with a set of columns with protein A (Amersham) and cialiswhat against the FBI. Further, as described in the Example, CF-1 cells are used as antigens for immunization; however, it is possible to use the same method of obtaining monoclonal antibodies in the use of other stromal cells, which are able to support the growth of hematopoietic stem cells, so that the present invention is not limited to the above-mentioned antibodies, but also includes the monoclonal antibodies having such characteristics, as well as all hybridoma producing monoclonal antibodies.

Hybridoma producing monoclonal antibody WMAR-1 of the present invention is a hybrid cell, obtained by merging the splenic cells from Wistar rats Imamah and myeloma cells from the cell line of mice S2/0-Ad, which was deposited on August 9, 1993, entitled WMAR-1 (hybridoma material of rats and mice) under the Deposit number FERM BP-4382 at the National Institute of Biological Sciences and Technology Human Research Agency of Industrial Science and Technology in Japan apan (1-3, Higashi 1-chome, Tsukuba-Shi, Ibaraki 305, Japan) international Depositary management, acting on the basis of the Budapest Treaty on the international classification of deposits of microorganisms for the purpose of their patenting.

5) Properties of antibodies
(i) Reactivity of antibodies
(Reactivity relative to CF-1 cells)
The results of studies using immunofluorescence analysis of the reactivity of monoclonal antibodies GSS-1 and UMAR relatively CF-1 cells is shown in Fig.1-3. So in Fig.1 shows the data for the study of control in the absence of antibodies, Fig.2 presents the results of research ability GSPST-1 to bind with CF-1 cells, and Fig.3 - results of analysis of binding properties of WMAR-1 with CF-1 cells. In these figures the vertical axis shows the relative number of cells, whereas the horizontal axis shows the intensity of fluorescence.

As follows from Fig.1-3, monoclonal antibodies GSS-1 and WMAR-1 are characterized by an ability to bind with CF-1 cells, and the recognition of surface antigens CF-1 cells.

(The relative reactivity of bone marrow cells)
Further, in Fig. 4-6 presents the results of a study primenumber-1 on bone marrow cells. So, in Fig.4 shows the data for the study of control in the absence of antibodies, Fig. 5 presents the results of research ability GSPST-1 for binding to bone marrow cells, and Fig.6 - the results of the analysis of binding properties of WMAR-1 with bone marrow cells. In these figures the vertical axis shows the relative number of cells, whereas the horizontal axis shows the intensity of fluorescence.

As follows from Fig.4-6, GSPST-1 completely lack the ability to bind to bone marrow cells, whereas WMAR-1 are characterised by their ability to bind with all bone marrow cells.

(Reactivity relative to cells miliitary leukemic cell lines (F-60))
The results of the study using the method of flow cytometry (Facscan, Becton Dickinson) reactivity GSPST-1 and WMAR-1 relative to NFS-60 cells (WGE. Natl. Acad. Sci, USA, 82, 6687-6691, 1985) is shown in Fig. 7-10. So, in Fig. 7 shows the data for the study of control in the absence of antibodies, Fig. 8 presents the results of research ability GSPST-1 to bind to NFS-60 cells, Fig. 9 - results of analysis control using a commercially available rat lgG1 [Zymed (Zymed)] whereas artikelnya axis shows the relative number of cells whereas the horizontal axis shows the intensity of fluorescence.

As follows from Fig.7-10, GSPST-1 does not interact with NFS-60 cells, whereas WMAR-1 has the ability to bind with NFS-60 cells.

(Method of testing the ability WMAR-1 to inhibit proliferation of NFS-60 cells)
In Fig.11 shows the results of the research steps WMAR-1 NFS-60 in the presence of 100 ng/ml CSF (G-GT) and 10-6M cycloheximide in accordance with the MTT test [quantitative method using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). Using a 96-microtiter cell plateau to each cell containing 100 ál 4103NFS-60 cells, add 10 ál of solution WMAR-1 at concentrations of 0, 10, 100 ng/ml, and 1, 10, 100 μg/ml and counted after two days using the MTT method, the number of living cells.

As follows from the data of Fig. 11, is pronounced inhibition of profilerate NFS-60 cells under the action of UMAR-1.

(ii) Typing antibodies
Next in the typing of lgG subclass derived monoclonal antibodies [c a set of Mono Abid-SP (Zymed) (Mono AbID - Sp, Zimed) and labeled with Biotin mouse anti rat lgG1 (Timed)] the situation of bone marrow
To further study the antibodies of the present invention were used in experiments on the inhibition of bone marrow transplantation. In Fig. 12 and 13 presents the results obtained here. As follows from the data shown in these figures, BMAP-1 shows the inhibitory bone marrow transplantation effect, whereas CSPST-1 such action is not typical. These results were obtained by introduction through the tail vein of irradiated with a lethal dose of radiation (900 cGy) mice C57BL/6J bone marrow cells in a quantity of 1.0105in per animal and monoclonal antibodies, followed by counting the number of cell colonies in the spleen. The option of "Not processed" in Fig.13 refers to the case in which bone marrow cells are not entered in the test animal.

As can be seen from Fig.13, in the described study on the inhibition of bone marrow transplantation was confirmed the idea that just because BMAP-1 can interact with bone marrow cells, ultimately leading to apoptosis, monoclonal antibody completely inhibits bone marrow transplantation in this test. When hybridoma producing WMAR-1, whodat concentrated in a small volume. In addition, it was shown that all the bone marrow cells are killed with intravenous WMAR-1 normal mice C57BL/6J at the rate of 50 μg per animal, while in Fig.14 shows a micrograph showing the death of bone marrow cells on day 6 after intravenous WMAR-1. As you can see from this photo, in this case die not only lymphoid cells, but also neutrophils, megakaryocytes, myeloblast, plasmic order has been revealed, mast cells, macrophages, monocytes and erythroblast (the so-called myeloid cells). Moreover, as follows from Fig. 15, in the study of the DNA of bone marrow cells of the mouse, which was introduced WMAR-1 antibody at a rate of 30 μg per animal, was demonstrated obvious education ladder-DNA sequence, it was found that the above reaction WMAR-1 with bone marrow cells is due to apoptosis.

PC region lgG antibodies WMAR-1 digested with pepsin (Sigma) and purified using the TRU column in the form F (AB')2, after which it is injected mice C57BL/6J per animal in the number 33.5 ág (equivalent to 50 μg per animal lgG); in this experiment it was shown that bone marrow cells die in the bone marrow. Ha is avisimas from antibody cytotoxicity, neither with the complement-dependent cellular cytotoxicity.

There is a message stating that as antigen, causes apoptosis, can function protein FAS antigen on the cell surface; however, the expression of corresponding Fas antigen mRNA was detected in thymus, heart, liver, lung and ovary, whereas in the bone marrow was demonstrated only low levels of mRNA (J. Immunol., 148, 1274-1279, 1992), and in this connection it is obvious that the antigens recognized WMAR-1, different from the usual well-known Fas antigen.

In addition, to ascertain if the antigen recognized WMAR-1, TNF receptor (TNF - necrosis factor tumor cells), investigated the functioning WMAR-1 using L-929 cells, responsive to TNF, which cause cell death. The final concentration of mouse, Fnaa (TNFa) [Gensym (Genzym)] was in experiment 0, 1, 10, 100 PG/ml, and 1, 10, 100 ng/ml and 1 μg/ml, whereas the concentration WMAR-1 was equal to 0, 10, 100 PG/ml, 1, 10, 100 ng/ml, 1, 10 μg/ml, and the number of live L-929 cells was measured by MTT method on the second day after adding Fnaa (NF) and WMAR-1. The results of the experiment, as follows from the data of Fig. 16 and 17 show that while under the influence of Fnoa, (NF) is expressed in the have, the antigen recognized WMAR-1, is not Fnoa, (TNF) receptor.

The results of the research using flow cytometry (Facscan, Becton, Dickinson) to determine, not whether the antigens recognized WMAR-1 antigens class 1 master system tissue compatibility (MHC), is shown in Fig.18-21.

So, in Fig. 18 shows the results of analysis control using rat lgG1 (Zymed), Fig.19 - the results of the analysis of the ability of the antibody to the main system tissue compatibility class (rat lqG2a, BMA) contact BWV1 cells (mouse lymphoma derived from BW 5147 cells), Fig.20 the data analysis control using rat lgG1 (Zymed), and Fig.21 shows the results of a study linking abilities WMAR-1 against BWV1 cells. In these figures the vertical axis shows the relative number of cells, whereas the horizontal axis shows the intensity of fluorescence. As follows from the above results, WMAR-1 does not recognize BWV1 cells, whereas the antibody of the class 1 master system tissue compatibility (MHC) reacts with BWV1 cells.

As mentioned above, was experimentally confirmed by the fact that Manastash of the invention, hitherto lacked any information about monoclonal antibodies capable of causing apoptosis on myeloid cells, the monoclonal antibodies of the present invention, having such a function are the new antibodies, open the real authors. In this case, since the monoclonal antibodies of the present invention, presents WMAR-1, can lead, on the basis of the ability of monoclonal antibodies to induce apoptosis of bone marrow cells, to the death of the considered miliitary leukemic cells expressing high levels of the antigens mentioned monoclonal antibodies suitable for use as a drug for the treatment of malacitana leukemia.

Monoclonal antibodies of the present invention have been described in detail above in the section of Example; however, despite the fact that monoclonal antibodies with in accordance with the present invention the ability to cause apoptosis of myeloid cells, can be illustrated by the above examples, the present invention is not restricted by them, and includes all cooked in the same way monoclonal antibodies having the same characteristics and funkciyasiga of the invention can be used as antibodies, specifically recognizing and identifying antigens that cause apoptosis of myeloid cells, and, in addition, have the ability to directly induce apoptosis of myeloid cells, they can on the basis of this property, to find application in medicine as a drug effective for treating malacitana leukemia.


Claims

1. Monoclonal antibody capable of recognizing the antigen, causing apoptosis of myeloid bone marrow cells, obtained by immunization of animals stromal cells of the spleen, pre-inoculated with recombinant granulocyte colony-stimulating factor (rG-CSF), and having a property to cause apoptosis of myeloma cells.

2. Monoclonal antibody under item 1, characterized in that belongs to the IgG class.

3. Monoclonal antibody under item 1, characterized in that obtained by the use of human splenocytes stromal cells as antigen.

4. Monoclonal antibody under item 1, characterized in that it is WMAR-1, produced by hybridoma FERM BP-4382.

5. Monoclonal antibody under item 1, characterized in that it is used for the treatment of Mielec the mA FERM BR-4382, producing a monoclonal antibody according to p. 4.

 

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The invention relates to veterinary Virology and biotechnology

The invention relates to immunology and can be used to generate antibodies, the diagnosis of autoimmune diseases and the treatment of rheumatoid arthritis

The invention relates to biotechnology and medicine and can be used for the treatment of AIDS
The invention relates to medicine and for the preparation of chromosomal preparations for karyotype analysis of bone marrow cells in diseases of the blood system

The invention relates to biotechnology, in particular genetic and protein engineering

The invention relates to genetic engineering, specifically to the use of chimeric somatostatinergic protein to increase the productivity of farm animals
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