RU2599423C1 - Recombinant single-domain antibodies specifically binding f4/80 protein, method for production thereof and use for detection of said protein - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: disclosed are recombinant single-domain nano-antibodies (VHH) obtained based on antibodies double-humbed camel (Camelus bactrianus) and specifically binding mouse protein F4/80. Nano-antibodies are characterised by amino acid sequences. Invention provides use thereof for detection of F4/80 protein on surface of monocytes, tissue macrophages, myeloid dendritic cells and eosinophils of mice, as well as use as a subunit of multi-domain protein for delivering selected multi-domain protein to F4/80-positive target mouse cells.

EFFECT: disclosed nano-antibodies are characterised by small size, which enables further use thereof in clinical applications.

11 cl, 6 dwg, 5 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of molecular immunology, biotechnology and medicine, namely to the production of recombinant single domain nanoantibodies (VHH) that selectively bind F4 / 80 protein.

State of the art

Surface cell glycoprotein F4 / 80 (mouse homologue of human EMR1) has been identified in monocytes, most tissue macrophages, myeloid dendritic cells and mouse eosinophils. It belongs to the EGF-TM7 family of receptors, which are fixed in the cell membrane using 7 transmembrane domains, and also include large extracellular regions containing a variable number of NH2 terminal EGF domains [Hamann, J. et al., EMR1, the human homolog of F4 / 80, is an eosinophil-specific receptor. Eur J Immunol; 2007.P. 2797-2802. - EMR1, the human homologue of F4 / 80, an eosinophil-specific receptor.]. The F4 / 80 gene is located on the 17th chromosome and encodes a 931 amino acid polypeptide, which is subsequently processed to form a mature protein of 904 amino acids [McKnight, A.J. and Gordon, S. The EGF-TM7 family: unusual structures at the leukocyte surface. J Leukoc Biol; 1998. P. 271-280. - TEGF-TM7 TEGF-TM7 family of proteins: unusual structures on the surface of leukocytes.].

Protein F4 / 80 attracts special attention, primarily, as a mouse macrophage marker constitutively expressed during embryonic development and during adulthood. Studying the F4 / 80 protein was key to characterizing the mouse mononuclear phagocyte system and to detect macrophages in numerous in vivo studies. Recent studies have demonstrated the role of F4 / 80 in the induction of tolerance [Lin, H.H. et al., The macrophage F4 / 80 receptor is required for the induction of antigen-specific efferent regulatory Τ cells in peripheral tolerance. J Exp Med; 2005.P. 1615-1625. - The macrophage receptor F4 / 80 is necessary for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.].

There is abundant evidence that the EMR1 protein, the human mouse homologue of mouse F4 / 80, is an important diagnostic marker and target for the development of new therapeutic drugs [Legrand, F. Et al., Eosinophil surface receptor EMR1: a novel therapeutic target for eosinophilic disorders. J Allergy Clin Immunol. 2014.P. 1439-1447. - Surface eosinophil receptor EMR1: a new therapeutic target for the treatment of diseases characterized by impaired eosinophil function.]. This fact justifies the relevance and feasibility of research in this direction, including through the development of functional analyzes on mouse models.

In recent years, more and more attention when creating new diagnostic and therapeutic drugs for the detection and treatment of many diseases has been given to drugs based on antibodies, usually monoclonal classical antibodies. An antibody is capable of specifically binding a specific antigen / protein, both free or complexed with other molecules, and a protein that is overexpressed and localized on the surface of a cell of a certain type. Such binding can block an important biological process, a cascade, and also stimulate the patient’s immune system to attack the cells that the antibody has bound to. An antibody may also be a specific delivery vehicle for other therapeutic molecules, substances or radioisotopes. Finally, it is possible to use bi- and poly-functional and multicomponent constructs based on the resulting antibodies or their derivatives.

As the closest analogue of the technical solution that forms the basis of the present invention, the following example can be given.

Patent US 20130064826 A1 "Anti-emr1 antibodies" - "Anti-emr1 antibodies" from 03/14/2013.

This patent claims the production of monoclonal antibodies to a specific peptide region of the EMR1 protein (human homologue of mouse F4 / 80), as well as the use of these antibodies for the diagnosis and treatment of EMR1-dependent human diseases.

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:

- Relatively expensive production of antibodies, the difficulty of maintaining and storing the producer, very high requirements for the quality of the reagents used and the cultivation conditions.

- The relatively large size of the resulting antibodies, which entails reduced tissue permeability.

- Structural features impose a restriction on the recognition of some "hidden" epitopes located, for example, in recesses, small gaps in the structure of proteins.

- The limited and relative complexity of genetic engineering manipulations, adaptations for specific tasks, the complexity of creating multivalent and multifunctional derivatives of the claimed antibodies.

Thus, in the prior art there is a need for the development of new antibodies (antigen-recognizing molecules) lacking the above disadvantages and specifically recognizing the EMR1 protein and its corresponding mouse homologue F4 / 80 (for functional assays in mouse models).

The present invention is the creation of new antibodies that can efficiently bind the homologue of the EMR1 protein in mice, known as protein F4 / 80, the production of these antibodies, the production and storage of which should be economical. New antibodies must be effective and relatively simple; their size should be several times smaller than that of classical antibodies.

The technical problem is solved due to the fact that single-domain nanoantibodies are obtained that specifically bind mouse F4 / 80 protein. Moreover, the method of producing single-domain nanoantibodies to F4 / 80 protein involves immunization of a two-humped camel (Camelus bactrianus) with mouse purified F4 / 80 protein, obtaining a library of sequences encoding nanoantibodies, a system for selecting specific nanoantibodies using phage display, production of nanoantibodies in prokaryotes expression and purification. And the method of detecting F4 / 80 protein on the cell surface is carried out using nanoantibodies.

The basis of the present invention are not classical bivalent antibodies, which are considered as a prototype, but small single-domain nanoantibodies, which have several advantages, which suggests a great potential for their future use in various studies and in the creation of new biotechnological devices, as well as for clinical purposes diagnosis and treatment of diseases. Since nanoantibodies (molecular weight of about 12-15 kDa) are an order of magnitude smaller in size of traditional classical antibodies, they acquire a number of new positive qualities of practical importance. Nanoantibodies may possess new structural features that, in principle, allow them to recognize some epitopes that are “hidden” for ordinary antibodies.

Disclosure of invention

The method of obtaining single-domain nanoantibodies that bind antigenic epitopes of the F4 / 80 protein includes the following principal steps:

1) the induction of the formation of specific antibodies as a result of immunization of a representative of the Camelidae family (in particular, the two-humped camel) with mouse affinity-purified protein F4 / 80;

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

3) selection by the phage display method (functional selection of phage particles carrying an expressed nanoantibody on the surface, and inside the DNA encoding this nanoantibody) of nanoantibody clones that bind to a given antigen, protein F4 / 80, from the obtained library of nanoantibodies;

4) carrying out the cloning and genetic engineering modifications of the coding sequences of the selected nanoantibodies with the aim of their effective production in the bacterial expression system and subsequent effective purification (E. coli bacteria are used as the producer of the active substance).

The extracellular part of the F4 / 80 protein specially synthesized for this purpose was used as an antigen for immunization (number in the EXPASY Q61549 database). Recombinant murine protein F4 / 80 was expressed in Chinese hamster ovary cells

The algorithm for producing recombinant protein F4 / 80 included the following steps.

DNA cloning

The extracellular part of the F4 / 80 Ν- and C- terminally bound to the affinity tag was optimized by codon composition for protein expression in the eukaryotic system. The resulting sequence was synthesized in GenScript (USA) and subcloned into the vector pOptiVec (Invitrogen). Plasmid DNA for transfection was purified using the Qiagen Plasmid Maxi kit, digested with Sail, and then purified with the QIAquick DNA extraction kit. A linearized vector was used to transfect DG44 cells (Invitrogen), as described below.

DG44 cell culture and transfection

The DG44 cell culture was cultured in Erlenmeyer flasks in CD-DG44 medium (Gibco) supplemented with 8 mM Glutamax-1 and hypoxanthine and thymidine (HT) additives in a shaker at 125 rpm, in a humidified incubator with 8% CO ₂ at 37 ° FROM. Before transfection, cells were harvested by centrifugation and resuspended in CHO-S SFMII medium (Invitrogen). Transfection was performed with PeiProTM (Polyplus Transfections) at a density of 4 million cells in 1 ml (4M). Six hours after transfection, three volumes of OptiCHO medium (Invitrogen) with HT supplementation were added, after which the cells were cultured for another 48 hours. Further, the medium was replaced with OptiCHO CD without HT; the culture was grown for 4 weeks, until it reached a sustainable viability of more than 95%.

Getting a stable cell pool

The cell pools obtained in the previous step were subjected to gene amplification in the presence of 500 nM methotrexate (Sigma). During this stage of selection, no visible changes in cell viability were observed.

The resulting cell population was used to produce protein, as described below.

Protein production

For the production of recombinant protein, cells were cultured in OptiCHO medium CD containing 20% Cell Boost5 (Hyclone). Cell culture was harvested after 6 days at a cell density of 8-9 million per ml. Cells were separated by centrifugation, the supernatant was filtered through a 0.45 μm filter and used to purify the protein.

Protein purification

The conditioned medium was applied to a column containing CL-4B Sepharose with attached antibodies. Then the carrier was washed successively with 20 mM Tris mM 150 mM NaCl, pH 7.5 (buffer A), 20 mM Tris, 700 mM NaCl, pH 7.5 (buffer B) and 5 mM Tris, 150 mM NaCl, pH 7.5 (buffer C). The bound protein was eluted with 0.1 M glycine pH 2.5, the eluate was immediately neutralized with Tris buffer. The eluted F4 / 80 protein was stored in aliquots at -70 ° C. Protein concentration was determined spectrophotometrically when absorbed at 280 nm against an elution buffer. The quality of the drug was checked using denaturing electrophoresis in PAAG (FIG. 2).

A method of obtaining single-domain nanoantibodies is described in examples 1 and 2. The possibility of using the obtained nanoantibodies for the detection of F4 / 80 protein and their functional characteristics are shown in examples 3-5.

Nanoantibodies (molecular weight of about 12-15 kDa) are an order of magnitude smaller in size of traditional antibodies and are fully functional antigen-recognizing units with a number of new positive qualities of practical importance. Nanoantibody is a single domain protein that is highly soluble and has increased stability (over a wide range of temperatures and pH). This avoids problems with the solubility and correct folding of antibody molecules during production by prokaryotic cells and leads to a significant reduction in the cost of their production compared to traditional methods for producing therapeutic monoclonal antibodies in eukaryotic expression systems. The procedure for storing and transporting antibodies is also significantly facilitated compared to markedly less stable traditional antibodies. Nanoantibodies make it easy to carry out genetic engineering manipulations for the purpose of subsequent production of bispecific nanoantibodies or chimeras, which in addition to the nanoantibodies include another protein with the desired properties.

For illustrative purposes, the authors of the present invention focuses on the production of antibodies using the E.coli bacterium, however, it is obvious that there are other options for implementation systems of the present invention that are not expressly stated in this document. For example, yeast cultures or any other systems obvious in the art can be used as a eukaryotic producer.

The choice of implementation paths in order to obtain nanoantibodies with the claimed properties from a prokaryotic expression system in accordance with one of the preferred embodiments of the present invention is due to the following factors:

1) High expression yield, cost-effective production in large quantities, provided by the expression of nanoantibodies in the periplasm of E. coli bacteria (in an amount of 1-10 mg from 1 liter of culture).

2) The simplicity of all kinds of genetic engineering manipulations, adaptations for specific tasks, the ability to create multivalent and multifunctional derivatives.

3) High economic profitability of production.

The authors of the present invention proceed from the fact that the primary, initially obtained sequence of single-domain nanoantibodies can then be adapted or "formatted" in various ways for subsequent practical use.

So, single-domain nanoantibodies can be the original modules (blocks) of more complex multimodule preparations. It is possible to combine in one multivalent derivative two, three or more monovalent primary single-domain nanoantibodies. These single domain nanoantibodies combined into a single construction 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 nanoantibodies 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 forming stable complexes. To modulate the properties of the preparation of a single-domain nanoantibody, for example, to increase the lifetime or to improve the purification method, additional amino acid sequences can be introduced into the final compound. Obviously, such modifications and other variants of the antibodies underlying the present invention fall within the scope of the present invention, since they are structural and functional variants of single domain nanoantibodies. Thus, the authors of the present invention understand the term "single-domain nanoantibodies" as the primary, initially obtained, "minimum" amino acid sequences of single-domain nanoantibodies, as well as their modifications resulting from the mentioned adaptations 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 initial single domain nanoantibody. A number of changes in a protein variant depend on the position or type of amino acid residue in the three-dimensional structure of the protein. 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 nanoantibody. 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 nanoantibody, provided 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.

The single-domain nanoantibody 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 antibodies of the invention and within the scope of the invention.

Brief Description of the Drawings

FIG. 1. Amino acid sequences of nine selected single domain antibodies (nanoantibodies) capable of specifically binding to F4 / 80 protein. In the indicated sequences, the hypervariable regions of CDR1, CDR2 and CDR3 are highlighted and underlined (respectively, from left to right, from N- to C-terminus).

FIG. 2. The results of the analysis of protein F4 / 80 by denaturing polyacrylamide gel electrophoresis.

FIG. 3. Purified formatted single-domain antibodies (odAT), specifically recognizing F4 / 80 protein, fractionated in 14% SDS-polyacrylamide gel.

FIG. 4. Results of an enzyme-linked immunosorbent assay recognizing the recombinant protein F4 / 80, immobilized in the wells of the immunological plate, selected nine single-domain formatted antibodies (indicated at the bottom of the figure with a number from 1 to 9). Control wells did not contain antigen, but were then blocked and processed in parallel with experimental cells (with antigen). The ordinate axis corresponds to the absorption data in the experimental cells at a wavelength of 405 nm. The averaged results of three repetitions of this analysis are presented (with the indicated ranges of scatter of the obtained values).

FIG. 5. Analysis of the binding of nine single-domain formatted nanoantibodies (indicated by numbers from 1 to 9) to F4 / 80 with the surface of macrophages by flow cytometry.

FIG. 6. Measurement of the binding and dissociation constants of single domain antibodies against F4 / 80 with the recombinant protein F4 / 80 by surface plasmon resonance.

The figure shows the sensograms obtained as a result of 5 interactions for each of the antibodies. The results were analyzed using ProteOn Manager Software (Bio-Rad) using the Langmuir model. For the experiment, biotinylated single-domain antibodies against F4 / 80 were used, which were immobilized on an NLC chip at a concentration of 5 nM. Antibodies were applied at a speed of 30 μl / min in a volume of 150 μl in a vertical orientation. The immobilization level of antibodies was 600-800 RU. After washing with a 1 M sodium chloride solution (100 μl / min, volume 50 μl), the orientation was changed to horizontal. Then it was washed with phosphate-buffered saline containing 0.005% Tween (100 μl / min, volume 10 μl, dissociation 3600 s), after which the baseline was established. Then, also in horizontal orientation, a preparation of recombinant antibodies against the F4 / 80 protein was applied in phosphate-buffered saline in twice decreasing concentrations of 200 - 12.5 nM.

Examples of the implementation of the present invention

Example 1

Obtaining a library of variable domains of single-chain antibodies

Immunization

To generate single-domain nanoantibodies that specifically recognize the F4 / 80 protein, a 5-fold subcutaneous immunization of a two-humped camel (Camelus bactrianus) was carried out. As an antigen for immunization, a specially synthesized recombinant protein F4 / 80, which was expressed in the eukaryotic system, was used.

The preparation of the recombinant protein F4 / 80 was divided into 6 equal parts of 0.5 mg (the first 5 parts for 5 consecutive injections, and also left an aliquot for the subsequent stages of selection and analysis of the specified nanoantibodies). All aliquots were frozen for storage at -70 ° C. Before each immunization step, one of the aliquots of the preparation stored at -70 ° C was thawed and immediately before injection, they were mixed in equal proportions with LQ adjuvant (Gerbu, Heidelberg, Germany).

Before the 1st immunization, camel was taken from the camel to obtain preimmune serum, which was subsequently used as a negative control. Five-fold immunization was performed by subcutaneous injection of the recombinant protein F4 / 80, mixed in equal proportions with adjuvant. The second immunization was carried out 4 weeks after the first, and the next 3 with an interval of 10 days. Blood sampling was performed 5 days after the last injection.

To prevent coagulation of the taken blood, 50 ml of standard phosphate-saline solution (PBS) containing heparin (50 units / ml) and EDTA (2 mM) was added.

Blood was diluted 2 times with a PBS solution 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 from total RNA, poly (A) -containing RNA was purified. 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 carried out according to the standard protocol [Sambrook et al., 1989] using reverse transcriptase HM-MuLV and onnro primer (dT) 15 as a seed.

Reverse transcription products were used as templates in a two-step polymerase chain reaction, and the resulting amplification products were cloned at the Ncol (Pstl) and Notl sites into the pHEN4 plasmid vector as previously described [Hamers-Casterman C, Atarhouch Τ, Muyldermans S, et al. Naturally occurring antibodies devoid of light chains, Nature, 1993; 363: 446-448. - Existing naturally occurring antibodies without light chains. Nguyen V.K., Desmyter Α., Muyldermans S. 2001. Functional heavy-chain antibodies in camelidae. Adv. Immunol. 79, 261-296. - Functional antibodies, consisting only of heavy chains, in Camelids.

Saerens D., Kinne J., Bosmans Ε., Wernery U., Muyldermans S., Conrath K. 2004. Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J. Biol. Chem. 279, 51965-51972. - Single domain antibodies, originating from the lymph node or lymphocytes of the peripheral blood of a camel, recognize conformational variants of a prostate-specific antigen.

Rothbauer U., Zolghadr K., Tillib S., Nowak D., Schermelleh L., Gahl Α., Backmann N., Conrath K., Muyldermans S., Cardoso MC, Leonhardt L. 2006. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nature Methods. 3, 887-889. - Targeting and observation of antigens in living cells using fluorescent nanobodies.].

Example 2

Selection of single domain nanoantibodies that specifically recognize F4 / 80.

Single domain nanoantibodies were selected by phage display using a recombinant protein F4 / 80 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 procedure and the subsequent amplification of the selected phage particles was repeated successively three times. All manipulations were performed as described in the publications [Tillib S.V., Ivanova T.I., Vasiliev L.A. 2010. Fingerprint analysis of the selection of "nanoantibodies" by the phage display method using two variants of assistant phages. Acta Naturae 2010; 2 (3): 100-108. Hamers-Casterman C. et al. Nature. 363, 446-448. Nguyen V.K. et al. Adv. Immunol. 2001; 79: 261-296. Saerens D. et al. J Biol Chem. 2004; 279: 51965-51972. Rothbauer U. et al. Nature Methods 2006; 3: 887-889. (A detailed description of the last four links has already been given above)]. The clone sequences of the selected nanoantibodies were grouped by the similarity of their fingerprints obtained by electrophoretic separation of the hydrolysis products of amplified sequences of single-domain nanoantibodies in parallel with three frequently digested restriction endonucleases (Hinfl, Mspl, Rsal). The cDNA sequences of single domain nanoantibodies to F4 / 80 were determined. The corresponding amino acid sequences of 9 clones of nanoantibodies to F4 / 80 (SEQ ID ΝΟ: 1-9) (FIG. 1) were deduced from them.

Nanoantibody Products

The cDNA sequences of nine selected nanoantibody clones were cloned into an expression modified plasmid vector pHEN6 [Conrath KE, Lauwereys M, Galieni M, Matagne A, Frere JM, Kinne J, Wyns L, Muyldermans S. Betalactamase inhibitors derived from single-domain antibody fragments eited Camelidae. Antimicrob Agents Chemother. 2001; 45: 2807-2812. - Beta-lactamase inhibitors, originating from single-domain camelid antibody fragments.], Which allows the addition of a single-domain nano-antibody (His) 6-epitope to the C-terminus. Due to the presence of the signal peptide sequence (peIB) at the N-terminus of the expressed sequence, the produced recombinant protein (single-domain nanoantibody) accumulates in the periplasm of bacteria, which allows it to be efficiently isolated by the osmotic shock method without destroying the bacterial cells themselves. The production of single domain nanoantibodies was carried out 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 29 ° C. A single domain nanoantibody was isolated from the periplasmic extract using affinity chromatography on Ni-NTA agarose using the QIAExpressionist purification system (QIAGEN, USA).

In FIG. Figure 3 shows the electrophoregram of a polyacrylamide gel with the developed and purified single domain antibodies (odAT) that specifically recognize F4 / 80 protein.

The structure of nine single-domain nanoantibodies to F4 / 80 is shown in FIG. 1. In the indicated sequences, the hypervariable regions of CDR1, CDR2 and CDR3 are highlighted and underlined (respectively, from left to right, from N- to C-terminus).

Example 3

Detection of F4 / 80 Recombinant Protein Using Selected Nanoantibody Clones

The periplasmic extract containing the mini-antibody with the HA tag obtained as a result of the described procedure was used to assess the specificity and binding efficiency of the corresponding mini-antibody with the preparation of the recombinant protein antigen using the immunofluorescence analysis method.

Recombinant F4 / 80 recombinant protein was applied in a volume of 50 μl in a volume of 50 μl into a standard 96-well immunological plate (from polystyrene, Microlon 600 hi-binding, from Greiner bio-one) in a volume of about 2 μg / ml. Immobilization was carried out overnight at + 4 ° C. Then the cells were washed (in PBS) from unbound antigen and the wells were blocked with a solution of 1% bovine serum albumin (BSA) in PBS for 2 hours at room temperature. Cells were washed in PBS and PBS with 0.05% Tween-20, after which verified single-domain formatted antibodies containing the HA tag at the C-terminus were added in an amount of about 500 ng in 50 μl of PBS containing 0.1% BSA. After incubation (with stirring) for 2 hours at room temperature, the cells were washed again, as described above, and then incubated with 2000-fold diluted PBS (0.1% BSA) secondary anti-HA antibody conjugated to horseradish peroxidase ( CHGT-45P-Z, ICL, Inc., USA). Incubation was carried out for 1 hour at room temperature. After thorough washing, the activity of the horseradish peroxidase enzyme was detected using ABTS (2,2′-azino-bis 3-ethylbenzothiazolin-6-sulfonate) as a chromogenic substrate. The optical density was measured at a wavelength of 405 nm using a flatbed fluorimeter. Control wells did not contain antigen, but were then blocked and processed in parallel with experimental cells (with antigen).

In FIG. Figure 4 shows the results of an enzyme immunoassay, from which it follows that all the studied clones of single domain nanoantibodies specifically bind to the recombinant protein F4 / 80. The size of the columns in the figure corresponds to the relative values of absorption at a wavelength of 405 nm, quantitatively reflecting the efficiency of binding of nanoantibodies to F4 / 80.

Example 4

Investigation of the ability of nanoantibodies to bind to F4 / 80 on the surface of macrophages using flow cytometry

The ability of single domain nanoantibody clones to bind to the surface of macrophages due to specific interactions with F4 / 80 was tested by flow cytometry. Primary cultures of bone marrow macrophages were isolated from the femur and tibia of C57BL / 6 mice. Macrophages were cultured for seven days in Petri dishes in macrophage culture medium (50% DMEM medium (containing 2 mML-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin), 30% conditioned medium obtained from supernatant-cell line L929 and 20% horse serum) at 37 ° С and 5% С02, then the cells were transplanted onto 96-well plates in the amount of 100 thousand cells per well and stained. Selected clones of single domain nanoantibodies to F4 / 80 were conjugated to biotin. Cells were stained with antibodies specific for the macrophage marker CD11b conjugated to a fluorescent marker, as well as selected biotinylated clones of single domain nanoantibodies to F4 / 80. Cells were then incubated with streptavidin conjugated to a fluorescent marker. After staining, cells were fixed with 4% paraformaldehyde solution.

The resulting samples were analyzed on flow cytometer Gallios ™ (Beckman Coulter Inc.) and FACSCanto II (BDBiosciences). The results were processed using the FlowJo program (Treestar Inc.).

In FIG. Figure 5 presents the results of flow cytofluorimetry, from which it follows that the obtained clones of single domain nanoantibodies to F4 / 80 are able to bind to the surface of macrophages. The data presented demonstrate that the ability to bind to the surface of macrophages in the selected clones of single-domain nanoantibodies to F4 / 80 is heterogeneous. Among the nine clones, there are several that show good binding to F4 / 80 (VHH1, 2, 3, 8, 9), and clones that practically or completely do not bind to the surface of macrophages (VHH4, 5, 6, 7).

Thus, for further studies, the most promising single domain antibody clones (VHH 1, 2, 3, 8, 9) were selected that can be used as potential fragments for the subsequent construction of bispecific antibodies.

Example 5

Measurement of the binding and dissociation constant of single domain antibodies against F4 / 80 with the recombinant protein F4 / 80 by surface-plasmon resonance.

For analysis, the most promising

Binding kinetics were measured: association rates, dissociation rates, and dissociation constants of single-domain antibodies with the recombinant protein F4 / 80 were carried out using a Proteon XPR36 surface plasmon resonance device (Bio-Rad). The measurements were carried out at 20 ° С. A phosphate-saline buffer pH 7.4, 0.005% Tween 20 was used as a buffer.

For this, biotinylated single domain anti-F4 / 80 antibodies in phosphate-buffered saline (pH = 8.0) were immobilized on an NLC chip (on this chip, a layer of alginic acid polymer was covalently coated on top of a gold layer in which the surface plasmon resonance effect occurs) attached molecules of neutravidin (neutravidin) - a recombinant analogue of biotin with a neutral charge) at a concentration of 5 nm. Antibodies were applied at a speed of 30 μl / min in a volume of 150 μl in a vertical orientation. The immobilization level of antibodies was 600-800 RU. After washing with a 1 M sodium chloride solution (100 μl / min, volume 50 μl), the orientation was changed to horizontal. Then it was washed with phosphate-buffered saline containing 0.005% Tween (100 μl / min, volume 10 μl, dissociation 3600 s), after which the baseline was established. Then, also in horizontal orientation, a preparation of recombinant antibodies against the F4 / 80 protein was applied in phosphate-buffered saline in twice decreasing concentrations of 200 - 12.5 nM.

Sensograms resulting from 5 interactions for each of the antibodies were analyzed using ProteOn Manager Software (Bio-Rad) using the Langmuir model.

In FIG. Figure 6 shows the interaction curves (sensograms) of recombinant F4 / 80 with sensor chips on which single domain antibodies against F4 / 80 were immobilized.

These examples with illustrations prove the achievement of the objectives of this invention. New special single-domain nanoantibodies capable of efficiently binding F4 / 80 protein have been created. These new specific nanoantibodies have the following advantages relative to the prototype (monoclonal antibodies to a specific peptide region of the EMR1 protein (human homologue of mouse F4 / 80)): their preparation, production and storage are more economical, efficient and relatively simple; their preparation includes the stage of immunization and affinity maturation in vivo, which usually contributes to higher affinity and a significant increase in the efficiency of selection of desired nanoantibodies, they are highly soluble, have new structural features that allow them to potentially recognize some of the epitopes that are “hidden” for ordinary antibodies. Thus, the problem posed in this invention is solved.

Claims (11)

1. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 1. 2. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 2. 3. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 3. 4. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 4. 5. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 5. 6. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 6. 7. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 7. 8. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 8. 9. Recombinant single-domain nanoantibodies that specifically bind mouse protein F4 / 80 and having the amino acid sequence of SEQ ID NO: 9. 10. The use of single-domain nanoantibodies according to any one of claims 1 to 9 for the detection of F4 / 80 protein on the surface of monocytes, tissue macrophages, myeloid dendritic cells and mouse eosinophils. 11. The use of single domain nanoantibodies to F4 / 80 according to any one of claims 1 to 9 as a subunit of a multi-domain protein for delivery of the selected multi-domain protein to F4 / 80-positive mouse target cells.

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