RU2319743C1 - Strain of hybrid cultured mammalian cells mus musculus as producer of monoclonal antibodies to hypoglycosidated and deglycosidated isoforms of tumor-associated human antigen muc i - Google Patents

Abstract

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-98 is prepared by the hybridoma technology method. This strain is a producer of monoclonal antibodies possessing individual specificity to hypoglycosidated and deglycosidated isoforms of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on the immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR₂₂-polypeptide; synthetic monomeric polypeptide (TR₁); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of antigen Muc I and allows assaying its concentrations in human serum blood in carrying out the early diagnosis of tumors. Invention can be used in preparing monoclonal antibodies to tumor-associated human antigen Muc I.

EFFECT: valuable properties of strain.

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Description

The strain of hybrid cultured animal cells of mus musculus is a producer of monoclonal antibodies to hypoglycosylated and deglycosylated isoforms, a tumor of the associated human Muc I antigen.

The invention relates to biotechnology and immunology, and relates to the production of monoclonal antibodies to a tumor associated with the human Muc I antigen (tumor marker Muc I) using hybrid cultured animal cells (hybridomas). The presence of a panel of monoclonal antibodies to the Muc I tumor marker is promising for the creation of various diagnosticums in clinical oncology, in particular for use in the diagnostic two-site immunometric method for determining the concentration of Muc I in human serum (Br. Cancer Res. And Treat. 81: 195-207, 2003 )

Of greatest interest from the point of view of early oncodiagnosis are hypoglycosylated and deglycosylated isoforms of the Muc I human antigen, characteristic of tumors of epithelial cell origin.

Some producers of monoclonal antibodies are known that are specific for various epitopes of the Muc I human tumor marker molecule (Cancer Res. 47: 5476-5482, 1986). The closest analogues of the claimed invention for the specificity of monoclonal antibodies are hybrid strains SM3 ISOBM TD-4 N165 and BC2 ISOBM TD-4 N138. The strains produce monoclonal antibodies specific for both the native tumor marker molecule and the peptide backbone of the Muc I molecule (VNTR) (Inf. J. Cancer: 43, 1072-1076, 1989; Br. Cancer Res. And Treat. 81: 195- 207, 2003). It is known that monoclonal antibodies SM3 and BC2 recognize a linear peptide region of an antigen molecule both in the form of a monomer (TR) and in the form of polymers with different degrees of polymerization (TR ₅ -TR ₁₀₀ ), while the degree of glycation of antigen is only insignificant degree affects the specific activity of these antibodies (Tumor.Biol.:19 (suppl 1), 111-117, 1998).

The spectrum of well-known producers of clinically significant monoclonal antibodies is insufficient to diagnose the early stages of epithelial cell tumors.

The task of the invention is to expand the arsenal of strains of hybridomas producing monoclonal antibodies to the tumor associated human Muc I antigen.

The problem was solved by obtaining strain M3B11 of hybrid cultured animal cells of Mus musculus animals producing monoclonal antibodies with selective specificity and high affinity for the hypoglycosylated and deglycosylated isoforms of the human tumor marker Muc I.

The strain is deposited in the All-Russian collection of industrial microorganisms and has the number VKPM N-98.

The strain was obtained by hybridizing the lymphocytes of an immunized mouse (Balb / c) with myeloma cells (Sp2 / 0 / Ag14) and using a subcutaneous immunization scheme, followed by isolation of mouse lymph node B lymphocytes. As the immunogen, a deglycosylated human Muc I (deMuc) was used (Analitical Biochemistry: 82.289-309.1977).

To assess the productivity of the obtained hybridomas and the specificity of the antibodies produced by them, an enzyme-linked immunosorbent assay (ELISA) (ELISA) was used (In the book "Antibodies", v.2 // ed. "Mir", edited by D. Catty, 223-230 1991) using several specificity markers, namely:

- natural purified Muc I antigen isolated from human milk (J. Immmol .:135, 3610-3616, 1985);

VNTR ₂₂ polypetide (J. Biochem .: 263, 12820-12823, 1988);

- synthetic monomeric polypeptide (TR ₁ ) (Encyclopedia of Polimer Science and Technology // by J. Wihey and S. Iuc, 3-49, 2004);

- deglycosylated antigen Muc I (de-Muc) obtained by chemical oxidation of natural Muc I;

- hypoglycosylated Muc I (o-Muc I) obtained by periodically oxidizing a natural antigen (J.immunol. Methods: 78, 143-153, 1985).

Naturally purified Muc I antigen, VNIR ₂₂ polypeptide, hypoglycosylated and deglycosylated antigens (o-Muc and de-Muc) are isoforms of the Muc I tumor marker with varying degrees of glycosylation. The Muc I antigen isolated from milk contains a complex of glycoproteins consisting of a polypeptide skeleton with a variable number of tandem repeats of 20 amino acids (VNTR) and oligosaccharide chains. This antigen was used as a control in assessing the interaction of monoclonal antibodies with less glycosylated antigens. Deglycosylated antigen (de-Muc) contains no more than 1% carbohydrate chains, and o-Muc I, obtained as a result of milder periodical oxidation, is characterized by aberrant glycosylation (hypoglycosylated antigen). Thus, the above specificity markers make it possible to establish the individual specificity of monoclonal antibodies against clinically significant hypoglycosylated and deglycosylated isoforms of the Muc I human tumor marker.

Final product.

Monoclonal antibodies of class IgG 1 (test system for isotyping - ISO 2-1KT, Sigma), which are highly specific for the Muc I antigen in hypoglycosylated and deglycosylated form.

Cultivation of the strain in a nutrient medium.

For cultivation using DMEM medium (Sigma), containing 10% fetal serum, 1% glutamine and pyruvate, 50 IU / ml penicillin and 25 μg / ml streptomycin. Cells were cultured at 37 ° C in an atmosphere containing 5-7% CO ₂ . For cultivation, use plastic or glass tablets or 50 ml vials (Lux). The nature of the growth is a stationary suspension. Passaging frequency is 2-3 days. Multiplicity of sieving 1: 3-1: 4. The antibody titer in the culture fluid is 1: 128.

The cultivation of the strain in the body of the animal.

To obtain ascites, mice are injected intraperitoneally with 0.5 ml of pristan (In the book "Monoclonal Antibodies" // ed. Medicine, under the editorship of R. Kenneth, 396-397, 1983). Prepared animals rest for 3 weeks. Hybridoma cells are washed from the serum contained in the medium and 4 · 10 ⁶ cells are injected in Hanks solution (manufacturer - Moscow Bacterial Preparations Plant). The growth of ascites tumors is observed on the 7-10th day. The antibody titer in the culture fluid is 1: 128, and in the ascitic one - 1: 10000.

Productivity.

The concentration of antibodies produced by the strain is 15 μg / ml in the culture fluid, and 8 mg / ml in the ascites fluid. Stability of antibody production is maintained for 20 passages in culture and 7 passages in animals if the hybridoma is interrupted.

Contamination.

Bacteria and fungi were not found in cultures during long-term observation and plating on standard nutrient media (MPA for bacteria and Saburo agar for fungi). Mycoplasma infection was not detected (test system Mycoplasma Stain Kit, Flow Lab.).

Cell preservation.

Cells are frozen after cloning 2 and 3 at 10 and 15 passages in culture and after growth in the form of an ascites tumor in mice. Cells in a cryoprotective medium of the following composition (wt.%): DMEM - 50, fetal serum - 40 and dimethyl sulfoxide - 10, placed in a refrigerator at - 70 ° C, and then transferred to liquid nitrogen. The survival rate after thawing is 70%.

The invention is illustrated by the following drawings.

Figure 1. Immunospecificity of monoclonal antibodies of the claimed strain to hypoglycosylated and deglycosylated isoforms of a tumor of the associated human Muc I antigen.

Figure 2. Immunospecificity of the monoclonal antibodies of the claimed strain to the monomeric peptide TR ₁ and the polypeptide VNTR ₂₂ tumor of the associated human Muc I antigen.

Example 1. Obtaining a strain of VKPM H-98.

Balb / c mice were immunized with de-Muc I in Freund's complete adjuvant in the pads of the hind legs (50 μg per mouse). On the 13th day, mice were immunized in the pads of the hind legs with the same amount of de-Muc I in Freund's incomplete adjuvant (In the book "Monoclonal Antibodies" // ed. Medicine, under the editorship of R. Kenneth, 371-382, 1983) . On day 16, mice were sacrificed, popliteal lymph nodes were isolated, and lymph node cells were poured into Sp2 / 0 / Ag14 myeloma cells using polyethylene glycol (PEG). The ratio of lymph node cells and myeloma cells is 5: 1. After fusion, the cells are seeded into the wells of a 96-well plate (Nunc) in the amount of 200 thousand cells per well, into which mouse peritoneal macrophages are previously seeded at 10 thousand cells per well. Hybridomas are selected in DMEM containing hypoxanthine, aminopterin and thymidine (GAT) (in the book "Monoclonal Antibodies" // ed. Medicine, under the editorship of R. Kenneth, 371-382, 1983). Hybrid growth in a culture medium containing 15% fetal serum was observed on days 3-7.

The selection of growing clones of cells secreting antibodies of a given specificity is carried out on days 10-12 based on the ELISA test using the previously mentioned specificity markers.

As a result, we obtained a strain of hybrid cultured animal cells of animals Mus musculus VKPM H-98, a producer of monoclonal antibodies to hypo- and deglycosylated isoforms, a tumor of the associated human Muc I antigen.

Twice the cloning of hybridomas obtained by the method of limiting dilutions by sieving into wells of a 96-well plate at the rate of 1 cell per well in DMEM and subsequent testing for antibody secretion showed that the proportion of clones that retain the production of antibodies of a given specificity is 100%.

Example 2. Obtaining, isolation and purification of monoclonal antibodies produced by strain VKPM H-98.

To obtain monoclonal antibodies, cells of the hybridoma strain are grown in 24-well plates (Nunc) on a culture medium. When the cell concentration in well 10 ^{6 is} reached, the culture fluid containing the cells is collected, centrifuged for 10 min at 2000 g and the supernatant is removed. The collected cells are transferred to serum-free medium (DMEM) and injected into mice intraperitoneally at a rate of 4 · 10 ⁶ cells / mouse. After 7-10 days, visual growth of ascites tumor is observed, mice are sacrificed and ascites from the abdominal cavity is collected with a syringe. The ascitic fluid is centrifuged for 10 min at 2000 g to clear the cells, and the supernatant is used to isolate monoclonal antibodies.

To this end, an equal volume of a saturated (4 M) solution of ammonium sulfate is added to the ascites fluid of the mouse containing monoclonal antibodies and centrifuged at 5000 g for 20 minutes. The precipitate was dissolved, carefully dialyzed against 0.5 M phosphate buffer pH 7.2 and purified on a column of DEAE cellulose (DE-52). (Immunology: 4, 40-44, 1980). From 1 ml of ascites fluid, 8 mg of monoclonal antibodies are obtained. The purity of thus isolated monoclonal antibodies is confirmed by electrophoretic and amounts to 90-95%.

Example 3. Determination of the specificity and concentration of monoclonal antibodies in culture and ascites fluids by ELISA.

100 μl of antigen solution (Muc I, VNTR ₂₂ polypeptide, monomer polypeptide TR ₁ , de-Muc I, o-Muc I) with a concentration of 10 μg / ml was added to the wells of a 96-well plate and incubated for 2 hours at 37 ° C . After 4 times washing the tablet from excess antigen, a working buffer (phosphate buffer with a pH of 7.2, containing 0.05% bovine serum albumin and tween 20) is added to the wells (In the book "Antibodies", v.2 // under Edited by D. Catty, 153-183, 1991). Incubate for 30 minutes. Prepare a series of 10-fold dilutions of culture or ascites fluids in a working buffer. The analyzed samples in a volume of 50 μl with a 2-fold repetition are introduced into the wells of the antigen plate and incubated for 2 hours at room temperature. After incubation, the contents of the wells are removed, the plate is washed and 50 μl per well of rabbit antibody to mouse immunoglobulin conjugate with peroxidase dilution 1: 3000 diluted. The plates are incubated for another 2 hours, washed, and 100 μl of substrate buffer (phosphate-citrate buffer with pH 4.5) are added to the wells (In the book "Antibodies", v.2 // edited by D. Catty, 153-183 , 1991) containing 4 mg / 10 ml of 0-phenylenediamine and 0.03% hydrogen peroxide. The plates are incubated for 10-15 minutes and the reaction is stopped by adding 10% sulfuric acid at 100 μl per well. The plates are scanned on a microplate reader (Labsystems) at a wavelength of 492 nm. The concentration of antibodies is determined by the calibration curve, which is obtained simultaneously with the test. To build a calibration curve, instead of samples, dilutions of standard preparations of purified antibodies of the inventive strain in a known concentration are introduced into the wells of the plate.

The concentration of antibodies in the culture fluid is 15 μg / ml, and in ascites - 8 mg / ml.

Figure 1 and 2 presents data on the glycopeptide specificity of the obtained monoclonal antibodies. Concentrations of monoclonal antibodies are plotted along the X axis, and optical density at a wavelength of 492 nm is plotted along the Y axis. The activity of the interaction of monoclonal antibodies with an antigen increases as the concentration of antibodies increases from 1 ng to 1 μg.

As follows from the data shown in FIG. 1, monoclonal antibodies of the claimed strain interact with a deglycosylated antigen (de-Muc I) at the same level as with the natural antigen (Muc I). These data indicate a high affinity of the obtained antibodies to the polypeptide region of the antigen molecule, which is confirmed by the results of the interaction of antibodies with VNTR in both monomeric and polymer form (Figure 2). When interacting with a hypoglycosylated antigen, which underwent a milder destruction of the carbohydrate structure, the obtained antibodies sharply increase the level of binding, which indicates the selective glycopeptide specificity of the obtained antibodies and high affinity for the hypoglycosylated form of the Muc I antigen.

Thus, a strain of hybrid cultured animal cells of animals Mus musculus VKPM H-98 was obtained - a producer of clinically significant monoclonal antibodies with individual specificity for hypo- and deglycosylated isoforms of the Muc I human tumor marker.

Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of the Muc I antigen and are suitable for determining its concentration in human serum during early diagnosis of tumors.

Claims (1)

The strain of hybrid cultured animal cells Mus musculus VKPM H-98 is a producer of monoclonal antibodies to hypoglycosylated and deglycosylated isoforms of the tumor-associated human Muc I antigen.

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