RU2533802C1 - Trimerised single-domain antibody that specifically binds with glycoprotein g of rabies virus, neutralising rabies virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: trimerised single-domain antibody is created, that binds specifically with the surface (capsid) protein - glycoprotein G of rabies virus, having an amino acid sequence of SEQ ID NO: 2, which amino acid sequence is encoded by the nucleotide sequence of SEQ ID NO: 1, and at that neutralising the infectious activity of rabies virus.

EFFECT: improvement of properties of the compound.

3 cl, 4 dwg, 1 tbl

Description

FIELD OF THE INVENTION

The present invention relates to the field of immunology and virology and involves the use of antibodies, which are trimerized single-domain antibodies, for the prevention and treatment of rabies.

State of the art

Rabies is a particularly dangerous disease of animals of all species and humans, characterized by severe damage to the central nervous system and death. Annually, 45-60 thousand people die from rabies in the world, and approximately 10 million people injured by animals seek rabies care. A particular danger of rabies is that to date, no effective means of treating an already developed disease has been found.

In Russia, the average rate of appeal for anti-rabies assistance in the country is on average 300.0 per 100 thousand of the population. Every year, more than 250 thousand people are at risk of rabies virus infection and require specific prophylaxis.

The trend of complications of the epizootic situation in Russia is still preserved and even intensifies. A simultaneous increase in the incidence of rabies in both wild and domestic carnivores is characteristic. The rise of natural type epizootics is undeniable. The epizootological and epidemiological significance of rabies of wolves, foxes, raccoon dogs is growing. Wild coons are increasingly included in epizootic chains; isolated cases of rabies of elks, beavers, muskrats, hamsters, hares, hedgehogs, gray rats and mice have been reported.

The main way to combat rabies in animals is specific prophylaxis with the use of vaccines and rabies immunoglobulin. According to the WHO treatment of rabies in the condition of contact category III (single or multiple transdermal bites or scratches, licking of damaged skin; mucosal contamination with saliva when licking, contact with bats), the administration of an anti-rabies immunoglobulin is prescribed along with the use of a vaccine. However, currently existing preparations containing rabies immunoglobulin (heterologous (equine) and homologous (human)) do not meet modern safety and efficacy requirements. Therefore, obtaining new innovative means of combating rabies virus is an urgent task to increase the level of protection of the population from rabies virus and reduce the number of deaths from infection.

One of the most effective approaches to date to create safe and effective drugs to combat viral infections is the use of virus-neutralizing antibodies. Currently, only one preparation of anti-rabies immunoglobulin from human blood serum manufactured by the Sichuan Yuanda Shuyan Pharmaceutical Company (China) is registered and purchased for state needs in Russia. There are no similar drugs of domestic production.

Compared to immunoglobulins obtained from human serum given a vaccine preparation of inactivated rabies virus, recombinant monoclonal antibodies or antibody fragments that can be obtained in cell cultures under strictly controlled and reproducible conditions are safer, better characterized and standardized and more suitable for production and use as therapeutic agents.

As the closest analogue of the technical solution that forms the basis of the present invention, the following example can be given. Patent application US 2013/0039926 A1 ("Human antibodies against rabies and uses thereof" - "Human antibodies against rabies and their use", published February 14, 2013) describes the preparation of recombinant monoclonal human antibodies and their antigen-binding fragments (Fab), capable of specifically interacting with the main surface viral antigen, glycoprotein G, rabies virus and, as a result of such an interaction, counteract the ability of the virus to infect cells.

This technical solution, as the closest to the claimed by the composition of the active substance and the method of its use, is selected by the authors of the present invention for the prototype.

The disadvantages of the prototype are:

- The use of mammalian cells as an producer of antibodies, in particular, myeloma of mice that are difficult to cultivate and store, are demanding on the quality of the reagents used, can be infected with pathogens that are dangerous to humans (viruses, mycoplasmas, etc.) and must be constantly monitored for their absence , which ultimately affects the high cost of monoclonal antibodies;

- The use of traditional hybridoma technology in mice necessitates additional genetic engineering manipulations for the humanization of immunoglobulin genes in order to bring the amino acid sequences of the claimed antibodies into human ones, however, this does not cancel out post-translational modifications specific for mouse immunoglobulins, and not for humans . Antibodies thus obtained can cause an undesirable immune response when they are introduced into the human body.

- Monoclonal antibodies have a large size of the resulting antibodies, which entails reduced permeability in the tissue;

- Structural features impose a restriction on the recognition of some “hidden” epitopes located, for example, in recesses, small gaps in the structure of proteins, which affects the decrease in the biological activity of monoclonal antibodies.

Thus, in the prior art there is a need for the development of new antibodies - antigen-recognizing molecules, devoid of the above disadvantages and specifically recognizing rabies virus, as well as capable of inhibiting its infectious activity.

Disclosure of invention

The present invention was the creation of a new single-domain antibody, which will effectively bind and neutralize the rabies virus.

The technical problem is solved due to the fact that a trimerized single-domain antibody is obtained that specifically binds the main surface (capsid) protein, glycoprotein G, rabies virus and is able to neutralize rabies virus.

In order to obtain a more stable, compact, economical in production, convenient for all possible subsequent modifications and various ways of combined use of antibodies against rabies virus, we used the technology of generating a library of single-domain antibodies and subsequent selection of sequences encoding a single-domain antibody with a given specificity using the phage display method .

The technology used is based on obtaining the nucleotide sequences of the genes of noncanonical antibodies of animals of the camelidae family (Camelidae). These antibodies are a dimer of only one shortened (first constant region of CH1 is absent) immunoglobulin heavy chain. For the specific recognition and binding of antigen proper, in this case, only one variable domain of this antibody is necessary and sufficient.

The full equivalent of the term “single-domain antibody” for the purposes of the present invention is the widely used designation “nanobody”, “NANOBODY”, as well as “nanoantibody” and “single-domain nanoantibody”.

Obtained by this technology single-domain antibodies have the following improved properties [Tillib S.V. Camel Mini Antibodies is an effective tool for research, diagnosis and therapy. Molecular biology. 2011; 45 (1): 77-85]:

1) The presence of a method developed by the applicant for the production and selection of single-domain antibodies, which is based on obtaining the nucleotide sequences of the genes of non-canonical antibodies of animals of the camelid family (Camelidae);

2) The possibility of economical operating time in large quantities, since the producer is the bacterium E. Coli, which indicates the industrial applicability of the invention;

3) The biological safety of antibodies, since the culture of E. coli bacteria cannot be contaminated by eukaryotic pathogens;

4) Low immunogenicity, since a single-domain antibody consists only of the variable fragment of the heavy chain responsible for binding to the antigen and does not contain other domains to which undesirable immune reactions are formed.

5) Single-domain antibodies have improved cell permeability due to their small size (~ 2 × 4 nm, 13-15 kDa), which is very important for rabies virus infection.

6) The high solubility and stability (over a wide range of temperatures and acidity of the medium) makes it possible to prepare preparations for medical use based on single-domain antibodies.

Although the single-domain antibodies obtained (in the bacterial expression system) do not contain post-translational modifications, another positive feature of these antibodies is the possibility of relatively “humanizing” them without appreciable loss of their specific activity by a small number of point substitutions of amino acids [Vincke C., Loris R. , Saerens D., et al. // J. Biol. Chem. 2009. V.284. No. 5. P.3273-3284]. This opens up the potential for the widespread use of single-domain antibodies as passive immunization agents to prevent the development of various dangerous infectious diseases [Wesolowski J., Alzogaray V., Reyelt J. et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009; 198, 157-174].

The problem is solved by the present invention by creating a trimerized single-domain antibody that specifically binds to a surface (capsid) protein - glycoprotein G, rabies virus and neutralizes the infectious activity of rabies virus.

A method for producing a trimerized single-domain antibody specifically binding to rabies virus glycoprotein G and neutralizing the infectious activity of rabies virus includes the following principal steps:

1) the induction of the formation of specific antibodies as a result of a 5-stage immunization of a representative of the Camelidae family (two-humped camel) with a substance prepared on the basis of an inactivated rabies virus mixed with adjuvant (Gerbu LQ);

2) cloning of sequences of antigen-recognizing domains - special single-chain antibodies synthesized in the lymphocytes of an immunized animal in the phagemid vector used in the subsequent phage display procedure, obtaining a specifically enriched library of sequences encoding single-domain antibodies;

3) selection by the modified phage display method (functional selection of phage particles carrying an expressed antibody on the surface, and inside the DNA encoding this antibody) antibody clones that bind to a given antigen, both with the whole rabies virus and with rabies glycoprotein G, from the resulting library of single-domain antibodies;

4) carrying out the cloning and genetic engineering modifications of the coding sequences of selected single-domain antibodies with the aim of their effective production in the bacterial expression system, subsequent effective purification (E. coli bacteria are used as the producer of the active substance), as well as to increase their biological activity (due to homotrimerization );

5) demonstration of the functional activity of selected and formatted trimerized antibodies, namely the ability to specifically recognize rabies virus glycoprotein G and rabies virus, as well as neutralize the rabies virus infectious activity.

A fundamentally important step in the preparation of modified derivatives with substantially higher biological activity (neutralization of rabies virus) from the monomer single-domain single-domain antibodies originally selected by phage display is the addition of additional sequences (domains) to the end of the primary antibody, which include: a) a linear region (linker region corresponding to the long hinged portion of the camelid antibody (“hinge”, h), which is important for separating antigen-recognizing function from function nd other domains, b) fundamentally important to strengthen the neutralizing function of the virus trimerizuyuschiysya domain (variant leucine zipper, ILZ), and (at the end) coding regions are two oligopeptide tag: in) the HA-tag and g) (a His) ₆ - tag. The last two sites significantly facilitate, respectively, the detection and purification of this trimerized single-chain antibody. A detailed description of the use of the trimerizing domain to increase the biological activity of a single chain antibody has been described in our recent work (Tillib S., Ivanova TI, Vasilev LA, Rutovskaya MV, Saakyan SA, Gribova IY, Tutykhina IL, Sedova ES, Lysenko AA, Shmarov MM, Logunov DY, Naroditsky BS, Gintsburg AL Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2). Antiviral Research 2013, 97: 245-254. // Formatted single-domain antibodies can protect the mouse from a lethal infection caused by influenza virus )

The authors of the present invention proceed from the fact that, as is well known to the average person skilled in the art, the primary, initially obtained sequences of single-domain antibodies can then be adapted or "formatted" in various ways for subsequent practical use.

So, single-domain antibodies can be the original modules (blocks) of more complex multimodular drugs. It is possible to combine in one multivalent derivative two, three or more monovalent primary single-domain antibodies. These single domain antibodies combined into a single construct can bind both to the same epitope of the target antigen, and to its different epitopes, or even to different target antigens. It is also possible combined combination into a single design of single-domain antibodies and other molecules or drugs to obtain multifunctional drugs; multimerization by introducing additional amino acid sequences, interacting protein domains, such as leucine zippers, or sequences of small proteins that form stable complexes. To modulate the properties of the preparation of a single domain antibody, for example, to increase the lifetime or to improve the purification method, additional amino acid sequences can be introduced into the final compound. It will be apparent to one of ordinary skill in the art that such modifications and other variants of the antibodies underlying the present invention fall within the scope of the present invention, as they are structural and functional variants of single domain antibodies. Thus, the author of the present invention refers to the term "single-domain antibodies" as the primary, initially obtained, "minimum" amino acid sequences of single-domain antibodies, and their modifications resulting from the above, adaptation or "formatting" and their variants. The term "antibody variant" for the purposes of the present invention means a polypeptide that contains changes in the amino acid sequence, namely deletion, insertion, addition or replacement of amino acids, provided that this maintains the necessary level of protein activity, for example, at least 10% of the activity original single domain antibody. A number of changes in the variant of the protein depends on the position or type of amino acid residue in the three-dimensional structure of the protein. For the variable domain of an antibody (or single domain antibody), these three key positions are three hypervariable regions, called CDR1, CDR2 and CDR3, flanked by more conservative framework regions. In this case, CDR3 plays a particularly important role for specific recognition, while all other regions are substantially less characteristic and reproduce with high homology in other genes of the variable domains of immunoglobulins.

The number of changes can be, for example, from 1 to 30, more preferably from 1 to 15, and most preferably from 1 to 5 changes in the sequence of the original single domain antibody. These changes can occur in areas of the polypeptide that are not critical to its function. This is possible due to the fact that some amino acids have high homology with each other and therefore the tertiary structure or activity of the protein is not violated by such a change. Therefore, as a variant of the protein can be a protein that is characterized by a homology of at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% with respect to the amino acid sequence of the original single domain antibody, while maintaining the activity of the polypeptide .

Homology between amino acid sequences can be established using well-known methods, for example, using sequence alignment in the BLAST 2.0 computer program, which calculates three parameters: count, identity, and similarity.

Substitution, deletion, insertion, addition or replacement of one or more amino acid residues will be a conservative mutation or conservative mutations, provided that the protein activity is maintained.

An example of a conservative mutation is a conservative substitution. A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acids having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, series, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine , fe nilalanine, tryptophan, histidine). Since the hypervariable regions of single-domain antibodies determine their specific interaction with the antigen, it is the homologous substitutions of amino acids in these regions that can lead to the production of single-domain antibodies that differ in sequence, which have identical or similar properties. Thus, it will be apparent to one of ordinary skill in the art that the scope of the present invention encompasses not only the single-domain antibody sequences indicated in the appendix, but also those that can be obtained by substituting amino acids in the hypervariable regions (indicated in the sequence listing as CDRs) with other, but very similar in properties, amino acids (conservative substitutions).

DNA fragments that encode essentially the same functional polypeptide can be obtained, for example, by modifying the nucleotide sequence of a DNA fragment encoding the original single domain antibody, for example, by the method of site-directed mutagenesis, so that one or more amino acid residues at a particular site will be deleted, replaced, inserted or added. DNA fragments modified as described above can be obtained using traditional processing methods to obtain mutations.

DNA fragments that encode essentially the same functional polypeptide of the original single domain antibody are obtained by expressing DNA fragments having the mutation described above and establishing the activity of the expressed product.

Substitution, deletion, insertion or addition of nucleotides described above also includes mutations that occur in nature and, for example, are due to variability.

The single domain antibody polypeptides of the present invention can be encoded by a large number of nucleic acid molecules, which is the result of the degeneracy of the genetic code well known in the art. The essence of the phenomenon is that any amino acid (with the exception of tryptophan and methionine) that is part of natural peptides can be encoded by more than one triplet nucleotide codon. Any of these degenerate coding nucleic acid molecules may be included in cassettes expressing single domain antibodies of the invention and falling within the scope of the invention.

Description of figures

Figure 1 presents:

SEQ ID NO: 2 is the amino acid sequence of a selected trimerized single domain antibody capable of specifically binding and neutralizing rabies virus,

SEQ ID NO: 1 is a nucleotide sequence encoding the amino acid sequence of a selected trimerized single domain antibody capable of specifically binding and neutralizing rabies virus.

In the indicated sequence, SEQ ID NO: 2 are highlighted in bold and underlined (left to right, from the N- to C-terminus) hypervariable regions of CDR1, CDR2 and CDR3, which are crucial (CDR3 is especially important) for specific recognition of the surface protein of the virus by this single-chain antibody rabies. Two cytosine residues are also identified in the amino acid sequence, which, based on many previous structural studies of such molecules, can form an additional noncanonical Cys-Cys bond and thus significantly stabilize the structure of the antibody.

The fragment added during the trimerization of the initial single domain antibody contains a linear region (the linker region corresponding to the long hinge region of the camelid antibody (“hinge”, h), which is crucial for enhancing the virus-neutralizing function, the trimerizing domain (leucine zipper variant, ILZ, is highlighted in gray), and and (at the end) portions encoding two oligopeptide tag:. HA-tag and (his) ₆ -tag at the end is the termination codon TAA (in the amino acid sequence it corresponds asterisk).

Figure 2 presents the analysis data of the purified trimerized single-domain antibody specifically binding to rabies virus using Laemmli electrophoresis in 14% polyacrylamide gel separating under denaturing and reducing conditions (with sodium dodecyl sulfate, SDS, and with dithiothreitol, DTT). A molecular weight marker is applied in the left lane.

A trimerized single-domain antibody was generated in the bacterial periplasm and then purified (including using affinity metal-chelate chromatography and subsequent gel filtration chromatography) to an almost individually protein product migrating in the gel in a protein zone of approximately 30 kDa (actual size is approximately 22 kDa; the antibody moves slowly in the gel due to structural features, in particular due to the presence of terminal positively charged six histidine residues (His tag)).

Figure 3 shows the gel filtration chromatography profile of purified trimerized single domain antibody passed through a Sephacryl S-100 HR 1.5 × 50 column in standard PBS buffered saline (under normal physiological conditions without denaturation). Proteins with known molecular weights were used as markers: Bovine serum albumin, BSA - 67 kDa; Ovalbumin, OVA - 45 kDa; Lysozyme, LC - 14.4 kDa. The dashed line indicates the profile of the purified antibody preparation obtained as a result of a repeated round of gel filtration. The antibody preparation elutes in fractions corresponding to the trimerized antibody.

Figure 4 illustrates the results of an enzyme-linked immunosorbent assay for the binding of trimerized single-domain antibodies to the inactivated rabies virus drug and rabies virus G protein glycoprotein G immobilized in the plate well. Bovine serum albumin immobilized protein (BSA) wells were used as a control. The trimerized single domain antibody was taken in serial dilutions with factor 5, starting at a concentration of 1 μg / ml. The obtained absorption data at a wavelength of 405 nm (ordinate axis), reflecting the efficiency of binding of trimerized single-chain antibodies to immobilized antigens, are presented. Averaged data are presented. The bars in the upper part of the columns show the ranges of deviations of the results in individual cells.

Examples of the implementation of the present invention

Example 1

Obtaining a library of variable domains of noncanonical single chain antibodies.

Bactrian camel Camelus bactrianus was subsequently immunized 5 times by subcutaneous administration of antigenic material. The second immunization was carried out 3 weeks after the first, then three more immunizations were carried out with an interval of 10-14 days.

As an antigen, the rabies vaccine from the Schelkovo-51 strain inactivated liquid culture (Rabikov) was used (FSUE Schelkovo Biocombine, Russia).

Blood sampling (150 ml) was performed 5 days after the last injection. To prevent coagulation of the taken blood, 50 ml of standard saline solution (PBS) containing heparin (100 units / ml) and EDTA (3 mM) was added.

Blood was diluted 2 times with standard saline (PBS) containing 1 mm EDTA. 35 ml of a diluted blood solution was layered on a step of a special medium (Histopaque-1077, Sigma) with a density of 1.077 g / ml and a volume of 15 ml and centrifugation was performed for 20 min at 800 × g. Mononuclear cells (lymphocytes and monocytes) were selected from the plasma / Histopaque interphase, and then washed with a PBS solution containing 1 mM EDTA.

Total RNA from B lymphocytes was isolated using TRIzol reagent (Invitrogen). Then, on a column of oligo (dT) cellulose, poly (A) -containing RNA was isolated from total RNA. RNA concentration was determined using a biophotometer (Eppendorf) and the quality of the isolated RNA was checked by electrophoresis in 1.5% formaldehyde agarose gel.

The reverse transcription reaction was performed using reverse transcriptase H ^- M-MuLV and oligo primer (dt) ₁₅ as a seed according to the standard protocol of the manufacturer of the enzyme (Fermentas / Thermo Fisher Scientific).

Reverse transcription products were used as a template in a two-step polymerase chain reaction, and the obtained amplification products were cloned at the Ncol (Pstl) and Notl sites into a phagemid vector (pHEN4) as previously described [Hamers-Casterman C., Atarhouch T., Muyldermans S. et al. Nature 1993; 363: 446-448 .; Nguyen V.K., Desmyter A., Muyldermans S. Adv. Immunol. 2001; 79: 261-296; Saerens D., Kinne J., Bosmans E., Wernery U., Muyldermans S., Conrath K. J Biol Chem. 2004; 279: 51965-51972; Rothbauer U., Zolghadr K., Tillib S., et al. Nature Methods 2006; 3: 887-889]. The selection procedure was also carried out similarly to those described in these works. It was based on the phage display method, in which the bacteriophage M13KO7 (New England Biolabs, USA) was used as an assistant phage.

As a result of the work, a library of single-domain antibodies was obtained.

Example 2

Selection of single-domain antibodies that specifically recognize rabies virus (glycoprotein G), their trimerization (modification, formatting).

Single domain antibodies were selected by phage display using an inactivated rabies virus preparation, and at the final stage, rabies virus glycoprotein G (from the commercial PLATELLATM RABIES II KIT kit, Bio-Rad, France) immobilized at the bottom of the wells of a 96-well ELISA plate . Used polystyrene immunological plates with high sorption MICROLON 600 (Greiner Bio-One). For blocking, 1% BSA (Sigma-Aldrich, USA) and / or 1% skim milk (Bio-Rad, USA) in PBS were used. The selection and subsequent amplification of the selected phage particles (containing the single-domain antibody gene inside, and the expressed single-domain antibody as part of the surface phage protein pIII) was repeated, as a rule, three times in succession. All manipulations were performed as described in publications Tillib S.V., Ivanova T.I., Vasiliev L.A. 2010. Fingerprint analysis of the selection of "single-domain mini-antibodies" by the phage display method using two variants of assistant phages. Acta Naturae 2010; 2 (3): 100-108; Hamers-Casterman C, Atarhouch T., Muyldermans S. et al. Nature 1993; 363: 446-448 .; Nguyen V.K., Desmyter A., Muyldermans S. Adv. Immunol. 2001; 79: 261-296; Saerens D., Kinne J., Bosmans E., Wernery U., Muyldermans S., Conrath K. J Biol Chem. 2004; 279: 51965-51972; Rothbauer U., Zolghadr K., Tillib S., et al. Nature Methods 2006; 3: 887-889].

Clone sequences of selected single-domain antibodies were grouped according to the similarity of their fingerprints obtained by electrophoretic separation of the hydrolysis products of amplified single-domain antibody sequences in parallel with three often-binding restriction endonucleases (Hinfl, Mspl, Rsal). Then, selected clones representing each of the fingerprint-different groups were used for the analytical production of single-domain antibodies, the activity of which was checked by enzyme-linked immunosorbent assay for effective binding of the inactivated bird flu virus A / Mallard duck / PA / 10218/84 immobilized in the cell of an immunological tablet (H5N2).

As a result, a sequence encoding a single domain antibody was selected (FIG. 1, SEQ ID NO: 2).

In order to increase the efficiency of isolation and detection of a single-domain antibody, as well as to significantly enhance the biological activity of the initially selected monovalent single-domain antibody, we used the earlier effective trimerization procedure (formatting, modification of the amino acid sequence of an antibody), described in detail by Tillib et al. (Tillib et al., 2013, Antiviral Res. 97: 245-254. Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2) // Formatted single-domain antibodies can protect the mouse from infection with influenza virus (H5N2)). This procedure included attaching to the C-terminus of the initially selected antibody four additional amino acid sites (domains) shown in FIG. 1 — a linear linker site (corresponding to the upper elongated hinge site of the camel’s special immunoglobulins) —a special trimerizing domain - “ILZ” (" isoleucine zipper domain ", or, in translation," isoleucine zipper "), - the N-tag (peptide from hemagglutinin protein, to which there are commercial antibodies) and His-tag (six histidine residues at the C-terminus of the protein for effective protein purification using metal chelate chromatography).

Example 3

The production of trimerized single-domain antibodies that specifically recognize rabies virus (glycoprotein G).

The coding DNA sequence of the selected and trimerized, as described above, single chain antibody shown in FIG. 1 was cloned into an expression plasmid vector — a modified pHEN6 vector [Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J, Wyns L, , Muyldermans S. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob Agents Chemother. 2001; 45: 2807-12]. Due to the presence of the signal peptide sequence (peIB) at the N-terminus of the expressed sequence, the produced recombinant protein (single-domain antibody) accumulates in the periplasm of bacteria, which allows it to be efficiently isolated by the osmotic shock method without destroying the actual bacterial cells. The production of single domain antibodies was performed in E. coli (strain BL21). Expression was induced by adding 1 mM indolyl-beta-D-galactopyranoside and the cells were incubated with vigorous stirring for 7 hours at 37 ° C or overnight at 28 ° C. A single domain antibody was isolated from the periplasmic extract using affinity chromatography on Ni-NTA agarose using the QIAExpressionist purification system (QIAGEN, USA).

The resulting protein fractions were concentrated (to a final concentration of about 1-5 mg / ml) using ultrafiltration devices (with a cut-off size of 10 kDa) from Millicore Amicon, and then layered in a volume of 0.5-2 ml onto a gel column filtration chromatography (Sephacryl S-100 HR; 1.5 × 50 cm). Chromatography was performed using a BioLogic LP system from Bio-Rad at a constant flow rate of 0.3 ml / min. A standard saline buffer solution (PBS) was used as the buffer solution. Fractions were collected according to the absorption profile of the eluted protein at a wavelength of 280 nm (figure 3). The size of the trimerized single chain antibody approximately corresponded to the size (and peak elution) of the marker protein, bovine serum albumin (BSA, 67 kDa). The combined selected trimerized antibodies (from the fractions of the corresponding elution peaks) were again concentrated (as described above), further purified from endotoxin on an Thermo Scientific affinity column (Detoxi-Gel Endotoxin Removing Gel), and then sterilized by filtration through a cellulose acetate membrane (Nalgene company) ) with pores of 0.2 μm. The resulting preparation of a trimerized single chain antibody at various stages was analyzed by electrophoresis in 14% SDS-polyacrylamide gel (figure 2). The purified antibodies were stored in aliquots at 4 ° C or, after adding 50% glycerol, at -20 ° C. For longer storage, antibodies were lyophilized.

Example 4

Demonstration of the specific binding of the trimerized single chain antibody obtained according to the invention to rabies virus and rabies virus glycoprotein G.

The ability of the obtained trimerized single-domain antibody to bind both the whole (inactivated but retained capsid integrity) rabies virus (Shchelkovo-51 vaccine strain) and its specific protein, glycoprotein G (from the commercial PLATELLATM RABIES II KIT kit, Bio-Rad, France), immobilized (from a solution with a concentration of approximately 4 μg of protein in 1 ml) in the wells of the immunological tablet, was checked using enzyme-linked immunosorbent assay using a standard protocol. As a control, wells with bovine serum albumin immobilized protein (non-specific protein) were used. The trimerized single domain antibody was taken in serial dilutions with factor 5, starting at a concentration of 1 μg / ml. A formatted single-domain antibody bound to glycoprotein G or rabies virus was detected using anti-HA mouse antibodies, secondary antibodies to mouse immunoglobulins conjugated to horseradish peroxidase, and the ABST chromogenic substrate (2,2′-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid, Sigma). Figure 4 presents the results of the analysis, from which it follows that the trimerized single-domain antibody specifically binds to both the immobilized rabies virus and the immobilized rabies virus glycoprotein G, both in the case of an isolated protein, and, apparently, in the case when this protein is part of a viral capsid, but does not bind to bovine serum albumin (BSA), used as a blocking agent.

Example 5

Demonstration of the ability of a trimerized single domain antibody to neutralize rabies virus in vitro (i.e., virus neutralizing activity in cell culture).

Determination of the neutralizing activity of the trimerized single-domain antibody was carried out according to the well-known approved methodology "Methodological guidelines for the determination of virus-neutralizing antibodies to rabies virus in animal blood serum in the neutralization reaction in the cell culture of BHK-21 (FAVN)", 2011.

To compare the neutralizing activity of the trimerized single-domain antibody of the invention, a Chinese commercial anti-rabies immunoglobulin (Sichuan Yuanda Shuyan Pharmaceutical Company, China), widely known in Russia, was used. To compare with the prototype was not possible due to the incomplete disclosure of the technology for producing antibodies by the authors of the patent, as well as the lack of such antibodies in the commercial domain.

Previously, the protein concentration in the preparation of Chinese immunoglobulin was determined spectrophotometrically and titration was performed on a cell culture according to the above standard method. According to the results of titration, it was found that when the immunoglobulin is diluted to 300 μg / ml and higher, the level of virus-neutralizing antibodies in the preparation is protective (0.5 IU / ml). Accordingly, this concentration was taken as the initial for both studied drugs.

Next, titration was carried out in the neutralization reaction of a trimerized single-domain antibody and Chinese immunoglobulin simultaneously. The titration results are shown in the table.

Table Titration results of a trimerized single-domain antibody and commercial Chinese anti-rabies immunoglobulin in the neutralization reaction in the cell culture of BHK-21 (FAVN) Test drug The concentration of protein in the drug, mcg / ml The level of neutralizing antibodies, ME / ml Commercial Chinese anti-rabies immunoglobulin 300 0.5 Trimerized single domain antibody 300 9720

Thus, the level of virus-neutralizing antibodies at a protein concentration of 300 μg / ml in Chinese immunoglobulin was 0.5 IU / ml, and for a trimerized single-domain antibody at the same concentration it was 9720 IU / ml, which is 19440 times higher than that of Chinese immunoglobulin.

As a result of the experiment, it was found that a trimerized single-domain antibody can neutralize rabies virus in vitro and protect cell culture from infection with rabies virus at a concentration of 300 μg / ml and above. Moreover, the virus-neutralizing activity of the trimerized single-domain antibody according to the invention is 19440 times higher than that of a commercial immunoglobulin.

Industrial applicability

All examples presented confirm the industrial applicability of the claimed trimerized single domain antibodies for the prevention and treatment of rabies and confirm the fulfillment of the technical task.

Claims (3)

1. Trimerized single-domain antibody specifically binding to rabies virus glycoprotein G, having the amino acid sequence of SEQ ID NO: 2. 2. The trimerized single domain antibody according to claim 1, the amino acid sequence of which is encoded by the nucleotide sequence of SEQ ID NO: 1. 3. The trimerized single-domain antibody according to claim 1, characterized in that it neutralizes the rabies virus.

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