Monoclonal antibody against human interleukin-6 and hybridoma producing this monoclonal antibody

FIELD: biotechnology.

SUBSTANCE: monoclonal antibody against human interleukin-6 is represented, which comprises hypervariable regions of the heavy chain CDRH-1: GFSLSTSGMGVG; CDRH-2: HIWWDDDKYYNPSLKS; and CDRH-3: RANYGTSYDYGMDY; and the hypervariable regions of the light chain CDRs CDRL-1: KASQSVSDVLT; CDRL-2: YASNRYT; and CDRL-3: QQGYRSPYT. In addition, the invention also relates to a hybridoma strain deposited in the Russian Collection of Cell Cultures under number RKKK(P) 751D, and producing the said antibody.

EFFECT: invention enables to expand the range of antibodies against human IL-6, having high ability to inhibit IL-6.

4 cl, 3 dwg, 2 tbl, 4 ex

 

The invention relates to biotechnology, in particular to antibodies that bind interleukin-6 and blocking its biological activity, as well as technologies of their production.

Interleukin-6 (IL-6) is a cytokine produced by many types of cells, stimulating proliferation of activated antigen of b-lymphocytes and T-lymphocytes, increases the activity of T-killer cells, stimulating granulocyte Rostock blood, the liver synthesis of most proteins of acute phase of inflammation, the growth of keratinocytes and nerve cells (S. A. state, Simbirtsev A. S. cytokines. Saint-Petersburg, 2008). However, in a number of diseases, such as rheumatoid arthritis, osteoporosis, the disease of Alzheimer, myocardial infarction, some cancers (renal cell carcinoma, prostate cancer and bladder cancer, malignant disease associated with proliferation of b-cells, in particular multiple myeloma, chronic lymphocytic leukemia) and others, there is an increase in the level of production of IL-6. Since IL-6 is involved in the pathogenesis of many of these diseases, the binding of excess IL-6 and/or block its biological activity are promising to treat them.

IL-6 can bind to a receptor of IL-6, expressed on mitogen-activated b-cells, T-cells, peripheral�die monocytes and certain tumors (Ishimi Y. et al., Immunology 145:3297-3303 (1990)). The receptor of IL-6 has at least two different forms and consists of an alpha chain called gp80, which is responsible for the binding of IL-6, and beta-chains, called gp130, which is necessary for signal transmission (O. Adebanjo et al., Cell Biology 142:1347-1356 (1998) and Poli V. et al., EMBO 13:1189-1196 (1994)).

Known at least two major biological functions of IL-6: mediation of acute phase proteins of inflammation and act as a factor of differentiation and activation (Avvisti G. et al., Baillieres Clinical Hematology 8:815-829 (1995) and Poli V. et al., EMBO 13:1189-1196 (1994)). It is known that acute phase proteins regulate immune responses that mediate inflammatory processes and play a role in tissue remodeling. IL-6, as a factor of differentiation and activation, induce the differentiation of b-cells and the secretion of antibodies, induce the differentiation of T cells into cytotoxic T cells, activates factor in the transmission of cellular signals and stimulates hematopoiesis (Y. Ishimi et al., J. Immunology 145:3297-3303 (1990)). It is obvious that IL-6 is involved in many important bodily functions and processes. As a result of physiological processes, including bone metabolism, neoplastic transformation, and immune and inflammatory responses can be amplified, suppressed or prevented by modification of the biological activity of IL-6 in vivo under the action of the special�quarter antibody (O. Adebanjo et al., J. Cell Biology 142:1347-1356 (1998)).

It was shown that the antibody against IL-6 can inhibit in vivo growth of prostate tumors (R. S. Smith & Keller E. T. The Prostate in press & Okatomo M. et al., Cancer Research 57:141-146 (1997), and carcinoma of the kidney (Weissglas M. et al., The Journal of Urology 153:554-557 (1995)). In addition to direct effects on tumor growth, blocking the production of IL-6 may also inhibit the degradation of bones and cachexia in cancer.

Were obtained from mouse, rat and rabbit monoclonal antibodies, and chimeric and humanized antibodies to IL-6 human, in particular, neutralizing its biological activity, but none of them is currently not implemented in clinical practice. Thus, the known murine monoclonal antibody against IL-6, for example, CLB-8, which has a high affinity ((Brakenhoff et al., J. Immunol. (1990) p.145-561); US 5618700, 1988). However, the murine antibody is highly immunogenic to humans, and therefore it has limited therapeutic use.

In U.S. patent No. 5856135, 1999 described the reconstructed human antibody against human IL-6, derived from mouse monoclonal antibody SK2 in which hypervariable region (CDR) of the variable regions of the mouse antibody SK2 were transferred to the variable region of human antibody and joined to the constant region of human antibodies�and. The disadvantage of these antibodies is their immunogenicity to humans.

The closest in technical essence to the claimed invention is a chimeric (mouse-human) antibody CLB8 to IL-6 of human rights and the technology of its receipt (RU2318829, 2008). Antibody CLB8 neutralizes 50% of the activity of IL-6 (measured in a cell proliferation b-cell myeloma mouse induced IL-6) at a molar ratio of antibody/IL-6, 1.8.

The object of the present invention to expand the range of antibodies to IL-6 for the purpose of obtaining antibodies with a higher ability to inhibition of the biological activity of IL-6 on the basis of which you can create antibodies suitable for clinical use, i.e., chimeric and humanized antibodies.

The technical result was achieved by receiving murine monoclonal antibodies (author's name antibody IL-9), with specific and high affinity binding to IL-6 and inhibiting its biological activity, as well as obtaining hybridomas mice IL-9 producing this monoclonal antibody.

It was found that the antibody binds to IL-6 and specific with high affinity and inhibits its biological activity.

Obtained a monoclonal antibody against IL-6 people� contains hypervariable sites of the heavy chain of sequence SEQ ID NO. 5 to 7, and hypervariable sections of the light chain sequence SEQ ID NO 8-10.

These hypervariable stations, in particular, to enter the sequence variable regions of the heavy chain according to SEQ ID NO. 3 and the sequence of variable region light chain according to SEQ ID NO. 4.

Sequence analysis of hypervariable sites (sites CDR) of the variable parts of the light and heavy chains obtained monoclonal antibodies IL-9 showed that they differ significantly from similar plots of all known CDR of monoclonal antibodies to IL-6 of human rights, including EN 2318829, 2008.

As a rule, the specified monoclonal antibody against interleukin-6 was produced using the DNA containing the sequence of nucleotides in SEQ ID NO. 1 and SEQ ID NO. 2. This DNA is expressed in cells of murine hybridomas, the strain of which (the author's name of the strain - IL-9) was deposited in the Russian collection of cell cultures under number RCCC(P) 751 D.

Pedigree strain: Parent cell hybridomas SP2/0, the antigen - interleu-kin-6, and merging agent - PEG/DMSO, the system of selection of hybrid cells-HAT, the multiplicity of cloning - 4, the percentage of positive clones - 100%.

Cultural properties: cultivation in mattresses, sowing dose of 200,000 cells/ml, the ratio of the sieving 1:3, feeder requires macrophages during defrosting.

Standard growing conditions: medium RPMI-1640, 10% fetal serum, 37°C, 5% CO2 .

Characterization of culturing the hybridomas in the body of the animal: Mice Balb/c or F2 (Balb/c×DBA) of 1.5-2.0×106 cells, 14-20 days, transplantable.

The strain produces a monoclonal antibody IL-9 to interleukin-6. The isotype IgG1, Kappa. The antibody specifically binds, neutralizes the biological activity of interleukin-6 in the test for cell lines 9. 50% neutralization of IL-6 is observed at a molar ratio of antigen and antibody 1 to 1,48.

Monoclonal antibody IL-9 accumulates in ascitic fluid in a concentration of at least 1.0 mg/ml. Stability of product antibody persists for over 25 passages in culture and 5 passages on animals.

Method of cryopreservation: fetal serum with 10% DMSO, freezing to -70°C at a rate of 1°C/min, then the rooms in liquid nitrogen, the viability after thawing and washing from cryoconserved 80% (with trypan blue).

The invention is illustrated by the following drawings and sequences.

Fig.1 shows the electrophoresis of the purified antibodies IL-9 polyacrylamide gel 4-20% polyacrylamide gel with SDS-Na, where paths:

1 - antibody IL-9 (1 μg/ml) 5 μl native conditions;

2 - protein markers of molecular weight 97, 66, 45, 30, 20, 14 kDa;

3 - antibody IL-9 (1 μg/ml) 5 μl restored conditions.

Fig.2 shows the binding of an antibody IL-9 interlay�other-6 (IL-6) human immunoblotting electrophoresis in 4-20% polyacrylamide gel with Na DDS, immunoblotting with an antibody to the IL-6-9). Tracks:

1 - IL-6 (5 μg), restored conditions;

2 - IL-6 (5 μg), native conditions;

3 - molecular weight markers, R250 Coomassie blue staining.

Fig.3 shows the determination of the isoelectric point of the antibody IL-9 (isfocusowner polyacrylamide gel Pharmalyte 3-10). The following expressions are used:

1 - isoelectric point markers.

2 - antibody IL-9 (1 μg/μl) 5 μl;

3 - antibody IL-9 (1 μg/μl) 15 ál.

The sequence listing:

Seq ID NO:1 a nucleotide Sequence that encodes the variable region of the heavy chain of monoclonal antibody IL-9

Seq ID NO:2 a nucleotide Sequence that encodes a variable region light chain antibodies IL-9

Seq ID NO:3 the amino acid Sequence of variable regions of the heavy chain of monoclonal antibody IL-9

Seq ID NO:4 amino acid Sequence of variable region of light chain of monoclonal antibody IL-9

Seq ID NO:5 the amino acid Sequence of plot CDRH-1 monoclonal antibody IL-9

Seq ID NO:6 Sequence aminocyclohexane CDRH-2 monoclonal antibody IL-9

Seq ID NO:7 amino acid Sequence of the plot CDRH-3 monoclonal antibody IL-9

Seq ID NO:8 the amino acid Sequence of cdrl stock footage-1 monoclonal antibody IL-9

Seq ID NO:9 Consistently�th amino acids cdrl stock footage-2 monoclonal antibody IL-9

Seq ID NO:10 the amino acid Sequence of cdrl stock footage-3 monoclonal antibody IL-9

The invention is demonstrated by the following examples.

Example 1. Immunization of mice and obtaining hybridomas

Mice of the Balb/c were immunized by purified recombinant IL-6 human, for which 15 μg of IL-6 in a volume of 50 μl was mixed with 50 μl complete adjuvant of freind and was injected into the paws of the hind limbs. After 30 days and 51 days produced the second and third immunization, different from the first replacement of the complete adjuvant of frand part. Even after 21 days spent bushiribana intravenous IL-6 at a dose of 10 μg/mouse, 4 days after busterboy animals were sacrificed, and their spleens and peripheral lymph nodes were isolated lymphocytes. The merger received lymphocytes with myeloma cells Sp2/0 were carried out using polyethylene glycol according to standard methods. After selection on selective medium containing HAT, 5300 cells growing cells were selected samples of the culture fluid for screening of antibodies to IL-6 using enzyme-linked immunosorbent assay. Received three positive clone was subjected to further cloning and reproduction. As a result of several cycles of cultivation and screening was selected a clone of hybrid cells IL-9, stably secreting vysokoaffinnye IL-9 to IL-6.

Example 2. Purification of monoclonal antibodies and its properties

Antibody IL-9 from ascitic fluid of mice of the line Balb/c inoculated intraperitoneally by hybridoma IL-9 was isolated using gel permeation and ion exchange chromatography for further characterization.

According to the results of electrophoresis in 4-20% polyacrylamide gel with SDS sodium molecular weight antibodies IL-9: native (unreduced) conditions expected for native mouse IgG (160 kDa), in restored conditions, the antibody dissociates into heavy (52 kDa) and light (28 kDa) chains (Fig.1). To study the binding of IL-6 antibody IL-9 drug IL-6 was subjected to polyacrylamide gel electrophoresis under the same conditions. After electrophoresis, the material was transferred to nitrocellulose, incubated with antibody solution IL-9, and then with goat antibodies against mouse immunoglobulins, conjugated with horseradish peroxidase. Track with protein molecular weight markers were stained Kumasi (Fig.2). Antibody IL-9 engages in the immunoblot with recombinant IL-6, which is in the restored condition has a molecular weight of 18 KD and unrestored conditions represented by two bands with molecular weights of 18 KD and 40 KD, which corresponds to the monomer and dimer of IL-6.

Using ELISA kit company SigmaISO-2 kit 072K4849 was set on the isotype of the heavy chain of the antibody IL-9 - G1 and light chains - λ. Using ELISA, it was found that the antibody IL-9 is not bound to proteins in serum and spleen of mice, as well as protein human serum and soluble protein homogenate of cultured cells Chinese hamster ovary (Cho) cells. Isoelectric point is at pH range between 6.4 and 6.8 (Fig.3).

The dissociation constant for IL-6 determined by ELISA and calculated by Scatchard is 1×10-10M.

Example 3. Inhibition of antibody IL-9 biological activity of IL-6 and the definition S%

Antibodies IL-9 in serial dilutions in the range from 1.25×10-7M to 1.25×10-12M were incubated with 8.4×10-12M IL-6 within 2 hours, after which 100 µl of a mixture of antibody-IL-6 contributed to the B9 cells (5×103cells in RPMI medium 1640 on trial, control cells without IL-6 and IL-6 without antibodies). Cells were cultured for 72 hours at 37º and 5% CO26 hours before the end of cultivation the cells were added H-thymidine. Cell proliferation induced by IL-6 was assessed by incorporation into acid-insoluble residue of H-thymidine and expressed by the number of pulses per minute, and the inhibition of the biological activity of IL-6 were expressed in percentage of inhibition of the incorporation of H-thymidine (table.1).

96,5
Table 1
Inhibition of antibody IL-9-induced IL-6 proliferation of cells B9
Concentration IL-9 (M)1,25×10-71,25×10-81,25×10-91,25×10-101,25×10-111,25×10-1200
The concentration of IL-6(M)8,4×10-128,4×10-128,4×10-128,4×10-128,4×10-128,4×10-128,4×10-120
Acid-insoluble radioactivity (counts/min)16184137721658434709596828831410782513690
Inhibition (%)97,399,877,651,1A 20.70100

From these data it follows that the 50% inhibition activity of IL-6 occurs at a molar ratio IL-9/IL-6, equal to 1.48, indicating a high neutralizing activity of antibodies IL-9.

Example 4. The synthesis and sequencing of cDNA encoding the variable light and heavy chains of monoclonal antibodies IL-9

From the cells of hybridomas IL-9 RNA was isolated on the matrix using sets of synthetic primers (Immunogenetics Information System http://www.imgt.org) fragments were amplified genes encoding variable regions of heavy (VH) and light (VL) chains of antibodies. The resulting gene fragments were cloned into the vector pALTA (Evrogen) and sequenced with the external primers (M13). Sequence fragments of genes encoding VH and VL represented in SEQ ID No:1 and SEQ ID No:2, the calculated amino acid sequences of VH and VL represented in SEQ ID No. 3 and SEQ ID No. 4. Analysis of the amino acid sequences of variable regions of the heavy and light chains of monoclonal antibodies IL-9 produced by the method of Cabot (Kabat E. A., Wu, T. T., Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins oflm-munological Interest, Fifth Edition. NIH Publication No. 91-3242) that allowed us to identify areas CDR, complementarity determining a�of tetela antigen (tab.2).

Table 2
The amino acid sequence of sections that define complementarity monoclonal antibody IL-9
Name plotThe amino acid sequence of CDR of the plotThe sequence number
CDRH-1GFSLSTSGMGVGSeq ID NO:5
CDRH-2HIWWDDDKYYNPSLKSSeq ID NO:6
CDRH-3RANYGTSYDYGMDYSeq ID NO:7
Cdrl stock-1KASQSVSDVLTSeq ID NO:8
Cdrl stock-2YASNRYTSeq ID NO:9
Cdrl stock-3QQGYRSPYTSeq ID NO:10

The amino acid sequence Seq ID NO:5-10 using the program BLAST (National Center for Biotechnology Information http://www.ncbi.rilm.nih.gov were compared with sequences of variable regions of heavy and light chains of the known monoclonal antibodies to IL-6. Parcel d�ogy is not found.

The hybridoma IL-9 was placed in the Russian collection of cell cultures under number RCCC(P)751 D.

1. A monoclonal antibody against interleukin-6, containing hypervariable sites of the heavy chain with the sequence SEQ ID NO: 5-7 and hypervariable sections of the light chain sequence SEQ ID NO: 8-10.

2. A monoclonal antibody against interleukin-6 according to claim 1 having the sequence of the variable regions of the heavy chain according to SEQ ID NO: 3 and a sequence of variable region light chain according to SEQ ID NO: 4.

3. A monoclonal antibody against interleukin-6 according to claim 2 encoded by the DNA containing the sequence of nucleotides in SEQ ID NO: 1 and SEQ ID NO: 2.

4. Strain of hybridomas producing monoclonal antibody against interleukin-6 according to claim 1, deposited in the Russian collection of cell cultures under number RCCC(P) D.



 

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FIELD: biotechnology.

SUBSTANCE: humanised monoclonal antibody against TNF and its application are described.

EFFECT: invention enables to reduce significantly the immunogenicity of murine antibody while maintaining the ability of the antibody to recognise the antigen as compared with the conservative chimeric murine antibody, increased antibody safety in clinical applications.

11 cl, 3 dwg, 5 tbl, 16 ex

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