LT5855B - Single stranded recombinant antibody fragment able to neutralize cytolytic activity of vaginolysin - Google Patents

Abstract

Recombinant antibody fragment scFv was obtained from hybridoma 9B4 (DSM ACC2991) by genetic engineering method. It was purified successfully and its specific activity towards VLY cytolysin from G. vaginalis was disclosed, as well as effective neutralization of cytolytic activity. As demonstrated by the invention the recombinant antibody fragment scFv may be used for therapy and prevention of pathology caused by G. vaginalis. Single stranded antibody fragments thus obtained reveals important advantages against traditionally obtained monoclonal antibodies: they may be prepared by simpler and cheaper process of bacterial biosynthesis; due to smaller molecular weight such proteins penetrates easier into tissues affected by infection.

Description

Field of the Invention

The invention relates to the field of protein biotechnology; it describes the preparation of a recombinant single-chain antibody fragment (scFv) which neutralizes the cytolytic activity of vaginolysin.

BACKGROUND OF THE INVENTION

Vaginolysin (VLY) is a major virulence factor in Gardnerella vaginalis bacteria. It belongs to the large pore-forming family of cholesterol-dependent toxin cytolysins (CDCs). Toxins of this family are found in more than 20 bacterial species including Clostridium, Streptococcus, Listeria, Arcanobacterium, Bacillus and others. These toxins (often called cytolysins or hemolysins) cause cell membrane lysis and perform several other functions including cytokine induction and immune system modulation. Thus, it is very likely that these cytolysins act as virulence factors in the infectious processes of the aforementioned bacteria. [Tweten R. K. Infect. Immun. 2005, 73, pp.6199-6209].

G.vaginalis bacteria are known to cause bacterial vaginosis (BV) and are likely to be secreted by the VLY toxin and are responsible for the progression of said disease [Cauci S, Monte M, Ropele C et al., Mol Microbiol, 9, 1993, p. 5358]. BV affects the health of millions of women because it is believed to be a premature causative agent of low birth weight and neonatal mortality and morbidity. In addition, BV was found to be the cause of postoperative endometritis and pelvic inflammation. It is conceivable that neutralizing VLY with antibodies could be a promising method for the treatment and prevention of pathogenic effects caused by G.vaginalis.

Antibodies that neutralize bacterial toxins were described in 1993 when new monoclonal antibodies against tetanus toxin were obtained and their protective effect was analyzed. [Lang et al., J. Immunol. 1993, 151, p. 466-472],

Monoclonal antibodies against botulism toxin were obtained using phage exposure libraries obtained from immunized mice and human [Brekke and Sandlie, Nat.Rev. Drug Discov. 2002, 2, p. 52-62]. Recently, murine monoclonal antibodies to pneumolysin have been obtained and their efficacy tested on animal models. [Garsia-Suarez et al., Infect. Immun. 2004, 72, p. 4534-4540]. The mouse monoclonal antibody PLY5, which neutralized various cytolysins, was identified. This monoclonal antibody has been shown to bind to a specific epitope, undecapeptide common to the entire cholesterol-binding toxin family, and this monoclonal antibody inhibits the binding of toxins to the cell membrane by blocking the undecapeptide epitope [Jacobs et al., FEBS Lett. 1999, 459, p. 463-466],

Advances in genetic engineering and antibody humanization techniques allow the widespread use of antibodies in biopharmaceuticals. Modern recombinant DNA technology allows the construction and expression of modified (altered) antibodies.

These new molecules are designed for a new specific application. Antibodies for biopharmaceutical use can be obtained in different forms - full-length chimeric forms, otherwise known as humanized immunoglobulins, or antibody fragments comprising only the antigen-binding antibody region (scFv). Genes encoding variable (variable) immunoglobulin fragments can be cloned from mouse or human B lymphocytes and expressed in bacteria, yeast, plant and mammalian cells, or in phage display systems. A number of chimeric antibodies are obtained by fusing the mouse V domain to the constant (constant) (C) human immunoglobulin domain. The new antibodies showed the expected binding and effector functions. Humanized antibodies have a reduced host (human) immune response and bifunctional immunoglobulins are used in cancer therapy and diagnostics.

Single-chain antibodies and fusion proteins with antibody specificity (variable regions) linked to non-immunoglobulin sequences are a promising source of modified antibodies with novel properties [Sandhu, Crit Rev Biotechnol.

1 992, Ί 2; pp. 437-462].

Construction of high affinity single-stranded antibody (scFv) neutralizing a Bacillus anthracis toxin has been reported by Maynard et al., Maynard et al., Nature Biotechnol. 2002, 30, p. 597-601]. By injecting these scFv mice, they became protected against the lethal effect of B.anthracis toxin. Neutralizing human antibodies to Shiga toxin 1 were obtained using transgenic mice [Mukherjee et al., Infect. Immun. 2002, 70, p. 5896-5899]. Neutralizing scFvs against scorpion toxin II were obtained and characterized [Mousli et al., FEBS Lett. 1999, 442 pp. 183-188],

Recently, recombinant vaginolysin from G.vaginalis was obtained using genetic engineering techniques [Gelber SE, Aguilar JL, Lewis KLT et al., J Bacteriol, 2008, 190, p. 3896-3903], Hybridomas producing monoclonal antibodies against VLY were obtained by the authors of this application [Sparrow A, Pleckitte M, Lasickien R, Sparrow G, 2010, 56, pp.19-28]. Several monoclonal antibodies produced by hybridoma clones 9B4 and 23A2 have shown neutralization of VLY cytolytic activity in vitro [WO 2010/095917].

Single-chain antibody fragments (scFv) specific for VLY cytolysin and neutralizing its activity have not been constructed and described previously. Although the single-stranded antibody fragments of the present invention are derived from a 9B4 hybridoma clone described previously [WO 2010/095917], the full-length murine monoclonal antibodies described differ from the scFv both structurally and functionally. At present, there is a clear need for effective means for neutralizing cytolytic VLY activity, the discovery of which is an object of the present invention. The problem can be solved by constructing a VLY-binding recombinant scFv that could recognize native vaginolysin from G.vaginalis and counteract the cell lysis it causes. The advantage of single-chain antibody fragments (scFv) over monoclonal antibodies is that scFVs are obtained significantly simpler and cheaper (by bacterial biosynthesis). In addition, the single-stranded antibody fragment has a lower molecular weight, which determines its more efficient delivery to infected tissues and a stronger therapeutic effect. The recombinant scFv described herein can be used as a potential therapeutic agent for the treatment of pathogenic effects caused by G.vaginalis bacteria.

The essence of the invention

It is an object of the present invention to provide a novel single-stranded recombinant antibody fragment scFv which neutralizes cytolytic activity of VLY. The present invention provides recombinant scFv produced using genetic material from hybridoma cell line 9B4 (DSM ACC2991) producing neutralizing monoclonal antibodies to VLY [WO 2010/095917].

The present invention demonstrates that the engineered scFv recognizes VLY and neutralizes the latter's biological activity, namely, inhibits the ability of VLY to induce hemolysis of human erythrocytes.

The present invention provides wherein the recombinant single-chain antibody fragment scFv recognizes the following amino acid sequence (SEQ ID: NO.1):

MNNTKFYRNAAMLLLAGATIIPOCLAAPAMAAPAAKDSEPTASCAAKKDSLNNYL

WDLQYDKTNILARHGETIDNKFSSDSFNKGDEFVWEHQKKNITNTTSNLSVTSA

NDDRVYPGALFRADQNLMDNMPSLISANRAPITLSVDLPGFHGGESAVTVQRPT KSSVTSAVNGLVSKWNAQYAASHHVAARMQYDSASAQSMSQLKAKFGADFAKI GVPLKIDFDAVHKGEKQTQIVNFKQTYYTVSVDAPDSPADFFAPCTTPESLKSRG VDSKRPPVYVSNVAYGRSMYVKFDTRSKSTDFOAAVEAAIKGVEIKPNTEFHRIL QNTSVTAVILGGSANGAAKVITGNVDTLKALIQEGANLSTSSPAVPIAYTTSFVKD

NEVATLQTNSDYVETKVSSYRDGYLTLDHRGAYVARYYIYWDEYGTEIDGTPYV RSRAWEGNGKYRTAHFNTTIQFKGNVHNLRIKLVEKTGLVWEPWRTVYDRSDLP LVRQ RTIKN WGTTLWPRVAETVKN D at least 95% amino acid.

Such an antibody fragment has the amino acid sequence (SEQ ID: No. 2):

DIVMTQTTSHLSVSLGDRVTIACKASAHINNWLAWYQQKPGNAPSLLIAGATGLET

GVPSRFSGSGSGKDFTLSISSLQTEDVATYYCQQYWDTPYTFGGGTKLEIRGGG

GSGGGGSGGGGSGGGGSQVQLEQSGPGLVAPSESLTITCTVSGFSLNSYGVH

WVRQPPGKGLEWVGVMWAGGNTSYNSALMSRLSISKDNSKSQVFLKMNSLQT

DDTAIYYCARARGAMDYWGQGTSVTVSS or a similar sequence with at least 95% homology.

The antibody fragment of the present invention is for use in inhibiting or neutralizing the biological activity of vaginolysin.

Another object of the invention is a preparation of a recombinant single-chain antibody fragment for use in inhibiting or neutralizing the biological activity of vaginolysin.

This product is for use in the suppression of infection such as bacterial vaginosis caused by Gardnerella vaginalis bacteria. That is, a possible field of application of the present invention is the use of scFv preparations for the treatment of bacterial vaginosis caused by G. vaginalis. Preparations having such activity are suitable for the production of biopharmaceutical recombinant antibodies and for potential therapeutic use.

Another object of the invention is pharmaceutical compositions comprising an effective amount of a formulation of the present invention in combination with pharmaceutically acceptable carriers, diluents, excipients and / or excipients for the prophylaxis and / or treatment of effects caused by G. vaginalis infection. possible applications include pathologies leading to preterm birth that lead to low birth weight, pelvic inflammatory diseases, endometritis and other diseases caused by G. vaginalis infections.

Brief description of the drawings:

The preparation of a recombinant antibody fragment (scFv) specific for cytolysin VLY is illustrated in Figs. 1-5:

Figure 1 illustrates a schematic representation of the construct VL- (G4S) ₄ -VH comprising an antibody fragment (scFv) specific for VLY cytolysin. Linker O’jungle) is (G ₄ S) ₄ .

FIG. 2 shows an electrophoresis image of a recombinant antibody fragment (scFv) in lysate of transformed E.coli cells. NDS-PAGE shows that the scFv protein was detected in an insoluble fraction of transformed E.coli cells.

lane - soluble fraction of E.coli cells;

lane - insoluble fraction of E.coli cells;

Lane 3 - total lysate of E.coli cells;

4- stained protein molecular weight standard (kDa).

FIG. 3 shows an electrophoresis image of a purified recombinant antibody fragment (scFv) in polyacrylamide.

lane 4 pg purified scFv protein.

Lanes 2, 3 - 10 pg of purified scFv protein from two different purifications.

M-molecular weight markers (15, 20, 25, 30, 40, 50 kDa from the bottom).

FIG. 4 shows the interaction of the recombinant antibody fragment scFv with cytolysin VLY by ELISA. Reactivity of recombinant antibody fragment scFv with cytolysin VLY by ELISA (squares); positive control reactivity of full-length monoclonal antibody 9B4 with cytolysin VLY (triangles).

FIG. 5 shows the hemolysis neutralization activity of the recombinant antibody fragment scFv erythrocytes in the presence of cytolysin VLY.

The scFv neutralizing activity was evaluated in a human erythrocyte hemolysis assay. The full-length 9B4 monoclonal antibody is a positive control. OD values are shown on the Y axis. On the X-axis, reactions are numbered:

1) 5 ng / ml VLY and 20 ng / ml 9B4 antibody in PBS;

2) 5 ng / ml VLY and 200 ng / ml 9B4 antibody in PBS;

3) 5 ng / ml VLY and 1000 ng / ml 9B4 antibody in PBS;

4) 5 ng / ml VLY and 500 ng / ml scFv in PBS;

5) 5 ng / ml VLY and 1000 ng / ml scFv in PBS;

6) 5 ng / ml VLY and 2000 ng / ml scFv in PBS;

7) 5 ng / ml VLY and 5000 ng / ml scFv in PBS;

8) 5 ng / ml VLY and 10,000 ng / ml scFv in PBS.

Detailed Description of the Invention

Construction of the recombinant antibody fragment scFv was initiated by isolating the genetic material (RNA) from hybridoma 9B4 (DSM ACC2991). The following procedures were used:

1) Isolation of RNA from Hybridoma 9B4 - Total RNA was isolated from Hybridoma 9B4 (DSM ACC 2991) cells using a single-step technique as described previously [Chomczynski and Sacchi, Anai Biochem, 1987, 162, p. 156159], Cell homogenate is prepared using guanidine thiocyanate and RNA is extracted with phenol-chloroform at acidic pH. The RNA is further precipitated with isopropanol.

2) First cDNA strand synthesis - The first cDNA strand is synthesized using RNA-spliced DNA polymerase (reverse transcriptase), together with a poly (A) ⁺ RNA template and primer oligo (dT) nucleotides.

3) The final cDNA was amplified using PCR and specific primers for immunoglobulin variable (variable) domain sequences.

Mouse immunoglobulin heavy chain constant region primer: 5'-TTAATAGACAGATGGGGGTGTCGTTTTGGC and mouse immunoglobulin heavy chain FR1 region heavily degenerate primer: 5'CATATGSARGTNMAGCTGSAGSAGTC (R - denotes A / G; . Mouse kappa chain immunoglobulin constant region primer: 5'TTAGGATACAGTTGGTGCAGCATC and mouse kappa chain immunoglobulin constant region FR1 universal degenerate primer: 5'15 CATATGGAYATTGTGMTSACMCARVVCTMCA (R - denotes A / G; S - Y / G; S - C; / T).

4) The PCR products were cloned into a vector plasmid and the nucleotide sequence was determined by sequencing. The sequences were analyzed using the BLASTN program and the GenBank and IgBLAST databases.

5) A scFv fragment was constructed by introducing a linker (linker) sequence (G4S) 4 between the domain VL (variable region L variable region of murine immunoglobulins) and the domain VH (variable region H region variable region of murine immunoglobulin). The final construct encoding VL- (G4S) 4-VH was cloned into the expression vector pET28a (+) [Merck, Darmstadt, Germany].

The scFv construct comprises an (His) ₆ anchor sequence at the N-terminus of the protein. The E. coli strain BL21 (DE3) was transformed with the final plasmid. Recombinant scFv biosynthesis was induced with 0.5 mM IPTG.

6) The amino acid sequence of scFv (SEQ ID: NO.2) is as follows:

DIVMTQTTSHLSVSLGDRVTIACKASAHINNWLAWYQQKPGNAPSLLIAGAT GLETGVPSRFSGSGSGKDFTLSISSLQTEDVATYYCQQYWDTPYTFGGGT KLEIRGGGGSGGGGSGGGGSGGGGSQVQLEQSGPGLVAPSESLTITCTVS GFSLNSYGVHWVRQPPGKGLEVWGVMWAGGNTSYNSALMSRLSISKDNS KSQVFLKMNSLQTDDTAIYYCARARGAMDYWGQGTSVTVSS

7) Following induction, the inclusion bodies with scFv protein were solubilized using chaotropic agents. Oxidative renaturation to form disulfide bonds was accomplished either by dilution or by column using a pair of reduced and oxidized glutathione or Cu (II) divalent copper as the oxidant. The renatured scFv protein was purified using metal chelate affinity chromatography. Some properties of the scFv protein are described below in the corresponding examples.

In the present invention, it was found that the recombinant antibody fragment derived from hybridoma 9B4 recognizes the amino acid sequence of SEQ ID NO: 1:

MNNTKFYRNAAMLLLAGATIIPOCLAAPAMAAPAAKDSEPTASCAAKKDSLNNYL

WDLQYDKTNILARHGETIDNKFSSDSFNKGDEFVWEHQKKNITNTTSNLSVTSA

NDDRVYPGALFRADQNLMDNMPSLISANRAPITLSVDLPGFHGGESAVTVQRPT

KSSVTSAVNGLVSKWNAQYAASHHVAARMQYDSASAQSMSQLKAKFGADFAKI

GVPLKIDFDAVHKGEKQTQIVNFKQTYYTVSVDAPDSPADFFAPCTTPESLKSRG VDSKRPPVYVSNVAYGRSMYVKFDTRSKSTDFQAAVEAAIKGVEIKPNTEFHRIL QNTSVTAVILGGSANGAAKVITGNVDTLKALIQEGANLSTSSPAVPIAYTTSFVKD NEVATLQTNSDYVETKVSSYRDGYLTLDHRGAYVARYYIYWDEYGTEIDGTPYV RSRAWEGNGKYRTAHFNTTIQFKGNVHNLRIKLVEKTGLVWEPWRTVYDRSDLP

LVRQRTIKNWGTTLWPRVAETVKND.

This amino acid sequence is fully consistent with the VLY cytolysin sequence from Gardnerella vaginalis, previously identified and published (GenBank No. EU697812, EU 697811). Different strains of Gardnerella vaginalis may express slightly different VLY variants, which may differ from one or more amino acids of SEQ ID: No.1.

Examples of implementation of the invention

Specific examples illustrating the manner in which the recombinant antibody fragment scFv against VLY cytolysin was obtained as well as examples for determining the properties of scFv are provided below. This information is provided for illustrative purposes only and is not intended to limit the scope of the invention.

example purification of mRNA from hybridoma 9B4 cells and cloning of DNA sequence encoding VLY-specific antibody fragment

Hybridoma 9B4 (DSM ACC 2991) cells (3x10 ⁶ cells) were washed with sterile cold PBS buffer and disrupted with D containing guanidine thiocyanate. RNA was purified from the homogenate using a phenol-chloroform mixture at pH

5. After adding each reagent, mix thoroughly by inverting the tube several times. The solution was held for 15 min. on ice. The tube was centrifuged and the lower aqueous phase containing RNA was collected. An equal volume of isopropanol was added, the solution well mixed, and the RNA was settled for 1 h. At -20 ° C. RNA was collected by centrifugation, washed with 75% ethanol, dissolved in water treated with DEPC, and stored at -70 ° C.

Total RNA purified from hybridoma 9B4 cells was used for first-strand cDNA synthesis. Traces of DNA in the RNA sample were removed by addition of enzyme

DNaseI (without RNase). Prepared DNA-free RNA (0.5 pg) was mixed with oligo (dT) ₁₈ primer and incubated with enzyme RNA-dependent DNA polymerase (reverse transcriptase). The reaction was quenched by heating the reaction mixture at 70 ° C for 5 min. The cDNA encoding the variable region of the monoclonal antibody from hybridoma 9B4 was obtained using the following specific primer pairs: 1) mouse heavy chain variable region primer: 5'TTAATAGACAGATGGGGGTGTCGTTTTGGC and mouse heavy chain FR1 domain AGGGAGGGGAGGGGAGGGGGAGGG G; S - C / G; N - A, C, G, T); 2) mouse kappa chain constant region primer: 5'TTAGGATACAGTTGGTGCAGCATC and mouse kappa chain FR1 domain universal abnormal primer 5'-CATATGGAYATTGTGMTSACMCARV \ / CTMCA (R - A / G; S - C / G; W - A / T; M - A / C; Y - C / T). Each PCR reaction mix contains: cDNA (first strand), 5 'and 3' primers, dNTP, Taq polymerase. The resulting PCR product was purified from an agarose gel and cloned into the pJET1.2 cloning vector (Fermentas, Vilnius, Lithuania). The ligated DNA was transformed

E. coli DH10B cells and stained on LB agar with 100 pg / ml ampicillin. Colonies were grown on agar plates, purified from plasmids, and checked by plasmid for the required DNA insert. The nucleotide sequence of the inclusions was determined by sequencing. For each IgG strand, a sequence of nucleotide inserts was identified from five clones and the sequences were found to be identical. The linker sequence encoding (G ₄ S) 4 and restriction endonuclease targets were inserted by PCR. The DNA fragments encoding VL and VH were fused to give the genetic construct encoding the amino acid sequence VL- (G4S) 4-VH, schematically shown in Figure 1.

example

Construction of expression plasmids and expression of recombinant scFv in E.coli cells.

The DNA fragment encoding VL- (G ₄ S) 4-VH was cloned into the expression vector pET28 (+). The resulting plasmid pET28 containing scFv was transformed with E. coli BL21 (DE3) cells and the transfected bacteria were deleted on LB agar plates with 25 pg / ml kanamycin. Cells transformed with expression plasmid were grown in 500 ml of LB medium with kanamycin. When the cell culture reached an OD _{6 of} 0.8-1.0, expression of the target gene was initiated by the addition of up to 0.5 mM IPTG (isopropyl β-D-thiogalactoside). Cells were cultured for 2.5 h. At 37 ° C and collected by centrifugation. Part of the cells were suspended in Tris-EDTA (TE) buffer and sonicated. The disrupted cells were centrifuged and the supernatant (soluble cell fraction) and sediment (insoluble cell fraction) analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (NDS-PAGE). The gel is painted with Coomassie Blue paint. The 27.5 kDa target protein scFv was detected in the insoluble E.coli cell fraction (Fig. (Fig.2). 2). The target protein accounted for about 35% of total cellular protein and about 80% of insoluble cellular protein. The obtained results show that scFv produced in E.coli cells is incorporated into the inclusion bodies.

example

Purification of recombinant scFv

The E. coli biomass with the target protein scFV was homogenized in 100 ml (ratio

1:10, biomass: buffer) 0.1 M Tris-HCl, pH 7.0 in 5 mM EDTA 0.1 g lysozyme, 0.1 ml Triton Χ-100, 1.0 ml 100 mM phenylmethylsulfofluoride (PMSF ) and 0.7 mL of 2-mercaptoethanol. The homogenate was stirred for 30 min. at room temperature, cells were sonicated and centrifuged for 25 min at 24,500 g. The residue was washed twice with 100 mL of 1 M NaCl, 0.1% Tween 80, and 100 mL of distilled water. After each wash cycle, the suspension was centrifuged for 25 min at 24.500 g.

Oxidative Renaturation: The precipitate obtained after the last wash and containing the scFv protein is dissolved in 10 mM Tris-HCl, pH 7.0 buffer containing 7M guanidine hydrochloride. The suspension was stirred overnight at 4 ° C, centrifuged for 25 min. 40,000g and the supernatant was diluted in 10mM Tris-HCl buffer (pH 7.0) containing 6M GuHCl to a total protein concentration of 1mg / ml, followed by the addition of CUSO4 solution to a concentration of 20µ.. Renovation is carried out for about 1 hour at room temperature. The reaction was stopped by adding EDTA solution to a final concentration of 10 mM. The protein solution was centrifuged for 25 min. 40,000 loaded onto a Sephadex G-25 column nupusiausvyrintos 25 mM Tris buffer, pH 8.0 containing 0.25M Na2S _4th Fractions containing the target protein were collected, pooled and sorbed onto a Ni (II) NTA sorbent loaded column. Sorption buffer was used in 25 mM TRIS, pH 8.0, and desorption was performed with the same buffer with 1M imidazole. Fractions containing the target protein were pooled, dialyzed against 20 mM TRIS, 50 mM NaCl, pH 8.0 and filtered with a 0.22 pm filter for further storage. NDS-PAGE electrophoresis of purified recombinant scFv is shown in Fig. 3.

Stable protein preparation obtained by transferring the purified antibody fragment scFv to a storage solution (formulation) containing a selected buffer system, salts, detergents, antioxidants and other auxiliaries necessary for protein stabilization and isotonicity.

The calculated molecular weight of the protein scFv is 25.182 kDa, the theoretical isoelectric point pi is 6.99. The molecular weight experimentally determined (by NDS-PAGE) is about 25.5 kDa. The fluorescence method used to determine the melting (denaturation) temperature of the scFv protein is about 55 ° C using a citrate buffer system. In addition, protein identity was confirmed by immunoblotting (WB) and immunoenzyme analysis (ELISA) using antigen interaction (VLY).

example

Determination of specificity of recombinant scFv

The specificity of recombinant scFv derived from hybridoma 9B4 was determined using an immunoenzyme assay (ELISA). Recombinant purified scFv was diluted and plated in a 96-well plate coated with recombinant VLY protein. After a while. at room temperature incubation, VLY-bound scFv was detected by incubation with mouse monoclonal antibody against (His) s sequence and horseradish peroxidase (HRP) -labeled secondary antibody (IgG conjugate against mouse immunoglobulins). The enzymatic reaction was eluted using the prepared TMB substrate. The optical density was measured at 405 nm. Diluted full-length monoclonal antibody 9B4, incubated in a 96-well plate coated with recombinant VLY protein, was used as a positive control. Plates were expressed by HRP-IgG conjugate against mouse immunoglobulins and TMB substrate. FIG. illustrates the titration curves for recombinant scFv and full length antibody 9B4 in microtiter plates coated with recombinant VLY. This experimental result demonstrates the specificity of recombinant scFv for VLY vaginolysin.

example scFv neutralizing activity assay

Erythrocyte hemolysis in vitro [Cauci S, Monte R, Ropele M et al., Mol Microbiol, 1,993, 9, p. 1143-1155.] Was used to assay the activity of recombinant scFv neutralizing VLY. Human erythrocyte suspension (1 ml) was incubated for 15 min. At 22 ° C, the following reaction mixtures:

1) Phosphate Buffer in PBS pH 7.4 (no red cell hemolysis);

2) Deionized water (100% erythrocyte hemolysis);

3) PBS with 5 ng / ml VLY (100% erythrocyte hemolysis);

4) 5 ng / ml VLY and 20 ng / ml 9B4 antibody in PBS;

5) 5 ng / ml VLY and 200 ng / ml 9B4 antibody in PBS;

6) 5 ng / ml VLY and 1000 ng / ml 9B4 antibody in PBS;

7) 5 ng / ml VLY and 500 ng / ml scFv in PBS;

8) 5 ng / ml VLY and 1000 ng / ml scFv in PBS;

9) 5 ng / ml VLY and 2000 ng / ml scFv in PBS;

10) 5 ng / ml VLY and 5000 ng / ml scFv in PBS;

11) 5 ng / ml VLY and 10,000 ng / ml scFv in PBS.

After incubation, the erythrocyte suspension was centrifuged for 3 min at 1000 x g and the solution was measured at 415 nm for OD. High optical density values (OD> 2.0) indicate erythrocyte hemolysis. Low OD values (OD <0.2) indicate that erythrocytes were protected from destruction. Addition of 5 ng / ml recombinant VLY to the erythrocyte suspension resulted in complete destruction of the erythrocytes (OD = 2.5). When 20 ng / ml of 9B4 antibody or 5000 ng / ml of recombinant scFv was added to the mixture, erythrocytes were completely protected from hemolysis (Fig. (Fig.5). 5). The results obtained indicate that both full-length 9B4 antibody and recombinant scFv neutralized the cytolytic activity of VLY. The 9B4 antibody exhibited higher neutralizing activity than scFv because it required a lower concentration to neutralize VLY.

The pharmaceutical composition with the scFv protein of the present invention may comprise one or more of the following components:

a carrier which may be polyhydric alcohols (eg mannitol, sorbitol), various monosaccharides (eg glucose), disaccharides (eg sucrose);

- buffer substance to maintain the required pH value (eg acetate, phosphate, TRIS, HEPES);

- salt additives to maintain the isotonicity of the composition (e.g., sodium chloride);

- a detergent which protects against protein degradation at the liquid-air separation (eg polysorbate 80, polysorbate 20, various compounds of the Pluronic type);

- stabilizing agents such as polyethylene glycols, polyvinylpyrrolidones, amino acids (e.g. L-methionine, L-arginine, L-histindine, L-glutathionic acid, Lasparaginic acid);

- a chelating agent such as EDTA, EGTA, IDA;

- antimicrobial agent (eg benzene derivatives such as cresol, benzyl alcohol);

- SH agent (eg glutathione, cysteine, acetylcysteine)

- other suitable excipients.

In conclusion, the recombinant antibody fragment scFv, derived from hybridoma 9B4, exhibits specificity for VLY cytolysin from G.vaginalis and effectively neutralizes the latter's cytolytic activity. Therefore, this recombinant antibody fragment scFv can be used for therapeutic and prophylactic purposes in the treatment or prevention of pathologies caused by G.vaginalis infection. The resulting single-chain antibody fragments (scFv) revealed advantages over monoclonal antibodies in the production of scFv by simpler and less expensive bacterial biosynthesis. In addition, the low molecular weight of scFv potentially leads to a stronger therapeutic effect due to more efficient delivery of scFv to infected tissues.

Moreover, the resulting DNA sequences encoding scFv can be used to construct novel humanized antibodies that will neutralize the biological activity of VLY cytolysin. Because VLY is a major virulence factor in G.vaginalis, its neutralization may be an effective pathway to treat pathology caused by infection.

Claims (7)

DEFINITION OF INVENTION 1. Recombinant single-chain antibody fragment recognizing the amino acid sequenceSEO ID: NO.1: 5 MNNTKFYRNAAMLLLAGATIIPOCLAAPAMAAPAAKDSEPTASCAAKKDSLNNYL WDLQYDKTNILARHGETIDNKFSSDSFNKGDEFVWEHQKKNITNTTSNLSVTSA NDDRVYPGALFRADONLMDNMPSLISANRAPITLSVDLPGFHGGESAVTVORPT KSSVTSAVNGLVSKWNAQYAASHHVAARMQYDSASAQSMSQLKAKFGADFAKI GVPLKIDFDAVHKGEKQTQIVNFKQTYYTVSVDAPDSPADFFAPCTTPESLKSRG 10 VDSKRPPVYVSNVAYGRSMYVKFDTRSKSTDFOAAVEAAIKGVEIKPNTEFHRIL ONTSVTAVILGGSANGAAKVITGNVDTLKALIOEGANLSTSSPAVPIAYTTSFVKD NEVATLQTNSDYVETKVSSYRDGYLTLDHRGAYVARYYIYWDEYGTEIDGTPYV RSRAWEGNGKYRTAHFNTTIQFKGNVHNLRIKLVEKTGLVWEPWRTVYDRSDLP LVRQRTIKNWGTTLWPRVAETVKND 15 or a similar sequence with at least 95% identity. The antibody fragment of claim 1, having the amino acid sequence SEQ ID: No. 2: DIVMTQTTSHLSVSLGDRVTIACKASAHINNWLAWYQQKPGNAPSLLIAGATGLET GVPSRFSGSGSGKDFTLSISSLQTEDVATYYCQQYWDTPYTFGGGTKLEIRGGG 20 GSGGGGSGGGGSGGGGSQVQLEQSGPGLVAPSESLTITCTVSGFSLNSYGVH WVRQPPGKGLEWVGVMWAGGNTSYNSALMSRLSISKDNSKSQVFLKMNSLQT DDTAIYYCARARGAMDVWGQ is similar to or equal to. 25th The antibody fragment of any one of the preceding claims for use in inhibiting or neutralizing the biological activity of vaginolysin. A preparation comprising a recombinant single-chain antibody fragment according to any one of claims 1-2 for use in inhibiting the biological activity of vaginolysin or 30 for neutralization. The preparation of claim 4 for use in inhibiting an infection such as bacterial vaginosis caused by Gardnerella vaginalis bacteria. 35 A pharmaceutical composition comprising an effective amount of a preparation according to claim 4 in combination with a pharmaceutically acceptable carrier, diluent, excipient and / or excipient. A pharmaceutical composition according to claim 6 for use in a condition caused by 40 Prevention and / or treatment of Gardnerella vaginalis infection.