Recombinant Bispecific Antibodies to the Human ErbB2 Receptor and Interferon-Beta.

The development of and research into new therapies that can selectively and effectively destroy tumor cells that overexpress the ErbB2 receptor is a pressing task. Recently, research into the use of type I interferons in the treatment of cancer has intensified. Cytokine therapy is aimed at activating the cells of the immune system to fight tumors, but it has drawbacks that limit its use because of a number of side effects the severity of which varies depending on the dosage and type of used cytokine. At the moment, a number of studies are being conducted regarding the use of IFNß in oncology. The studies are aimed at mitigating the systemic action of this cytokine. The immunocytokine complex made of a bispecific antibody against the ErbB2 receptor and recombinant IFNß developed in this study underlies the mechanism meant to avoid the systemic action of this cytokine. Part of this study focuses on the development of full-length antibodies that bind to the ErbB2 receptor on the one hand, and bind and neutralize IFNß, on the other hand, which allows us to consider the antibodies as a means of cytokine delivery to tumor cells.

The Generation of the Human mAb RabD4 Specific to the Rabies Virus Glycoprotein and Characterization Thereof.

We generated a novel human neutralizing human mAb RabD4 against rabies virus glycoprotein using in vitro stimulation of human peripheral B cells produced by immunized donor. The human mAb RabD4 showed a high antigen-binding activity and virus-neutralizing activity in the FAVN test with the CVS-11 rabies virus.

Structure of the Anti-C60 Fullerene Antibody Fab Fragment: Structural Determinants of Fullerene Binding.

The structure of the anti-C60 fullerene antibody Fab fragment (FabC60) was solved by X-ray crystallography. The computer-aided docking of C60 into the antigen-binding pocket of FabC60 showed that binding of C60 to FabC60 is governed by the enthalpy and entropy; namely, by π-π stacking interactions with aromatic residues of the antigen-binding site and reduction of the solvent-accessible area of the hydrophobic surface of C60. A fragment of the mobile CDR H3 loop located on the surface of FabC60 interferes with C60 binding in the antigen-binding site, thereby resulting in low antibody affinity for C60. The structure of apo-FabC60 has been deposited with pdbid 6H3H.

The Development and Study of Recombinant Immunoglobulin A to Hemagglutinins of the Influenza Virus.

We obtained recombinant variants of human antibody FI6 broadly specific to hemagglutinins of the influenza A virus. On the basis of a bi-promoter (CMV, hEF1-HTLV) vector, we developed genetic constructs for the expression of the heavy and light chains of the immunoglobulins of IgA1-, IgA2m1-, and IgG-isotypes. Following transfection and selection, stable Chinese hamster ovary (CHO) cell lines were produced. The antibodies of IgA1-, IgA2m1-, and IgG-isotypes were purified from culture media. We performed an immunochemical characterization and studied their interactions with influenza A strains of the H1N1- and H3N2-subtypes. It was shown that recombinant FI6 variants of the IgA-isotype retain the properties of the parental IgG antibody to demonstrate specificity to all the strains tested. The strongest binding was observed for the H1N1 subtype, which belongs to hemagglutinins of phylogenetic group I.

The Development and Inmmunochemical Properties of the Dimer of Immunoglobulin A Specific to the Influenza Virus A Hemagglutinin.

We obtained dimeric forms of IgA1- and IgA2m1-isotypes of FI6 antibody broadly specific to hemagglutinins of different subtypes of influenza A virus. It was shown that the dimers of IgA1 isotype are characterized by a higher antigen-binding activity compared to the IgA2m1 dimers. The affinity of IgA1 dimers to the strains of the H1N1 subtype is higher than that of the H3N2 subtype, which correlates with the properties of the parental human FI6 antibody.

[In vitro Antiviral Activity of Recombinant Antibodies of IgG and IgA Isotypes to Hemagglutinin of the Influenza A Virus].

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

Recombinant Antibodies to the Ebola Virus Glycoprotein.

Currently, there are no approved therapies for targeted prevention and treatment of Ebola hemorrhagic fever. In the present work, we describe the development of a eukaryotic expression system for the production of three full-length chimeric antibodies (IgG1-kappa isotypes) GPE118, GPE325, and GPE534 to the recombinant glycoprotein of the Ebola virus (EBOV GP), which is a key factor in the pathogenicity of the disease. The immunochemical properties of the obtained antibodies were studied by immunoblotting and indirect, direct, and competitive ELISA using the recombinant EBOV proteins rGPdTM, NP, and VP40. The authenticity of the antibodies and the absence of cross-specificity with respect to the structural proteins NP and VP40 of the Ebola virus were proved. The epitope specificity of the resulting recombinant antibodies was studied using commercial neutralizing antibodies against the viral glycoprotein. The recombinant antibodies GPE118, GPE325, and GPE534 were shown to recognize glycoprotein epitopes that coincide or overlap with the epitopes of three well-studied neutralizing anti-Ebola virus antibodies.

New monoclonal antibodies to the Ebola virus glycoprotein: Identification and analysis of the amino acid sequence of the variable domains.

We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained.

[Production of humanized F(ab')2 fragment of rabies blocking antibodies in Pichia pastoris yeast].

The yeast strain Pichia pastoris, a producer of humanized F(ab')2 fragments of rabies-blocking antibodies, has been obtained. Human chaperone BiP coexpression caused a twofold increase of the immunoglobulins secretion level. The use of Fos and Jun zippers in the composition of heavy chains facilitated the dimerization of F(ab')2 fragments of the shared pool of secreted immunoglobulins up to 75%.

[Development of an Immunochromatographic Test System for the Detection of Helicobacter pylori Antigens].

A test system based on immunochromatography in the sandwich format and intended for express detection of Helicobacter pylori antigens has been developed. Contact of a sample with a test strip coated with immunochemical reagents triggers the movement of the liquid along the membrane components of the test strip, immunochemical interactions, and the formation of detection zones stained by gold nanoparticles. The concentration and kinetic dependences of the immunochemical interactions have been characterized. The reagent and membrane composition of the test system has been selected to provide a minimal detection limit. The detection of H. pylori cell wall antigens at concentrations as low as 0.3 µg/mL in aqueous solution and a suspension of a clinical sample of feces has been demonstrated; the assay duration was 10 minutes. Staining enhancement by the addition of silver salts allowed for a further reduction of the detection limit to 0.03 µg/mL. The developed test system can be used for field diagnostics.

[Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

Application of gold nanoparticles produced by laser ablation for immunochromatographic assay labeling.

Nanodispersed gold is widely used as a marker in different analytical systems. For such purposes, it is usually obtained by the reduction of salts. This work studied the potential analytical applications of nanodispersed gold obtained by laser ablation because gold produced with this method has no chemical coating. The nanoparticles produced were characterized by transmission electron microscopy and spectrophotometry. The average size of the particles was 24.5 nm. Concentration dependences of antibody immobilization on ablative gold were obtained. With the use of antibody-conjugated nanoparticles, an immunochromatographic system was constructed for the detection of zearalenone mycotoxin. This immunoassay was characterized by a detection limit of 0.1 ng/ml antigen with an assay duration of only 15 min, which is on par with current test systems comprising nanodispersed gold obtained by chemical reduction. The simplicity of ablative dispersing makes this a prospective method for the labeling of various antibodies for analytical use.

[Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

Conformational Differences between Active Angiotensins and Their Inactive Precursors.

The peptide conformation in the context of a protein polypeptide chain is influenced by proximal amino acid residues. However, the mechanisms of this interference remain poorly understood. We studied the conformation of angiotensins 1, 2 and 3, which are produced naturally in a sequential fashion from a precursor protein angiotensinogen and contain an identical peptide core structure. Using the example of angiotensins 1, 2 and 3, it was shown that similar amino acid sequences may have significant conformational differences in various molecules. In order to assess the conformational changes, we developed a panel of high-affinity mouse monoclonal antibodies against angiotensins 1, 2 and 3 and studied their cross-reactivity in indirect and competitive ELISAs. It was found that the conformations of inactive angiotensin1 and the corresponding fragment of angiotensinogen are similar; the same is true for the conformations of active angiotensins 2 and 3, whereas the conformations of homologous fragments in the active and inactive angiotensins differ significantly.

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products.

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR-protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL(-1)) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16-250 ng mL(-1) its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR-protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).

[Heat-shock protein enhances immunogenicity of E7 oncoprotein of human papillomavirus included in chimeric construction (E7 HPV-HSP70)].

AIM: To study the effect of chimeric E7 protein of human papillomavirus type 18 on activation of adaptive immunity in absence of adjuvant. MATERIALS AND METHODS: Chimeric protein was genetically engineered and represents the protein molecule consisting of full-size E7 oncoprotein and heat-shock protein 70 (HSP70) of Mycobacterium tuberculosis in one polypeptide chain. Antibody titers as well as isotypes and subisotypes of immunoglobulins were measured by ELISA in sera of immunized animals. RESULTS: It was shown that studied construction E7 (HPV-18)-HSP70 significantly increases titers of antibodies to E7 protein of HPV type 18 and have cross-reactive antigenic activity with E7 protein of HPV type 16. Immunization with chimeric protein resulted in increase of IgG1 and IgG2b levels and decrease of IgG2a and IgM levels. CONCLUSION: . Oncoprotein E7 included in chimeric construction with HSP70 could be used for further studies on development of therapeutic vaccine for treatment of cervical cancer and precancerous lesions. Skew of immune response to Th2 type after intraperitoneal administration of the studied construction points to necessity for control of immunity during such studies.

[Production of antibodies to aflatoxins].

A panel of ten monoclonal antibodies to aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA. The structures of monoclonal antibody genes were comprehensively studied and the variable and constant domains of the antibody genes were cloned and sequenced. Sequencing analysis confirmed the results of isotyping the light and heavy antibody chains obtained by ELISA. Variable and constant fragments of the antibody genes were cloned into a bicistron expression vector for the recombinant Fab' fragment for one of the antibodies expressed in Escherichia coli and purified. Thus, data were obtained that can be useful for the development of an aflatoxin detection system on the basis of the described monoclonal antibodies and the creation of recombinant antibodies with changed parameters of specificity using protein engineering methods.

[Assessment of protective effect of heat shock protein-lypopolysaccharide of Salmonella typhimurium recombinant construction in mice].

AIM: To study protective activity of recombinant construction of heat-shock protein with lypopolysaccharide (rcHSP-LPS) as well as its variants (with destroyed protein or bounded LPS) against Salmonella typhimurium. It was also planned to study the ability of rcHSP-LPS to interact with toll-like receptors (TLRs) expressed on continuous cell lines. MATERIALS AND METHODS: One of the following preparations was administered to outbred mice: rcHSP-LPS; rcHSP-LPS treated by polymyxin B (PMB) for bounding of LPS - rc(HSP-LPS)PMB; rcHSP-LPS in which protein was treated by boiling during 30 min--rc (HSP-LPS)B; LPS (E. coli K-235); polymyxin B (PMB). Twenty-four hours after single or last administration of rcHSP-LPS, each mice was intraperitoneally inoculated with 63 LD50 of S. typhimurium 415 contained in 0.5 ml of physiologic solution. Antibody titer to LPS of Salmonella typhimurium was measured by immunoenzyme assay. RESULTS: It was demonstrated that rcHSP-LPS administered 24 hours before inoculation induced resistance to S. typhimurium infection. Protection formed after 3 injections of rcHSP-LPS with 10 mcg in each or single injection with 100 mcg/mouse. Forty to eighty percent of immunized mice survived after challenge while 90% of control animals died. Destroy of the HSP by boiling of the construction led to loss of protective effect. Bounding of LPS by PMB did not lead to loss of protective properties of the construction but they expressed only after its multiple administration with 10 mcg per mouse. LPS of E. coli in dose 0.0266 mcg per mouse as well as PMB did not influence the course of S. typhimurium infection in mice. CONCLUSION: It was shown that rcHSP-LPS effectively protects mice from S. typhimurium infection by activating innate immunity; one of the possible mechanisms for such protection determined by interaction with TLRs 2 and 4 was considered. Other studies are needed in order to elucidate other mechanisms of innate immunity, which can be activated by rcHSP-LPS.

[Recombinant heat-shock protein (rHSP70) boosts activation of innate and adaptive immunity during simultaneous administration with bacterial antigens in experiment].

Experimental study of adjuvant effect of recombinant mycobacterial heat-shock protein rHSP70 on immune response to antigens of opportunistic microorganisms was performed. Therapeutic poly-component vaccine Immunovac-VP-4, containing ligands for binding with Toll-like receptors (TLRs) 2, 4 and 9, was used as a source of bacterial antigens. Obtained data showed that administration of rHSP70 mixed with bacterial antigens of opportunistic microorganisms leads to maturation of mice dendritic cells obtained from bone marrow precursors, increased expression of TLRs 2, 4, 9 on their surface and production of cytokines IL-1beta, IL-6, IL-10, TNFalpha, as well as to activation of innate immunity in experiments in vivo resulting to increased resistance of mice to experimental Salmonella infection. Simultaneous administration of rHSP70 with bacterial antigens increased titers of antibodies to vaccine's antigenic components in direct hemagglutination reaction. Thus, adjuvant effect of rHSP70 was confirmed on the used model as well as increase of stimulation of innate and adaptive immunity was shown.

[Effect of recombinant heat-shock protein (rHSP70) of Mycobacterium tuberculosis on immunogenicity of Haemophilus influenzae type B capsular polysaccharide].

AIM: To study effect of recombinant heat-shock protein (rHSP70) of Mycobacterium tuberculosis on immunogenicity of Haemophilus influenzae type b capsular polysaccharide (CPSHib). MATERIALS AND METHODS: Capsular polysaccharide was obtained by precipitation with cetavlon, antibody titers and Toll-like receptors (TLRs) were detected by enzyme immunoassay and flow cytometry respectively. RESULTS: rHSP70 modified immune response to chemically conjugated and unconjugated CPSHib. rHSP70 enhanced expression of TLRs 2, 4, 9 on mice splenocytes; increased levels of CD3+, CD8+, NK, CD3/NK (NKT) lymphocytes. Levels of CD4+, CD25+ (markers of early activation of T-helpers) as well as MHC class II molecules were increased that could be appraised as a shift from T-independent to T-dependent immune response. Difference in antibody titers after 2- or 3-dose immunization of mice with 5 mcg/dose of CPSHib in mixture or conjugated with rHSP70 was not revealed. Level of antibodies to rHSP70 in serum samples of mice immunized with CPSHib conjugated with rHSP70 was 6.55 - 8.4 times higher compared to unimmunized animals. Antibodies, which have common antigenic epitopes to human organs and tissues, were not detected. CONCLUSION: rHSP70 modifies immune response to CPSHib.