RU2644334C2 - Monoclonal antibody, linking with e-bir virus globe-protein, dna fragments coding this antibody and antigen-binding fragment - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a monoclonal antibody selectively binding the Ebola virus glycoprotein with a dissociation constant of 0.8×10^-9 M, as well as an isolated DNA fragment encoding the light and heavy chain regions of the said antibody, and an antigen-binding fragment of the said monoclonal antibody. The specificity of the monoclonal antibody to the Ebola virus glycoprotein was confirmed by immunoassay and immunoblot analysis. An amino acid and nucleotide sequences of the variable domains of the monoclonal antibody are determined.

EFFECT: invention allows to prepare new monoclonal antibodies selectively binding the Ebola virus glycoprotein.

4 cl, 4 dwg, 2 tbl, 3 ex

Description

Technical field

The invention relates to biotechnology and biochemistry, in particular to the production of a monoclonal antibody that binds to the Ebola virus glycoprotein.

State of the art

The Ebola virus (EBOV) first appeared in 1976 in the Sudan and the Democratic Republic of the Congo (at that time, Zaire). The initial outbreaks were not massive, but attracted attention due to the high mortality rate reaching up to 90%. A new variant of the Ebola virus appeared in the spring of 2014 in Guinea, where no such disease had previously been observed (Baize S., Pannetier D., Oestereich L. et al. Emergence of Zaire Ebola virus disease in Guinea // N. Engl. J. Med . 2014. Vol. 371. N 15. P. 1418-1425). Despite the efforts of international organizations, the Ebola epidemic has spread to Sierra Leone, Liberia and Nigeria.

Over the past 10 years, several approaches have been proposed for the treatment of Ebola. Intravenous administration of blood coagulation inhibitors, such as recombinant human activated protein C (Geisbert T.W., Young N.A., Jahrling R.V., Davis K.J., Kagan E., Hensley L.E. Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes / macrophages is a key event // J. Infect. Dis. 2003. Vol. 188. N 11. P. 1618-1629), increases survival by 18%. Another approach is the use of synthetic antisense analogues of oligonucleotides that form duplexes with specific RNA viruses (Geisbert T.W., Hensley L.E., Jahrling R.V., Larsen T., Geisbert J. B., Paragas J., Young HA , Fredeking T. M., Rote WE, Vlasuk GP Treatment of Ebola virus infection with a recombinant inhibitor of factor Vila / tissue factor: a study in rhesus monkeys // Lancet. 2003. Vol.362. N 9400. P. 1953- 1958). Intravenous administration of small interfering RNAs resulted in increased survival rates ranging from 66% to 100% depending on the number of injections (Warren T.K., Warfield K.L., Wells J., Swenson DL, Donner KS, Van Tongeren SA, Garza NL, Dong L., Mourich DV, Crumley S., Nichols DK, Iversen PL, Bavari S. Advanced antisense therapies for postexposure protection against lethal filovirus infections // Nat. Med. 2010. Vol.16. N 9. P. 991 -994). However, it must be emphasized that all the above methods require the start of treatment within the first 30-60 minutes from the moment of infection.

The development of antibody therapy for the Ebola virus has been under way since the 1990s. Of the 8 proteins of the virus, the key target for immunotherapy is viral glycoprotein (GP), which is a decisive factor in pathogenicity. This protein is located on the surface of the viral capsid and serves to attach the virus and fuse with the membrane. GP undergoes post-translational cleavage and forms two subunits linked by a disulfide bond: GP1 and GP2. The GP1 subunit is responsible for the attachment of the virus to the host cells, and GP2 affects the fusion of the virus and cell membranes (Volchkov VE, Feldmann N., Volchkova VA, & Klenk HD Processing of the Ebola virus glycoprotein by the proprotein convertase furin // Proc. Natl. Acad. Sci. USA. 1998. Vol. 95. N 10. P. 5762-5767). GP is a central component of vaccines, as well as a target for neutralizing antibodies and inhibitors of addition and fusion (Lee J E, Saphire E O. Ebolavirus glycoprotein structure and mechanism of entry // Future Virol. 2009. Vol. 4. N6. P. 621- 635).

However, in experiments on primates, it was shown that, in contrast to the use of various non-specific antiviral therapy, passive immunization with antibodies allows to achieve a therapeutic effect when administered 24 hours after infection [Olinger G.G., Pettitt J., Kim D. et al. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques // Proc. Natl. Acad. Sci. USA 2012. Vol. 109. N 44. P. 18030-18035; Qiu X., Audet J., Wong G. et al. Successful treatment of Ebola virus-infected cynomolgus macaques with monoclonal antibodies // Sci. Transl. Med. 2012. Vol. 4. N 138. P. 138ra81).

Monoclonal antibodies to the Ebola virus glycoprotein are known (US 6,630,144 B1), characterized by a unique amino acid sequence and in that they neutralize the Ebola virus and bind to glycoprotein epitopes.

Monoclonal antibodies to the Ebola virus glycoprotein are known and their antigen-binding fragments (WO 2015/127136 A2), characterized by a unique amino acid sequence. 7 antibodies are described, of which CAN9G1 recognizes the mucin domain of the Ebola virus glycoprotein and has a pronounced protective effect on the lethal mouse model.

Disclosure of invention

An object of the present invention was to provide a monoclonal antibody capable of binding to Ebola glycoprotein and differing in amino acid sequence of the light and heavy chain variable domains from monoclonal antibodies to Ebola virus proteins known from the prior art. As well as obtaining isolated DNA fragments encoding sections of the light and heavy chains of the indicated antibodies and antigen-binding fragments of the specified monoclonal antibodies.

The technical result is to obtain a new monoclonal mouse antibody IgG2b isotype capable of binding to the Ebola glycoprotein and characterized by a dissociation constant of the antigen-antibody complex of 0.8 nM.

Also the technical result is the expansion of the arsenal of funds for similar purposes, namely the receipt of a new monoclonal antibody to Ebola virus proteins.

The problem is solved by obtaining a monoclonal antibody that selectively binds the Ebola virus glycoprotein, including the variable region of the heavy chain (VH) of the indicated antibody containing an amino acid sequence of at least 90% homologous to SEQ ID NO: 1; and the variable region of the light chain (VL) of the indicated antibody contains an amino acid sequence that is not less than 90% homologous to SEQ ID NO: 2.

The problem is also solved by the fact that installed an isolated DNA fragment encoding the VH of the indicated antibodies with the nucleotide sequence of SEQ ID NO: 3.

The problem is also solved by the fact that the obtained isolated DNA fragment encoding the VL of the indicated antibodies with the nucleotide sequence of SEQ ID NO: 4.

The problem is also solved by the fact that the obtained antigen-binding fragment of the specified monoclonal antibodies containing the variable region of the heavy chain (VH) of the indicated antibodies with an amino acid sequence of at least 90% homologous to SEQ ID NO: 1, and the variable region of the light chain (VL) of the specified antibodies with an amino acid sequence of at least 90% homologous to SEQ ID NO: 2.

A monoclonal antibody that selectively binds an Ebola virus glycoprotein of the present invention is characterized in that the variable region of the heavy chain (VH) of said antibody contains

(i) CDR1 with the amino acid sequence GYRVTDYV of SEQ ID NO: 1;

(ii) CDR2 with the amino acid sequence IYPGGGNI of SEQ ID NO: 1;

(iii) CDR3 with the amino acid sequence AAAFDY from SEQ ID NO: 1.

Moreover, the variable region of the light chain (VL) of the indicated antibody contains

(i) CDR1 with the amino acid sequence QSLVHSNGNTY of SEQ ID NO: 2;

(ii) CDR2 with the KVS amino acid sequence of SEQ ID NO: 2;

(iii) CDR3 with the amino acid sequence SQSTHVPYT from SEQ ID NO: 2.

A monoclonal antibody was obtained by immunizing Balb / C mice with a conjugate of recombinant Ebola virus GP with mycobacterial heat shock protein HSP65, obtaining and selection of hybridoma lines producing monoclonal antibodies to recombinant Ebola virus GP, analysis of the affinity and specificity of its selected MoAb amino acid sequence, and its determination domains.

Antibodies typically consist of two heavy chains linked together by disulfide bonds, and light chains associated with the N-terminus of each of the heavy chains. Each heavy chain contains at the N-terminus a variable domain with a constant domain at the other end. Each light chain contains at the N-terminus a variable domain with a constant domain at the other end. The variable domains of each pair of light and heavy chains form an antigen-binding site. The variable domains of light and heavy chains have a similar overall structure, and each domain includes a framework of four regions, the sequences of which are relatively conservative, linked through three regions that define complementarity (complementarity determining regions, CDRs). Four frame sections form a beta-fold type conformation. Plots of CDRs are located in close proximity to each other due to the skeleton plots and contribute to the formation of antigen-binding plot. Plots of CDRs and frame regions of antibodies can be determined by reference to the Kabat numbering system (Kabat numbering system, Kabat et al., 1987 "Sequences of Proteins of Immunological Interest", US Dept. of Health and Human Services, US Government Printing Office) in combined with X-ray diffraction analysis data, as described in WO 91/09967. Plots of CDRs and frame regions of antibodies can also be determined by the nomenclature of the International Immunogenetics Information System, www.imgt.org.

Kohler and Milstein (Kohler et al., (1976) Nature 256: 495-497) are usually used to produce antibodies that can bind to any specific antigen. Monoclonal antibodies are obtained by fusion of spleen cells from an immunized animal and myeloma cells to produce a hybridoma. Hybridomas can be tested for the ability to produce the desired antibody, then hybridomas can be grown, and these antibodies can be isolated. The term "grown cells", as used here, means hybridomas or other cell lines that produce antibodies. Methods for obtaining and testing such grown cells are described by Harlow et al. (Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press, 1988). The preparation of material used as an antigen for injection of animals includes techniques well known in the art, for example using a full-sized protein, using a peptide selected from immunogenic regions of the protein, as well as any other methods known in the art. See Harlow et al. (See above).

A suitable method for isolating antibodies effective for use in the framework of the present invention includes (a) administering to the animal an effective amount of a protein or peptide to produce antibodies, (b) isolating said antibodies, (c) determining the sequence of antibodies.

To increase the efficiency of immunization and the quality of the obtained monoclonal antibodies, a recombinant Ebola GP glycoprotein immunoconjugate and mycobacterial heat shock protein HSP65 were obtained.

Then immunized Balb / c mice with the resulting conjugate in two ways - in the pads of the hind legs and intraperitoneally. The resulting lymphocytes hybridized with SP2 / 0 myeloma cells. Hybridoma supernatants were tested by indirect enzyme-linked immunosorbent assay (ELISA) using biotinylated GP. All primary cultures that showed activity in ELISA were cloned using the limiting dilution method. Groups of clones, descendants of one primary culture, were selected. The cells of each of the subclones were accumulated in culture in an amount sufficient to produce ascites fluids. Monoclonal antibodies isolated from ascites fluid were compared by indirect ELISA, which resulted in the selection of the GPE534 antibody.

Then, the subisotype of the obtained antibody was determined, the specificity and immunoreactivity of the obtained antibody were evaluated by immunoblotting and ELISA with a biotinylated antigen in solution. The dissociation constant of the monoclonal antibody GPE534 was determined.

Next, cloned and sequenced cDNA sequences encoding the variable domains of the murine GPE534 anti-Ebola virus glycoprotein. For this, total RNA was isolated from hybridoma cells, and a reverse transcription-amplification reaction was performed. The resulting DNA fragments were cloned and sequenced. The DNA sequencing results of various clones were compared with each other and with GeneBank (IgBlast) databases. As a result, the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 were found, based on which the amino acid sequences of SEQ ID NO: 1 (Fig. 1) and SEQ ID NO: 2 (Fig. 1) of the variable domains of the indicated antibody were determined . Plots of CDRs were identified by the nomenclature of the International Immunogenetics Information System, www.imgt.org.

In particular, the antibody of the present invention is a monoclonal antibody having the ability to bind a recombinant Ebola glycoprotein.

Such an antibody is an antibody containing an amino acid sequence of at least 90% homologous to SEQ ID NO: 1 as a variable region of the heavy chain (VH) of the indicated antibody, an amino acid sequence of at least 90% homologous to SEQ ID NO: 2 as a variable region the light chain (VL) of the specified antibodies and conservative sites of both chains necessary for the functioning of the specified antibodies. Such a new humanized antibody selectively binds to the Ebola virus glycoprotein. Conservative sections of the heavy chain of the human immunoglobulin IgG, IgM, IgA, IgD or IgE can be used as conservative sections for the heavy chain according to the present invention. Conservative regions of the light chain of the human immunoglobulin kappa or lambda can be used as conservative regions for the light chain of an antibody of the present invention.

In the present invention, the term "antibody" is used to describe immunoglobulins or fragments thereof, monomers or dimers of a light chain or heavy chain, single chain antibodies, such as Fv's single chain antibodies, in which the variable domains of the heavy and light chains are connected by a peptide linker, as well as natural, and obtained by recombinant DNA methods or in another way, provided that the antibody contains at least one antigen binding site. The rest of the antibody does not need to include only the sequence derived from immunoglobulin. For example, a gene can be constructed in which a part of a chain, a DNA sequence encoding a part of a chain of human immunoglobulin, is connected to a DNA sequence encoding the amino acid sequence of an effector polypeptide or reporter molecule. As used herein, the term “antibody” is intended to refer to immunoglobulin molecules composed of four polypeptide chains, with two heavy (H) chains and two light (L) chains linking to each other by disulfide bonds. Each heavy chain contains a variable region of the heavy chain (abbreviated herein as VH) and a constant region of the heavy chain. The constant region of the heavy chain consists of three domains: CH1, CH2, and CH3. Each light chain contains a variable region of the light chain (abbreviated herein as VL) and a constant region of the light chain. The constant region of the light chain consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariability regions, which are called complementarity determining regions (CDRs), interspersed by regions with a higher level of conservatism, called framework regions (FR). Each VH and VL region is formed by three CDRs and four FRs, located from the amino-terminal end to the carboxy-terminal end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

In the present invention, the phrase “antibody having the ability to bind to recombinant Ebola GP GP” means a molecule that binds to GP and forms a stable complex. The ability of an antibody to bind to antigen can be determined by a person skilled in the art using methods including, but not limited to limited by enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, using plasmon-surface resonance. Methods for determining affinity are well known to the person skilled in the art iki are detailed Janeway et al (Immunobiology: The Immune System in Health and Disease (Garland Publishing Company, 1996))..

An isolated DNA fragment encoding an antibody according to the present invention is a DNA fragment encoding the variable light or heavy chains of a monoclonal antibody that selectively binds an Ebola virus glycoprotein containing an amino acid sequence of at least 90% homologous to SEQ ID NO: 1 and an amino acid sequence, not less than 90% homologous to SEQ ID NO: 2.

Due to the degeneracy of the translation code, there may be differences in the DNA sequence. The DNA fragments according to the present invention are not limited to the fragments shown in SEQ ID NO: 3 or 4, provided that they encode chain segments of an antibody containing an amino acid sequence of at least 90% homologous to SEQ ID NO: 1, and an amino acid sequence not less than 90% homologous to SEQ ID NO: 2.

As used herein, the term “antigen binding region” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments included in the term “antigen binding region” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab ′) 2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single region of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241: 544-546) consisting of the VH domain; and (vi) a separate complementarity determining region (CDR). Moreover, despite the fact that the two domains in the Fv, VL, and VH fragments are encoded by different genes, they can be combined using recombinant technologies using a synthetic linker that allows them to build a single protein chain in which the VL and VH regions bind to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also included within the scope of the term “antigen binding region” of an antibody.

Brief Description of the Drawings

In FIG. 1 shows the amino acid (SEQ ID NO: 1 and 2) and nucleotide (SEQ ID NO: 3 and 4) MoAt sequences of GPE534.

In FIG. 2 shows a gel electrophoregram of MoAt GPE534 in a 10% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and β-mercaptoethanol; M - protein markers of molecular weight, 1 - MoAT GPE534.

In FIG. 3 shows an immunoblot of recombinant GP Ebola virus stained with MoAt GPE534; M - protein markers of molecular weight, 1 - MoAt GPE534; 2 - control antibody Ru52; 3 - control anti-species peroxidase conjugate.

In FIG. Figure 4 shows the location of CDRs in the variable domain of light GPE534L and heavy GPE534H MoAt chains of GPE534. Sites of CDRs are underlined.

The implementation of the invention

The following examples are provided for purposes of explanation and do not in any way limit the scope of the present invention.

Previously, it was shown that conjugation of recombinant antigens with mycobacterial heat shock proteins (M. tuberculosis HSP65) significantly increases their immunogenicity, and as a result of immunization of Balb / c mice, it is possible to obtain high-affinity MoABs directed against epitopes of natural proteins (Sveshnikov P.G., Malaytsev VV, Kiselev VI The role of heat shock proteins in the development of innate immunity reactions, Journal of Microbiology, Epidemiology and Immunobiology, No. 5, 2007, pp. 108-117; Sveshnikov PG, Malaytsev VV , Kiselev V.I., Functions of thermal proteins shock in the adaptive immunity system. Vaccine Construction, Journal of Microbiology, Epidemiology and Immunobiology, No. 6, 2007, pp. 96-106; P. Sveshnikov and V. Kiselev, "METHODS, KITS AND COMPOSITIONS FOR THE DEVELOPMENT AND USE OF MONOCLONAL ANTIBODIES SPECIFIC TO ANTIGENS OF LOW IMMUNOGENICITY ", PCT / RU 2004/000373, Filing date: 09.24.2004).

Example 1. Obtaining a mouse monoclonal antibody against Ebola glycoprotein

To prepare immunoconjugates, the recombinant Ebola GP glycoprotein GP (IBT Bioservices, USA) and HSP65 were dialyzed against 0.1 M sodium phosphate buffer pH 6.8 overnight in the cold. Mycobacterial HSP65 was activated by adding adenosine diphosphate mixed with MgCl ₂ to a concentration of 1 mM and incubated at 37 ° C for 1 h. Activated HSP65 (150 μg, 2 nmol) was added to 50 μg (0.8 nmol) GP and incubated on a shaker with stirring at room temperature temperature for 3 hours. The resulting complexes were fixed by adding glutaraldehyde to a concentration of 0.05% (v / v) and incubated for 30 min at 37 ° C. The reaction was stopped by adding glycine to a concentration of 0.1 M. It was incubated on a shaker at room temperature for 30 minutes, and then the resulting conjugates were transferred to phosphate-buffered saline (PBS) using dialysis. The degree of conjugation of immunogens was checked by electrophoresis in 10% SDS-PAGE.

For immunization, 2 two-month-old Balb / c mice were taken. One of them was injected immunoconjugate HSP65-GP in the pads of the hind legs. Another was given an immunoconjugate intraperitoneally. The immunogen was prepared as follows: 20 μg of conjugate per mouse was brought to a volume of 50 μl using PBS, then an equal volume of Freund's adjuvant was added and suspended until a homogeneous emulsion was formed. For the first immunization, Freund's complete adjuvant was used; for the second immunization, carried out on day 14 according to the same scheme, incomplete. Two days after the second immunization, the mice that injected the antigen into the paw pads were blocked, and splenocytes from the popliteal lymph nodes were used to hybridize with mouse myeloma cells. The final immunization of the remaining mouse was carried out two weeks after the second intraperitoneal conjugate of HSP65-GP in FSB. Two days after the final immunization, the mice were sacrificed to extract the corresponding cells.

The obtained lymphocytes were hybridized with SP2 / 0 myeloma cells according to the standard method using 50% PEG 4000 (Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity // Nature. 1975. Vol. 256. N 5517. P . 495-497). Cells were plated in 96-well culture plates (6 plates for each hybridization) in DMEM growth medium supplemented with fetal bovine serum to a concentration of 8%, L-glutamine, pyruvate, gentamicin, as well as hypoxanthine, thymidine and aminopterin (HAT) in concentrations recommended by manufacturers of additives (complete selective growth medium). The growth of cell colonies was evaluated visually using a microscope. In most wells of 96-well plates, growth of 1-2 primary cultures was observed on the seventh to tenth day after hybridization. About 4,000 NAT-resistant primary cultures were obtained from each hybridization. Hybridoma supernatants were tested by indirect enzyme-linked immunosorbent assay (ELISA) using biotinylated GP. GP biotinylation was carried out using activated biotin ester in the ratio of biotin / GP in the reaction mixture equal to 15: 1.

Monoclonal antibodies were generated in ascites fluids of Balb / c mice. Ascitic fluids were obtained by introducing 1 million hybridoma cells into the peritoneal cavity of BALB / c mice. Mice were pre-injected with a 0.5 ml pristan per mouse intraperitoneally one week before the introduction of the cells. 11-14 days after the introduction of cells, ascites fluid was collected.

The results of titration in indirect ELISA of ascites from a group of clones, conventionally designated GPE 5XX, are presented in Table 1. Based on the obtained data, clone GPE534 was selected.

The antibody subisotype was determined by ELISA using commercial antisera in accordance with the manufacturer's recommendations (Mouse Typer Isotyping Panel # 1722055 Isotyping Kit # 1722055, Bio-Rad, USA). The monoclonal antibody from the GPE534 hybridoma clone was found to be IgG2b-H30THny. The antibody of the same name was isolated from ascites fluid by affinity chromatography on G-Sepharose protein (GE Healthcare, USA) in accordance with the protocol recommended by the manufacturer and transferred by dialysis to FSB. The purity of MoAt was monitored by electrophoresis on a 10% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and β-mercaptoethanol in a Laemmli step buffer system (SDS-PAGE) (Fig. 2), as well as by high performance liquid chromatography on a chromatograph “ACTA explorer / purifier / basic” (GE Healthcare, USA) in gel filtration mode using a Superdex 200 column (1 × 40 cm). According to electrophoresis in reducing conditions, 2 bands are present on the gel, corresponding to the heavy and light chains of the mouse IgG2b isotype immunoglobulin.

Example 2. Analysis of mouse monoclonal antibodies against Ebola glycoprotein.

To determine the specificity and immunoreactivity of mouse MoAt against the Ebola virus, the ability of its binding to the antigen in solution using the biotinylated GP Ebola virus was studied. For this, in a 96-well plate with high binding capacity (Corning-Costar, Netherlands, cat. No. 9018), 5 μg / ml mouse MoAt GPE534 was sorbed in PBS at 4 ° C. overnight. The tablet was washed 5 times with PBS with 0.05% Tween-20. Then, the conjugate GP-biotin was introduced into the FSB with 2% BSA in a culture of 1 μg / ml to 0.3 ng / ml, incubated for 1 h at 37 ° C, washed 5 times with FSB with 0.05% Tween-20. The next stage is incubation with ExtraAvidin-peroxidase at a concentration of 0.15 μg / ml (FSB, 2% BSA) for 1 h at 37 ° C. To manifest the reaction, the wells were washed 5 times with PBS with 0.05% Tween-20 and 100 μl TMB substrate (3,3,5,5-Tetramethyl-benzidine) was added, incubated for 15 min at room temperature on a shaker. The reaction was stopped with 0.5 M H ₂ SO ₄ and the absorbance was determined at a wavelength of 450 nm. The results of the binding of MoAt GPE534 to the biotinylated GP of the Ebola virus are presented in Table 2.

The study showed the presence of specific binding of GPE534 to GP in solution.

To confirm the specificity of the obtained MoAt GPE534, the method of immunoblotting was used. At the first stage, the Ebola virus GP antigen was electrophoretically separated in 12% polyacrylamide gel under reducing conditions. Then, electrophoretic transfer (electroblotting) of proteins from the gel to a nitrocellulose membrane (Membrane filters cellulose nitrate; pore size 0.45 μm, S045A330R, Advantec MFS, Inc., USA) was performed. Transferred proteins were detected on a nitrocellulose membrane using indirect ELISA (immunoblot). For this, the membrane was blocked with a solution of 5% casein in PBS overnight at + 4 ° C and washed three times with FSB buffer containing 0.05% Tween20. The nitrocellulose membrane was cut into strips and placed in a solution of antibodies GPE534 and Ru52 (anti-Rubella virus antibody as a negative control), 100 μg / ml each, incubated for 1 h at + 37 ° С. After washing three times, the strips were incubated with anti-mouse peroxidase conjugate of anti-mouse IgG antibodies for 1 h at 37 ° C. After repeated washing three times in an FSB containing 0.05% Tween20, a substrate of 3,3-diaminobenzidine, 4-chloro-1-naphthol and hydrogen peroxide was added, incubated for 4 to 10 min and the reaction was stopped by washing the strips with water (Fig. 3 ) According to immunoblot data, GPE534 interacts intensively with the full-sized GP molecule of 94 kDa, with its fragment with a molecular mass of ~ 67 kDa, as well as with a minor fragment of 20 kDa, which confirms the specificity of GPA534 MoAt to the Ebola virus glycoprotein.

MoAT dissociation constants were determined in accordance with the method proposed by Klotz I.M. (The Proteins. Ed. H. Neurath and K. Bailey Academic Press, New York. 1953. VlP727) with modifications from B. Friguet et al. (B. Friguet et. Al. Measurements of the True Affinity Constant in Solution of Antigen-Antibody Complex by Enzyme-Linked Immunosorbent Assay. Journal of Immunological Methods, 77 (1985) 305-319). At the first stage, MoAb is incubated at a constant concentration of 1 nM (150 ng / ml) with the GP antigen in the concentration range of 0.1-10 nM (10-1000 ng / ml) for 2 hours at room temperature with constant stirring on a shaker to achieve thermodynamic equilibrium in a three-component system: free antigen, free antibody and antigen-antibody complex. At the second stage, the concentration of free antibodies is measured by the method of solid-phase ELISA immobilized on a tablet GP. At the final stage, the cd is calculated according to the Klotz equation:

Ao / Ao-A = 1 + 1 / a Cd,

where Ao is the optical density measured for antibodies in the absence of antigen;

A is the optical density measured for free antibodies in an antigen-antibody mixture;

and - antigen concentration.

For MoAt GPE534, the antigen-antibody complex dissociation constant was 0.8 nM.

Example 3. Cloning and sequencing of cDNA sequences encoding the variable domains of the mouse antibody GPE534 to the Ebola virus glycoprotein.

The genes of the variable domains of the light and heavy chains of MoAT GPE534 were obtained using the reverse transcription-amplification reaction of total GPE534 RNA. RNA was isolated from 1-2 × 10 ⁶ cells of the GPE534 hybridoma using Trizol reagent (Life Technologies, USA) according to the protocol recommended by the manufacturer. Reverse transcription was performed using reverse transcriptase MMuLV (Eurogen, Russia) and oligo (dT ₁₈ ) primer. The obtained cDNA was amplified using Tersus DNA polymerase (Evrogen, Russia) using a set of primers complementary to the regions of the constant domains of MoAt:

SMRibo 5'-AAGCAGTGGTATCAACGCAGAGTACGCrGrGrG

RT-KAP 5'-GAGTCAGCACACGAAGAACTTG

NesG 5'-CAGGGGCCAGTGGATAGAC

The resulting PCR fragments with a length of 600-650 bp cloned into the pALT2 vector (Eurogen, Russia) and at least 10 positive clones were selected for both the light and heavy chains of MoAT, after which nucleotide sequences encoding variable fragments were determined by sequencing. Then, nucleotide sequences were compared using the Chromas, CLC Sequence Viewer, and GeneBee programs. As a result of the analysis, the consensus nucleotide sequences of the variable domains of the heavy (SEQ ID NO: 3) and light (SEQ ID NO: 4) MoAt chains of GPE534 were determined. Based on them, taking into account the translation code, the amino acid sequences of the variable domains of the heavy (SEQ ID NO: 1) and light (SEQ ID NO: 2) MoAt chains of GPE534 were determined. To confirm the reliability of the determination of the amino acid sequences of MoAt GPE534, a mass spectrometric analysis of trypsin hydrolysis fragments of light and heavy chains of the initial monoclonal MoAt GPE534 was performed. The obtained sequences were evaluated by comparison with homologous sequences located in the GeneBank database (IgBlast), which confirmed their belonging to the genes of the variable domains of the heavy and light chains of Mus musculus antibodies. Plots of CDRs VL and VH (Fig. 4) were identified by the nomenclature of the International Immunogenetics Information System, www.imgt.org).

Thus, the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, as well as the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, were obtained.

Claims (4)

1. A monoclonal antibody that selectively binds an Ebola virus glycoprotein, comprising a variable region of the heavy chain (VH) of the indicated antibody, containing an amino acid sequence of at least 90% homologous to SEQ ID NO: 1, and the variable region of the light chain (VL) of the indicated antibody contains amino acid sequence not less than 90% homologous to SEQ ID NO: 2. 2. An isolated DNA fragment encoding a VH antibody according to claim 1, with the nucleotide sequence of SEQ ID NO: 3. 3. An isolated DNA fragment encoding a VL antibody according to claim 1, with the nucleotide sequence of SEQ ID NO: 4. 4. The antigen-binding fragment of the monoclonal antibody according to claim 1, selectively binding an Ebola virus glycoprotein containing a variable region of the heavy chain (VH) of the indicated antibody with an amino acid sequence of at least 90% homologous to SEQ ID NO: 1, and the variable region of the light chain ( VL) of the indicated antibody with an amino acid sequence not less than 90% homologous to SEQ ID NO: 2.

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