RU2395521C1 - Vegf binding v9 nanoantibody and method of obtaining said antibody, v9-coding nucleotide sequence and vector containing said sequence, endothelial cell proliferation inhibition method - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention describes a nucleotide sequence which codes the V9 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V9 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V9 nanoantibody, as well as use of the V9 nanoantibody for qualitative and quantitative determination of VEGF in a sample. Use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

EFFECT: invention discloses a monomer single-strand V9 nanoantibody which can bind and inhibit the human vascular endothelial growth factor.

7 cl, 7 dwg, 7 ex

Description

The technical field of the present invention

The present invention relates mainly to the field of molecular medicine and is associated with monomeric single chain nanoantibodies. In particular, the present invention relates to a new nanoantibody capable of inhibiting the action of vascular endothelial growth factor (isoform A-165) of a person, and to the use of this nanoantibody for diagnostic and therapeutic purposes.

BACKGROUND OF THE INVENTION

Vascular endothelial growth factor (VEGF) is an important signaling protein involved in vasculogenesis (the formation of a de novo circulatory system of the embryo) and angiogenesis (growth of blood vessels from preexisting vessels). Based on the name of this protein, VEGF activity is best studied on vascular endothelial cells, although it has an effect on some other cells. For example, VEGF stimulates the migration of monocytes / macrophages, neurons, cancer cells, kidney epithelial cells. In vitro experiments have shown that VEGF stimulates mitogenesis of endothelial cells and cell migration. VEGF is also a vasodilator and increases capillary permeability, for which it was originally called the vascular permeability factor.

The VEGF family includes a prototype member of VEGF-A, placental growth factor (PIGF), VEGF-B, VEGF-C and VEGF-D. Convincing evidence suggests that while the construction and maturation of the vascular wall are very complex processes that require the combined effects of angiopoietins, platelet-derived growth factor B (PDGF-B) and other factors, the action of VEGF-A is a rate-limiting stage of normal and pathological growth of blood vessels vessels. It is important that VEGF-C and VEGF-D regulate angiogenesis of the lymphatic vessels, which emphasizes the unique role of this family of genes in controlling the growth and differentiation of some anatomical components of the vascular system.

VEGF is characterized by significant homology with chains A and B of PDGF. The gene encoding human VEGF-A consists of eight exons separated by seven introns. Alternative exon splicing leads to the formation of four main isoforms - VEGF121, VEGF165, VEGF189 and VEGF206, consisting after cleavage of the signal sequence of 121, 165, 189 and 206 amino acids, respectively. Alternative splicing regulates the bioavailability of VEGF. To date, a wealth of evidence has accumulated that VEGF165 is the most physiologically competent isoform. Extracellular proteolysis also plays an important role in the regulation of the bioavailability of VEGF. Plasmin is able to cleave VEGF165 and VEGF189 and release a biologically active product consisting of the first 110 amino-terminal amino acids.

The main biological effects of VEGF in vitro and in vivo are mediated by its interaction with specific receptors. Currently, two main types of receptor tyrosine kinases acting as a receptor have been described: VEGFR1 (aka FLT-1) and VEGFR2 (aka FLK-1 or KDR). In addition, it is believed that neuropilin can perform the functions of VEGFR3. Currently, VEGFR2 plays a central role in ensuring the biological functions of VEGF. Signaling through VEGFR2 triggers proliferation, migration, increases endothelial cell survival, stimulates angiogenesis and vascular permeability [Ferrara N., Hillan K.J., Gerber H.P., Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat. Rev. Drug Discov. 2004; 3: 391-400].

In view of the fundamental role of growth and proliferation of endothelial cells in various human pathologies and the critical role of VEGF in this process, it is desirable to develop a method for controlling the activity of VEGF. This strategy is indeed practically useful and is currently implemented in several different ways based on blocking the mitogenic VEGF signal in endothelial cells. These methods [Ellis L.M. & Hicklin D.J. VEGF-targeted therapy: mechanisms of anti-tumor activity. Nature Reviews Cancer. 2008; 8: 579-591] provide for the use of:

- antibodies that neutralize the action of VEGF or its receptor by binding them. In particular, (1) the use of humanized anti-VEGF monoclonal antibodies [international patent publication WO 98/45331] or their Fab'fragments (bevacizumab (Avastin) and ranizumab (Lucentis) preparations), the binding of which to VEGF blocks the ability of VEGF initiate signal transmission through an appropriate receptor on the surface of endothelial cells, or (2) the use of intrinsic antibodies to VEGF, induced by immunization with an antigen containing VEGF (kinoid vaccine), the principle of which is similar to the principle of action of humanizir monoclonal antibodies [Rad FH, Le Buanec H., Paturance S., Larcier P., Genne P., Ryffel B., Bensussan A., Bizzini B., Gallo RC, Zagury D., Uzan G. VEGF kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases. Proc. Natl. Acad. Sci. USA 2007; 104: 2837-42];

tyrosine protein kinase inhibitors with selectivity for VEGF receptors (e.g., sorafenib and sunitinib). Their action blocks the action of VEGF by inhibiting receptor activation and subsequent intracellular signal transduction;

- soluble forms of VEGF receptors or their hybrids. The action of such agents (e.g., VEGF-trap) is based on the binding of VEGF, which leads to the depletion of free VEGF, which can interact with receptors on the cells.

As the closest analogue of the technical solution that forms the basis of the present invention, humanized antibodies against VEGF bevacizumab and lucentis can be cited.

However, the present invention is based not on classical bivalent antibodies, such as bevacizumab, but on small single-chain nanoantibodies, which have several advantages over classical antibodies for practical use in the field of diagnosis and treatment of diseases. Since nanoantibodies (molecular weight of about 17 kDa) are an order of magnitude smaller in size of traditional antibodies, they acquire a number of new positive qualities of practical importance. The polypeptide chain in the composition of nanoantibodies forms only one domain that does not contain hydrophobic sites. This avoids problems with the solubility and proper folding of nanoantibody 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. Due to the smaller size of the polypeptide chain, nanoantibodies are more resistant to temperature and pH, stored at room temperature, while traditional multi-domain antibodies require special storage and transportation conditions at 4 ° C. Due to their smaller size, nanoantibodies are characterized by better ability to penetrate tissues. Finally, 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 nanoantibodies, include another protein with the desired properties.

The possibility of obtaining recombinant nanoantibodies with a given specificity is determined by the existence of functional and possessing a fairly wide recognition spectrum of noncanonical antibodies in representatives of the Camelidae family. Noncanonical antibodies consist of a dimer of only one shortened immunoglobulin heavy chain (without light chains), the specificity of recognition of which is determined by only one variable domain [Hamers-Casterman C., Atarhouch T., Muyldermans S., Robinson G., Hamers C., Bajyana Songa E., Bendahman N., Hamers R. Naturally occurring antibodies devoid of light chains. Nature. 1993; 363: 446-448]. The technical implementation of the selection of nanoantibodies, which are genetically engineered derivatives of antigen-recognizing domains of single-chain camel antibodies, is based on a highly efficient selection procedure for antigen-recognizing polypeptides exposed on the surface of a filamentous phage particle (“phage display”).

The phage display method is a very effective and widely used technology for the selection from large recombinant libraries of peptides and proteins expressed in the surface protein of filamentous phages [Brissette R. & Goldstein N.I. The use of phage display peptide libraries for basic and translational research. Methods Mol. Biol. 2007; 383: 203-13; Sidhu S.S. & Koide S. Phage display for engineering and analyzing protein interaction interfaces. Curr. Opin. Struct. Biol. 2007; 17: 481-7]. One of the most important applications of this technology is the generation of specific recombinant antibodies for a wide variety of antigens [Hoogenboom H.R. Selecting and screening recombinant antibody libraries. Nat. Biotechnol. 2005; 23: 1105-16].

Usually, instead of large whole molecules of classical antibodies, hybrid recombinant single-stranded proteins are used to display on the phage surface, which are random combinations of cloned sequences of the variable regions of the heavy and light chains of immunoglobulins connected by a short linker-rich serine and glycine linker. Such a chimeric molecule, in the case of the correct combination of domains, is able to maintain the specificity of the initial immunoglobulin, despite the changes introduced in comparison with the native antibody molecule. One of the problems of traditional recombinant technologies is the need to work with very large libraries of recombinant antibodies, in which all possible combinations of two random variable regions (heavy and light chains of immunoglobulins) connected by a linker sequence should be presented. In addition to the problem of representation, the problem of forming the correct relative conformation of these two domains is also obvious here, as well as the solubility of individual variable domains, which often tend to aggregate. The mentioned problems can be avoided when using nanoantibodies, since practically every cloned variable domain of single-chain antibodies in this case will have a certain antigen-knowing specificity corresponding to one of the antibodies of the immunized animal, and selection from relatively small libraries of such domains can be effectively carried out.

Nanoantibodies with a given specificity or their derivatives can be used, like classical antibodies, in various applications, including, without limitation, the detection of antigens (both for research and diagnostic purposes), blocking antigen protein activity, specific delivery due to binding to the antigen of the desired molecules conjugated to the antibody.

Brief Description of the Drawings

Figure 1. Amplification products of cDNA fragments encoding the variable domains of antibodies (shown by arrows), separated in an agarose gel containing ethidium bromide (lane P). On the right (lane M), molecular weight markers are shown with their lengths in pairs of nucleotides (bp).

Figure 2. A typical cleavage pattern of the plasmid pHEN4 containing the sequence encoding the nanoantibody V9, restriction endonuclease HinfI (lane V). On the right (lane M), molecular weight markers are given indicating their lengths in pairs of nucleotides (bp).

Figure 3. Schematic representation of the recombinant V9 nanoantibody encoded by a plasmid based on a modified pHEN6 vector. The leader sequence of periplasmic localization (peIB), nanoantibody (V9), and HA and (His) 6 epitopes are indicated.

Figure 4. Purification of Nanoantibodies V9. Polyacrylamide gel gel electrophoresis with addition of SDS of the periplasmic extract of E. coli cells producing V9 nanoantibodies with HA and (His) 6 epitopes (1), proteins of the extract (2) not bound to Ni-NTA agarose, proteins, 1) the dissociated with Ni-NTA agarose during washing (3) and the purified V9 nanoantibody eluted from Ni-NTA agarose in an amount of 1 μg (4). The position of the V9 nanoantibody in the gel is shown by the arrow. On the right are 1 μg of the commercial lysozyme preparation (5) and protein standards for molecular weights (M). The gel is stained with Coomassie Brilliant Blue.

Figure 5. V9 nanoantibody specifically binds VEGF in an enzyme immunoassay. Wells are shown after addition of a chromogenic substrate. 1 - a well without immobilized proteins (only blocking BSA solution), 2 - a well with immobilized VEGF, 3 - a well with immobilized immunoglobulins.

6. Evidence of a strong relationship between luminescence intensity and HUVEC cell count.

7. V9 nanoantibody blocks VEGF-dependent proliferation of HUVEC endothelial cells. HUVEC cells were scattered into the wells of a 96-well plate (2000 cells per well) in basal medium for growth of EBM-2 endothelial cells (Lonza, USA) containing 2% fetal bovine serum. After cell attachment (after 4 hours), VEGF was added to the indicated concentrations (A) or to a concentration of 100 ng / ml (B) and nanoantibodies to a concentration of 12.5 μg / ml or Avastin (Ava) to a concentration of 6 μg / ml. Cell proliferation was determined after 96 hours using a CeliTiter Glo Luminescent Cell Viability Assay kit (Promega, USA). Data are given in relative luminescence units as the average of three independent wells ± standard deviation.

Disclosure of the present invention

The present invention relates to a recombinant V9 nanoantibody that is capable of specifically binding to VEGF and neutralizing its mitogenic activity. The term "VEGF" refers to the 165-amino acid growth factor of human vascular endothelium A. The term "nanoantibody" means that the protein of the invention is a recombinant variable domain of single-chain camel antibodies.

To obtain a library of the variable domains of single-chain antibodies of a two-humped camel, VEGF or a mixture of proteins containing VEGF was immunized.

After immunization, B lymphocytes were isolated from the peripheral blood of the immunized camel and used as an RNA source for cloning the entire repertoire of the variable domains of camel specific immunoglobulins consisting of only one heavy chain dimer.

Isolated RNA was used as a template in reverse transcription polymerase chain reaction (PCR) with primer pairs corresponding to conserved RNA sequences of immunoglobulin heavy chains between the signal peptide and the constant CH2 domain region [Hamers-Casterman et al., 1993; Nguyen V.K., Desmyter A., Muyldermans S. Functional heavy-chain antibodies in Camelidae. Adv. Immunol. 2001; 79: 261-96; Saerens D., Kinne J., Bosnians E., Wernery U., Muyldermans S., Conrath K. Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen. J. Biol. Chem. 2004; 279: 51965-72; Rothbauer U., Zolghadr K., Tillib S., Nowak D., Schermelleh L, Gahl A., Backmann N., Conrath K., Muyldermans S., Cardoso MC, Leonhardt H. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 2006; 3: 887-9].

The obtained amplification products were separated on an agarose gel and 600-800 bp PCR products corresponding to non-canonical antibodies were isolated from the gel [Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Edition 1989, Cold Spring Harbor: CSHL Press]. The isolated amplification products were used as a template in PCR with primer pairs corresponding to conserved sequences at the beginning and at the end of this intrinsically variable (antigen-recognizing) domain [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006}. The primers used in this reaction also contained additional sequences corresponding to recognition sites for restriction endonucleases, respectively, Ncol and Notl.

The amplification products obtained were cloned at the Ncol and Notl sites into the phagemid vector pHEN4 [Ghahroudi M.A., Desmyter A., Wyns L., Hamers R., Muyldermans S. Selection and identification of single-domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 1997; 414: 521-526], also encoding ampicillin resistance protein, for phage display [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006].

The selection of nanoantibodies that specifically recognize VEGF was carried out by phage display using immobilized recombinant VEGF (Peprotech), as described [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006]. The selection and subsequent amplification of the selected phage particles (containing the nanoantibody gene inside, and the expressed nanoantibody as a part of the surface phage protein pIII) was repeated, as a rule, three times in succession. Clones of nanoantibodies resulting from this procedure were examined for their diversity and required specificity.

The sequences of the clones of the selected nanoantibodies contained in the plasmid pHEN4 were grouped according to the similarity of the patterns of their electrophoretically separated products of the hydrolysis of often sprinkling restriction endonucleases. 2-3 representatives of each group were used for cloning into another expression plasmid vector, ensuring the attachment of the (His) 6-epitope to the C-terminus.

Thus, the present invention also relates to an expression vector containing a nucleotide sequence encoding a V9 nanoantibody and providing its expression with additional epitope (s) at the C-terminus for detection and isolation and a signal peptide at the N-terminus.

Due to the presence of the signal peptide sequence (pelB) at the N-terminus of the expressed sequence, the produced recombinant protein (nanoantibody) accumulates in the periplasm of E. coli, which makes it possible to efficiently isolate it by the osmotic shock method without destroying the actual bacterial cells. Recombinant nanoantibodies were isolated from the periplasmic extract using affinity chromatography on a column of Ni-NTA agarose.

Thus, the present invention also relates to a method for producing V9 nanoantibody, comprising the following steps:

a) transfection of the producer cell with an expression vector containing the nucleotide sequence encoding the V9 nanoantibody, and ensuring its expression with additional epitope (s) at the C-terminus for detection and isolation and a signal peptide at the N-terminus;

b) expression of V9 nanoantibody in the producer cell;

c) isolation of V9 nanoantibody from the producer cell.

Isolated nanoantibodies were used to analyze their ability to recognize VEGF, preferably by enzyme-linked immunosorbent assay with immobilized recombinant VEGF. VEGF-bound nanoantibodies were detected using mouse anti-HA antibodies, secondary antibodies to mouse immunoglobulins conjugated to horseradish peroxidase, and the ABTS chromogenic substrate (2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid, Sigma) .

In another preferred embodiment, the V9 nanoantibody is neutralizing. The term "neutralizing" means that the nanoantibody is able to specifically bind to VEGF and significantly inhibit or cancel the mitogenic activity of VEGF on endothelial cells. The neutralizing nanoantibody of this invention is particularly useful for the prophylactic or therapeutic treatment of unwanted proliferation of endothelial cells and neoangiogenesis by delivering the nanoantibody to endothelial cells whose proliferation is desired to be inhibited. The pharmacologically acceptable form, delivery method and dose of V9 nanoantibody administered for prophylactic or therapeutic purposes will be determined by a combination of factors, including the type of disease and its severity, tolerance of the administered nanoantibody and the required amount of nanoantibody to neutralize VEGF with the chosen route of administration. Confirmation of the existence of this possibility is the effectiveness of the drug Avastin, which is a neutralizing VEGF classic antibody.

The plasmid encoding the V9 nanoantibody was isolated from E. coli cells and the nucleotide sequence (SEQ ID NO: 1) encoding the nanoantibody was determined by sequencing.

Based on the nucleotide sequence encoding the V9 nanoantibody, the amino acid sequence of the V9 nanoantibody was determined (SEQ ID NO: 2).

Thus, the present invention also relates to a method for inhibiting endothelial cell proliferation, the method comprising delivering V9 nanoantibodies to endothelial cells whose proliferative activity is desired to be inhibited. Such inhibition may be necessary for the prophylactic or therapeutic treatment of diseases in which excessive proliferation of the endothelium is observed.

For diagnostic applications, which are another object of the present invention, the V9 nanoantibody containing the HA epitope for detection can be detected directly with anti-HA antibodies if the antibodies are conjugated to horseradish peroxidase or other component that allows detection, including other enzymes ( for example, phosphatase) and fluorophores, or anti-HA antibodies can be detected using suitable secondary antibodies for detection. V9 nanoantibodies can be used in any known detection method, including direct and indirect ELISA, competitive binding, immunofluorescence detection, immunoprecipitation, Western blotting.

Examples

Example 1

Obtaining a library of variable domains of single-chain antibodies

Immunization

Bactrian camel Camelus bactrianus was subsequently immunized 5 times. A concentrated conditioned medium of CHO line cells transiently producing VEGF was used as antigen. For this, cells were transfected using the Unifectin-56 reagent (Transfection Group, Russia) with a plasmid containing VEGF cDNA under the control of the CMV promoter. 24 hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and incubated in serum-free medium for 3 days to obtain a conditioned medium containing VEGF. The conditioned medium was collected, filtered through a 0.2 μm PES filter (Corning-Costar, USA) and concentrated using an Immersible CX-10 submersible cartridge (Millipore, USA) with a nominal pore size of 10 kDa. The amount of VEGF in concentrated conditioned medium was determined by protein staining in SDS-polyacrylamide gel with Coomassie dye. For each injection, a conditioned medium containing approximately 300 μg of antigen in a final volume of 7.5 ml adjusted with PBS was taken. The first injection was performed with antigen mixed with Freund's complete adjuvant in a 1: 1 ratio. Then the antigen was mixed with incomplete Freund's adjuvant (1: 1) and another 4 injections were performed sequentially, respectively after 1 month and three times after 2 weeks. Blood sampling (150 ml) was performed 5 days after the last injection. To prevent coagulation of the taken blood, heparin 35 units / ml and EDTA (2 mM) were added.

Isolation of B-lymphocytes

Blood was diluted 2 times with standard saline (PBS) containing 1 mm EDTA. 35 ml of diluted blood solution was layered on a 15 ml step of a special medium (Histopaque-1077, Sigma) with a density of 1.077 g / ml and centrifuged for 800 minutes 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.

Isolation of RNA from B-lymphocytes

Total RNA from B lymphocytes was isolated using TRIzol reagent (Invitrogen). Then, poly (A) -containing RNA was isolated 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.

Reverse transcription reaction: cDNA synthesis on a poly (A) + RNA template isolated from B-lymphocytes

The reverse transcription reaction was carried out in 40 μl according to the standard protocol [Sambrook et al., 1989] using reverse transcriptase H-M-MuLV, 1 μg RNA and 1 μg oligo primer (dT) 15 as a seed.

Amplification of cDNA fragments encoding antibody variable domains

Reverse transcription products (1 μl) were used as a template in a 50 μl polymerase chain reaction containing two primers CALL001 (5'-gtcctggctgctcttctacaagg-3 ') and CALL002 (5'-ggtacgtgctgttgaactgttcc-3') in the following conditions : 95 ° C, 90 s, (95 ° C - 30 s, 59 ° C - 120 s, 72 ° C - 90 s) × 30 cycles, 72 ° C, 300 s. The amplification products were separated on an agarose gel containing ethidium bromide (Fig. 1). Amplification products 600-800 bp in size corresponding to noncanonical antibodies were isolated from the gel using a QIAEX II kit (QIAGEN, USA) and were used as matrices in a similar amplification reaction with primers 5'-ccagccggccatggctgatgtgcagctggtggagtctgg-3 'and 5'- ggactagtgcggccgcttgaggagacggtgacctgggt-3 'containing additional sequences corresponding to recognition sites of restriction endonucleases, respectively, Ncol and Notl.

Creating a library of variable domains of single-chain antibodies

The obtained amplification products were cloned at the Ncol and Notl sites into the pHEN4 phagemid vector and, using the bacteriophage M13KO7 (New England Biolabs, USA) as a phage helper, a phage library was obtained with surface expression of the variable domains of single chain antibodies as described by [Hamers-Casterman et al al ., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006].

Example 2

Selection of nanoantibodies that specifically recognize VEGF

Nanoantibodies were selected using phage display using recombinant VEGF (Peprotech) immobilized at the bottom of the wells of a 96-well ELISA plate. Used polystyrene immunological tablets 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 (containing the nanoantibody gene inside, and the expressed nanoantibody as a part of the surface phage protein pill) were repeated, as a rule, three times in succession. All manipulations were performed as described [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006].

Example 3

V9 Nanoantibody Identification

The sequences of clones of the selected nanoantibodies contained in the plasmid pHEN4 were grouped according to the similarity of the patterns of their electrophoretically separated products of the hydrolysis of restriction endonuclease Hinfl. One of the clone groups was characterized by the restriction pattern shown in FIG. 2. The cDNA sequences of nanoantibodies were determined for three representatives of this group and were found identical and encoding the same nanoantibody, named V9 (SEQ ID NO: 1 and 2).

Example 4

Nanoantibody Products V9

V9 nanoantibody cDNA was cloned into an expression plasmid vector — a modified pHEN6 vector [Conrath KE, Lauwereys M., Galleni M., Matagne A., Frère 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], which ensures the attachment of the polyhistidine (His) 6-epitope to the C-terminus of the nanoantibody (immediately after the HA epitope encoded in the pHEN6 vector). Due to the presence of a signal peptide sequence (pelB) at the N-terminus of the expressed sequence, the produced recombinant protein (nanoantibody) accumulates in the periplasm of bacteria, which allows it to be efficiently isolated by the osmotic shock method without destroying the bacterial cells proper (Fig. 3). The production of 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. V9 nanoantibodies were isolated from the periplasmic extract using affinity chromatography on Ni-NTA agarose using the QIAExpressionist purification system (QIAGEN, USA). The cleaning results of the nanoantibody V9 are shown in Fig.4.

Example 5

Nanoantibody V9 Binds to VEGF

The ability of V9 nanoantibodies to bind VEGF was tested by enzyme-linked immunosorbent assay with immobilized recombinant VEGF (Peprotech) according to a standard protocol. Wells without immobilized VEGF and with immobilized immunoglobulins (non-specific protein) were used as a control. VEGF-bound nanoantibodies were detected using mouse anti-HA antibodies, secondary antibodies to mouse immunoglobulins conjugated to horseradish peroxidase, and the ABTS chromogenic substrate (2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid, Sigma) .

Figure 5 presents the results of the analysis, from which it follows that the V9 nanoantibody specifically binds VEGF, but not the immunoglobulins or BSA used as a blocking agent.

Example 6

V9 Nanoantibody Inhibits VEGF-Induced VEGFR2 Receptor Phosphorylation

The action of VEGF is realized through its binding to the main receptor, VEGFR2, which leads to its autophosphorylation and activation [Meyer M., Clauss M., Lepple-Wienhues A., Waltenberger J., Augustin HG, Ziche M., Lanz C., Büttner M., Rziha HJ, Dehio C. A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases. EMBO J. 1999; 18: 363-74]. VEGFR2 receptor activation is necessary for VEGF-stimulated proliferation, chemotaxis and endothelial cell survival in vitro and in vivo angiogenesis [Karkkainen M.J. & Petrova T.V. Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis. Oncogene. 2000; 19: 5598-605; Rahimi N., Dayanir V., Lashkari K. Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. J. Biol. Chem. 2000; 275: 16986-92; Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors. Biochem. Soc. Trans. 2003; 31: 20-4]. Therefore, autophosphorylation of VEGFR2 in response to VEGF treatment can be considered as a measure of the pro-angiogenic effect of VEGF, and the blocking of VEGF-dependent autophosphorylation by V9 nanoantibody as evidence of its neutralizing VEGF activity. Human VEGFR2 was transiently expressed in HEK293 cells by transfection with an appropriate expression vector containing full-length human VEGFR2 cDNA under the control of the CMV promoter. 18 hours after transfection, the cells were washed with PBS and incubated in serum-free medium for 24 hours. The cells were then treated with 30 ng / ml VEGF in serum-free medium, 30 ng per point) or VEGF at the same concentration pre-incubated for 60 min with 10 μg of V9 nanoantibody. After 5 min, the cells were lysed and the degree of VEGFR2 activation was evaluated by the amount of tyrosine-951 VEGFR2 phosphorylated by Western blotting with antibodies specifically recognizing tyrosine-951 VEGFR2 phosphorylated (Cell Signaling, USA). Figure 6 shows that the pre-incubation of VEGF with V9 nanoantibody leads to significant suppression of VEGFR2 phosphorylation by tyrosine-951, which indicates the ability of V9 nanoantibodies not only to bind, but also inhibit the action of VEGF.

Example 7

Nanoantibody V9 blocks VEGF-dependent proliferation of endothelial cells

HUVEC endothelial cells were seeded into the wells of a 96-well plate (2000 cells per well) in basal medium for the growth of EBM-2 endothelial cells (Lonza). After cell attachment (after 4 hours), VEGF was added to a concentration of 1, 1.5, 3 or 100 ng / ml and V9 nanoantibody to a concentration of 12.5 μg / ml or, as a control, bevacizumab (Avastin) to a concentration of 6 μg / ml The number of cells was determined after 96 hours. To determine the number of HUVEC cells, the CellTiter Glo® Luminescent Cell Viability Assay kit manufactured by Promega (Madison, Wl, USA) was used in accordance with the kit manufacturer's protocol (Promega Technical Bulletin Part # TB288). The luminescent signal generated by the reaction is directly proportional to the number of cells in the well (Promega Technical Bulletin Part # TB288). In addition, we studied the linearity of the relationship between the number of HUVEC cells in the well and the generated luminescent signal for a range of HUVEC cells over the number of cells in the well at the time of analysis. As can be seen in Fig.6, in the range from 250 to 5000 cells in the well, the luminescence signal is linearly (r ² = 0.9935) associated with the number of cells in the well. It can be seen (Fig. 7A) that, at VEGF concentrations of 1-3 ng / ml, the V9 nanoantibody, like Avastin, used as a control, is able to completely inhibit HUVEC cell proliferation. Moreover, the effect of V9 nanoantibody on the proliferation of HUVEC cells is not due to their nonspecific toxicity or toxicity of impurities contained in the nanoantibody preparation, since under similar conditions with an excess of VEGF (100 ng / ml) in the presence of nanoantibodies, there was no change in the proliferative activity of HUVEC cells (Fig. 7B).

Claims (7)

1. Nanoantibody V9, characterized by the amino acid sequence of SEQ ID NO: 2 and able to bind human vascular endothelial growth factor and block its effect. 2. The nucleotide sequence encoding the nanoantibody V9 according to claim 1. 3. The nucleotide sequence according to claim 2, which is SEQ ID NO: 1. 4. The expression vector containing the nucleotide sequence according to claim 2 and providing for the expression of V9 nanoantibody with additional epitope (s) at the C-terminus for detection and isolation and a signal peptide at the N-terminus. 5. The method of producing nanoantibody V9 according to claim 1, comprising the following steps:
a) transfection of the producer cell with the vector according to claim 4;
b) expression of V9 nanoantibody in the producer cell;
c) isolation of V9 nanoantibody from the producer cell. 6. A method of inhibiting the proliferation of endothelial cells,
providing for the delivery of nanoantibodies V9 according to claim 1 to endothelial cells. 7. The use of nanoantibodies V9 according to claim 1 as a diagnostic reagent for the qualitative and quantitative determination of VEGF in a sample.

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