RU2550262C1 - Monoclonal antibody against human interleukin-6 and hybridoma producing this monoclonal antibody - Google Patents

Abstract

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.

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Description

The invention relates to biotechnology, in particular to antibodies that bind interleukin-6 and block its biological activity, as well as to the technology for their preparation.

Interleukin-6 (IL-6) is a cytokine produced by many types of cells, stimulating the proliferation of antigen-activated B-lymphocytes and T-lymphocytes, enhancing the activity of T-killers, stimulating the granulocyte hematopoiesis, synthesis of most proteins of the acute phase of inflammation in the liver, growth keratinocytes and nerve cells (Ketlinsky S.A., Simbirtsev A.S. Cytokines. St. Petersburg, 2008). At the same time, in a number of diseases, such as rheumatoid arthritis, osteoporosis, Alzheimer's disease, myocardial infarction, a number of oncological diseases (renal cell carcinoma, prostate and bladder cancer, malignant diseases 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 the above diseases, the binding of excess IL-6 and / or blocking its biological activity seems promising for their treatment.

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

At least two main biological functions of IL-6 are known: mediation of acute inflammatory phase proteins and action as a differentiation and activation factor (Avvisti G. et al., Baillieres Clinical Hematology 8: 815-829 (1995) and Poli V. et al ., EMBO 13: 1189-1196 (1994)). The acute phase proteins are known to regulate immune responses, mediate inflammatory processes, and play a role in tissue remodeling. IL-6, as a differentiation and activation factor, induces B-cell differentiation and antibody secretion, induces T-cell differentiation into cytotoxic T-cells, activates cellular signaling factors and stimulates hematopoiesis (Ishimi Y. et al., J. Immunology 145: 3297-3303 (1990)). It is obvious that IL-6 is involved in many important functions of the body and in the processes occurring in it. As a result, physiological processes, including bone metabolism, tumor transformation, and immune and inflammatory responses, can be enhanced, suppressed, or prevented by modifying the biological activity of IL-6 in vivo by a specific antibody (Adebanjo O. et al., J. Cell Biology 142: 1347-1356 (1998)).

An anti-IL-6 antibody has been shown to inhibit in vivo growth of prostate tumors (Smith P.C. & Keller ET The Prostate in press & Okatomo M. et al., Cancer Research 57: 141-146 (1997) and carcinomas kidney (Weissglas M. et al., The Journal of Urology 153: 554-557 (1995)). In addition to directly affecting tumor growth, blocking the production of IL-6 can also inhibit bone degradation and cachexia in cancer.

Murine, rat and rabbit monoclonal antibodies were obtained, as well as chimeric and humanized antibodies to human IL-6, in particular, neutralizing its biological activity, however, none of them are currently introduced into clinical practice. Thus, murine monoclonal antibodies against IL-6 are known, for example, CLB-8, which has high affinity ((Brakenhoff et al., J. Immunol. (1990) p. 145-561); US 5618700, 1988). However, a murine antibody is highly immunogenic to humans, and therefore has limited therapeutic use.

US Pat. No. 5,856,135, 1999 describes reconstructed human anti-human IL-6 antibodies derived from a murine monoclonal antibody SK2 in which hypervariable regions (CDRs) from the variable region of murine SK2 antibodies were transferred to the variable region of a human antibody and attached to a constant region of a human antibodies. The disadvantage of these antibodies is also their immunogenicity to humans.

The closest in technical essence to the claimed invention is a chimeric (mouse-human) antibody CLB8 to human IL-6 and the technology for its production (RU2318829, 2008). The CLB8 antibody neutralizes 50% of human IL-6 activity (measured by proliferation of mouse B-cell myeloma cells induced by human IL-6) with an antibody / IL-6 molar ratio of 1.8.

The objective of the present invention was to expand the spectrum of antibodies to human IL-6 in order to obtain antibodies with a higher ability to inhibit the biological activity of human IL-6, on the basis of which antibodies suitable for clinical use can be created, i.e. chimeric and humanized antibodies.

The technical result was achieved by obtaining a murine monoclonal antibody (the author’s name is antibody 6IL-9), which specifically and with high affinity binds to human IL-6 and inhibits its biological activity, as well as obtaining a hybridoma of 6IL-9 mice producing this monoclonal antibody.

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

The resulting monoclonal antibody against human IL-6 contains hypervariable heavy chain regions with the sequences of SEQ ID NO 5-7 and hypervariable light chain regions with the sequences of SEQ ID NO 8-10.

Said hypervarial regions may in particular be included in the sequences of the variable region of the heavy chain according to SEQ ID NO 3 and the sequences of the variable region of the light chain according to SEQ ID NO 4.

The analysis of the sequences of the hypervariable regions (CDR regions) of the variable parts of the light and heavy chains of the obtained 6IL-9 monoclonal antibody showed that they differ significantly from the similar CDR regions of all known human monoclonal antibodies to IL-6, including RU 2318829, 2008.

Typically, this monoclonal antibody against human interleukin-6 is obtained using DNA containing the nucleotide sequences of SEQ ID NO 1 and SEQ ID NO 2. This DNA is expressed in cells of a murine hybridoma, the strain of which (the copyright name of the strain is 6IL-9), was deposited in the Russian collection of cell cultures under the number RKKK (P) 751 D.

The pedigree of the strain: Parent cells of the hybridoma SP2 / 0, antigen - human interleukin-6, the merging agent - PEG / DMSO, hybrid cell selection system -HAT, cloning ratio - 4, the percentage of positive clones - 100%.

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

Standard growing conditions: RPMI-1640 medium, 10% fetal serum, 37 ° C, 5% CO ₂ .

Characterization of hybridoma cultivation in the animal: Balb / c or F2 mice (Balb / c × DBA) 1.5-2.0 × 106 cells, 14-20 days, transplantable.

The strain produces monoclonal antibody 6IL-9 to human interleukin-6. Isotype IgG1, kappa. The antibody, specifically binding, neutralizes the biological activity of human interleukin-6 in a test on the B 9 cell line. 50% neutralization of human IL-6 is observed at a molar ratio of antigen to antibody of 1 to 1.48.

Monoclonal antibody 6IL-9 accumulates in ascites fluid at a concentration of not less than 1.0 mg / ml. Stability of antibody production is maintained for 25 passages in culture and 5 passages in animals.

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

The invention is illustrated by the following drawings and sequences.

Figure 1 shows the electrophoresis of purified antibody 6IL-9 in a polyacrylamide gel 4-20% polyacrylamide gel with DDS-Na, where the tracks:

1 - antibody 6IL-9 (1 μg / ml) 5 μl, native conditions;

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

3 - antibody 6IL-9 (1 μg / ml) 5 μl, restored conditions.

Figure 2 shows the binding of antibody 6IL-9 with interleukin-6 (IL-6) of a person in an immunoblot (electrophoresis in 4-20% polyacrylamide gel with Na DDS, immunoblot with antibody IL-6-9). Tracks:

1 - human IL-6 (5 μg), restored conditions;

2 - human IL-6 (5 mcg), native conditions;

3 - molecular weight markers, Coomassie R250 stain.

Figure 3 shows the determination of the isoelectric point of antibody 6IL-9 (isofocusing in Pharmalyte 3-10 polyacrylamide gel). The following notation is used:

1 - markers of an isoelectric point;

2 - antibody 6IL-9 (1 μg / μl) 5 μl;

3 - antibody 6IL-9 (1 μg / μl) 15 μl.

Sequence listing:

Seq ID NO: 1 The nucleotide sequence encoding the variable region of the heavy chain of the monoclonal antibody 6IL-9

Seq ID NO: 2 The nucleotide sequence encoding the variable region of the light chain of the antibody 6IL-9

Seq ID NO: 3 The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 6IL-9

Seq ID NO: 4 The amino acid sequence of the variable region of the light chain of the monoclonal antibody 6IL-9

Seq ID NO: 5 The amino acid sequence of the CDRH-1 site of monoclonal antibody 6IL-9

Seq ID NO: 6 amino acid sequence of the CDRH-2 monoclonal antibody 6IL-9

Seq ID NO: 7 The amino acid sequence of the CDRH-3 site of monoclonal antibody 6IL-9

Seq ID NO: 8 The amino acid sequence of the CDRL-1 region of monoclonal antibody 6IL-9

Seq ID NO: 9 The amino acid sequence of the CDRL-2 region of the monoclonal antibody 6IL-9

Seq ID NO: 10 The amino acid sequence of the CDRL-3 region of monoclonal antibody 6IL-9

The invention is demonstrated by the following examples.

Example 1. Immunization of mice and obtaining hybridomas

Balb / c mice were immunized with purified recombinant human IL-6, for which 15 μg of IL-6 in a volume of 50 μl was mixed with 50 μl of Freund's complete adjuvant and injected into the pads of the hind limbs. After 30 days and after 51 days, the second and third immunizations were performed, which differed from the first replacement of Freund's complete adjuvant with incomplete. After another 21 days, boosting was carried out by intravenous administration of IL-6 at a dose of 10 μg / mouse, 4 days after the boosting, animals were killed 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, culture fluid samples were taken from 5300 cells with growing cells for screening antibodies to human IL-6 using enzyme-linked immunosorbent assay. The resulting three positive clones were subjected to further cloning and reproduction. As a result of several growth and screening cycles, a clone of 6IL-9 hybrid cells stably secreting a high-affinity 6IL-9 anti-human IL-6 antibody was selected.

Example 2. Purification of monoclonal antibodies and its properties

The 6IL-9 antibody from the ascites fluid of Balb / c mice was intraperitoneally inoculated with the 6IL-9 hybridoma and was isolated by gel permeation and ion exchange chromatography for its further characterization.

According to the results of electrophoresis in a 4-20% polyacrylamide gel with sodium DDS, the molecular weight of the 6IL-9 antibody under native (unreduced) conditions corresponds to that expected for native mouse IgG (160 kDa), under reduced conditions, the antibody dissociates into heavy (52 kDa) and light ( 28 kDa) chains (Fig. 1). To study the binding of IL-6 to antibody 6IL-9, the human IL-6 preparation was subjected to polyacrylamide gel electrophoresis under the same conditions. After electrophoresis, the material was transferred to nitrocellulose, which was incubated with a solution of antibody 6IL-9, and then with goat antibodies against mouse immunoglobulins conjugated to horseradish peroxidase. A lane with molecular weight marker proteins was stained with Coomassie (FIG. 2). Antibody 6IL-9 interacts in an immunoblot with recombinant IL-6, which under reduced conditions has a molecular weight of 18 kD, and under unreduced conditions, is represented by two bands with molecular weights of 18 kD and 40 kD, which corresponds to human IL-6 monomer and dimer.

Using the Sigma enzyme immunoassay kit ISO-2 kit 072K4849, the heavy chain isotype of antibody 6IL-9, G1, and the light chain, λ, were determined. Using an enzyme-linked immunosorbent assay, it was found that the 6IL-9 antibody does not bind to the blood serum and spleen proteins of mice, as well as to human serum proteins and soluble homogenate proteins of cultured Chinese hamster ovary cells (CHO). The isoelectric point is in the range of pH 6.4-6.8 (figure 3).

The dissociation constant for human IL-6, determined using enzyme-linked immunosorbent assay and calculated according to Scatchard, is 1 × 10 ^-10 M.

Example 3. Inhibition by antibody 6IL-9 of the biological activity of IL-6 and determination of 1C50%

Antibodies 6IL-9 in serial dilutions ranging from 1.25 × 10 ^-7 M to 1.25 × 10 ^-12 M were incubated with 8.4 × 10 ^-12 M IL-6 for 2 hours, after which 100 μl of the antibody-IL-6 mixture was added to B9 cells (5 × 10 ³ cells in RPMI 1640 medium per sample; control, cells without IL-6 and IL-6 without antibodies). Cells were cultured for 72 hours at 37 ° C and 5% CO ₂ ; 6 hours before the end of cultivation, H-thymidine was added to the cells. Cell proliferation induced by IL-6 was evaluated by the inclusion of H-thymidine in the acid-insoluble residue and expressed by the number of pulses per minute, and the inhibition of the biological activity of IL-6 was expressed as a percentage of inhibition of H-thymidine incorporation (Table 1).

Table 1 Inhibition of 6-IL-9 antibody-induced IL-6 cell proliferation of B9 Concentration 6IL-9 (M) 1.25 × 10 ^-7 1.25 × 10 ^-8 1.25 × 10 ^-9 1.25 × 10 ^-10 1.25 × 10 ^-11 1.25 × 10 ^-12 0 0 The concentration of IL-6 (M) 8.4 × 10 ^-12 8.4 × 10 ^-12 8.4 × 10 ^-12 8.4 × 10 ^-12 8.4 × 10 ^-12 8.4 × 10 ^-12 8.4 × 10 ^-12 0 Acid Insoluble Radioactivity (cpm) 16184 13772 16584 34709 59682 88314 107825 13690 Inhibition (%) 97.3 99.8 96.5 77.6 51.1 20.7 0 one hundred

From the above data it follows that 50% inhibition of the activity of IL-6 occurs with a molar ratio of 6 IL-9 / IL-6 equal to 1.48, which indicates a high neutralizing activity of the antibody 6 IL-9.

Example 4. Synthesis and sequencing of cDNA encoding the variable parts of the light and heavy chains of monoclonal antibodies 6IL-9

RNA was isolated from hybridoma 6IL-9 cells, on the matrix of which using gene primers (Immunogenetics Information System http://www.imgt.org), fragments of genes encoding the variable regions of the heavy (VH) and light (VL) chains of the antibody were amplified . The resulting gene fragments were cloned into the pALTA vector (Eurogen) and sequenced from external primers (M13). The sequences of the fragments of the genes encoding VH and VL are presented on SEQ ID No: 1 and SEQ ID No: 2, the calculated amino acid sequences of VH and VL are presented on SEQ ID No 3 and SEQ ID No 4. Analysis of amino acid sequences of the variable regions of the heavy and light chains 6IL-9 monoclonal antibody was produced by the Cabot method (Kabat EA, Wu, TT, Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins oflm-munological Interest, Fifth Edition. NIH Publication No.91 -3242), which allowed us to identify sections of CDR that determine the complementarity of the antibody to the antigen (table 2).

table 2 The amino acid sequences of the sites determining the complementarity of monoclonal antibodies 6IL-9 The name of the site The amino acid sequence of the CDR plot Sequence number CDRH-1 GFSLSTSGMGVG Seq ID NO: 5 CDRH-2 HIWWDDDKYYNPSLKS Seq ID NO: 6 CDRH-3 RANYGTSYDYGMDY Seq ID NO: 7 CDRL-1 KASQSVSDVLT Seq ID NO: 8 CDRL-2 YASNRYT Seq ID NO: 9 CDRL-3 QQGYRSPYT Seq ID NO: 10

The amino acid sequences of Seq ID NO: 5-10 using the BLAST program (National Center for Biotechnology Information http://www.ncbi.rilm.nih.gov) were compared with the sequences of the variable regions of the heavy and light chains of known monoclonal antibodies to human IL-6 . No homology sites were found.

Hybridoma 6IL-9 was placed in the Russian collection of cell cultures under the number RKKK (P) 751 D.

Claims (4)

1. Monoclonal antibody against human interleukin-6, containing the hypervariable regions of the heavy chain with the sequences of SEQ ID NO: 5-7 and the hypervariable regions of the light chain with the sequences of SEQ ID NO: 8-10. 2. The monoclonal antibody against human interleukin-6 according to claim 1, having the sequence of the variable region of the heavy chain according to SEQ ID NO: 3 and the sequence of the variable region of the light chain according to SEQ ID NO: 4. 3. The monoclonal antibody against human interleukin-6 according to claim 2, encoded by a DNA containing the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2. 4. The hybridoma strain producing a monoclonal antibody against human interleukin-6 according to claim 1, deposited in the Russian collection of cell cultures under the number RKKK (P) 751D.

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