RU2070928C1 - Strain of hybrid cultured mammalian cells mus musculus - a producer of monoclonal antibody to human platelet glycoproteins iib-iiia and monoclonal antibody framon to human platelet glycoproteins iib-iiia - Google Patents

Abstract

FIELD: biotechnology, medicine. SUBSTANCE: method involves preparing high-productive strain of murine hybrid cells producing monoclonal antibodies to the human platelet glycoproteins IIb-IIIa. Strain FraMon is obtained by step-by-step recloning and selection from the primary population of hybrid cells CR C64. It shows enhanced stability and high growth rate. Concentration of monoclonal antibodies in cultural fluid is 60-80 mcg/ml. FraMon antibodies inhibit human platelet aggregation completely at concentration 1-3 mcg/ml. Antibodies can be used for treatment of thrombosis, myocardium infarction, for vessel prosthesis operations. EFFECT: enhanced effectiveness of antibody production. 3 cl, 3 dwg

Description

 The invention relates to biotechnology, in particular to monoclonal antibodies (MoATs) and elements of the technology for their preparation by hybridoma technology for further use in medicine, veterinary medicine and research.

 It is known that the main processes occurring with the participation of platelets are mediated by membrane glycoproteins (GPs), primarily the IIb-IIIa complex, which is a fibrinogen receptor. It is assumed that fibrinogen forms bridges between platelets or serves as a cofactor in the process of aggregation.

 MoATs and their producing strains are known that are capable of specifically binding to the GP IIb-IIIa complex, inhibiting the binding of fibrinogen and other proteins to platelets, mimicking the state of platelets similar to that observed with Glanzmann thrombasthenia. In this case, platelets lose the ability to aggregate induced by most agonists (ADP, thrombin, collagen, adrenaline, etc.).

 Antibodies with similar properties are promising for use in clinical practice as antithrombotic agents, in particular in the treatment of myocardial infarction and in vascular prosthetics.

 However, the known MoATs are able to completely inhibit platelet aggregation only at an antibody concentration of more than 5 μg / ml, which may be their disadvantage when used in clinical practice.

 The aim of the invention is to obtain more effective antibodies and hybridomas that produce them.

 This is achieved by creating a hybridoma strain of FRaMon (6C4.2F5.1B5) and antibodies of the same name.

 The FRaMon strain was obtained by stepwise reclamation and selection from the primary 6C4 population of СRC64 hybridoma obtained on the basis of the BALB / C mouse immune spleen cells and the X63-Ag8-653 mouse myeloma line.

 The preparation of CRC64 strain has been described previously and includes immunization of BALB / C mice with freshly washed platelets in a Tyrode-HEPS buffer and subsequent hybridization of immune mouse spleen cells with murine myeloma X63-Ag8-653 cells in the presence of 45% RPMI 1640 medium (volume / volume) PEG (mol. weight 3350) and 15% DMSO. After hybridization and PEG washing, cells were plated in 96-well plates.

 To obtain the FRaMon strain, CRC64 cells were cloned at a rate of 1.5 cells / well in RPMI 1640 medium with the addition of 20% cow fetal serum, 4 mM glutamine, 10 mM sodium pyruvate, amino acids and vitamins.

 After a week of cultivation, the selection of primary hybrid producer clones was carried out, determining the content of antiplatelet antibodies by standard enzyme immunoassay for testing immunoglobulins (test a) and an aggregation test to determine the specific activity for inhibiting human platelet aggregation (b).

Platelet binding MAAT was screened by ELISA using intact platelets. Washed platelets (10 ppm in 100 μl of Tyrode-HEPES solution) were added to the wells of a 96-well plate and pelleted for 5 min at 1000 g. For better platelet attachment, the plates were incubated for 30 min at 37B198JUS and then non-adherent platelets were removed by washing the wells with Tyrode-HEPES solution. After that, the plastic was blocked with 1% BSA (150 μl / well, 30 min, at 37 ^o C). The culture fluid or purified antibodies were added to the wells of 50 μl and incubated for 40 min at 37 ^° C. After washing with Tyrode-HEPES solution (5 times), 50 μl of goat anti-mouse Ig antibodies conjugated to horseradish peroxidase were introduced into the wells. After incubation (30 min) and washing (7 times), bound peroxidase was determined by color reaction with a chromogen (0.1 mg / ml 1,2-phenylenediamine and 0.006% H ₂ 0 ₂ in 50 mM citrate buffer, pH 4.5) . Chromogen was added 100 μl. After 5-10 minutes, the reaction was stopped by adding 25 μl of 50% H ₂ S0 ₄ . The optical density was determined spectrophotometrically at 492 nm. In the positive control, the serum of immunized mice was used, and in the negative, culture fluid from myeloma cells and the serum of intact mice.

The ability of MoAT to inhibit platelet aggregation (test b) was tested as follows. ADP-stimulated aggregation was studied in platelet-rich plasma (OTP, 3 × 10 ⁸ platelets / ml), and thrombin-induced aggregation in a suspension of washed platelets (3 × 10 ⁸ platelets / ml). Aggregation was determined in an aggregometer at 37 ^{° C.} with stirring at 900 rpm.

Producing hybrids were cloned by the method of limiting dilutions on the feeder layer of mouse splenocytes. The efficiency of clone formation was 70-90%. The number of productive clones was 80-90%.
As a result of four step-wise reclamation and selection, a strain was obtained that significantly exceeded the primary population in terms of stability, growth rate, and level of synthesis of highly effective antibodies.

 Conditional name of strain FRaMon (6С4.2F5.1В5). The strain is deposited in the Cell Culture Collection of the Institute of Cytology RAS under number 644D and is characterized by the following properties.

 Morphology: FRaMon strain cells are transparent, well refracting light, round or ellipsoidal, growing in the culture in the form of aggregates (3-5 cells) and isolated cells. The homogeneity of the population in morphology and size is at least 85%. Polymorphic cells make up no more than 15% of the composition of the population.

 Standard conditions for the cultivation of the strain: medium RPMI 1640 with 20% fetal bovine serum, 4 mM glutamine, 10 mM sodium pyruvate, amino acids and vitamins, penicillin and streptomycin, 100 U / ml and 100 μg / ml, respectively.

 Cryopreservation: in the fetal serum of a cow with 10% DMSO at a concentration of 1 million / ml.

 When grown on RPMI 1640 medium with 20% serum of a cow fetus, the saturating population density in static culture reached 1.34 million / ml with a viability of 80-90% for 24 hours after reaching the maximum density.

 The standard growth curve of the strain is shown in figure 1.

The level of antibody synthesis determined by test a was 60-80 μg / ml of culture fluid. In test b for specific activity, the synthesis level was 60-72 μg / ml. The freeze-thaw procedure has practically no effect on the cell growth rate. The level of antibody synthesis for 3 days after thawing is reduced by 10-15 μg, then within 7-10 days is restored to its previous level (figure 2). Cell viability after thawing not less than 75%
Characterization of the cultivation of hybridomas in the animal.

To obtain preparative amounts of antibodies, cells of the FRaMon strain are cultured in the abdominal cavity of BALB / C mice. 7 days before cell injection, 0.5 ml of pristan is administered intraperitoneally to mice. Cells are injected intraperitoneally at a dose of 4x10 ⁶ . Ascites is formed after 7-10 days. Compared to the initial 6C4 population, the FRaMon strain is characterized by the following properties (Table 1).

 The monoclonal antibody produced by the FRaMon strain received the same code name FRaMon.

 The FRaMon antibody is an IgG1 class immunoglobulin. The concentration of MoAT in ascites fluid, determined by ELISA, is 10-20 mg / ml.

Isolation of MoF FRaMon from ascitic fluid is carried out using precipitation with sodium sulfate, followed by chromatography on DEAE-TouoreaI. The purified antibody is electrophoretically homogeneous and has a mol.v. 150 kDa. It specifically binds to human platelet GP IIb-IIIa. Determination of the nature of the antigen is carried out by the method of its precipitation MoAT FRaMon from a lysate of ¹²⁵ J-labeled platelets. In the precipitates, two proteins with mol. with weights of 130 kDa and 90 kDa in non-reducing conditions and 100 kDa in reducing conditions, which corresponds to electrophoretic characteristics of GP IIb and IIIa. Dissociation of the GP IIb-IIIa complex with EDTA prevents the binding of MoAT FRaMon.

The specificity of MoAT. To characterize specificity, binding parameters of the FRaMon antibody to platelets in the presence of divalent cations or EDTA were determined. In the presence of EDTA, the antibody practically did not bind to platelets, which is consistent with immunoprecipitation data and indicates the complex-dependent nature of the MoAT FRaMon epitope. In a saturating concentration, about 48,000 ₁₂₅ J-FRaMon molecules were bound to a single platelet. This corresponds to the number of copies of the GP IIb-IIIa complex on the platelet surface (40-50 thousand / cell). Comparison of binding parameters and aggregometry results shows that saturation of binding sites with antibodies and complete blocking of platelet aggregation occur at the same antibody concentrations.

 These data suggest that the antigen of the FRaMon antibody is the GP IIb-IIIa complex, and the antibody recognizes determinants only on the complex of these proteins.

 Moat FRaMon is intended to inhibit human platelet aggregation. This property of the antibody was tested on platelets of 25 donors. Complete inhibition of ADP-induced aggregation in OTP is observed at an antibody concentration of 3 μg / ml. In some donors, complete inhibition of aggregation was observed at lower antibody concentrations (the lowest value is 0.5 μg / ml). Thrombin-induced aggregation was completely inhibited in the presence of 3 μg / ml, and incomplete at 1 μg / ml.

The effect of F (ab ') ₂ fragments of the FRaMon antibody on aggregation was studied on platelets of 15 donors. F (ab ') ₂ - fragments at a concentration of 5 μg / ml completely blocked the induced aggregation.

 Thus, MoAT FRaMon, like most antibodies with similar effects described in the literature, is complex-specific and does not interact with dissociated proteins. Its specific effect is due to the blocking of the fibrinogen receptor of the GP IIb-IIIa complex.

 The main properties of Moat FRaMon are: belongs to class I g G1; molecular weight 150 kDa; produced by FRaMon hybridoma deposited HSCV (P) under the number 644D; binds on the surface of platelets a complex of membrane glycoproteins IIb-IIIa; inhibits fibrinogen-dependent platelet aggregation.

 The essence of the claimed group of inventions is illustrated by an example of a specific embodiment of the invention.

Cloning of the primary population of CRC64 hybridoma selects 600 cells of the primary CRC64 hybridoma in a logarithmic growth phase in 40 ml of complete culture medium (see copy above) and seeded in four 96-well plates with a feeder layer of 100 μl / moon, which is 1.5 cells / moon The day before cloning, a feeder is prepared from intact mouse splenocytes. To do this, a day before cloning, a suspension of splenocytes is scattered at the rate of 10 ⁵ cells per well in a volume of 100 μl / moon. The plates are incubated in a humid atmosphere of 5-10% CO ₂ at 37 ^o C.

 Accounting for clone formation is carried out after 10 days. Cloning efficiency is 80%. The level of antibody synthesis in clones is monitored by standard enzyme-linked immunosorbent assay (method a). Clones with the maximum level of synthesis are selected and the entire procedure is repeated 4 times in full. The synthesis level of the FRaMon antibody after 4 reclations is 60-80 μg / ml.

 The accumulation of FRaMon antibodies in ascites fluid.

 Clone cells with a synthesis level of 70 μg / ml are injected into BALC / C mice at a dose of 4 million intraperitoneally. 7 days before cell injection, 0.5 ml of pristan is administered intraperitoneally to mice. Ascites are collected after 10 days. The concentration of antibodies in ascites is 15 mg / ml.

 Purification of FRaMon antibody from ascites fluid.

To one volume of ascitic fluid containing FRaMon antibody, 0.9 drops of a volume of 2.53 M Na ₂ SO ₄ solution are added dropwise and stirred for 90 minutes. The precipitate is precipitated at 5000 g for 30 minutes and then dissolved in 10 mM phosphate buffer (pH 8.0) for subsequent purification on an ion-exchange sorbent. A solution of FRaMon antibody in 10 mM phosphate buffer is applied to a DEAE-ToyoPearl sorbent column. After applying MoAT, the column is washed with 10 mM phosphate buffer (3 column volumes) and then the sorbed antibody is eluted with a gradient of phosphate buffer 10-150 mM (total eluent volume is 500 ml for column V = 30 ml). The content of antibodies in the fractions is determined by ELISA. Fractions containing antibodies are pooled, concentrated and dialyzed against FSB. The purity of the MoAT preparation is monitored by electrophoresis in 7.5% PAG according to Laemmli. The purified antibody is electrophoretically homogeneous and has a molecular weight of 150 kDa.

 Inhibition of human platelet aggregation by FRaMon antibody.

Platelet aggregation was measured by changing the light transmission of the platelet suspension using an Aggregation Analaizer aggregometer (BIOLA, Russia) at a platelet concentration of 2-3x10 / ml, a temperature of 37 ^° C and stirring at 900 rpm.

For this, 400 μl of platelet suspension was added to the cuvette of the aggregometer, the test antibody (50 μl) was added at a dilution of 1: 8 (1-3 μg / ml), or the control antibody VM 64, which did not affect platelet aggregation. After a three-minute incubation, aggregation was stimulated by the addition of 10 μM ADP. Aggregation was recorded within 3 min after addition of the inductor and was estimated by the level of light transmission. In the control, the light transmittance increased to 100%. When inhibiting the aggregation of MoAT FRaMon, the level of light transmission did not change (see Fig. 3, where 1 platelet-rich plasma + MoAT FRaMon 3 μg / ml; 2 platelet-rich plasma + F (ab ') ₂ moAT fragment FRaMon 2 μg / ml; 3 platelet-rich plasma + MoAT FRaMon 2 μg / ml; 4 control).

Claims (2)

 1. The strain of hybrid cultured animal cells of Mus musculus BCCK (P) N 644D producer of monoclonal antibodies to glycoproteins IIb IIIa of human platelets.  2. Monoclonal antibody FRaMon to glycoproteins IIb IIIa of human platelets, having the following properties: produced by a strain of hybrid cultured FRaMon cells; refers to Ig G1; possesses a pier. m. 150 kDa; inhibits fibrinogen-dependent platelet aggregation; binds on the surface of platelets a complex of membrane glycoproteins IIb IIIa.

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