RU2513689C1 - Antibody to human erythropoietin (versions) and hybridome strain producing monoclonal antibody to erythropoietin - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10^-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10^-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

Description

Field of Invention

The invention relates to biotechnology, in particular to the production of antibodies to human erythropoietin, and can be used to clean it.

State of the art

Erythropoietin is the main regulator of erythropoiesis and stimulates the formation of red blood cells from late progenitor cells. With pathologies of the kidneys and in the late stages of a number of oncological diseases, its synthesis decreases, which leads to severe anemic conditions. Erythropoietin deficiency is compensated by the introduction of recombinant erythropoietin, which is produced using genetic engineering technologies. To obtain drugs, recombinant human erythropoietin obtained by culturing producer cells is subjected to purification, which may include the step of immunochromatography using immobilized monoclonal antibodies to erythropoietin.

Monoclonal antibodies to erythropoietin are known (US 4,558,005 A, 12/10/1985; EP 0116446 B1, 08.22.1990; RU 2151184 C1, 06/20/2000; RU 2145610 C1, 02/20/2000, RU 2451071 C1, 05/02/2012). These antibodies can be used in the purification of erythropoietin, however, they are all murine.

The use of murine monoclonal antibodies for the purification of erythropoietin has two drawbacks, namely, the potential for contamination of the erythropoietin preparation with viruses of mouse origin, as well as the possibility of developing an immune response to murine antibodies or their fragments, which can contaminate the erythropoietin preparation due to the proteolysis of immobilized antibodies used to purify it.

Thus, there is a need for improved antibodies to erythropoietin.

SUMMARY OF THE INVENTION

The objective of the invention is to obtain a recombinant antibody to erythropoietin, in particular a chimeric (mouse-human) antibody.

This problem is solved by the fact that the proposed chimeric recombinant antibody that specifically binds to human erythropoietin, characterized by the following features:

A) Kd = 2.4 × 10 ^-9 M, isoelectric point in the pH range 7.5-8.0;

B) the sequence of the heavy chain Seq ID NO: 12;

B) the sequence of the light chain Seq ID NO: 14.

Also proposed is a strain of mouse hybridoma, which is a producer of a monoclonal antibody to human erythropoietin, deposited in the CJS RAS under No. 84, and a mouse monoclonal antibody that specifically binds to human erythropoietin produced by this hybridoma, characterized by the following features:

A) Kd = 0.95 × 10 ^-9 M, molecular weight = 160 kDa, isochka in the pH range of 6.8-7.1;

B) the sequence of the variable region of the light chain Seq ID NO: 1;

B) the sequence of the variable region of the heavy chain Seq ID NO: 2;

D) the sequence of sites that determine the complementarity of antibodies:

CDRH-1 - Seq ID NO: 5, CDRH-2 - Seq ID NO: 6, CDRH-3 - Seq ID NO: 7,

CDRL-1 - Seq ID NO: 8, CDRL-2 - Seq ID NO: 9, CDRL-3 - Seq ID NO: 10.

The resulting antibody effectively binds human erythropoietin. Its structure is close to the structure of human antibodies, due to the replacement of the constant regions of the light and heavy chains of mouse IgG with similar regions of human IgG. The recombinant chimeric antibody of the present invention can be expressed in cultured Chinese hamster ovary cells (CHO), widely used to produce therapeutic recombinant proteins.

A brief description of the graphic materials

Figure 1 represents the immunoblot data for the binding of antibodies 6-4A3 and 6-4A3-him with human erythropoietin.

Figure 2 represents the electrophoresis of antibody 6-4A3 in a polyacrylamide gel (4-20% polyacrylamide gel with sodium DDS).

Figure 3 is an isofocusing data of 6-4A3 and 6-4A3-him antibodies to human erythropoietin in a polyacrylamide gel (Pharmalyte 3-10).

DETAILED DESCRIPTION OF THE INVENTION

To create these antibodies, a mouse hybridoma strain was obtained, which was deposited in the Specialized Collection of Transplantable Somatic Cell Cultures of Agricultural and Commercial Animals RKKK (P) (CXF RAAS) at the All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after Ya.R. Kovalenko (VIEV) under No. 84 dated May 24, 2012. The indicated hybridoma is a producer of a monoclonal antibody (working title 6-4A3) to human erythropoietin. CDNAs encoding the variable regions of the light and heavy chains of this antibody were isolated from the cells of this strain. The cDNA data were fused to sequences encoding, respectively, the constant regions of the light and heavy chains of human immunoglobulin G to produce a chimeric antibody (working name 6-4A3-him).

It has been shown that the expression of chimeric (mouse-human) chains of an antibody of the invention in CHO cells results in secretion of an antibody that efficiently binds human erythropoietin. The recombinant antibody according to the invention has an affinity for the antigen (erythropoietin), comparable with the affinity of the original monoclonal antibody, that is, when creating the recombinant antibody, it was possible to avoid the possible loss of both affinity, which in some cases decreases by several orders of magnitude, and selectivity. Thus, the objective of the invention is solved.

On the basis of the monoclonal antibody 6-4A3 and the chimeric antibody 6-4A3-him, single chain and humanized antibodies to human erythropoietin can also be obtained.

The invention is illustrated by the following examples:

Example 1. Obtaining and characterization of clones of a cultured mouse hybridoma.

To obtain a hybridoma producing a monoclonal antibody against human erythropoietin, Balb / c mice were immunized with purified recombinant human erythropoietin. Erythropoietin (10 μg of erythropoietin in a volume of 50 μl + 50 μl of Freund's complete adjuvant) was injected subcutaneously: half the dose into the paw pads of the hind limbs and the other half into the withers of the animals. After 30 days and after 51 days, the second and third immunizations were performed, which differed from the first use of Freund's incomplete adjuvant. After another 21 days, an intravenous injection of erythropoietin at a dose of 10 μg / mouse was performed, after which the animals were sacrificed and lymphocytes were isolated from their spleens and peripheral lymph nodes. The fusion of the obtained lymphocytes with Sp2 / 0 myeloma cells was carried out using polyethylene glycol according to the standard method. After selection on a selective nutrient medium containing HAT, samples of culture fluid were taken from cells with growing cells (200 primary clones) for screening antibodies to human erythropoietin using enzyme-linked immunosorbent assay. The resulting positive clones were subjected to further cloning and reproduction. As a result of several growth and screening cycles, a clone of hybrid cells was selected that stably secreted a high-affinity human erythropoietin antibody, suitable for diagnostic purposes, as well as for isolation of EPO from various biological fluids. This clone was designated as 6-4A3.

The resulting strain of cultured hybridoma of mice 6-4A3 was deposited in the Union of Agriculturalists of the Russian Academy of Agricultural Sciences No. 84 dated May 24, 2012.

The strain is characterized by the following features:

1. Morphological signs.

Cells of regular round shape, single or clustered.

2. Cultural traits.

Type of growth - suspension.

3. The method of hybridization.

The hybridoma was obtained by fusion of SP 2/0 mouse myeloma with lymphocytes of Balb / c mice using polyethylene glycol.

4. Resistance to selective factors.

Grows on a HAT environment.

5. Cryopreservation.

Cryopreservation medium - 90% fetal serum and 10% dimethyl sulfoxide. Storage temperature - 196 ° C (liquid nitrogen).

6. Control of species identity.

The hybridoma is capable of culturing syngeneic mice in the abdominal cavity without the use of immunosuppression and produces mouse type G2A immunoglobulins.

7. Contamination.

Hybridoma cells are free of mycoplasma.

Example 2. Purification and characterization of antibodies 6-4A3.

Antibody 6-4A3 was isolated from the ascites fluid of mice using gel permeation and ion exchange chromatography for its further characterization.

Using the Invitrogen enzyme immunoassay kit, the heavy chain isotype G2A and the light chain isotype λ were determined. Using immunoblot and enzyme immunoassay it was found that the antibody (6-4A3) binds to human erythropoietin (Figure 1) and does not bind to blood serum and spleen proteins of mice, as well as to human serum proteins and soluble homogenate proteins of cultured ovarian cells Chinese hamster (SSS). The molecular weight of the antibody 6-4A3 is 160 kilodaltons according to the results of polyacrylamide gel electrophoresis in non-reducing conditions, under reducing conditions, the antibody dissociates into the heavy (52 kDa) and light (28 kDa) chains (Figure 2), the isoelectric point is in the pH range of 6 8-7.1 (Figure 3).

The dissociation constant (Kd) of the purified antibody for human erythropoietin, calculated on the basis of the results of the Scatterwork enzyme-linked immunosorbent assay, is 0.95 × 10 ^-9 M. The properties of antibody 6-4A3 are shown in Table 1.

Table 1 Immunological characteristics of antibodies to human erythropoietin Antite
lo Ig type Kd by EPO The presence of non-specific binding Human serum Mouse serum Mouse spleen proteins Proteins SNO 6-4A3 IgG2A Isotype CL λ 0.95 × 10 ^-9 M     -     -     -     - 6-4A3-him IgGγ1 Isotype CL k 2.4 × 10 ^-9 M     -     -     -     -

Example 3. Isolation of cDNA and sequencing of the variable regions of the light and heavy chains.

RNA was isolated from hybridoma 6-4A3 cells, on the matrix of which gene fragments encoding the variable regions of the heavy and light chains of the antibody were amplified using synthetic primer sets (Immunogenetics Information System http://www.imgt.org).

The resulting fragments were cloned into the pALTA vector and Sanger sequenced from external primers (M13dir-M13rev) using an AB3500 automatic sequencer. The nucleotide sequences encoding the variable regions of the heavy and light chains of the antibody 6-4A3 are presented in the sequence list under the numbers Seq ID NO: 1 and Seq ID NO: 2, the calculated amino acid sequences of the variable regions of the heavy and light chains of the antibody 6-4A3 are presented under the Seq numbers ID NO: 3 and Seq ID NO: 4.

Analysis of the obtained sequences, performed using the IMGT / V-QUEST program (Immunogenetics Information System http://www.imgt.org), revealed CDR portions that determine the complementarity of the antibody to the antigen. Plots CDRH-1, CDRH-2, CDRH-3, heavy chain antibodies 6-4A3 and sections CDRL-1 CDRL-2 CDRL-2 CDRL-3 light chain antibodies 6-4A3 are presented in table 2.

table 2 The sequence of sections of the CDRs of the heavy and light chains of antibodies 6-4A3 The name of the site The amino acid sequence of the plot N → C Sequence List Number CDRH-1 GYTFTDYE Seq ID NO: 5 CDRH-2 IYPGSGGT Seq ID NO: 6 CDRH-3 TRIGIMTGYFDV Seq ID NO: 7 CDRL-1 DHINNW Seq ID NO: 8 CDRL-2 Gat Seq ID NO: 9 CDRL-3 QQYWSTPT Seq ID NO: 10

Example 4. Construction of a chimeric antibody 6-4A3-him (mouse-human) to human erythropoietin.

Using the polymerase chain reaction, DNA sequences encoding the variable regions of the heavy and light chains of the monoclonal antibody 6-4A3 were docked with DNA sequences encoding, respectively, the constant regions of the heavy chain of human immunoglobulin G1 (γ1) and the constant region of the light chain of human immunoglobulin G (k ) The resulting genes encoding the heavy and light chains of the recombinant chimeric (mouse-human) antibody 6-4A3-him to human erythropoietin are presented under the numbers:

The nucleotide sequence of the heavy chain gene of the chimeric antibody 6-4A3-him-Seq ID NO: 11;

The nucleotide sequence of the light chain gene of the chimeric antibody 6-4A3-him-Seq ID NO: 13 .;

The amino acids sequences of the heavy and light chains of the chimeric antibody 6-4A3-him are presented under the numbers Seq ID NO: 12 and Seq ID NO: 14, respectively. Using standard genetic engineering techniques, DNA sequences encoding the heavy and light chains of the chimeric antibody 6-4A3-him were inserted into the expression vectors pOptiVec and pCDNA3.3 to obtain expression plasmid DNAs p6-4A3-him-H and p6-4A3-him -L, with which CHO cells of the DG-44 line were transformed. After 7 days of culturing the transformed cells, production of an antibody that binds human erythropoietin was shown using an immunoblot.

Example 5. Purification of the chimeric 6-4A3-him antibody, determination of its properties and the use of immobilized monoclonal and chimeric antibodies for the purification of erythropoietin.

Preliminarily selected on OptiCHO (-HT) medium with the necessary additives, CHO cells transformed with vectors expressing the heavy and light chains of the chimeric 6-4A3-him antibody were cultured in MEM medium supplemented with 10% fetal calf serum and necessary additives at 37 ° C and 5% CO ₂ . The 6-4A3-him antibody was purified from the culture medium using gel permeation and ion exchange chromatography, after which its properties were examined. The 6-4A3-him antibody binds to recombinant human erythropoietin in an immunoblot (Figure 1), its isoelectric point is in the pH range of 7.5-8.0 (Figure 2), Kd calculated by Scatchard is 2.4 × 10 ^-9 M.

Purified 6-4A3 and 6-4A3-him antibodies — 10 mg of each antibody — were immobilized in 5 ml of a Sepharose FF CNBr-activated matrix according to the manufacturer's procedure and packed into chromatographic columns. For each column balanced with phosphate buffer, 100 ml of culture fluid containing human erythropoietin was applied. After washing the columns with a phosphate buffer containing sodium chloride (1M) and a phosphate buffer containing 0.02% nonionic detergent, the sorbed protein was eluted with a 0.1 M citric acid solution, neutralized, and then its properties were analyzed. According to polyacrylamide gel electrophoresis, the purity of erythropoietin purified using immobilized antibodies 6-4A3 and 6-4A3-him is 95%, with a yield of at least 80%, the biological activity was 1.3 × 10 ⁵ IU / mg protein for erythropoietin purified using an antibody 6-4A3, and 0.98 × 10 ⁵ IU / mg protein for erythropoietin purified using an antibody 6-4A3-him.

Claims (3)

1. Chimeric recombinant antibody that specifically binds to human erythropoietin, characterized by the following features:
A) Kd = 2.4 × 10 ^-9 M, isoelectric point in the pH range 7.5-8.0;
B) the heavy chain sequence of SEQ ID NO: 12;
B) the sequence of the light chain of SEQ ID NO: 14. 2. The strain of mouse hybridoma, which is a producer of a monoclonal antibody to human erythropoietin, deposited in the Union of Agricultural Sciences under No. 84. 3. A mouse monoclonal antibody that specifically binds to human erythropoietin, produced by the hybridoma according to claim 2 and characterized by the following features:
A) Kd = 0.95 × 10 ^-9 M, molecular weight = 160 kDa, isochka in the pH range of 6.8-7.1;
B) the sequence of the variable region of the light chain of SEQ ID NO: 1;
B) the sequence of the variable region of the heavy chain of SEQ ID NO: 2;
D) the sequence of sites that determine the complementarity of antibodies:
CDRH-1 - SEQ ID NO: 5, CDRH-2 - SEQ ID NO: 6, CDRH-3 - SEQ ID NO: 7, CDRL-1 - SEQ ID NO: 8, CDRL-2 - SEQ ID NO: 9, CDRL-3 - SEQ ID NO: 10.

Images