Monoclonal antibody reactive surface agent stromal cells and producing his hybridoma

 

(57) Abstract:

The invention relates to a monoclonal antibody having the ability to inhibit homing hematopoietic stem cells and to identify surface antigen stromal cells, having the ability to maintain homing hematopoietic stem cells, as well as to hybridoma producing monoclonal antibody. Monoclonal antibodies of the invention are characterized by their ability to inhibit homing hematopoietic stem cells and can be used as drugs that enhance the action of anti-cancer drugs used to treat leukemia. 3 S. and 1 C.p. f-crystals, 8 ill.

The technical field to which the invention is applicable

The present invention relates to novel monoclonal antibodies, which have the ability to inhibit homing hematopoietic stem cells and to identify stromal cells of the spleen, which, in turn, can support homing hematopoietic stem cells, in addition, the invention relates also to hybridoma producing monoclonal antibodies.

Since the monoclonal antibodies of the present invention can recognize the gene and characterized by their ability to inhibit homing hematopoietic stem cells, they can be considered on the basis of their characteristic functions enhance the effectiveness of anticancer funds as a promising drug, in particular, by strengthening the action of the anticancer drug for treatment of leukemia through the release myeloid leukemia cells from the bone marrow.

Background of the invention

Granulocyte colony-stimulating factors, such as 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 ICRI is ishushim hematopoietic potential.

On this basis, the authors present invention has marked stromal cells of the spleen, which was introduced WG-CSF, in order to clarify 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, as well as the ability to support hematopoietic stem cells in vivo (Blood, 80, 1914, 1992).

Because supporting the ability of hematopoietic stem cells in vivo is realized in the case when CF-1 cells transplanted with bone marrow cells in the bone marrow transplant, it was considered that the above-mentioned hematopoietic stromal cell line (CF-1 cells) expresses a specified function in the form factor cell adhesion, allowing hematopoietic stem cells to make homing on hematopoietic tissue.

However, despite the fact that Volovich cells, managed to get a stable cell line (CF-1 cells) and to determine their cytological characteristics, still not received specific antibodies that recognize cell surface antigens, as known up to the present time and the characteristics of such antibodies.

In this regard, the authors of the present invention undertook painstaking research with the purpose 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 that can recognize the stromal cells of the spleen, with the ability to maintain homing hematopoietic cells, creation of cell lines based on the stromal cells of the spleen, with the ability to maintain homing hematopoietic cells, and also for preparation of monoclonal antibodies using stromal cell lines from spleen as antigens for immunization, resulting in the obtained new, previously unknown monoclonal antibodies.

In the study of the properties of the obtained monoclonal antibodies, the authors of the present invention showed that the antibody characterizes the poetic stem cells, and also to inhibit homing hematopoietic stem 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 preparation of new monoclonal antibodies that recognize surface antigens stromal cells of the spleen, which have a function of maintaining homing hematopoietic stem cells, and characterized by the ability to inhibit homing hematopoietic stem cells. In addition, another objective of the present invention is to obtain hybridoma capable of producing monoclonal antibodies.

Monoclonal antibody of the present invention relates to a new monoclonal antibody to surface antigen stromal cells of the spleen, with the ability to maintain homing hematopoietic stem cells, the antibody is characterized by the ability to recognize stromal cell of the spleen, which is associated with increased hematopoietic potential of the spleen, these antibody appeared to be extremely useful from the point of view of its application as vasectomise homing hematopoietic stem cells is a process in the course of which under the influence of stromal cells hematopoietic tissue occurs adhesion of hematopoietic stem cells, and the cells differentiate and proliferate in the form of CFU-C colony forming units in the spleen, CFU-S) colonies.

In General, the monoclonal antibody of the present invention can be obtained by the following procedure.

Namely, the monoclonal antibody of the present invention can be obtained, for example, using as antigen stromal spleen cells taken from animals that were administered RG-CSF, in particular, CF-1 cells (stromal cells of the spleen), established by the authors of the present invention in the form of cell lines, then their application is carried out with the traditionally accepted methods of immunization, and then spend the merger immunizing cells using standard procedures merge and then clone merged cells using standard cloning techniques.

As the preferred method of obtaining monoclonal antibodies of the present invention may be a method comprising using as antigen CF-1 cells, representing stromal cells culturally 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, the target antibody can be used not only the above-mentioned CF-1 cells, but also other stromal cells hematopoietic tissue obtained by the method similar applied in the case of CF-1 cells, or stromal cells hematopoietic tissue obtained from spleen cells 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 specific restrictions, however, it is preferable to carry out the selection from the point of view of usability myeloma cells in the merger"ptx2">

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 P3(Rad.653) (J. Immunol. , 123, 1548, 1978), P3-U1 (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), Sp2/0-Agl4 (Nature, 276, 269-270, 1978), F O (J. Immunol. Meth. , 35, 1-21, 1980), S194 (J. Exp. Med., 148, 313-323, 1978) and R210 (Nature, 277, 131-133, 1979).

Merge cells using the above immunocytes and myeloma cells can be performed in General by using one of the traditional ways, nab, the cells 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 Sendai Virus (HVJ), and furthermore, 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 medium RPMI-1640 and MEM medium (minimum essential medium), 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 the above environment, adding in the environment, usually at a concentration of 30-60% (weight/volume) solution cularly weight in the range from 1000 to 6000, with further 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). Culture grow on 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 with the conventional method limited dilutions 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 hybridomas according to the usual method of obtaining them from supernatants, or method, vkljuchajuwih is. 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 the technique of vysalivaniya, gel filtration and affinity chromatography.

Since the monoclonal antibody of the present invention has the ability to inhibit homing hematopoietic stem cells, they may find use as a medicinal drug that enhances the effect of anti-cancer tool in the treatment of leukemia, in particular, in connection with the above-mentioned function of the bone marrow is the release of myeloid leukemia cells, which affects the efficiency of action of anti-cancer drug for the treatment of leukemia.

No need to say that creation on the basis of the monoclonal antibodies of the present invention are specific systems for use in medicine based on its properties to enhance the action of medicines in the treatment of leukemia, as well as all kinds of modifications and applications of this system are included in the region of the nogo for each specialist with an intermediate level of knowledge in this field.

Brief description of figures

In 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 HMS-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 the study of binding capacity GSPST-1 antibodies with bone marrow cells on the basis of immunofluorescence analysis.

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

In Fig. 7 illustrates a method of quantitative determination of monoclonal antibodies (GSPST-1), inhibiting bone marrow transplantation.

In Fig. 8 illustrates a method of quantitative determination of monoclonal antileishmania in accordance with the reference procedure and Example, non-limiting scope of the present invention.

Reference Procedure

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

1) creating a line of stromal cells of the spleen

Line stromal cells of the spleen, with the ability to support hematopoietic stem cells, created based on primary cultures of spleen cells of the mouse C57BL/6J, which was administered RG-CSF at a dose of 100 µg/kg for 5 days.

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 cm2/Corning, Co. (Corning Co. )/, using the environment Dulbecco modification Iscove (IMDM) /Boehringer-Mannheim, Co. (Boehringer-Mannheim Co.)/, which made inactivated by heating 10% fetal calf serum /TCF (F BS)/ /SANCO Unico, Tokyo (Sanko Junyaku, Tokyo)/, 100 U/ml penicillin and 100 μg/ml streptomycin, with the specified medium replaced with fresh growth medium twice a week.

In confluentes culture populations of fused cells (stromal cells) are selected from the bulb with a new bulb. This procedure 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 homogeneous and fibroblastoid.

By the 20th time of the above procedure stromal cells are selected as indicated above, and sent for cell cloning using technology limited dilution; 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 cm2(Corning Co. ) in 5 ml of IMDM medium with 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 adenoviral vector SV-40 (J. Cell. Physiol., 148, 245, 1991).

2) Characteristic of CF-1 cells

CF-1 cell is bound cytochemical technology for alkaline phosphatase, acid phosphatase-glucuronidase, -naphthyl-acetate esterase and oil red O. In the study of CF-1 cells by the methods of enzyme histochemistry used the following monoclonal and polyclonal antibodies: mac1 /Gray Tech. (Sero Tec.)/; the antigen associated with the factor VIII /Dakopatts (Dacopatts)/; collagen type I, collagen type III and fibronectin /Chemicon international Inc. (Chemicon International Inc.)/. Phagocytosis was investigated by absorption of latex granules (particle size of 1.09 μm; Sigma), and the ability of the CF-1 cells to become adipocytes was determined under the influence of hydrocortisone phosphate (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 10-6mol/l, although the CF-1 cells soda is present properties preadipocytes, macrophages and endothelial cells, and in this connection it is obvious they were derived 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 the technique of tilley and Makaleha (Till and McCulloch) testing was conducted on the formation of CFU-C (test for colony-forming ability of splenic cells). During testing, a group of 10 mice was irradiated with a dose of 900 cGy (MBR-1520R; Hitachi, Tokyo), after which animals were injected with intravenous menagerie bone marrow cells (BM cells) (1,0 105/the body, 5,0 104/the body or 2.5 104/the body) and CF-1 cells (1,0 105/the body), and on the 12th day in the spleen were counting the number of formed colonies in CFU-C 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 the village is 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.

The optimal way of carrying out the invention

Below is a detailed description of this version of the present invention.

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 37oC and a content of 5% CO2in the environment of Dulbecco modification Iscove (IMDM) (Boehringer-Mannheim Co.), 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 1 to 107/ml, after which the resulting suspension is injected Wistar rats Imamah (Wistar Imamich) /rat 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, and the Ziya, containing about 1 to 107cells/ml Then with an interval of one month enter a few more times 1 ml of cell suspension containing approximately 1 to 107cells/ml, and then, after observing the reaction between antibody immunized rats and CF-1 cells, administered the final dose immunization in 1 ml of cell suspension containing about 1 to 108cells/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 IMDM medium (Boehringer-Mannheim Co. ) and thoroughly washed. On the other hand, cells obtained by cultivation of the myeloma cell line of mouse Sp2/O-Aql4 (Nature, 276, 269-270, 1978) and in IMDM medium (Boehringer-Mannheim Co.) with the addition of 10% fetal calf serum (FCS; Sanko Junyaku), also washed in the above IMDM medium, where they are taken 1 to 108cells, as described previously splenic cells are selected respectively 2 108cells and placed both aliquots in a centrifuge tube for implementation according to traditional methods, the process of merging cells under the influence of the polyethylene is really on 96-alopecia tablet in IMDM medium, containing 20% FCS, and cultured at a temperature of 37oC and a content of 5% CO2. The next day they carefully transferred to selective 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 and maintenance of cell proliferation.

After that obtained after fusion hybrid cell clone according to traditional methods by using method of limited dilution. Namely, in accordance with the above conventional method with the method of the limited dilution 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 (which of peratata from growing hybridomas (approximately 1 to 106cells/100 μl) and conducting the reaction at a temperature of 4oC for 1 hour. After that, the cells are washed again above the buffer add-labeled fluorescent goat antibody to IqG rats (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 hybridoma 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 37oC and a content of 5% CO2and then purified by the usual methods for obtaining monoclonal antibodies GSPST-1 and HMS-1.

To highlight GSPST-1 and HMS-1 antibodies derived cells injected into the abdominal cavity deprived hairy Nude mice (nude) BALB/cAJc1-nu /mouse males, 8 weeks of age, Nippon Course (Nippon Kurea)/, and is.

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 of the spleen, which include stromal cell lines of hematopoietic tissue derived from stromal cells in the spleen of a person, so that the present invention is not limited to the above monoclonal antibody, but also includes all cooked in the same way monoclonal antibodies having the same characteristics, as well as all hybridoma producing monoclonal antibodies.

Hybridoma producing monoclonal antibody HMS-1 of the present invention is a new hybrid cell, obtained by merging the splenic cells from Wistar rats Imamah and myeloma cells from the cell line of mouse Sp2/0-Agl4, which was deposited on August 9, 1993, under the name of HMS-1 (hybridoma material of rats and mice) under Deposit number FPRM BP-4383 at the National Institute of Biological Sciences and Technology Human Research Agency of Industrial Science and Technology in Japan (address: 1-3, Higashi 1-Hom, Tsukubai, Ibar is Narodnogo Depository 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) Results of studies using immunofluorescence analysis of the reactivity of monoclonal antibodies GSPST-1 and HMS-1 relative to CF-1 cells is shown in Fig. 1 to 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 HMS-1 with CF-1 cells. In Fig. 1-3, 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 GSPST-1 and HMS-1 are characterized by an ability to bind with CF-1 cells, and the recognition of surface antigens CF-1 cells.

(Reactivity relative to bone marrow cells Further in Fig. 4 - 6 presents the results of a study using the method of flow cytometry /Facscan, Becton, Dickinson (FACS for the study of control in the absence of antibodies, in 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 HMS-1 with bone marrow cells. In Fig. 4-6 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 cells in the bone marrow, whereas for HMS-1 are characterised by their ability to bind with certain bone marrow cells.

(ii) Typing antibodies

Next in the typing of IgG subclass derived monoclonal antibodies /using a set of Mono ABID-SP (Zymed) (Mono AbID-Sp, Zymed) and labeled with Biotin mouse antibodies to lgGl rats (Timed)/ it was shown that GSPST-1 is an IgG2a, a HMS-1 is IgG2b.

(iii) the Ability to inhibit bone marrow transplantation

To further study the antibodies of the present invention were used in experiments on the inhibition of bone marrow transplantation. In Fig. 7 and 8 show the results obtained. As follows from the data shown in Fig. 7 and 8, HMS-1 played what s the results obtained by introduction through the tail vein of irradiated with a lethal dose of radiation (900 cGy) mice C57BL/6J bone marrow cells in the amount of 1.0 to 105in per animal and monoclonal antibodies, followed by counting the number of cell colonies in the spleen. The option of "Not processed" in Fig. 8 relates to the case in which bone marrow cells are not entered in the test animal. Accordingly, since the monoclonal antibody HMS-1 of the present invention inhibits bone marrow transplantation, it is obvious that the said antibody inhibits homing hematopoietic stem cells and the formation of CFU-C colonies.

In this case, each specialist with an intermediate level of knowledge in this area, it should be obvious that the inhibitory effect of monoclonal antibodies against bone marrow transplantation corresponds to those activities, which are aimed at the inhibition of homing hematopoietic stem cells and formation of CFU-C colonies. For example, there was a report that a monoclonal antibody (ASC-2 antibody, part C-kit antibodies obtained using as antigen known in the art of fat cells, capable, similarly, the monoclonal antibodies of the present invention, to inhibit bone marrow transplantation in mice (Blood, 78, 1706, 1991). In ukazannom conclusion about that the said antibody inhibits homing hematopoietic stem cells and the formation of CFU-C colonies.

The above-mentioned known technique of antibody ASC-2 is obtained using as antigen the fat cells, the fat cell is a differentiated proliferating hematopoietic cell, which has evolved from hematopoietic stem cells. On the other hand, used in the present invention stromal cells hematopoietic tissue are not hematopoietic cells, and cells that support the differentiation and proliferation of hematopoietic cells, and in this regard both types of cells differ significantly from each other.

Further, when the introduction of hybridoma producing HMS-1 in the peritoneal cavity of Nude mice, it was shown that the mouse does not die from the resulting ascites. In this regard, there is reason to believe that the action of HMS-1 by inhibition of bone marrow transplantation does not derive so-called apoptosis /this phenomenon is also called the self-destruction of cells in which the DNA of the nuclear chromatin is cleaved in the nucleosome (the formation of the so-called "ladder-sequence"), ultimately leading to GI homing hematopoietic stem cells in hematopoietic tissue. Thus, it is obvious that the antigen recognized by the HMS-1, is a substance that is important for the implementation of homing hematopoietic stem cells hematopoietic tissue.

As was found in the above-described experiment, a monoclonal antibody HMS-1 of the present invention recognizes a surface antigen stromal cells of the spleen, which has the ability to support homing hematopoietic stem cells, as well as its characteristic property to inhibit homing hematopoietic stem cells.

So, HMS-1 has the ability to inhibit homing bone marrow cells in hematopoietic tissue; there is a message stating that the leukemia cells are released from bone marrow in connection with a reduction in the level of expression of adhesion molecules VLA4 and VLA5 hematopoietic cells /expression of VLA molecules can be associated with the process of release from the bone marrow myeloid leukemia cells (Leuk Res., 16, 5, 469-474, 1992)/, it should be assumed that VLA4 and VLA5 enhance the effect of drugs used to treat leukemia, due to the release of leukemic cells from the bone marrow. Similarly, we can conclude that the above-mentioned antibody HMS-1 Titel of the present invention have been described in detail above, in the Example section; however, despite the fact that monoclonal antibodies 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 functions, regardless of the type of antigens.

Industrial application

Since the monoclonal antibodies of the present invention is able to detect both antigen a substance needed for homing bone marrow cells in hematopoietic tissues and are characterized by their ability to inhibit homing hematopoietic stem cells, they can, on the basis of their characteristic functions to enhance the action of anticancer means, in particular, to improve the action used for the treatment of leukemia cancer funds due to the release of bone marrow myeloid leukemia cells, to find application in medicine as an effective drug.

1. Monoclonal antibody inhibitory homing hematopoietic stem cells and recognizes a surface antigen stromal cells of the spleen, obtained from spleen cells mlekovita is in immunoglobulin G.

3. Monoclonal antibody described in paragraph 1, used to treat myelocytes leukemia.

4. Hybridoma producing monoclonal antibody described in paragraph 1.1

 

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