RU2603269C1 - Recombinant single-domain antibodies specifically binding human interleukin-6, method for their preparing and using for detection of this protein - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention relates to molecular immunology, biotechnology and medicine. Recombinant variants of a single-domain antibody (VHH) are presented, which specifically binds human interleukin-6 (IL-6) and is characterized by amino acid sequences. Single-domain antibodies disclosed herein are obtained on basis of antigen-recognizing antibody domains of lamp (Lama glama).

EFFECT: present invention can find further application in diagnosing and treating IL-6-mediated diseases.

3 cl, 4 dwg

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 interleukin-6 (IL-6).

State of the art

Interleukin-6 (IL-6) is a multifunctional pro-inflammatory cytokine with a wide range of immunoregulatory properties, as well as a mediator of the formation, growth and progression of tumors of various nature and localization. IL-6 was discovered in the 80s in several laboratories under different names (factor-2, stimulating B cells; interferon-beta-2; growth factor hybridomas; hepatocyte stimulation factor), which reflects the pleiotropy of this cytokine [Hirano T. et al., Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature, 324: 73-76, 1986. - Complementary DNA for the new human interleukin (BSF-2), which induces the production of immunoglobulin B-lymphocytes; Zilberstein A. et al., Structure and expression of cDNA and genes for human interferon-beta-2, a distinct species inducible by growth-stimulatory cytokines. EMBO J. 1986 Oct; 5 (10): 2529-37. - The structure and expression of cDNA and interferon-beta-2 genes, various species are induced by growth-stimulating cytokines; Lansdorp, P. M. et al., A Growth-Factor Dependent B-Cell Hybridoma. Mechanisms in B-Cell Neoplasia. Current Topics in Microbiology and Immunology Volume 132, 1986, pp. 105-113- B cell hybridoma dependent on growth factor. Mechanisms in B-cell neoplasia].

The human IL-6 gene is located on the short arm of the 7th chromosome [Sehgal R.V. Regulation of IL-6 gene expression. Res. Immunol., 1992. P. 724-734. - Regulation of gene expression of IL-6]. Human IL-6 is a glycoprotein with a molecular weight of 26 kDa, consisting of 184 amino acids [Yao X. et al., Targeting interleukin-6 in inflammatory autoimmune diseases and cancers. Pharmacol Ther. 2014, 141 (2), 125-139. - IL-6 - therapeutic target for inflammatory autoimmune and oncological diseases].

IL-6 is produced by macrophages, T and B lymphocytes, fibroblasts, endothelial, epidermal and microglial cells, chondrocytes, osteocytes, as well as some tumor cells [Ohsugi Y. Recent advances in immunopathophysiology of interleukin-6: an innovative therapeutic drug, Tocilizumab (recombinant humanized anti-human interleukin-6 receptor antibody), unveils the mysterious etiology of immune-mediated inflammatory diseases. Biol. Pharm. Bull., 2007. P. 2001-2006. - Recent advances in the immunopathophysiology of interleukin-6: an innovative therapeutic drug, Tocilizumab (a recombinant humanized antibody to the human receptor for interleukin-6), reveals the secret of the etiology of immune-mediated inflammatory diseases].

The biological activity of IL-6 is realized through interaction with a membrane-bound subunit of the receptor, which does not have a signal function, followed by signal transmission through the transmembrane glycoprotein gp130. This is a classic IL-6 signaling pathway for cells expressing the membrane-bound subunit of the IL-6 receptor. There is an alternative signal transmission mechanism in which the soluble subunit of the IL-6 receptor is involved. The complex of IL-6 with the soluble subunit of the IL-6 receptor can activate cells that do not express the membrane-bound subunit of the IL-6 receptor, but have gp130 glycoprotein on their surface. Such an alternative signal transmission mechanism significantly increases the number of IL-6 target cells [Scheller J., Interleukin-6: from basic biology to selective blockade of pro-inflammatory activities. Semin Immunol. 2014.P. 2-12. - Interleukin-6: from the basics of biology to the selective blocking of pro-inflammatory activity; Astrakhantseva I.V. et al. Modern anticytokine therapy of autoimmune diseases. Biochemistry, 2014, 79 (12), 1607-1618].

IL-6 is one of the key factors in the development of some autoimmune diseases, as well as some types of neoplasia. Overexpression of this cytokine leads to the launch of an inflammatory cascade and tissue damage by cells of the immune system. Blocking IL-6 using recombinant monoclonal antibodies has shown high clinical efficacy and is a component of the treatment of IL-6 dependent diseases (rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, ulcerative colitis, multiple myeloma, Castleman’s disease, malignant lymphoma, kidney cancer, etc. ) [Montero-Julian FA Pharmacokinetic study of anti-interleukin-6 (IL-6) therapy with monoclonal antibodies: enhancement of IL-6 clearance by cocktails of anti-IL-6 antibodies. Blood 1995. P. 917-924. - Pharmacokinetic study of monoclonal antibodies for anti-IL-6 therapy: increased clearance of IL-6 using a cocktail of anti-IL-6 antibodies; Trikha, M. et al., Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence. Clin Cancer Res .; 2003. P. 4653-4665. - The use of monoclonal antibodies to IL-6 for targeted cancer therapy: a review of clinical data; Woodrick, R. and Ruderman, E.M. Anti-interleukin-6 therapy in rheumatoid arthritis. Bull NYU Hosp Jt Dis. 2010. P.211-217. - Anti-interleukin-6 therapy for rheumatoid arthritis].

A number of recombinant IL-6 blockers are successfully undergoing clinical trials in the treatment of autoimmune and neoplastic diseases: aglycosylated, humanized monoclonal antibodies to IL-6 Clazacizumab (ALD518 / BMS-945429), human monoclonal antibodies to IL-6 subunit SARilab Sarilum α6 subunit (Sarilab 1REG), human monoclonal antibodies to soluble IL6 Sirukumab (Sirukumab, CNTO 136), high-affinity monoclonal antibodies IL-6, which block the final stage of assembly of the IL6 / IL6P Olokizumab complex (Olokizumab, CD6038). It is successfully used in clinical practice for the treatment of rheumatoid arthritis (with insufficient efficacy or intolerance to name inhibitors), a recombinant humanized monoclonal antibody to the human IL-6 receptor Tocilizumab (Actemra / Actemra).

The main limitation to the widespread use of anti-IL-6 therapy in the Russian Federation is its high cost. The absence of domestic production of IL-6 blockers on the pharmaceutical market leads to the fact that therapy with IL-6 inhibitors remains unavailable to most patients.

Examples of IL-6 blocking antibodies are widely reflected in international and Russian patents, for example, in patent WO / 2010/056948 "Humanized anti-IL-6 antibodies" - "Humanized anti-IL-6 antibodies"; in US patent 8992920 B2 "Anti-IL-6 antibodies for the treatment of arthritis" - Anti-IL-6 antibodies for the treatment of arthritis "; in US Pat. No. 8623362 B2 "Anti-IL-6 antibodies" - "Anti-IL-6 antibodies"; in patent RU 2550262 "Monoclonal antibody against human interleukin-6 and a hybridoma producing this monoclonal antibody", in patent RU 2532826 "Humanized antibodies against IL-6".

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

Patent WO / 2010/056948 "Humanized anti-IL-6 antibodies" - "Humanized anti-IL-6 antibodies."

This patent claims the production of monoclonal humanized antibodies against IL-6. 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, high requirements for cultivation conditions and the quality of the reagents used, the complexity of maintaining and storing the producer.

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

- The structural features of antibodies impose a restriction on the recognition of certain "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, there is a need for the development of new antibodies (antigen-recognizing molecules) lacking the above disadvantages and specifically recognizing IL-6.

Disclosure of invention

The task to which the invention is directed is to create new antibodies that can specifically bind to human IL-6 without significantly affecting its functioning, 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. In the future, such antibodies can find application in the bioengineering of molecular sensors IL-6, which will allow for systemic and local monitoring of the level of IL-6 in living systems.

The method of producing single-domain antibodies to the IL-6 protein includes immunization of a llama (Lama glama) with recombinant human affinity-purified IL-6, cloning the entire repertoire of cDNA sequences of antigen-recognizing domains of the llama antibodies into a phagemid vector, selection of a library of sequences encoding single-domain antibodies that specifically bind to the recombinant protein IL-6 using the phage display method, the production of antibodies in the bacterial expression system and subsequent purification.

The present invention is based not on classical bivalent antibodies, which are considered as a prototype, but on small single-domain antibodies, 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 single domain antibodies (molecular weight of about 12-15 kDa) are an order of magnitude smaller in size than traditional classical antibodies, they acquire a number of new positive qualities that are of practical importance. Single domain antibodies may possess new structural features that, in principle, allow them to recognize some epitopes that are “hidden” to ordinary antibodies.

The method of obtaining single-domain antibodies that bind antigenic epitopes of the human IL-6 protein includes the following principal steps:

1) the induction of the formation of specific antibodies as a result of immunization of the llama with the recombinant protein IL-6;

2) cloning of the sequences of antigen-recognizing domains 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 phage display of clones of nanoantibodies that bind to a given antigen, protein IL-6, from the resulting antibody library;

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).

As an antigen for immunization and selection, the recombinant protein IL-6 obtained in E. coli bacteria was used. Colonies of E. coli strain BL21-DE3-pLysS were transformed with pRSET expression vector containing human IL-6 cDNA. Gene expression and protein isolation were carried out according to the previously described protocol [van Dam et al. Structure-function analysis of interleukin-6 utilizing human / murine chimeric molecules. Involvement of two separate domains in receptor binding. J Biol Chem 1993, 268 (20), 15285-15290. - Structural and functional analysis of interleukin-6 using human / mouse chimeric molecules. The participation of two separate domains in receptor binding]. The purity of the recombinant protein IL-6 was more than 95%. The purity of the IL-6 protein was checked by denaturing PAGE and gel filtration (The described recombinant protein was kindly provided by Prof. Rose-John S., Department of Biochemistry, University of Kiel, Germany).

A method for producing single domain antibodies is described in examples 1 and 2. The possibility of using the obtained antibodies to detect IL-6 protein and their functional characteristics are shown in examples 3-5.

Brief Description of the Drawings

FIG. 1. Purified formatted single-domain antibodies that specifically recognize human IL-6 protein, fractionated in 14% SDS-polyacrylamide gel.

FIG. 2. The results of an enzyme-linked immunosorbent assay for recognition of the recombinant human IL-6 protein immobilized in the wells of an immunological plate, selected by three single-domain formatted antibodies (indicated at the bottom of the figure, numbered 1 to 3). 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 the scatter of the obtained values).

FIG. 3. Measurement of the binding and dissociation constants of single domain antibodies to IL-6 with recombinant human IL-6 protein 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.

FIG. 4. Selected single domain antibodies (VHH 44, VHH 87) do not affect cell proliferation dependent on IL-6. For the third antibody (VHH 49), the data were similar (not shown). The Ba / F3-gp130-hIL-6R cell line was stimulated with recombinant human IL-6 (10 ng / ml) in the presence of various concentrations of anti-IL-6 antibodies. Proliferative activity was evaluated using the MTT test. MTT activity in the presence of IL-6 without inhibitor was taken as 100%. As a positive control, commercial anti-hIL-6 antibodies (MQ2-13A5, eBioscience), which block IL-6 and inhibit cell proliferation, were used.

The implementation of the present invention

Example 1. Obtaining a library of variable domains of single-chain antibodies.

To generate in vivo single domain antibodies that specifically recognize the IL-6 protein, immunization was carried out with Lama (Lama glama).

As an antigen for immunization, a recombinant protein IL-6, specially synthesized for this purpose, was expressed, which was expressed in the prokaryotic system. The preparation of recombinant protein IL-6 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, blood was taken from the llama to obtain preimmune serum, which was subsequently used as a negative control. Five-fold immunization was carried out by subcutaneous injection of a recombinant human protein IL-6, 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. Final blood sampling was performed 5 days after the last immunization. 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, a poly (A) -containing RNA was purified on a column of oligo (dT) cellulose 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 according to the standard protocol [Sambrook et al., 1989] using reverse transcriptase HM-MuLV and oligo 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 NotI sites into the pHEN4 plasmid vector, as previously described [Hamers-Casterman C. et al. Naturally occurring antibodies devoid of light chains, Nature, 1993; 363: 446-448. - Existing naturally occurring antibodies without light chains. Nguyen V.K. et al. Functional heavy-chain antibodies in camelidae. Adv. Immunol., 2001, 79, 261-296. - Functional antibodies, consisting only of heavy chains, in Camelids. Saerens D. et al. Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J. Biol. Chem., 2004, 279, 51965-51972. - Single-domain antibodies derived from a lymph node or camel's peripheral blood lymphocytes recognize conformational variants of a prostate-specific antigen. Rothbauer U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nature Methods., 2006, 3, 887-889. - Targeting and observation of antigens in living cells using fluorescent nanobodies].

Example 2. Selection of single-domain antibodies that specifically recognize IL-6. Single domain antibodies were selected by phage display using a recombinant IL-6 protein immobilized on 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 publications [Tillib C.B. et al. Fingerprint analysis of the selection of “nanoantibodies” by the phage display method using two variants of assistant phages. Acta Natura, 2010, 2 (3), 100-108. Hamers-Casterman C. et al. Naturally occurring antibodies devoid of light chains, Nature, 1993; 363, 446-448. - Existing naturally occurring antibodies without light chains. Nguyen V.K. et al. Functional heavy-chain antibodies in camelidae. Adv. Immunol., 2001, 79, 261-296. - Functional antibodies, consisting only of heavy chains, in Camelids. Saerens D. et al. Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J. Biol. Chem., 2004, 279, 51965-51972. - Single-domain antibodies obtained from the lymph node or lymphocytes of the peripheral blood of a camel recognize conformational variants of a prostate-specific antigen]. Clone sequences of the selected antibodies were grouped by the similarity of their fingerprints obtained by electrophoretic separation of the hydrolysis products of amplified single domain antibody sequences in parallel with three frequently digested restriction endonucleases (Hinfl, Mspl, Rsal). The cDNA sequences of single domain antibodies to IL-6 were determined. The corresponding amino acid sequences of 3 clones of antibodies to IL-6 were deduced from them: SEQ ID NO: 1 (VHH 44); SEQ ID NO: 2 (VHH 49) and SEQ ID NO: 3 (VHH 87).

Nanoantibody Products

The cDNA sequences of the 3 selected nanoantibody clones were cloned into the expression modified plasmid vector pHEN6 [Conrath K.E. et al. Betalactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob Agents Chemother., 2001, 45, 2807-2812. - Beta-lactamase inhibitors isolated from single-domain camelid antibody fragments], allowing 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 antibodies was carried out in E. coli (strain BL21). Expression was induced by adding 1 mM indolyl beta-O-galactopyranoside and the cells were incubated with vigorous stirring for 7 hours at 37 ° C or overnight at 29 ° 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).

In FIG. Figure 1 shows the electrophoregram of a polyacrylamide gel with accumulated and purified single domain antibodies (odAT) that specifically recognize human IL-6.

The structure of three single-domain antibodies to IL-6 is given in the section "List of sequences". 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 recombinant protein IL-6 using selected clones of single domain antibodies

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 IL-6 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, 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. 2 presents the results of an enzyme immunoassay, from which it follows that all the studied single-domain antibody clones specifically bind to the recombinant protein IL-6. 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 IL-6.

Example 4. Measurement of the binding and dissociation constants of single domain antibodies against IL-6 with recombinant protein IL-6 by surface plasmon resonance.

Binding kinetics were measured: association rates, dissociation rates, and dissociation constants of single-domain antibodies with the recombinant protein IL-6 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, the recombinant human IL-6 preparation in acetate buffer (pH = 5.0) was immobilized on a GLM chip (Bio-Rad) (on this chip, a layer of alginic acid containing a polymer of alginic acid was deposited on top of the gold layer in which the effect of surface plasmon resonance occurs) free carboxyl groups) at a concentration of 1 μg / ml. Before immobilization, the surface of the chip was activated with a mixture of 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide at a concentration of 6.66 mM and N-Hydroxysuccinimide at a concentration of 1.33 mM for 30 s at a flow rate of 30 μl / min. The recombinant human IL-6 preparation was applied in a vertical orientation in two stages of 100 s and 300 s at a speed of 30 μl / min. The remaining free carboxyl groups were inactivated by ethanolamine. The total level of immobilization amounted to 800 RU. After washing with phosphate-buffered saline containing 0.005% Tween (100 μl / min, 660 s), the orientation was changed to horizontal. Then, an additional washing with phosphate-buffered saline was carried out (100 μl / min, volume 100 μl, dissociation 600 s), after which a baseline was established. Then, in a horizontal orientation, a preparation of single domain antibodies against IL-6 was also applied in phosphate-buffered saline in twice decreasing concentrations of 200 - 12.5 nM for 245 s at a speed of 100 μl / min. Dissociation was measured for 2000 s. 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 3 shows the interaction curves (sensograms) obtained as a result of 5 interactions with recombinant IL-6 for each of the antibodies.

The data presented demonstrate that the binding and dissociation constants of single domain antibodies VHH 49 and VHH 87 are markedly different from VHH 44, which is possibly due to the recognition by these antibodies of the less available (using the antigen immobilization method) epitope IL-6. However, experiments on the comparative parallel binding of all three antibodies to an immobilized antigen in ELISA indicate a comparable strength of antigen binding for all three antibody variants.

Example 5. Determination of the ability of single-domain antibodies to neutralize the biological activity of IL-6 person.

An analysis of the possible neutralizing activity of the obtained antibodies was determined by blocking cell proliferation on an IL-6-dependent Ba / F3-gp130-hIL-6R cell line in the presence of 10 ng / ml recombinant human IL-6. As a result of the studies, it was found that single-domain antibodies VHH 44, VHH 49, VHH 87 do not block the biological activity of human IL-6 (FIG. 4). This fact testifies to the ability of single-domain antibodies to recognize protein epitopes “hidden” for classical antibodies.

The above examples with figures prove the fulfillment of the task posed by this invention, namely the creation of new single-domain antibodies capable of efficiently binding human IL-6. These antibodies have the following advantages relative to the prototype (monoclonal humanized antibodies against human IL-6): 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 given single-domain antibodies, they are highly soluble, have new structural features that allow them to potentially recognize some of the epitopes that are “hidden” for ordinary antibodies. Since these antibodies do not neutralize the biological activity of human IL-6, the balance associated with the effects of this cytokine will not be distorted when used. Antibodies VHH44, VHH49, VHH87 are antigen-binding modules suitable for the creation of molecular sensors IL-6, which in the future will allow for systemic and local monitoring of the level of IL-6 in vivo.

Thus, the problem posed in this invention is solved.

Sequence listing

Amino acid sequences of three selected single domain antibodies (nanoantibodies) capable of specifically binding to human IL-6 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). The following is an additional sequence (*) containing a linear linker region, as well as the sequences of the HA tag and His tag added (as a result of “formatting”) to the C-terminus of the coding sequence of the originally selected antibody clone for its more efficient purification and detection .

SEQ ID NO: 1

Malqevqlvesggglvqpggslrlscaas gniariavmg wyrqapgkqrelva hifsggntiyvdsvkg

rftisranaqntvylqmnslkpedtavyyc narvvtsgtyny wgqgtqvtvss

SEQ ID NO: 2

Malqevqlvesggglvqaggslrlscaas gsirsiirma wyrqapgkrrelva hiisggstnvadsvkg

rftisrdgakntvhlqmnslkpedtaiyyc nvrdlysttyey wgqgtqvtvss

SEQ ID NO: 3

Malqevqlvesggglvqaggslrlscaas gtirsiirva wyrqapgkqrelva hiisggstgyadsvkg

rftisrdgakntvylqmnslkpedtaiyyc nvrdlystsyey wgrgtqvtvss

* epkipqpqpkpqpqpqpqpkpqpkpepesgrypydvpdyasgrgshhhhhh

Claims (3)

1. Recombinant single-domain antibody specifically binding to human IL-6 and having the amino acid sequence of SEQ ID NO: 1. 2. Recombinant single domain antibody specifically binding to human IL-6 and having the amino acid sequence of SEQ ID NO: 2. 3. Recombinant single domain antibody specifically binding to human IL-6 and having the amino acid sequence of SEQ ID NO: 3.

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