EA003318B1 - Monoclonal antibody which recognizes the oligosaccharide n-glycolylated-galactose-glucose sialic acid in malignant tumors, and composition containing them - Google Patents

Abstract

1. Hybrid cell culture of mouse 14F7 ECACC 98101901, generating monoclonal antibody 14F7 generated against the NGcGM3 ganglioside. 2. Monoclonal antibody 14F7, generated by hybrid cell culture according to claim 1, specifically recognizes the N-glycolylated-galactose-glucose sialic acid olygosaccharide sequence in ganglioside NGcGM3 and having cytolytic activity of the tumoral cells bearing said sequence. 3. Monoclonal antibody according to claim 2 wherein said cytolytic activity of the tumoral cells bearing the N-glycolylated-galactose-glucose sialic acid olygosaccharide sequence can be direct or complement mediated. 4. A pharmaceutical composition containing an amount of the monoclonal antibody of claims 2 to 3 and that additionally contains a diluent or a suitable excipient. 5. A pharmaceutical composition according to claim 5 where said antibody can be bound to therapeutic agents such as drugs, radioisotopes, immunomodulators, lectins and toxins. 6. A pharmaceutical composition according to claims 4-5 to be used in the treatment of malignant neoplasms. 7. A pharmaceutical composition according to claim 6 for the treatment of cancer of breast, renal, of digestive system and human melanoma. 8. A reagent containing the monoclonal antibody of claims 2 to 3 bound to markers such as enzymes, chromophors, chimioluminiscent materials and radionucleotides. 9. A reagent according to claim 8 for the detection of tumoral cells. 10. A reagent according to claim 9 for the detection of human tumoral cells of melanomas, breast, kidneys and digestive system such as liver, colon, stomach and rectum.

Description

Technical field

This invention relates to the field of human immunology and medicine, especially with respect to the preparation and selection of a monoclonal antibody (MAT) against the oligosaccharide sequence of --glycolylated galactose - glucose - sialic acid, which can be used to diagnose and treat certain tumor diseases.

State of the art

Oligosaccharide structures are involved in the formation of glycoproteins and glycolipids. Both those and others are present in normal and pathological tissues.

In approximately 100% of cases of malignant neoplasms, aberrant glycolylation is described. Common changes in aberrant glycolylation are, among other things, expression of neoantigens, changes in the composition of oligosaccharide sequences, an increase or decrease in the number of sialic acid molecules in the oligosaccharides, and an increase in the density of molecules on the cell surface (Nakotop 8.N. e! A1 .: Sigg. Ορίη ίη 1ttypo1. 1991, (3) 646653). In addition to the changes that can be found in the mechanism of sialyl transferases, there are changes in hydroxylases dependent on the activated Ν-acetylated form of sialic acid.

Gangliosides are glycosphingolipids that contain sialic acid in their structure and are characterized by what are present in most vertebrate cells. These molecules are found in normal tissue and they can be expressed more strongly in tumors with excellent organization and conformation on the surface of malignant cells (Nakotop 8.N., 1985, Sapseg Vek. 45: 2405-2414; M1ga1y1, P., 1989, 8th step ίη M .1c1eag Meiuchet, XIX, 282-294).

As a rule, immunoglobulins of the 1dM isotype are responsible for the humoral immune response against carbohydrate antigens. Oligosaccharide sequences associated with lipids are in most cases less immunogenic than glycoproteins. Therefore, the use of glycolipids as an immunogen requires its binding to carrier proteins or incorporation into liposomes or bacteria, such as Mucobacterium bacillus or Bacterium bacillus b595.

The response induced against gangliosides is thymus independent. This has been repeatedly reported by Mpdkip e! A1., (LMpdk! op, Ρ.Ο. e! a1., 1982, Proc. No. 11, Asai. 8c1. I8A 84: 2911-2915; b1utdk! op, Ρ.Ο. e! a1., 1989, Sapseg Vek. 49: 7045-7050). The main features of antibodies produced against gangliosides in the study of various types of serum are their low affinity, significant cross-reactivity, and short life span (L1utdk! Op, Ρ.Ο. 1991, 1 type 1 apy A11egdu Syssk o! No. P11 Atensa, 11: 401423 , RoyoikaLap, 1. e! A1., 1991, 1p !. 1. Sapseg, 49: 893-899).

The expression of the гли-glycolylated form of oligosaccharides occurs in normal and pathological tissues of all vertebrate species, with the exception of chicken and humans, in which it is found only in embryonic and tumor tissues. The normal tissues of these last two species contain only the acetylated variant (ShkKtaky e! A1., 1979, 1. 1 type 1: 122: 2314; Shdaky N. e! A1., 1985, Sapseg Vek., 45: 3796).

The study of the oligosaccharide composition in some human tumors shows the presence of the гли-glycolylated form both in the glycolipids and in the glycoproteins of tumor cells of melanoma (Shtauakuy, Υ. E! A1., 1987, 1. Sapseg Vek., 78, 614-620; 8a1ya T. e! a1., 1991 Atsy. Eegta! o1. Century 282 (3): 179-182; Ka ^ akIta I. e! a1., 1993, 1. Vusyet (Tokyu) (2) 186-193; Ka ^ acs 8. e! a1., 1992, 1. Eegta1o1 (11): 827-830), as well as in colon tumors, especially in glycolipids (M1uokY, I., e! a1., 1986, Mo1. 1tipo1. 23 (6): 631; Shdaky N., e! A1., 1985, Sapseg Vekeagsy, 45: 3796-3802).

In addition, studies were carried out indicating the presence of the Ν-glycolylated form of gangliosides in samples of liver tumors, teratomas, lymphomas, etc. (Ka ^ a1 T. e! A1., 1991, Sapseg Vek. (51) 1242-1246).

Although in the first cases the concentration of the ли glycolylated version of glycolipids was less than 0.05% of the total sialic acid content, Mags. found in lactic acid tumors approximately 10% of such sialic acid associated with lipids (Magtsspa, O. E! a1., 1996, Sapseg Vek. 56: 5165).

The formation of monoclonal antibodies against the гли-glycolylated variant of gangliosides has so far made it possible to obtain antibodies of the 1dM isotype, which usually recognize more than one ganglioside molecule, for example, human antibodies 2-39M and 32-27M (Hrithika ^ a K., e! A1., 1988 , 1. Vyuododka1 Sjet1k! Tu, 263: 18507) and mouse antibodies OMV8 and OMV3 (Ο / a ^ a N. e! A1., 1992, Vyusyet. Vyuryuk., 2 (294): 427). Other authors reported the receipt of specific antibodies to Ν-glycolylated gangliosides, always of the 1dM isotype, among which Υ-2-ΒΩ1 monoclonal antibodies against N6c6M ₂ (8ata1 е. E! A1., 1988, Vyusy Vyryuk. As!., 958, 368 ) and MK2-34 against the same molecule (M1uake, M. e! a1., 1990, Sapseg Vek. 48, 6154).

However, \ Wa1aga1 (\ Wa1agat 8. e!

Vusyet. 117, 1062) received monoclonal antibody 8H8-1 against ί-active Ν glycolylated ganliosides, and No. 1 kitaga received monoclonal antibody ΥΧ-3 against (ΝΟο-ΝΟο) OE1s (No. 1 kitaga e! A1., 1995, 1. o!

Βίο1ο§. Sztkk, 8 (270): 3876). Recently, νάζιιικζ, e1 a1., (Vaζ ^ ez, A.M. e1 a1., 1995, Nibbota, 14, 6, 551) have reported the preparation of a monoclonal antibody P3 that recognizes most ganglioside molecules containing a β-glycolylated form of sialic acid , as well as sulfated glycolipids.

No.shch; n e1 a1. received monoclonal antibody HMA1 against gangliosides (Ν; · ι§; · ιί е. e1 a1., US patent No. 4965198). They received a specific monoclonal antibody against mouse ganglioside ΝΟεΟΜ2, suffering from an autoimmune disease. Although they reported several of these antibodies that additionally recognize Ν-glycolylated gangliosides, designated as RuK, ΥΗ02, ΥΗ03, ΥΗ04, ΥΗ05, ΥΗ06 and ΥΗ07.

In addition, Hatakact M. et al. In US Pat. No. 4,942,131 report obtaining MAT ΥΗ08, ΥΗ09, ΥΗ10 and ΥΗ11 against 4-O-Ae1u1LCeCM3 ganglioside also in mice suffering from an autoimmune disease.

Monoclonal antibodies against gangliosides are prepared using these molecules as lactones or using cell lines containing gangliosides (US Patent Nos. 5308614, 5240833, 5389530 and 5500215).

In the same way, various monoclonal antibodies, murine and human, were obtained against gangliosides ΟΌ3, ΟΌ2 and OM2 in Ν-acetylated form, most of which belong to subclasses 1dM and 1dO3 (Rike1, C.8. E1 a1., 1982, 1. Exp.Meb., 115: 11331147; Shtauwaky, Υ. E1 a1., 1985, 1. Βίο1. Set., 260: 13328-13333; Pa1ep1 arrysayop GSO 86/00909; M1uake, M. e1 a1. 1988, Sapseg Vek. ., 48: 6154-6160; KatakYta, I. e1 a1., 1992, Mo1eci1ag 1tipododu, 29, 625-632; Ko1at, M. e1 a1. 1992, Vusytuya e1 Vyurkuya Ac1a, 1117: 97-103).

Passive immunotherapy using monoclonal antibodies against gangliosides is used in clinical practice to treat certain tumors, such as melanomas and neuroblastomas. In the case of melanoma, administration is carried out inside the affected area, or systemic treatment of the whole organism is used, and although the results seem encouraging, only a small number of patients undergo complete or partial remission (идοидЬΐοη, Α.Ν. e1 a1., 1985, Proc. No. I. Asab. .8sk I8A, 82: 1242; I NrrokT GS.O. e1 a1., 1988, 1. Sapseg Syp. Opso1; 24: 865; Vabbap-Baa, 8. e1 a1., 1988, 1. Syb. Ops1. 6: 1636; 8a1eB M / Ν. E1 a1., 1992, Sapseg Vek., 52: 4332-4347).

These antibodies show an effect in the study of cytotoxicity mediated by complement or cells (Wautbattat1 MH e1 a1., 1991, 1p1et. Beu. 1tipo1., 7, 303).

All monoclonal antibodies obtained so far against Ν-glycolylated gangliosides are of the 1dM isotype, and the toxicity that they cause is mediated by complement.

1dM, as a rule, have low affinity and antigen, and it is difficult to use them for diagnosis or treatment in the form of radioactively labeled monoclonal antibodies. Although they fix complement well and guarantee adequate cytotoxicity, large-scale isolation is much more difficult compared to that of the 1dC isotype immunoglobulins.

In addition, there is little data in the literature on monoclonal antibodies against Ν-glycolylated glycoproteins, and most of them report diagnostic purposes.

Eute and others described the monoclonal antibody 3E1.2, which recognizes ли glycolylated mucin (glycoprotein), expressed in 90% of breast tumors studied immunohistochemically (Oeute, R.E., e1 a1., 1991, Sapseg Vek. 51 (21) : 5826-36).

There is a publication on a monoclonal antibody, designated 1AM3, which recognizes the ацет-acetylated and ли glycolylated forms of a protein with a molecular weight of 250 kDa, which is present on the surface of the cysts formed during the infection with Epilatea (Autop, B., e1 a1., 1987, Mo1. Visus. Ratako 1. (3): 257-266).

Description of the invention

The novelty of this invention is to obtain a monoclonal antibody, highly specific for the oligosaccharide sequence of Ν-glycolylated galactose glucose - sialic acid, present both in the ganglioside and in glycoproteins. In addition, the characteristics of the resulting 1dC isotype immunoglobulin make it more specific and therefore have a high affinity for the molecules that it recognizes, thus improving its biological activity. Unexpectedly, this antibody showed the ability to cause the death of cells directly producing the indicated oligosaccharide sequence.

DETAILED DESCRIPTION OF THE INVENTION

Obtaining ganglioside HScSM3

To obtain the ganglioside HSCSM3, a modification of the Kakoto ^^ methodology was used (Kotok 8. E1 A1., 1974, Mölöbk and Edenzuto 32, Paradise B, 350) using a natural source such as horse red blood cells. The yield of ganglioside N6c6Μ3 was 180-300 mg / l of horse erythrocytes with a purification degree higher than 90%, confirmed by high performance liquid chromatography according to the 6aζζοΐ method (6aζζοй, О. е1 а1., 1985, 1. о £ Стота1дарьу 348: 371-378) .

Immunogen Production

To get an immunogen, ganglioside

HSCSM3 was hydrophobically associated with very low density human lipoproteins (UBITs) obtained according to Etop1e1 et al. (CytoShe !, S. e! A1., 1994, Sapseg 1ttypo1 1ttypoyeg. 38: 311-318).

Immunization schedule

In order to obtain antiganglioside monoclonal antibodies of type 1dC, the following immunization method was used. Mice or other mammals were immunized with vaccines containing 0.03 and 0.5 mg of U6c6M3 ganglioside bound to UBEB per dose, as well as an adjuvant selected from the following: albumin, Freund's complete or incomplete adjuvant or Mop1ashbe Ι8Ά 51.

Blood samples were taken from animals before and after the immunization period in order to obtain serum and control the antibodies appearing in animals against the ganglioside used as an antigen. For this purpose, all known methods of immunoassay were used to determine the antigenantibody (Ar - At) reaction.

Animals were immunized with various doses from 2 to 8 with time intervals ranging from 7 to 14 days. Injections were performed subcutaneously or intramuscularly in volumes of 0.1 and 0.2 ml.

Other possible methods of immunization are intravenous and intraperitoneal. Animals receiving this dose range show a specific response to the ganglioside used as an immunogen. Between 70 and 100% of immunized animals had a specific 1dC type immune response to I6c6M3 ganglioside.

Obtaining monoclonal antibodies

To obtain specific mAbs against IScSM3 ganglioside, mice with antibodies against this ganglioside were again immunized with a vaccine 3 days before receiving antibody-producing cells. It is preferable to use spleen cells, although you can use other cells.

These cells merge with myeloma cells, resulting in the formation of hybrid cells or hybridomas with the ability to multiply unlimited ίη νί \ Ό and ίη У11to. For this purpose, you can use any of the known methods of cell fusion. An enzyme-linked immunosorbent assay is preferably used to determine the antibodies formed by the hybridoma. Other immunoassay methods can also be used. The procedure of this analysis is the recognition of gangliosides by hybridoma in the supernatant, and the antigen-antibody reaction can be detected using a secondary antibody labeled with an enzyme that binds to the antibody formed by the hybridoma under adequate conditions, the determinations are carried out simultaneously.

A hybridoma once selected is cloned at least twice, for example, by mitigation dilution. The MAT synthesized by the cells can be produced ίη removed in the appropriate culture medium, any one known in the art, and then isolated from the indicated culture supernatant. In this case, from 1 to 8% of secreting cells were immunospecific to CM3 гли-glycolylated ganglioside.

Another method for producing antibodies is to inject hybridomas into animals (e.g., syngeneic animals). The hybridoma causes the formation of a non-solid tumor, which provides a high concentration of the desired antibody in the blood and peritoneal exudate of the host animal.

Monoclonal antibody purification

Monoclonal antibodies are purified using ascites fluid obtained after inoculation of 0.2 X 10 ⁶ cells of a mAb-producing hybridoma into the abdominal cavity of Ba1b / C mice pretreated with incomplete Freund's adjuvant as an ascitogenic substance.

Ascites liquid is diluted twice with glycine buffer 1.5 M, No. C1, 3M; pH 8.9 and subjected to affinity chromatography, where protein A — sepharose serves as a matrix, with an elution rate of 60 ml / h. The elution of the MAT is carried out using a citrate buffer of 0.14 M; pH 6.

The concentration of purified MAT is determined by the Lowry method (Bo ^ tu, S. N., 1951, 1. Vu1. Siet., 193: 256) and using the coefficient. absorption of mouse 1dC1 at 280 nm. Specificity is confirmed by ELISA.

Received from 2 to 5 mg of antibodies per ml of ascites fluid with a percentage of purity above 95%. This was confirmed by low pressure liquid chromatography.

Specificity studies

To determine the specificity of the obtained monoclonal antibodies, an enzyme-linked immunosorbent assay is performed on ELISA plates using thin-layer chromatography using ацет-acetylated (CM1, CM2, CM3, CM1, 6I1a, C1B, ΟΌ3, CT1b) and Ν-glycolylated (CM3, CM2 , CM1a, CM1b, SI1c, CE3) gangliosides.

When analyzing glycolipids using high pressure thin-layer chromatography, the solvent system includes chloroform: methanol: KC1 0.25% and 2.5 M Ν ^ (5: 4: 1) (volume: volume). For the manifestation of bands use orsine.

The tablets are coated with a solution of polyisobutyl methacrylate and dried in air at room temperature. The blocking is carried out for approximately 30 minutes with a 1% solution of bovine serum albumin dissolved in saline phosphate buffer (FSB), pH 7.4. Then the monoclonal antibodies are incubated in a blocking solution.

After that, the plates are washed with a FSB solution and peroxidase conjugated with mouse anti-immunoglobulin is added for 1 hour. The plates are washed again and the enzyme substrate solution is added until streaks appear. In conclusion, the obtained chemical and immunochemical data are compared. As a result, 1dC recognized only the ganglioside ISSCMZ.

Cytotoxicity determination

To determine whether the obtained antibodies cause cell death directly or in some other cytotoxic way, 10 ⁷ cells / ml of mouse P3X63 mouse myeloma containing SOCOM3 are incubated at 4 and 37 ° C, respectively, with a monoclonal antibody at a concentration of 0.01-1 mg / ml within 30 minutes Then trypan blue staining is used to determine viability. The number of dead cells as a result of the action of the antibody can be determined using propidium iodine or any other marker of viability.

To study complement-mediated cytotoxicity, a suspension containing 10 ⁷ cells / ml was used; monoclonal antibodies are added at a concentration of from 0.01 to 0.5 mg / ml. Rabbit serum, which contains a high concentration of complement proteins, is added in dilutions from 1/20 to 1/2 and the incubation is carried out for 1 h at 37 ° C. The cytotoxicity due to complement was determined by counting living cells as described above, or using the Cr ⁵¹ chromium isolation method, in which the radiolabeled P3X63 myeloma cells, when they die, release the isotope into the culture supernatant.

Direct cytotoxicity was measured using various methods that detected values from 50 to 85% of dead cells relative to the total number of cells studied.

Monoclonal body biodistribution

Obtained MAT can be used both for diagnosis and treatment by introducing a label of a radioactive isotope such as 99tTc, Be186 and Sun 188. 8c11 \\ 'agh apb 81s1p51ga55sg (8c11 \\' agkh A., apb 81e1p51ga55eg, A. 1987, 1. Iisl. Meb. 28: 721) described a method for producing radioisotope-labeled monoclonal antibodies that was modified by Mosheg at EShkop (Mosheg 8.1. Apb EShkop Ό., 1990, 1. Jesus1. Meb. 31: 692-697). Label quality control is carried out using Whatman 3MM chromatography. The percent inclusion label was 98 and 100%.

To determine the possible use of MAT, 10 mice were inoculated with a PXX63 tumor, and another 10 mice were used as a normal control (no tumor was inoculated). Expected for the length of time necessary for tumor growth, and then 20 mice were injected intravenously with MAT 14E7 labeled with 99tTc.

4 and 24 hours after the injection, the biodistribution of MAT against the oligosaccharide sequence was monitored in groups of 5 mice (5 healthy and 5 with a tumor). Animals were sacrificed, the main organs and tumor were weighed, and gamma radiation was quantified at the end of the experiment.

In healthy animals, monoclonal antibodies were distributed mainly in the blood, liver and kidneys, and in animals with a tumor, MAT was found in the first organs and, preferably, in the tumor after 24 hours.

Monoclonal Antibody Recognition of Normal and Embryonic Tissues Radioactive MATs, as described in the prior art section in the art, can be used to determine a tumor in which expression of the oligosaccharide sequence is observed.

Whole body radioactivity can be studied using the Gamma Camera. Pictures in development are obtained after 5 minutes and after 1, 3, 5, 24 and 48 hours after the injection of MAT. MAT was found only in the tumor and excretory organs.

MAT can also bind directly and indirectly to other therapeutic substances, such as drugs, radioisotopes, immunomodulators, lectins and toxins. Among the immunomodulators of the biological response, which in some way can enhance the destruction of the tumor using the MAT of the present invention, there are lymphokines such as tumor necrosis factor, macrophage activator factor, colony stimulation factor, interferons, etc.

Immunohistochemical studies were performed for diagnostic purposes. Tissue sections were fixed in a 10% buffered formalin solution, then they were dehydrated, clarified and embedded in paraffin. To study histopathology, tissue sections were stained with hematoxylin-eosin.

Serial sections of paraffin blocks used for histopathological purposes were immunostained by the biotin streptavidin peroxidase complex previously described (Nki, 8. M. vashi, b., 1981, 1. Noschoshet Sufset. 29: 1349-1353).

Deparaffined and dehydrated sections were treated with a solution of 3% methanol with hydrogen peroxide for 30 min to remove endogenous peroxidase activity. Tissue sections were incubated with purified MAT. Then anti-mouse antibodies and streptavidin peroxidase complex (IacoraSh) followed; the reaction was carried out at room temperature.

Between incubations, sections were washed with saline Tris-HCl buffer. A peroxidase reaction was carried out with 30% H ₂ O ₂ and 3-3 diaminobenzidine.

The sections were washed with tap water, stained with Mayer hematoxylin, enclosed in a balm and covered with a coverslip. The reaction with the enzyme gave a brownish red color.

Tumor tissues of the mammary gland, lung, skin, and nervous system were also studied as embryonic and adult tissues.

Fresh biopsy of the abnormal tissue was taken within the first hour after surgery. The biopsy pieces were frozen, then sliced, and the slices remained frozen until the start of the study.

The use of embryonic tissues is due to the fact that the gangliosides are associated with oncoembryonic antigens several times, as well as the similarity of these molecules in human embryonic and tumor tissue (Sayap, L. c1 a1., 1982, Prog. N311. Asab. 8c1. IZA. 79: 7629-7633).

Sections of the embryonic tissue were obtained from the embryo at the age of 12 to 18 weeks.

Pieces of adult tissue were obtained from people who died in accidents and / or as a result of brain death during the first hour after death.

Among the studied tumors, tumors of the lung and central nervous system of various etiologies gave a negative response, as well as sections of normal human tissues. Whereas sections of melanoma tissue and mammary gland all showed a positive reaction, as well as sections of embryonic tissue obtained from the digestive system (liver, stomach, small and large intestine, and renal system).

Antitumor effect

In order to show the antitumor effect of monoclonal antibodies against NСССΜΜ3, animals inoculated with a tumor bearing a ganglioside target (P3X63 myeloma) were treated with the obtained antibodies. The dose may vary from 0.01 mg / kg body weight to 200 mg / kg body weight in one or more treatments for one or more days. Antibodies can be used as parenteral injection (intravenous, intraperitoneal, intramuscular, subcutaneous, intracavitary or transdermal).

In a typical experiment, the percentage of survival of the mice treated with the antibody was 30-80% compared with the mice of the control group, which was confirmed by the Lod Kash test (Cox apb Oax5 (1984) Ll1yL5 o £ 8igl1a1 Ea1a ebike. Syartap Na11). Statistically significant differences (<0.05%) were found between the MAT-treated group and the control group.

Examples

Example 1. Specific 1dC response to NСССМ3 in mice immunized with the vaccine preparation NСсСМ3 / V ^^^ / Freund adjuvant, measured by enzyme immunoassay.

Female 6–8-week-old Ba1b / C mice were injected intramuscularly with 0.2 mg of the human vaccine NСССМ3 / V ^^^ with complete Freund's adjuvant in the first dose and incomplete Freund's adjuvant in subsequent doses (manufactured by Sigma) mixed in equal volumes. Each animal received 6 doses. The first 4 doses are weekly and the remaining 2 every 14 days. Blood sampling was carried out before the first dose and then every 2 weeks.

Antibody levels were measured in the serum of animals using an indirect ELISA test on Polysorp brand plates (Shp 1 gage tag), on which gangliosides were immobilized in the following manner described below.

Gangliosides NACMC3 and NCCMC3 were separately dissolved in methanol (4 μl / ml) and 50 μl were added to each well. The tablet was placed at 37 ° C for an hour and a half to evaporate methanol. Then, 100 μl / well Tris-HCl 0.05M pH 7.8 buffer containing 2% bovine serum albumin (BSA) was added and incubated for 1 h at room temperature. Then 50 μl of serum was diluted with the same buffer and incubated overnight at room temperature.

The wells were washed 4 times with 200 μl of phosphate-buffered saline (PBS) and 50 μl of antisera of mouse anti-immunoglobulins conjugated with biotin in the appropriate dilution were added for 1.5 hours at 37 ° C.

After further washing with PBS, 50 μl of a correspondingly diluted alkaline phosphatase conjugated with streptavidin was added. At the end of the well, the wells were washed one last time and 100 μl of p-nitrophenylphosphatasubstrate was dissolved in diethanolamine buffer, pH 9.8 (1 mg / ml). The absorption was measured at 405 nm on an ELISA counter.

FIG. 1 shows the optical density data Ο.Ό. at 405 nm in the serum of each animal diluted 1/80 on the 56th day of the experiment. The response to NСССМ3 and СМ3 was determined using the ELISA test using mouse anti-1dC conjugated with biotin and streptavidin with alkaline phosphatase from Jackson.

More than 70% of the animals immunized with the vaccine preparation had Ο.Ό. at

405 nm, exceeding 0.5 in response to NСССМ3.

All immunized animals showed

The 1dC response against NСССМ3 and no response was observed to NАССМ3, despite the minimal differences between the two molecules.

FIG. Figure 2 shows a long-term specific antibody response (1dC isotype against LybsSMZ ganglioside, three months after the last dose of immunization, with no response against ILsSMZ.

Example 2. Obtaining monoclonal antibodies against SCSSMZ.

Antibodies were obtained by immunization of Ba1b / C mice using the procedure described in Example 1.

Three days before the confluence, the animals were reimmunized with the YbbsbMZ / BNB immunogen using Freund's complete adjuvant. After that, a mouse spleen was obtained and a cell suspension was prepared by passing the tissue through a steel sieve or by perfusion. Cell fusion was carried out as described in Klbeg M11k! Es (Larche, 1975, Part 256, 495-497) with some modifications.

Cells of non-secreting mouse P3 / X63 Ad8 6.5.3 myeloma were merged with mouse spleen cells in a ratio of 1:10 in 0.5 ml of fusion medium containing 42% polyethylene glycol (3000-3600, Sigma) in BPM1 1640 medium.

After fusion, the cells were cultured in a selective NAT medium (hypoxanthine / aminopterin / thymidine) at 37 ° C in a humid atmosphere of 5% CO ₂ .

Between 10 and 15 days after the cell fusion was conducted, an experiment was started to determine the presence of antibodies in the supernatant of the hybridoma cell culture using the ELISA technique described in Example 1.

Hybridoma culture cells that responded to the ganglioside of interest were selected and cloned twice by limiting dilution in the presence of conditioning cells.

The specificity of the antibodies formed by the selected hybridomas was determined using an indirect ELISA with a set of glycolipids.

The number of specific clones against ganglioside NСССМ3 was 5.5%. One of the obtained clones was designated as clone 14P7.

Example 3. The definition of a subclass of monoclonal antibody 14P7.

To determine which subclass of the immunoglobulins the monoclonal antibody of the invention belongs to, an indirect ELISA test was used with NccCM3 coated plates as described in Example 1, but the serum was replaced with dilutions of the hybridoma supernatant or purified MAT.

Mouse Apb 1dS1, 1dS2a. 1dC2b and 1dC3 conjugated with biotin and obtained in rats (Farmingen) were diluted with incubation buffer and introduced into wells. After 1 h of incubation at 37 ° С, the plates were washed and alkaline phosphatase diluted in incubation buffer conjugated with streptavidin was added. As control of each subclass, murine MATs previously characterized were used.

At the end, a substrate solution was added. Determination of absorption was carried out as described previously. FIG. 3 shows that MAT 14P7 belongs to subclass 1dC1.

Example 4. The study of the specificity of monoclonal antibodies 14P7 using immunostaining in combination with high-resolution thin-layer chromatography.

High resolution thin layer chromatography was used to separate glycolipids. The solvent system used was chloroform: methanol: KC1 0.25% and 2.5 M ΝΗ ₃ (5: 4: 1) volume: volume. The bands were shown with the help of orsine (8eppeblob. 1964, 1. Lyrb. Century., 5, 145), while the Kagakysh method Υ was used for immunostaining. with sotr. 1993 (1. Wüssbesch 114, 186).

The plates on which thin-layer chromatography was performed were coated with plastic, immersed for 75 s in a solution of 0.1% polyisobutyl methacrylate (PIBM) in Ν-hexane. The plates were then dried at room temperature for 30 minutes. The edges of the plate were treated with a 1% PIBM solution, leaving them overnight at room temperature.

Blocking of nonspecific interactions was carried out using a solution of 1% bovine serum albumin dissolved in PBS for 30 minutes, pH between 7.2 and 7.4. Immediately after this, the plates were incubated with MAT 14P7 at a concentration of 0.01 to 0.02 mg / ml in the blocking solution.

The plates were washed with FSB and incubated with horseradish peroxidase conjugated with rabbit antiserum against mouse immunoglobulins diluted with blocking solution.

After one hour of incubation with stirring at room temperature, the plates were washed again and a substrate solution of 0.4 mg / ml orthophenyldiamine (ί.'6Η8Ν2) Sigma in 80 mM citrate-phosphate buffer, pH 5 with 0.12% hydrogen peroxide was added. H ₂ O ₂ (B1eb1be Naep). The reaction was stopped by repeated washes with phosphate buffer.

The reaction revealed specificity only with respect to the ganglioside NСсСМ3 and no reaction was observed with respect to other β-glycolylated gangliosides designated as CM 1a, CM1b, CM2, and β-acetylated (Figs. 4 and 5).

Example 5. Recognition by monoclonal antibody 14P7 of tumor and embryonic tissues.

Tissue sections were fixed in a 10% buffered formalin solution, then dehydrated, clarified and embedded in paraffin. Histopathology was examined on sections of tissue stained with hematoxylin-eosin.

Serial sections obtained from paraffin blocks used in histopathological studies were immunostained by the biotin streptavidin peroxidase complex previously described (Nki, 3. M. U., B., 1981, 1. Ηίκ1set Su1set. 29: 1349-1353).

Deparaffinized and dehydrated sections were treated with a 3% solution of methanol with hydrogen peroxide for 30 minutes to remove endogenous peroxidase activity. Tissue sections were incubated with purified MAT 14P7 for 1 h at room temperature. Biotin-conjugated anti-mouse antibodies and streptavidin peroxidase complex (EacoraOc) at room temperature followed.

Between incubations, sections were washed with saline Tris-HC1 buffer. The peroxidase reaction was carried out with 5 ml of solution buffered with Tris buffer. Also added were 0.005 ml of 30% H ₂ O ₂ and 3 mg of 3-3 diaminobenzidine.

The sections were washed with tap water, stained with Mayer hematoxylin, enclosed in a balm and covered with a coverslip. The reaction with the enzyme gave a brownish red color.

Pieces of adult tissue were obtained from people who died in accidents and / or as a result of brain death during the first hour after death. Fresh biopsy of the abnormal tissue was taken within the first hour after surgery. Slices of embryonic tissue were obtained from the embryo at the age of 12 to 18 weeks during the first hour after abortion. All biopsy pieces were washed in saline and immediately frozen in liquid nitrogen and stored frozen at -80 ° C.

Serial sections with a thickness of 5 μm were obtained from pieces of frozen tissue in a Leys cryostat at -25 ° C. Slices were dried in air and used immediately or stored at -20 ° C wrapped in aluminum foil. In all cases, sections were fixed at the time of use in 4% paraformaldehyde for 20 minutes.

FIG. 6 shows the results of an immunohistochemical study of MAT 14P7 in tissues of a healthy person. The reactivity of MAT in the membrane and in the cytoplasmic region was not observed.

FIG. 7 shows the results of the same study on pathological tissues. All studied breast tissue (33/33) and melanoma (20/20) gave a positive reaction, while 70 lung tumors of various etiologies gave a negative reaction as well as 33 different tumors of the central nervous system.

FIG. 8 shows recognition of MAT 14P7 in the case of the digestive system and embryonic renal tissue.

Example 6. Recognition using MAT 14P7 of the ganglioside YCeCM3 in cells studied by flow cytometry.

The studied cell lines included murine P3X63 myeloma expressing CM3 and LSCCM3, described by 1. Mi1Yid et al. (MiSchd, 1. e! A1., 1994, 1. Vyusyes 116: 64-73) and B16 myeloma, which expresses CM3. Cells were cultured in BPM1 medium with 8% bovine fetal serum. The cell concentration was adjusted to 10 ⁷ cells / ml of phosphate buffered saline pH 7.4, containing 0.02% sodium azide and 1% bovine serum albumin. To each tube containing 0.1 ml of cell suspension, 0.05 ml of a solution of MAT 14P7 dissolved in saline phosphate buffer was added to a final concentration of 0.1 mg / ml, and then incubated for 30 min at 4 ° C. The cells were then washed with a solution in which they were suspended.

Cell cultures were then centrifuged for 5 minutes at low speed to precipitate. The anti-mouse (1dC + 1dM) conjugate of biotin (Jackson) was added and washed after 30 minutes of incubation at 4 ° C. Finally, 0.002 mg of fluorescein streptavidin P1TC (Jackson) was added and incubated under the same conditions as previously described. The last wash was performed with saline phosphate buffer.

The supernatant after the last centrifugation was poured and the cells were resuspended in 0.6 ml of the solution for the last wash.

80% of P3X68 myeloma cells gave a positive color with 14P7 monoclonal antibody (Fig. 9).

Example 7. The study of direct cytotoxicity in monoclonal antibodies 14P7.

The mouse P3X63 mouse myeloma cell line was incubated with MAT 14P7 as described in Example 6. After washing, 0.01 ml of a solution of propidium iodine in saline phosphate buffer was added to the cells to determine cell viability using flow cytometry.

The results showed that 78% of the cells are dead (Fig. 10).

Example 8. The study of the biodistribution of 99tTc - labeled monoclonal antibody 14P7 in Ba1b / C mice, healthy and tumor bearing P3X63 myeloma.

Twenty female mice of the line

Ba1b / C weighing from 20 to 22 g (10 healthy and 10 peroconeally inoculated with P3X63 myeloma tumor) received an intravenous injection of MAT 14P7 labeled with 99tTc.

The label concentration was 0.03 mg MAT

14P7 / 60 μCi 99tTs.

The results of the determination of radioactivity in various organs were obtained on 5 animals in each group, 4 and 24 hours after injection. The animals were killed and the weight of each organ was determined on a Sartorius scale. The radioactivity of all tubes was determined simultaneously, approximately 25 hours after the start of the experiment, on a gamma counter of the ABBAS GS (model GSEAEE 1470).

The labeling method was previously described in 8c11 \\ 'agx 81esh51ga58eg (1987) and modified by MiShet and Yoshkoi in 1990.

Labeled MAT 14P7 was removed from the body of a healthy mouse using the kidneys and liver (Fig. 11).

In mice with P3X63 myeloma, binding of MAT 14P7 was detected after 4 hours (12% of the total injected radioactivity per gram of tissue) and after 24 hours (35% of the total injected radioactivity per gram of tissue). Removal of MAT from the body occurred mainly through the kidneys (Fig. 12).

Example 9. Antitumor effect of monoclonal antibody 14P7 in Ba1b / C mice bearing ascitic P3X63 myeloma.

Female Ba1b / C mice weighing 20-22 g were injected intravenously with 6 doses of 14P7 monoclonal antibody (one group 0.1 mg and another 0.2 mg) every 2 days, the monoclonal antibody was dissolved in saline phosphate buffer. Three days before the experiment, inflammation was caused in the peritoneal cavity using Freund's incomplete adjuvant to stimulate the moment when the tumor can be measured. 10,000 P3X63 murine myeloma cells were inoculated intraperitoneally on day 0 of the experiment, at the same time that passive therapy with 14P7 monoclonal antibody was started, only in the case of monoclonal antibody, intravenous inoculation was performed. The control group with a good prognosis (the best treatment) used for comparison was the third group, which was treated intravenously with cyclophosphamide (8yida1 Nia B1ai Riagshaseiisa 1 Co.) With a dose of 20 mg / kg body weight, which was a weekly dose throughout the experiment. Intravenous treatment with a solution of saline phosphate buffer, pH 7.4, served as a control of the experiment.

FIG. 13 shows survival results in the 4 groups previously described. In the groups treated with 14P7 (0.1 and 0.2 mg), as well as 20 mg / kg of cyclophosphamide, no measurable tumor was found in individual animals.

Survival results were in favor of groups treated with MAT 14P7. On the 30th day of the experiment, when no animals from the control group remained alive, 6 animals were still alive, both from the first group (0.1 mg MAT) and from the group treated with cyclophosphamide, as well as 7 animals from the second group (0 , 2 mg MAT). On the 60th day of treatment, 2 animals of the first group and 2 animals treated with cyclophosphamide remained alive, while in the second group 5 animals were still alive.

Example 10. Inhibition of tumor growth of dense P3X63 myeloma in mice without thymus.

On day 0 of the experiment, 10 female mouse individuals lacking a thymus from the oi line! Bteab IMK1, weighing from 20 to 22 g, was subcutaneously inoculated with 10 ⁶ cells of the tumor mouse line of P3X63 myeloma. Animals were divided into two groups of 5.

One group was started to be treated intraperitoneally with purified MAT 14P7, 0.15 mg per dose (6 doses) every 2 days. While the other group served as a control and received in the same way the same number of doses with the same frequency equal to the volume of saline phosphate buffer.

FIG. 14 shows the inhibition of tumor growth in mice treated with MAT 14P7, compared with the control group. Statistically significant differences were observed in these groups.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows serum antibody levels obtained against IScSM3, but not against CM3, on day 56 of the experiment in mice immunized with a vaccine preparation containing IScSM3 / UBPS / Freund's complete adjuvant.

FIG. 2 - determination of antibody response isotypes using ELISA test of IScSM3 ganglioside in mouse serum 3 months after receiving a fourth dose of 0.2 mg of IScSM3 / UBBP / Freund's complete adjuvant.

FIG. 3 - determination using an ELISA test of a subclass of immunoglobulin, which includes a monoclonal antibody 14P7.

FIG. 4 - recognition by immunostaining with thin-layer chromatography of Ν-glycolylated and Ν-acetylated gangliosides, which were used to study the specificity of monoclonal antibody 14P7.

FIG. 5 - ganglioside recognition

NСсСМ3 monoclonal antibody 14P7 using immunostaining with thin layer chromatography.

FIG. 6 - negative response in recognition of normal tissues of an adult organism during treatment with monoclonal antibody 14P7 and subsequent immunohistochemical study.

FIG. 7 - immunohistochemical recognition of certain malignant and benign human tumors by monoclonal antibody 14P7.

FIG. 8 - immunohistochemical recognition of normal human embryonic tissue by monoclonal antibody 14P7.

FIG. 9 - recognition of the cell line of myeloma PXX63 expressing ganglioside ΝΟοΟΜ3, monoclonal antibody 14P7 using flow cytometry.

FIG. 10. - complement independent cytotoxic effect of ΜΑΤ 14Р7 when using the P3X63 myeloma cell line and propidium iodine technique in conjunction with flow cytometry.

FIG. 11 - biodistribution of monoclonal antibody 14P7 labeled with 99tTc. Results in percent gamma radiation per gram weight in the organ under study in normal Ba1b / C mice.

FIG. 12 - biodistribution of monoclonal antibody 14P7 labeled with 99tTc. The results in percent of gamma radiation per weight in grams of the organ under study in Ba1b / C mice bearing tumor P3X63 myeloma.

FIG. 13 shows the antitumor effect of passive therapy with 14P7 monoclonal antibody in groups of Ba1b / C mice inoculated with mouse ascitic tumor of myeloma PXX63 and treated with 0.1 and 0.2 mg of the indicated antibody in comparison with the control group treated with cyclophosphamide (20 mg / kg) and a control group treated with saline phosphate buffer.

FIG. 14 - inhibition of tumor growth ίη νινο on the example of dense mouse myeloma P3X68 in mice without a thymus, line oi! Bgeab ΝΜΚΙ.

Claims (10)

CLAIM 1. The strain of hybrid cells of a mouse 14R7 ESACS 98101901, producing monoclonal antibody 14R7 against gangliosides ΝΟοΟΜ3. 2. The 14P7 monoclonal antibody produced by the hybrid cell strain according to claim 1, specifically recognizes the oligosaccharide sequence Ν-glycolylated galactose - glucose - sialic acid in the ganglioside ΝΟοΟΜ3 and has cytolytic activity against tumor cells containing the specified sequence. 3. The monoclonal antibody according to claim 2, characterized in that its cytolytic activity against tumor cells containing the oligosaccharide sequence Ν-glycolylated galactose - glucose-citric acid, can be direct or mediated by complement. 4. A pharmaceutical composition containing a monoclonal antibody, as described in any one of claims 2, 3, and a solvent or excipient. 5. The pharmaceutical composition according to claim 4, wherein the antibody may be associated with a therapeutic agent, such as a drug, a radioisotope, an immunomodulator, a lectin and a toxin. 6. The pharmaceutical composition according to any one of claims 4, 5, characterized in that it is used for the treatment of malignant tumors. 7. The pharmaceutical composition according to claim 6, characterized in that the malignant neoplasm is a malignant tumor of the mammary gland, kidney, organs of the digestive system or human melanoma. 8. A reagent containing a monoclonal antibody, as described in any of paragraphs 2, 3, associated with such markers as enzymes, chromophores, chemiluminescent substances and radionuclides. 9. The use of reagent, characterized in paragraph 8, to identify tumor cells. 10. The use according to claim 9, wherein the tumor cells are cells of human melanoma, breast, kidney, or organs of the digestive system, such as the liver, colon, stomach, and rectum.