LT6389B - Dimeric antibody fragment neutralizing cytolytic activity of vaginolysin - Google Patents

Abstract

New dimeric antibody fragments against vaginolysin were constructed by genetic methods and purified. They effectively neutralize the cytolytic effect of vaginolysin on human cells and can be used for prophylaxis and/or treatment of bacterial vaginosis caused by Gardnerella vaginalis.

Description

Field of the Invention

The present invention relates to the field of biotechnology and biopharmaceutical production and characterizes the production and properties of a novel recombinant covalently linked dimeric (tandem) antibody fragment (di-scFv) which neutralizes the cytotoxic activity of vaginolysin.

BACKGROUND OF THE INVENTION

Advances in genetic engineering and antibody humanization techniques allow the widespread use of antibodies in biopharmaceuticals. Modern recombinant DNA technology makes it possible to construct and isolate purposefully modified (modified) antibodies suitable for neutralizing bacterial toxins. Antibodies for biopharmaceutical use can take different forms - full-length chimeric forms, otherwise known as humanized immunoglobulins, or antibody fragments comprising only the region of the antibody that binds the antigen (Fv). Genes encoding variable (variable) fragments of immunoglobulins, or various combinations thereof, can be cloned from mouse or human B lymphocytes and expressed in bacteria, yeast, plant and mammalian cells. A number of chimeric antibodies have been obtained by fusing the murine V domain to the 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. To increase the stability and affinity of scFv, the Fv fragments of the antibody are fused to non-covalently linked homodimers, homotrimers, or homotetramers (diabodies, triabodies, tetrabodies) by genetic engineering. In order to obtain the optimal structure, it is very important to select the appropriate polypeptide linker (linker) between the variable domains of the antibody [Todorovska A. et al., Journal of Immunological Methods, vol. 248 (2001), 47-66]. The dimeric structure (both homodimeric and heterodimeric) of linear, covalently linked scFv (by attaching the N-terminus of the second (or more) monomers to the C-terminus of the first monomer via a selected linker) is significantly more stable than the non-covalent .Diabody) with higher affinity (avidity) than the monomeric variant of scFv. Recombinant antibodies (or fragments thereof) may also have additional functions. Recombinant bispecific antibody prototypes were developed more than two decades ago. Such antibodies recognize two different antigens. , which enables them to be more widely and efficiently applied in diagnostics or pharmacy [Kontermann, R. and Brinkmann, U., Drug Discovery Today, Vol. 20, No. 7, 2015, 838847],

Single-chain antibodies, or various combinations thereof, having antibody specificity sequences (variable regions) and linked to sequences of a different nature are promising sources of modified antibodies with novel properties [Sandhu, Crit Rev Biotechnol. 1992; 12; pp. 437-462].

Toxin (cytolysin) vaginolysin (VLY) is a major virulence factor in Gardnerella vaginalis. 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 Streptococcus, Clostridium, Listeria, Arcanobacterium, Bacillus and others. Such toxins (often called cytolysins or hemolysins) induce cell membrane lysis and perform several other functions, including cytokine induction and immune system modulation. These cytolysins are very likely to act as virulence factors in the infectious processes caused by the aforementioned bacteria. [Tweten R. K. Infect. Immun. 2005, 73, pp.6199-6209].

G.vaginalis bacteria are known to cause bacterial vaginosis (BV) and their secreted VLY toxin has been confirmed to be responsible for the progression of the disease. [Cauci S, Monte M, Ropele C et al., Mol Microbiol, 9, 1993, p. 53-58]. BV affects the health of millions of women by being thought 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 of pathogenic effects caused by G.vaginalis, which has been confirmed in subsequent publications.

Construction of high affinity single-stranded antibody (scFv) neutralizing Bacillus anthracis toxin has been described 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].

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 the VLY toxin have been obtained [Žvirbliene A. et al., Toxicon, 56, pp. 19-28, 2010]. Several monoclonal antibodies produced by hybridoma clones 9B4 and 23A2 have demonstrated neutralization of cytolytic activity of VLY in vitro [WO 2010/095917 A1].

The monomeric form of single-chain antibody fragments, which is specific for VLY cytolysin and neutralizes its activity, has been constructed and described previously [Pleckaitte M. et al. BMC Biotechnology 2011, 11: 100]. Full-length mouse monoclonal antibodies and monomeric scFvs described previously differ from dimeric covalently linked scFvs, both structurally and functionally, although in both cases the single-stranded antibody fragments are derived from the same 9B4 hybridoma clone described previously [WO 2010/095917 A1j.

At present, there is a great need for alternative means of neutralizing the cytotoxic activity of VLY, which have sufficient efficacy and are simple and economical to obtain. The problem can be solved by constructing a VLY-binding recombinant dimeric scFv that could recognize native vaginolysin from G.vaginalis and effectively neutralize the cell lysis it causes.

The essence of the invention

It is an object of the present invention to provide a novel dimeric scFv fragment of a single-stranded recombinant antibody capable of neutralizing cytolytic activity of VLY (vaginolysin).

The present invention provides the structure of a recombinant dimeric scFv obtained by using two covalently linked monomeric scFv molecules linking the C-terminus of one scFv molecule and the N-terminus of another molecule through a flexible linker (linker) sequence.

The main object of the invention is a dimeric covalently linked antibody fragment recognizing the amino acid sequence of SEQ ID NO. 1

MNNTKFYRNAAMLLLAGATIIPQCLAAPAMAAPAAKDSEPTASCAAKKDSL

NNYLWDLQYDKTNILARHGETIDNKFSSDSFNKGDEFVWEHQKKNITNTTSNLSVT

SANDDRVYPGALFRADQNLMDNMPSLISANRAPITLSVDLPGFHGGESAVTVQRPT

KSSVTSAVNGLVSKWNAQYAASHHVAARMQYDSASAQSMSQLKAKFGADFAKIGV

PLKIDFDAVHKGEKQTQIVNFKQTYYTVSVDAPDSPADFFAPCTTPESLKSRGVDSK

RPPVYVSNVAYGRSMYVKFDTRSKSTDFQAAVEAAIKGVEIKPNTEFHRILQNTSVT

AVILGGSANGAAKVITGNVDTLKALIQEGANLSTSSPAVPIAYTTSFVKDNEVATLQT

NSDYVETKVSSYRDGYLTLDHRGAYVARYYIYWDEYGTEIDGTPYVRSRAWEGNG

KYRTAHFNTTIQFKGNVHNLRIKLVEKTGLVWEPWRTVYDRSDLPLVRQRTIKNWG

TTLVVPRVAETVKND or similar amino acid sequences having at least 95% identity.

The structure of this dimeric antibody fragment is represented by the formula (VL-L4-VH) -SL- (VL-L4-VH) or (VL-L4-VH) -SL- (VH-L4-VL), wherein VL is a variable immunoglobulin light chain. L fragment, VH is a variable H chain fragment of immunoglobulins, L4 and SL are linker sequences.

In one embodiment of the invention the L4 linker sequence is (G4S) 4 and the SL linker sequence is SGLEA (EAAAK) ₄ ALEA (EAAAK) ₄ ALEGS; and the dimeric antibody fragment has the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO: 3 or similar sequences having at least 95% identity.

The dimeric antibody fragment of the present invention is characterized by inhibition or neutralization of vaginolysin biological activity.

The objects of the present invention are also a recombinant DNA molecule encoding said dimeric antibody fragment and a vector comprising such a DNA molecule.

In a particular embodiment of the invention, said recombinant DNA molecule comprises the DNA sequence of SEQ ID NO. 4 or SEQ ID NO: 5

An important object of the present invention is a preparation comprising said dimeric antibody fragment which is characterized by inhibition or neutralization of vaginolysin biological activity.

Another object of the invention is a pharmaceutical composition comprising the active component and a pharmaceutically acceptable carrier, diluent, excipient and / or excipient, the active component comprising a therapeutically effective amount of said preparation.

The pharmaceutical composition is for use in the prophylaxis and / or treatment of conditions caused by Gardnerella vaginalis infection. Optimally, the pharmaceutical composition is in the form of a gel, ovule or detergent.

The invention is described below with details and accompanying drawings and examples.

Brief description of the pictures:

The generation and properties of dimeric antibody fragments (di-scFv) specific for cytolysin VLY are illustrated in Figures 1-8.

Fig. provides a schematic diagram of the construction of di-scFv genes in (A) and (B), which illustrates the process of constructing recombinant DNA encoding fragments of the di-scFv antibody. (A) 1- and 2-synthesized VL-L4-VH-SL and VH-L4-VL-SL genes were obtained in pUC57 and pET28a, respectively. Labeled primers are used for scFv gene amplification and selection of positive clones. Targeted DNA hydrolysis targets for restriction endonucleases used for cloning and sequence analysis. (B) l and II - Two genes constructed: (VL-L4-VH) -SL- (VH-L4-VH) and (VL-L4-VH) -SL- (VL-L4-VH), derived from pUC57 plasmid and recloning into pET28a expression plasmid. The labeled primers were used for scFv gene amplification and selection of positive clones. Restriction endonuclease targets were used for cloning and sequence analysis.

Fig. shows the result of analysis of PCR products of di-scFv genes on agarose gel. Colony PCR is performed on randomly selected colonies with construct I VL-L4-VH-SL-VL-L4-VH (clones 1-10) and construct II VL-L4-VH-SL-VH-L4-VL (clones 11-19) ). M - MassRuler DNA Ladder Mix (ThermoFisher).

Fig. shows the result of restriction endonuclease analysis of pUC57-di-scFv plasmids. Analysis of pUC57-di-scFv plasmids by BamHI / HindIII restriction endonucleases.

Construction of Ia from clones 1, 3, 4 and 5 (VL-L4-VH-SL-VL-L4-VH) and Il-a from clones 11, 12, 13 and 14 (VL-L4-VH-SL- VH-L4-VL); M - MassRuler DNA Ladder Mix (ThermoFisher).

Fig. demonstrates results of restriction endonuclease plasmid analysis of selected clones of pET-28a-di-scFv. Clone plasmids tested: 11-3.11.11-5.13-8 and

14-12 VL-L4-VH-SL-VL-L4-VH, and 1111-19, 1111-20, 1112-22 and 1113-29 VL-L4-VH-SLVH-L4-VL

Gel samples: 1 - non-hydrolyzed DNA; 2 - BamHI hydrolyzed; 3 hydrolysed with Ndel / Hindlll; M - MassRuler DNA Ladder Mix (ThemoFisher).

Fig. the sequences of the analyzed genes are presented.

Fig. reports the results of an electrophoretic analysis of di-scFv expression in E. coli BL21 (DE3) sodium dodecyl sulfate polyacrylamide gel (NDS-PAAG). S protein standard. 0 h = total protein sample before induction; 1h, 2h, and 3h protein samples at 1, 2, and 3 hours after induction. T - soluble and N - insoluble protein fractions after 3 h induction.

Fig. The results of the purification of the di-scFv proteins by NDS-PAAG analysis are presented.

Lanes 1, 2, a variant of the dimeric anti-VLY di-scFv protein histag- (VL-L4-VH) -SL- (VLL4-VH), respectively, before and after dialysis. Lanes 3, 4, a variant of the dimeric anti-VLY di-scFv protein histag- (VL-L4-VH) -SL- (VH-L4-VL), respectively, before and after dialysis. Lanes 5, 6, a variant of the monomeric anti-VLY scFv protein histag-VH-L4-VL, respectively, before and after dialysis. Lane 7 - Control protein sample from biomass without target protein that has undergone all renaturation and purification steps.

M - Molecular weight markers (10, 15, 25, 35, 55, 70, 100, 130, and 250 kDa from the bottom).

Fig. demonstrating the cytolysin neutralizing activity of the di-scFv protein VLY as assessed by hemolysis assay. The neutralizing activity of scFv dimers in vaginolysin was evaluated in a human erythrocyte hemolysis assay. A positive control is the activity of the scFv monomer. The Y-axis shows the relative value (%) of erythrocyte lysis. The X-axis shows the concentration (ng / ml) of scFv protein. During the experiments, the same concentration of vaginolysin (VLY) was used in all samples - 3 ng / ml.

Detailed Description of the Invention

Construction of the recombinant dimeric scFv fragment was initiated by introducing the synthetic monomeric scFv gene variants VL-L4-VH and VH-L4-VL with the prepared linkers and cloning positions (Fig. 1A) by introducing the linker (linker L4) sequence encoding 20 amino acids. acids (G ₄ S) ₄ between the VL domain (variable region L of the murine immunoglobulin light chain) and the VH domain (variable region H of the murine immunoglobulin heavy chain) and the linker (linker SL) 54 amino acids SGLEA (EAAAK) 4 ALEA (EAAAK) ) 4 ALEGS between two scFv monomeric fragments (Figure 1 B). From the synthesized genes VL-L4-VH-SL and VHL4-VL-SL (GeneScript), polymerase chain reaction (PCR) was performed by introducing BamHI and HindIII restriction endonuclease recognition sequences at the 3 'and 5' ends, respectively. The PCR fragments are purified on GeneJet (ThermoFisher) DNA purification columns according to the protocol for DNA purification from a PCR mixture. The concentration of the purified DNA is evaluated by analyzing the electrophorogram and comparing the intensity of the target DNA strands with the DNA standard gel loaded. The final constructs (VL-L4VH) -SL- (VH-L4-VL) and (VL-L4-VH) -SL- (VL-L4-VH) were cloned into the expression vector pET28a (+) [Merck, Darmstadt, Germany ]. Constructs with di-scFv also include the (His) ₆ anchor sequence at the N-terminus of the protein. The final plasmids were transformed into E. coli BL21 (DE3) strain.

The following genetic engineering procedures were used:

1) The genes VL-L4-VH-SL and VH-L4-VL-SL (GeneScript) were synthesized.

2) The polymerase chain reaction (PCR) was performed by constructing the di-scFv genes with the following structure: (VL-L4-VH) -SL- (VH-L4-VH) and (VL-L4-VH) -SL- (VL-L4) -VH) and introducing BamHI and HindIII restriction endonuclease recognition sequences at the 3'- and 5'-ends, respectively. The following primers were used for construction and analysis:

M13-rev: 5'-CAGGAAACAGCTATGAC-3 ';

T7 forw: 5'-TAATACGACTCACTATAGGG-3 ';

T7 rev: 5'-GCTAGTTATTGCTCAGCGG-3 ';

VHVL-fw: 5'-AATGGATCCCAGGTTCAGCTGGAGCAG-3 'underlined BamHI recognition sequence, the highlighted sequence being complementary to the 3' end of the VH fragment;

VHVL-rev: 5'-ATAAAGCTTATTACCGTATTTCCAGCTTGGTCC-3 ^, underlined HindIII recognition sequence, the highlighted sequence is complementary to the 5 'end of the VL fragment. This primer also introduces a terminating codon at the 3 'end of the TAA gene.

VLmega-fw: 5'-ACCCATATGGATATTGTGATGACACAGTCTACATCCC3 ';

VLVH_fw: 5'-AATGGATCCGATATTGTGATGACACAGTCTACATCCC-3 ':

underlined BamHI recognition sequence, bold sequence complementary to the 3 'end of the VL fragment.

VLVH-rev: 5'-ATCAAGCTTATTAGGAGGAGACGGTGACTGAGG-3 'Underlined HindIII recognition sequence, the highlighted sequence being complementary to the 5' end of the VH fragment. This primer also introduces a terminating codon at the 3 'end of the TAA gene.

3) The PCR products were cloned into the expression plasmid and the nucleotide sequence of the fragments was determined by DNA sequencing. The amino acid sequences encoded by the di-scFv genes in SEQ ID NO: 2 and SEQ ID NO: 3 are as follows:

(VL-L4-VH) -SL- (VL-L4-VH), SEQ ID NO: 2:

MGSSHHHHHHSSGLVPRGSHMDIVMTQSTSHLSVSLGDRVTIACKASAHIN

NWLAWYQQKPGNAPSLLIAGATGLETGVPSRFSGSGSGKDFTLSISSLQTEDVATY

YCQQYWDTPYTFGGGTKLEIRGGGGSGGGGSGGGGSGGGGSQVQLEQSGPGLV

APSESLTITCTVSGFSLNSYGVHWVRQPPGKGLEWVGVMWAGGNTSYNSALMSR

LSISKDNSKSQVFLKMNSLQTDDTAIYYCARARGAMDYWGQGTSVTVSSSGLEAEA

AAAAAAAAAAAAAAAAKALEAAAAAAAAAAAAAAAAKALEGSDIVMTQSTSH

LSVSLGDRVTIACKASAHINNWLAWYQQKPGNAPSLLIAGATGLETGVPSRFSGSG

SGKDFTLSISSLQTEDVATYYCQQYWDTPYTFGGGTKLEIRGGGGSGGGGSGGGG

SGGGGSQVQLEQSGPGLVAPSESLTITCTVSGFSLNSYGVHWVRQPPGKGLEWV

GVMWAGGNTSYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCARARGAMD

YWGQGTSVTVSS (VL-L4-VH) -SL- (VH-L4-VL), SEQ ID NO: 3:

MGSSHHHHHHSSGLVPRGSHMDIVMTQSTSHLSVSLGDRVTIACKASAHIN

NWLAWYQQKPGNAPSLLIAGATGLETGVPSRFSGSGSGKDFTLSISSLQTEDVATY

YCQQYWDTPYTFGGGTKLEIRGGGGSGGGGSGGGGSGGGGSQVQLEQSGPGLV

APSESLTITCTVSGFSLNSYGVHWVRQPPGKGLEWVGVMWAGGNTSYNSALMSR

LSISKDNSKSQVFLKMNSLQTDDTAIYYCARARGAMDYWGQGTSVTVSSSGLEAEA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAALEGSQVQLEQSGP

GLVAPSESLTITCTVSGFSLNSYGVHVWRQPPGKGLEWVGVMWAGGNTSYNSAL

MSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCARARGAMDYWGQGTSVTVSSGGG

GSGGGGSGGGGSGGGGSDIVMTQSTSHLSVSLGDRVTIACKASAHINNWLAWYQ

QKPGNAPSLLIAGATGLETGVPSRFSGSGSGKDFTLSISSLQTEDVATYYCQQYWD

TPYTFGGGTKLEIR

4) The final plasmids were transformed into E. coli BL21 (DE3) strain. Selected and analyzed clones were cultured in LB medium and induced using IPTG. Analysis of the samples by electrophoresis in a polyacrylamide gel under denaturing conditions revealed the accumulation of the di-scFv protein in the inclusion bodies after induction, which migrated as an insoluble protein of ~ 58 kDa.

5) After induction, the resulting inclusion bodies with di-scFv protein were solubilized using chaotropic agents. The renatured di-scFv protein was purified using metal chelate affinity chromatography.

6) Validated anti-cytolytic activity of the di-scFv protein upon exposure of human erythrocytes to vaginolysin (hemolysis assay).

In the present invention, the recombinant antibody di-scFv fragment was found to recognize the following amino acid sequence, SEQ ID NO: 1:

MNNTKFYRNAAMLLLAGATIIPOCLAAPAMAAPAAKDSEPTASCAAKKDSL

NNYLWDLQYDKTNILARHGETIDNKFSSDSFNKGDEFVWEHQKKNITNTTSNLSVT

SANDDRVYPGALFRADQNLMDNMPSLISANRAPITLSVDLPGFHGGESAVTVQRPT

KSSVTSAVNGLVSKWNAQYAASHHVAARMQYDSASAQSMSQLKAKFGADFAKIGV

PLKIDFDAVHKGEKQTQIVNFKQTYYTVSVDAPDSPADFFAPCTTPESLKSRGVDSK

RPPVYVSNVAYGRSMYVKFDTRSKSTDFQAAVEAAIKGVEIKPNTEFHRILQNTSVT

AVILGGSANGAAKVITGNVDTLKALIQEGANLSTSSPAVPIAYTTSFVKDNEVATLQT

NSDYVETKVSSYRDGYLTLDHRGAYVARYYIYVVDEYGTEIDGTPYVRSRAVVEGNG

KYRTAHFNTTIQFKGNVHNLRIKLVEKTGLVWEPWRTVYDRSDLPLVRQRTIKNWG

TTLVVPRVAETVKND

The indicated amino acid sequence corresponds to the sequence of VLY cytolysin obtained from Gardnerella vaginalis, which was previously identified and published (GenBank No. EU697812, EU 697811). Different strains of Gardnerella vaginalis may express slightly different variants of VLY that differ from SEQ ID NO: 1 by one or more amino acids.

Examples of the invention

Specific examples showing how the recombinant antibody fragment di-scFv against VLY cytolysin was obtained, as well as examples for determining the properties of di-scFv are given below. This information is provided for illustrative purposes only and is not intended to limit the scope of the present invention.

Example Preparation of DNA constructs encoding di-scFv antibody fragments

The DNA constructs and the final expression plasmids are prepared according to the construction scheme shown in Figures 1A and 1B, which show the sequence of the fragment fusion, the vectors used and the enzymes used.

The sequence of the synthesized DNA fragments VH-L4-VL-SL and VL-L4-VH-SL (GeneScript) was modified by PCR by introducing BamHI and HindIII restriction endonuclease recognition sequences into the 3'- and 5'-ends of the fragment, respectively. Used primers VHVLfw, VHVL-rev, VLVH-fw, VLVH-rev.

The purified PCR products were hydrolyzed with BamHI and HindIII restriction endonucleases and the resulting 715 bp fragments were purified from an agarose gel by GeneJet (ThermoFisher) DNA purification columns using a gel extraction protocol.

A plasmid vector for construction of dimeric antibody fragments is prepared by hydrolysis of pUC57-VL-L4-VH-SL plasmid (GeneScript) with BamHI and HindIII restriction endonucleases. The ends of the plasmid DNA are dephosphorylated by FastAP (ThermoFisher) alkaline phosphatase. The purified vector is about 3300 bp long.

The prepared VL-L4-VH and VH-L4-VL genes (PCR fragments) and pUC57-VL-L4VH vector are mixed in a 3: 1 molar ratio and ligated using T4 DNA ligase ((ThermoFisher). The reaction mixture is transformed into prepared competent E. .coli DH10B bacteria were seeded on agarized LB medium by thermal shock and after half an hour incubation in liquid LB medium. The resulting target product is about 1660 bp in length, other fragments are generated by repetition in the sequence and the ability to hybridize to one of the primers with multiple sequences in the cloned gene (about 800 bp for the ll construct and about 400 bp for the ll construct). clonal plasmid DNA was further analyzed by restriction endonucleases.

From Figure 2. it can be seen that PCR amplification yields DNA fragments of the predicted size constructs VL-L4-VH-SL-VL-L4-VH and VL-L4-VH-SL-VH-L4-VL.

DNA from selected clones (colonies) is purified and plasmid DNA is analyzed by hydrolysis of the BamHI / HindIII pair of restriction endonucleases to yield 720 bp and 3300 bp DNA fragments, respectively, if the DNA sequence after cloning is of appropriate structure (Fig. 3). From Figure 3. it is seen that the resulting fragment sizes correspond to the theoretical ones.

The plasmid pET-28a vector for the recloning of di-scFv fragments is prepared by hydrolysis with NdeI and HindIII restriction endonucleases. The ends of the plasmid DNA are dephosphorylated using FastAP alkaline phosphatase (ThermoFisher) in the reaction mixture. The resulting DNA fragments are fractionated on an agarose gel and the 5306 bp fragment is purified from GeneJet (ThermoFisher) gel on a DNA purification column according to the gel extraction protocol.

The dimerized scFv genes are excised from selected clones 1, 3, 4 (construct I-a), 11, 12, and 13 (construct II-a) by hydrolysis of pUC57 with Ndel and HindIII restriction endonucleases. Approximately 1600 bp of DNA fragments are gel purified using GeneJet (ThermoFisher) DNA purification columns according to the gel extraction protocol.

Purified di-scFv genes were ligated with the prepared pET-28a vector and transformed into prepared competent E.coli DH10B bacteria. Colonies containing plasmid DNA with di-scFv constructs are selected by colony PCR and by screening the plasmid DNA structure of selected clones by restriction endonuclease hydrolysis assay.

From Figure 4. analysis of the resulting plasmids by restriction endonucleases results in DNA fragments of the required size.

The sequences of the selected plasmid pET-28a-di-scFv constructs are confirmed by DNA sequencing. The gene sequences are presented in Fig. 5.

example Di-scFv expression of dimeric antibody fragments in E.coli

The expression of dimeric single-chain antibody fragments in E. coli was confirmed by selecting plasmid constructs from clones 14-12 and 1111-20 and transforming them into the expressing strain of E.coli BL21 (DE3) (Novagen, Merck). The controls were used

E. coli BL21 (DE3) strain was transformed with pET-28a without cloned fragments. The bacteria were cultured at 37 ° C with constant shaking of flasks and cultures reaching 0.6-1.2 o.v. (At 600 nm), optical density of the target protein was induced by adding IPTG (isopropyl β-D-thiogalactoside, Sigma) to a final concentration of 1 mM. Biomass samples were taken before induction (0 h) and 1, 2 and 3 hours after induction.

After three hours of induction, all biomass was collected by centrifugation. Bacteria are resuspended in digestion buffer and sonicated. Soluble proteins are separated from the insoluble fraction by centrifugation. Protein composition in soluble and insoluble fractions was analyzed by polyacrylamide gel electrophoresis under denaturing conditions (PAAG method). Samples taken before and after induction are tested for total protein content by equilibrating the biomass content with the established optical density of the culture grown.

After analysis of the samples, electrophoresis in polyacrylamide gel under denaturing conditions determined the concentration of the target protein after induction (~ 58 kDa protein was observed). No similar protein accumulation was found before induction and in controls lacking the cloned di-scFv gene. The analysis of soluble and insoluble fractions of the digested biomass showed that the major part of the synthesized protein was accumulated in the insoluble fraction (inclusions).

From Figure 6. it can be seen that expression experiments yield predicted size proteins in bacterial lysates after induction.

In the course of this work, genes encoding dimerized, also known as tandem, single-stranded antibody fragments with a histidine (6xHis) anchor coding sequence at the start of the gene were constructed using genetic engineering techniques. The gene sequences were verified by sequencing. During recombinant protein expression in E. coli, accumulation of target proteins in the insoluble protein fraction - inclusion bodies - was observed under standard, non-optimized conditions.

Example 4. Purification of dimeric antibody frameworks di-scFv

E.coli biomasses with expressed target recombinant proteins scFV and di-scFV were resuspended in buffer (0.1 M Tris-HCl, pH 7.0 in 5 mM EDTA, 0.1% Triton Χ-100 and 300 mM 2 mercaptoethanol) in a 1:10 ratio (biomass: buffer) and sonicated. For control, E.coli biomass transformed with pET28a vector without the recombinant gene cloned and lacking expression of scFv. The homogenate was centrifuged for 60 min at 10000xg. The residual precipitate was washed twice with 20 mL of 20mM Tris-HCl pH7.0 with 1 M NaCl and 0.1% Tween 80 and once with 20 mL of 20mM Tris-HCl pH7.0 with 30 min centrifugation at 10000xg + 4 ° C. at temperature.

Renaturation. The precipitate obtained after the final wash and containing scFv and discFV proteins and control is dissolved in 20 mM Tris-HCl, pH 8.5 buffer containing 7 M guanidine hydrochloride and DTT is added to a final concentration of 10 mM. The suspensions were stirred for 2 hours. 4 ° C, centrifuged for 30 min. 10304xg and the supernatants were slowly diluted with 20 mM Tris-HCl buffer pH 8.5 to a final concentration of 3.5 M guanidine hydrochloride. Oxidized glutathione is added to a final 10 mM. The suspensions are stirred for 1 hour. 4 ° C, centrifuged for 30 min. 10304xg and the supernatants were slowly diluted with 20 mM Tris-HCl buffer pH 8.5 to a final concentration of 1.75 M guanidine hydrochloride. The suspensions are stirred for 1 hour. 4 ° C, centrifuged for 30 min. 10304xg and supernatants were slowly diluted with 20 mM Tris-HCl buffer pH 8.5 to a final concentration of 0.8 M guanidine hydrochloride. The suspensions were stirred overnight at 4 ° C, centrifuged for 30 min. 10304xg. NaCl was added to the supernatants to a final concentration of 0.3 M and the pH adjusted to pH 8.0.

Protein purification. Protein solutions were applied to 1 ml nickel (Ni ²⁺ ) loaded IMAC Sepharose 6 FF sorbent columns equilibrated in 50 mM Tris-HCl buffer, pH 8.0 containing 300 mM NaCl and 10 mM imidazole. The sorbents were washed with 10 volumes of equilibration buffer and 5 volumes of analogous buffer with 20 mM imidazole. Elution was performed with 50 mM Tris-HCl buffer, pH 8.0 containing 300 mM NaCl and 500 mM imidazole. Fractions containing the target protein are pooled and pooled. Dialysis against 20 mM Tris-HCl pH 8.0, 300 mM NaCl and centrifugation for 30 min at 29000xg at 4 ° C. Protein is stored at 4 ° C.

The calculated molecular weight of the protein monomeric scFv is 27.35 kDa, the theoretical isoelectric point pi is 7.76, the molecular weight of the dimeric di-scFv is 57.68 kDa and pi is 6.67. Experimentally (NDS-PAAG) the molecular weight of the monomeric scFv is about 27 kDa and that of the dimeric di-scFv is about 60 kDa. Figure 7 shows that the purified di-scFv proteins are suitable for further hemolysis experiments.

Example 3D-scFv activity assay of dimeric antibody fragments

Erythrocyte hemolysis in vitro [Cauci S, Monte R, Ropele M et al., Mol Microbiol, 1993, 9, p. 1143-1155.] Was used to test the activity of recombinant scFv neutralizing VLY. Human erythrocyte suspension (1 ml) was incubated for 30 min. At a temperature of 20 to 25 ° C in the following reaction mixtures:

Phosphate buffer, PBS, pH 6.5 (no red cell hemolysis);

Deionized water (100% erythrocyte hemolysis);

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

ng / ml VLY and 25 ng / ml scFv antibody monomer in PBS, pH 6.5;

ng / ml VLY and 50 ng / ml scFv antibody monomer in PBS, pH 6.5;

ng / ml VLY and 100 ng / ml scFv antibody monomer in PBS, pH 6.5;

ng / ml VLY and 200 ng / ml scFv antibody monomer in PBS, pH 6.5;

ng / ml VLY and 25 ng / ml scFv antibody dimer in PBS, pH 6.5;

ng / ml VLY and 50 ng / ml scFv antibody dimer in PBS, pH 6.5;

ng / ml VLY and 100 ng / ml scFv antibody dimer in PBS, pH 6.5;

ng / ml VLY and 200 ng / ml scFv antibody dimer in PBS, pH 6.5.

After incubation, the erythrocyte suspension was centrifuged for 3 min at 800 * g and the solution was measured at 417 nm for OD. High values of optical density (OD> 3.0) indicate erythrocyte hemolysis. Low OD values (OD <0.4) indicate that erythrocytes were protected from destruction. Addition of 3 ng / ml recombinant VLY to the erythrocyte suspension resulted in complete destruction of the erythrocytes (OD ~ 3.0). When 200 ng / ml scFv dimer antibody antibody was added to the mixture, erythrocytes were completely protected from hemolysis (Fig. 8). The obtained results show that both recombinant scFv monomer and recombinant scFv dimer neutralized the cytolytic activity of VLY. The recombinant scFv dimer (di-scFv) exhibited higher neutralizing activity than the scFv monomer because it was required for lower concentrations to neutralize VLY.

Fig. demonstrates that the neutralizing activity of cytolysin VLY is increased by increasing the concentration of di15 scFv protein and that di-scFv activity is higher than that of the monomeric scFv protein.

Thus, the present invention demonstrates that the constructed dimeric scFv recognizes VLY and effectively neutralizes the biological activity of the latter, i. inhibits the ability of VLY to induce pathogenic human erythrocyte hemolysis.

The advantage of single-chain antibody fragments (scFvs) linked to a covalent dimeric structure over monoclonal antibodies is that scFVs are obtained significantly simpler and cheaper (by bacterial biosynthesis), and that dimeric scFvs have a high affinity and relatively low molecular weight, which determines delivery of scFv to infected tissues and enhanced therapeutic effect. The recombinant dimeric scFv molecule can be used as a potential therapeutic agent for the treatment of pathogenic effects caused by G.vaginalis bacteria.

In conclusion, the novel dimeric antibody fragment scFv is specific for VLY cytolysin from G.vaginalis and effectively neutralizes the latter's cytolytic activity. Therefore, this dimeric antibody fragment scFv can be used for therapeutic and prophylactic purposes in treating or preventing pathologies caused by G.vaginalis infection. The resulting dimeric antibody fragments (di-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 di-scFv potentially results in a stronger therapeutic effect due to its more efficient delivery to infected tissues.

Industrial applicability

Applications of the dimeric scFv protein are related to its use in the prophylaxis and treatment of bacterial vaginosis caused by G.vaginalis. Pharmaceuticals and medical devices (gels, ovules or detergents) having anti-cytolytic activity are suitable for the production and widespread prophylactic use of biopharmaceutical recombinant antibodies.

The pharmaceutical compositions are comprised of a dimeric scFv preparation in combination with pharmaceutically acceptable carriers, diluents, excipients or excipients for the prophylaxis or treatment of bacterial vaginosis, and for the treatment of the consequences of G.vaginalis infection. The present invention encompasses some pathologies of bacterial vaginosis resulting in preterm birth leading to low birth weight, pelvic inflammatory diseases, endometritis and other diseases caused by G.vaginalis infections.

Moreover, the resulting DNA sequences encoding di-scFv can be used to construct novel humanized antibodies that are likely to 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 (12)

DEFINITION OF INVENTION A dimeric covalently linked antibody fragment having the amino acid sequence of SEQ ID NO: 1 or a similar sequence having at least 95% identity. The dimeric antibody fragment of claim 1 having the structure represented by the formula (VL-L4-VH) -SL- (VL-L4-VH) or (VL-L4-VH) -SL- (VH-L4-VL), wherein VL is a variable immunoglobulin light chain L fragment, VH is a variable immunoglobulin heavy chain H fragment, L4 and SL are linker sequences. The dimeric antibody fragment of claim 2, wherein the L4 linker sequence is (G ₄ S) ₄ and the SL linker sequence is SGLEA (EAAAK) ₄ ALEA (EAAAK) ₄ ALEGS. The dimeric antibody fragment of claim 2 or 3 having the amino acid sequence set forth in SEQ ID NO. 2 or SEQ ID NO: 3 or similar sequences having at least 95% identity. The dimeric antibody fragment of any one of the preceding claims, characterized by inhibition or neutralization of vaginolysin biological activity. A recombinant DNA molecule encoding a dimeric antibody fragment according to any one of the preceding claims. The recombinant DNA molecule of claim 6 having the DNA sequence of SEQ ID NO. 4 or SEQ ID NO: 5. A vector comprising a DNA molecule according to claim 6 or 7. A preparation comprising a dimeric antibody fragment according to any one of claims 1 to 5, characterized by inhibition or neutralization of vaginolysin biological activity. A pharmaceutical composition comprising the active component and a pharmaceutically acceptable carrier, diluent, excipient and / or excipient, wherein the active component comprises a therapeutically effective amount of a preparation according to claim 9. A pharmaceutical composition according to claim 10 for use in the prophylaxis and / or treatment of conditions caused by Gardnerella vaginalis infection. A pharmaceutical composition according to claim 10 or 11 in the form of a gel, ovule or detergent.