Antibody to human erythropoietin (versions) and hybridome strain producing monoclonal antibody to erythropoietin

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

 

The scope of the invention

The invention relates to biotechnology, in particular the production of antibodies to erythropoietin person, and can be used for cleaning.

The level of technology

Erythropoietin is the main regulator of erythropoiesis and stimulates the formation of erythrocytes from a late precursor cells. When abnormalities of the kidneys and in the later stages of some cancers its synthesis is reduced, which leads to severe anemic conditions. Deficiency of erythropoietin is compensated by the introduction of recombinant erythropoietin, which is produced using genetic engineering technology. To get drugs recombinant human erythropoietin obtained by culturing cells-producers, purified, which may include phase chromatography using immobilized monoclonal antibodies to erythropoietin.

Known monoclonal antibodies to erythropoietin (US 4558005 And 10.12.1985; EP 0116446 B1, 22.08.1990; EN 2151184 C1, 20.06.2000; EN 2145610 C1, 20.02.2000, EN 2451071 C1, 02.05.2012). These antibodies can be used in the purification of erythropoietin, but they are all a mouse.

The use of mouse monoclonal antibodies for the purification of erythropoietin has two disadvantage in potential contamination of the drug erythropoietin what hirosumi mouse origin and opportunities for the development of immune response to murine antibodies or their fragments that can contaminate the drug erythropoietin due to proteolysis used for cleaning the immobilized antibody.

Thus, there is a need to create a superior antibodies to erythropoietin.

The invention

The task of the invention is the preparation of recombinant antibodies to erythropoietin, in particular chimeric (mouse-human) antibodies.

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

A) Kd=2.4 x 10-9M, isoelectric point in the range of pH 7.5 to 8.0;

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

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

A strain hybridoma mice, which is the producer of monoclonal antibodies to human erythropoietin, deposited in she RAAS at number 84, and mouse monoclonal antibody specifically binding to human erythropoietin produced this hybridomas described by the following attributes:

A) Kd=0,95×10-9M, molecular weight = 160 KD, iStock in the range of pH 6.8 to 7.1;

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

B) posledovatel is of variable regions of the heavy chain Seq ID NO:2;

G) the sequence of sections that define complementarity 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 obtained antibody effectively binds human erythropoietin. Its structure is similar to the structure of human antibodies by replacing the constant regions of light and heavy chains of mouse IgG in a similar field of human IgG. Recombinant chimeric antibody of the present invention can be Express in cultured cells Chinese hamster ovary (Cho)widely used for production of recombinant proteins for therapeutic purposes.

A brief description of graphic materials

Figure 1 is an immunoblot data for binding of the antibody 6-A and 6-4A3-him with erythropoietin person.

Figure 2 represents the data of electrophoresis antibodies 6-A polyacrylamide gel (4-20% polyacrylamide gel with DDS sodium).

Figure 3 represents the data isfocusable antibodies 6-A and 6-4A3-him-to-human erythropoietin in polyacrylamide gel (Pharmalyte 3-10).

Detailed description of the invention

To create these antibodies was obtained strain mouse hybridoma deposited in the special collection transplantable somatic cell cultures of agricultural and game animals RCCC(P) (she RAAS) at User syscom scientific research Institute of experimental veterinary medicine named. Arrowlink (VIEW) No. 84 dated 24 may 2012. This hybridoma is the producer of monoclonal antibodies (working title 6-A) to human erythropoietin. Cells of this strain were isolated cDNA encoding variable region light and heavy chains of this antibody. Data cDNA was fused with sequences, encoding, respectively, the constant region of the light and heavy chains of human immunoglobulin G with obtaining chimeric antibodies (working title 6-4A3-him).

It was shown that the expression of chimeric (mouse-human) chains of the antibody according to the invention in cells SNO leads to the secretion of antibodies, effectively linking human erythropoietin. Recombinant antibody according to the invention has an affinity to the antigen (erythropoietin), comparable to the affinity of the original monoclonal antibodies, i.e. when creating recombinant antibodies managed to avoid any possible loss as affinity, which in some cases is reduced by several orders of magnitude and selectivity. Thus, the objective of the invention is resolved.

On the basis of monoclonal antibodies 6-A and chimeric antibodies 6-4A3-him can be also obtained single-stranded and humanized antibodies to erythropoietin person.

The invention is illustrated by the following examples:

Example 1. Fabrication and characterization of clones cultiv the alignment of hybridoma mouse.

To obtain hybridoma producing monoclonal antibody to human erythropoietin, strain Balb/c were immunized with purified recombinant human erythropoietin. Erythropoietin (10 μg erythropoietin in an amount of 50 μl +50 μl complete adjuvant's adjuvant) was injected subcutaneously: half-dose - in the pads of the hind limbs, and the other half in the withers of the animal. After 30 days 51 days produced the second and third immunization, different from the first use of incomplete adjuvant's adjuvant. Even after 21 days were botirovna intravenous erythropoietin at a dose of 10 μg/mouse, after which the animals were killed and their spleens and peripheral lymph nodes were isolated lymphocytes. The merger received the lymphocytes with myeloma cells Sp2/0 was performed using polyethylene glycol according to standard methods. After selection on selective nutrient medium containing HAT, from cells from growing cells (200 primary clones) were selected samples of the culture fluid for screening of antibodies to erythropoietin person using enzyme-linked immunosorbent assay. The positive clones were subjected to further cloning and reproduction. As a result of several cycles of cultivation and screening was selected clone hybrid cells stably secreting high-affinity antibody to erythropoietin che is oweka, suitable for diagnostic purposes, but also for the allocation of work items from various biological fluids. This clone was identified as 6-A.

The resulting strain cultivated hybridoma mice 6-A deposited in she RAAS No. 84 dated 24 may 2012.

The strain is characterized by the following features:

1. Morphological features.

Cells correctly rounded, solitary or grouped in clusters.

2. Cultural characteristics.

The type of growth - suspension.

3. The hybridization method.

Hybridoma obtained by fusion of myeloma mice SP 2/0 with lymphocytes of mice of Balb/c using peg.

4. Resistance to selective factors.

Grows on the environment HAT.

5. Cryopreservation.

Environment for cryopreservation - 90% fetal serum and 10% dimethyl sulfoxide. Storage temperature - 196°C (liquid nitrogen).

6. Control of species identity.

Hybridoma capable of cultivation in the peritoneal cavity of syngeneic mice without the use of immunosuppression and produces immunoglobulin type mouse G2A.

7. The contamination.

Cell hybridoma free from Mycoplasma.

Example 2. Purification and characterization of antibodies 6-A.

Antibody 6-A from ascitic fluid of mice was isolated using gel permeation and ion exchange chromatography for further characterization.

Using an enzyme immunoassay the company Invitrogen was installed isotype heavy chain - G2A and light chain - λ. Using immunoblot and ELISA analysis revealed that the antibody (6-A) binds to the human erythropoietin (Figure 1) and is not associated with blood serum and spleen of mice, as well as protein human serum and soluble protein homogenate of cultured cells ovaries Chinese hamster (Cho). Molecular weight antibodies 6-A is 160 kilodaltons on the results of polyacrylamide gel electrophoresis in non conditions reducing conditions, the antibody dissociates on heavy (52 kDa) and light (28 kDa) chains (Figure 2), the isoelectric point is at pH range of 6.8 to 7.1 (3).

The dissociation constant (Kd) of purified antibodies to human erythropoietin, calculated on the basis of the results of enzyme immunoassay for Scatcherd, is 0.95×10-9M. Properties of antibodies 6-A presented in table 1.

Table 1
Immunological characterization of antibodies to erythropoietin man
The antibodies
lo
Type IgKd on EPOThe presence of nonspecific binding
Serum human bloodSerum mouseProteins of mouse spleenProteins SNO
6-AIgG2A an Isotype CL λ0,95×10-9M----
6-4A3-himIgGγ1 the Isotype CL k2.4 x 10-9M----

Example 3. The selection of cDNA and sequencing of variable regions of light and heavy chains.

Of cells hybridoma 6-A were isolated RNA, the matrix of which use sets of synthetic primers (Immunogenetics Information System http://www.imgt.org) were amplified fragments of the genes encoding the variable regions of the heavy and light chains of the antibody.

The resulting fragments were cloned in the vector pALTA and sequentially on Sangero with external primers (M13dir-M13rev) with automatic sequentia AV. Nucleotide sequences coding for variable regions of the heavy and light chains of antibodies 6-A represented in the list of sequence numbers Seq ID NO:1 and Seq ID NO:2, the calculated amino acid members is Telenesti variable regions of the heavy and light chains of antibodies 6-A presents for rooms Seq ID NO:3 and Seq ID NO:4.

The analysis of the obtained sequences is performed using IMGT/V-QUEST (Immunogenetics Information System http://www.imgt.org), found areas CDR, complementarity determining antibodies to the antigen. Lots CDRH-1, CDRH-2, CDRH-3, heavy chain antibodies 6-A and plots CDRL-1 CDRL-2 CDRL-3 light chain antibody 6-A presented in table 2.

Table 2
Sequence plots CDRs of the heavy and light chains of antibodies 6-A
The name of the siteThe amino acid sequence of plot N→CThe number in the list of sequences
CDRH-1GYTFTDYESeq ID NO:5
CDRH-2IYPGSGGTSeq ID NO:6
CDRH-3TRIGIMTGYFDVSeq ID NO:7
CDRL-1DHINNWSeq ID NO:8
CDRL-2GATSeq ID NO:9
CDRL-3QQYWSTPTSeq ID NO:10

Example 4. konstruirovanie chimeric antibodies 6-4A3-him (mouse-human) erythropoietin person.

Using polymerase chain reaction DNA sequences encoding the variable regions of the heavy and light chains of the monoclonal antibodies 6-A were connected with DNA sequences, encoding, respectively, the constant region of the heavy chain of immunoglobulin G1 person (γ1) and the constant region of the light chain of human immunoglobulin G (k). Received the genes encoding the heavy and light chain recombinant chimeric (mouse-human) antibodies 6-4A3-him-to-human erythropoietin, presents under the numbers:

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

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

Amino acid sequence of the heavy and light chains of the chimeric antibody 6-4A3-him presents under the numbers Seq ID NO:12 and Seq ID NO:14, respectively. Using standard techniques of genetic engineering, DNA sequences encoding the heavy and light chain of the chimeric antibody 6-4A3-him, were inserted into the expression vector pOptiVec and pCDNA3.3 obtaining expression plasmid DNA p6-4A3-him-H and p6-4A3-him-L, which were transformed cells SNO line DG-44. After 7 days of culturing the transformed cell production of antibodies that bind human erythropoietin was shown by immunoblot.

Example 5. Clear the and chimeric antibodies 6-4A3-him, the definition of the properties and the use of immobilized monoclonal and chimeric initial for the purification of erythropoietin.

Pre-selected on the OptiCHO medium(-HT) with necessary additives cells SNO transformed with vectors expressing the heavy and light chain of the chimeric antibody 6-4A3-him, were cultured in MEM medium with addition of 10% fetal calf serum and necessary supplements at 37°C and 5% CO2. Antibody 6-4A3-him was purified from the medium of cultivation using gel permeation and ion-exchange chromatography, and then investigated its properties. Antibody 6-4A3-him associated with recombinant human erythropoietin in the immunoblot (Figure 1), its isoelectric point is in the range of pH 7.5 to 8.0 (Figure 2), Kd, was calculated from Scatchard, is 2.4×10-9M

Purified antibodies 6-A and 6-4A3-him - 10 mg of each antibody - immobilizovana 5 ml of CNBr-activated Sepharose matrix FF according to the method of the factory and Packed in a chromatographic column. For each column, equilibrated phosphate buffer, was applied in 100 ml of culture fluid containing human erythropoietin. After washing the column with phosphate buffer containing sodium chloride (1M), and phosphate buffer containing 0.02% of nonionic detergent, adsorbed protein was suirable 0.1 M solution of citric acid, neutralize, p is the following which analyzed its properties. According to polyacrylamide gel electrophoresis the purity of erythropoietin purified using immobilized antibodies 6-A and 6-4A3-him, is 95%, with a yield of not less than 80%, the biological activity was 1.3×105IU/mg protein for erythropoietin purified using antibodies 6-A, and 0.98×105IU/mg protein for erythropoietin purified using antibodies 6-4A3-him.

1. Chimeric recombinant antibody specifically binding to human erythropoietin, which is characterized by the following features:
A) Kd=2.4 x 10-9M, isoelectric point in the range of pH 7.5 to 8.0;
B) sequence of the heavy chain SEQ ID NO:12;
B) sequence of the light chain of SEQ ID NO:14.

2. Strain hybridoma mice, which is the producer of monoclonal antibodies to human erythropoietin, deposited in she RAAS at number 84.

3. Mouse monoclonal antibody specifically binding to human erythropoietin produced by hybridomas according to claim 2 and characterized by the following features:
A) Kd=0,95×10-9M, molecular weight = 160 KD, iStock in the range of pH 6.8 to 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;
G) the sequence of sections that define complementarity 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 - EQ ID NO:9, CDRL-3 - SEQ ID NO:10.



 

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7 cl, 12 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for producing a polyclonal antiserum which is immunospecifically bound to a biologically active parathyroid hormone (PTH) in a part of three or four N-terminal amino acid residues of (1-84) PTH. The first peptide antigen is introduced in a host animal other than a human. A titre of produced antibodies is monitored. Antiserum is extracted. At least one antibody is separated and recovered from antiserum by affine chromatography with using the second peptide antigen. It is followed by removing at least one antibody possessing specificity with respect to the peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH by affine chromatography with using the third peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH respectively. There is recovered polyclonal serum possessing binding affinity with respect to no more than the first three or four amino acid residues of the N-terminal part of PTH and possessing no affinity with respect to any amino acid residues in the fifth amino acid residue or after the fifth amino acid residue of the N-terminal of PTH.

EFFECT: determining the level of biologically active intact PTH in serum, plasma or cell culture medium.

6 cl, 4 dwg

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