Recombinant Hybrid Protein Research Articles

SAFETY AND SPECIFIC ACTIVITY OF THE RECOMBINANT SARS-COV-2 (CORONADERM-PS) ALLERGEN BASED ON PHASE I-II CLINICAL TRIAL RESULTS

Abstract The evaluation of cellular immunity to SARS-CoV-2 is a critical tool for assessing vaccination efficacy and development of post-infectious immunity. According to available studies, virus-specific T cells persist longer than antigen-specific antibodies and play a pivotal role in the effective virus elimination from human body. Current methodologies for assessing virus-specific T cells are mostly based on flow cytometry, which require specialized laboratory equipment and highly trained personnel. An alternative approach involves a skin test conducted to assess delayed-type hypersensitivity (DTH) reactions. For this, there has been developed the "CoronaDerm-PS" substance, a sterile isotonic solution for intradermal administration, containing a recombinant hybrid protein that incorporates regions of SARS-CoV-2 structural proteins S, M, N, and E, produced by a genetically modified E. coli BL21 strain cell culture. Following preclinical studies that demonstrated the safety of the above-noted medication, Phase I and Phase II clinical trials were initiated to evaluate safety and specific activity of "CoronaDerm-PS" in apparently healthy volunteers with SARS-CoV-2 post-vaccination and post-infection immunity. In Phase I clinical trial with COVID-19 unexposed or vaccinated volunteers, the data were obtained demonstrating the safety and good tolerability of the medication, thus enabling the progression to Phase II. The Phase II results provided additional evidence on the preparation’s safety in individuals with SARS-CoV-2 post-vaccination and post-infection immunity, as assessed by clinical and laboratory data. No serious adverse events were observed during the study, and in 93.5% cases, adverse events required no therapeutic or diagnostic intervention. To assess the specific activity of the preparation, the skin test data were compared with the IFNγ production stimulation index for CD4+ T lymphocytes assessed by flow cytometry. The ROC analysis revealed sensitivity magnitude ranging from 76.6% to 84%, and specificity level ranging from 80% to 87.5%. Based on ROC analysis results, "CoronaDerm-PS" can be an informative diagnostic tool (AUC = 0.795), demonstrating high sensitivity (79.8%) and specificity (80.8%), with consistent results across different volunteer cohorts. Analyzing the collected data suggests that "CoronaDerm-PS" is a robust alternative to laboratory methods for evaluating SARS-CoV-2-specific T-cell immunity, with high sensitivity and specificity, suitable for large-scale screening.

Experimentally investigated “CoronaDerm-PS”-driven SARS-CoV-2-specific cellular immunity and safety

Novel coronavirus disease 2019, caused by the SARS-CoV-2, initiate humoral and cellular immune responses against diverse virus antigens. The assessment of SARS-CoV-2-specific is mainly carried out in routine practice by determining specific immunoglobulins. However, high variability in S-protein structure in new genovariants of SARS-CoV-2 virus and the lack of correlation between specific antibodies and CD8+ T-lymphocytes underlie false negative responses, and mass assessment of cellular immunity is complicated due to the complexity of applying ELISPOT and cytofluorometry techniques. To solve this issue, a diagnostic method was developed for assessing SARS-CoV-2-specific cellular immune response, which is based on a skin test followed by evaluating a delayed-type hypersensitivity reaction involving antigen-specific memory T-lymphocytes. A diagnostic preparation CoronaDerm-PS is based on Cord_PS, which is a hybrid recombinant protein consisting of parts of the SARS-CoV-2 structural proteins S, M, N, E. The specific activity of this chimeric antigen was analyzed in cultured T-lymphocyte activation test by assessing interferon-γ production using cytofluorometry. To investigate the chimeric antigen specific activity, a preclinical safety study with CoronaDerm-PS preparation in experimental animals was conducted. A dose-dependent developing skin reaction was observed in 90–100% of guinea pigs vaccinated by EpiVacCorona, CoviVac, Gam-COVID-Vac, which confirms a potential for assessing post-vaccination cellular immunity using CoronaDerm-PS preparation. Upon this, the presence of functionally active T-cell-antigenic epitopes in the recombinant polypeptide allows to evaluate SARS-CoV-2-specific response illustrated by detected response after Gam-COVID-Vac (S-protein) and EpiVacCorona (N-protein) vaccination. Thus, a skin test based on CoronaDerm-PS preparation may be a promising diagnostic tool for rapid mass screening requiring no specialized laboratory equipment for assessing populational SARS-CoV-2-specific immunity. Such a test is distinguished by advantages such as ease of analysis, high specificity and sensitivity. The final decision-making on using this test in a real-world practice may achieved after conducting further clinical safety and effectiveness trials.

Efficient Expression in the Prokaryotic Host System, Purification and Structural Analyses of the Recombinant Human ACE2 Catalytic Subunit as a Hybrid Protein with the B Subunit of Cholera Toxin (CTB-ACE2).

Angiotensin-converting enzyme 2 (ACE2) has a specific interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identified with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ~ 100kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25mM IPTG and 1% sucrose for 10h. Finally, the protein structural features were assessed using several biophysical methods. The fluorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The β-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the efficient expression and successful purification of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.

Preclinical study of mutagenicity and safety of a vaccine against pneumococcal infection based on a recombinant hybrid protein

Pneumococcal infection is the cause of pneumo-nia. Vaccination can prevent the severe course of this disease. The pneumococcal vaccine considered in the work was created by a genetically modified method based on a hybrid recombinant protein. The aim of the work was preclinical investigation of the mutagenic, genetic safety of the new pneumococcal vaccine, Irivax, against pneumococcal infection in rats. Materials and methods. Studies of the Irivax vaccine were carried out in male Wistar rats’ cell populations of the bone marrow and peripheral blood mononuclear cells. The karyoty-pic stability of cells and myelogram of the bone mar-row were studied. Results. After the introduction of the vaccine, the content of the lymphoid germ cells in the blood of rats was increased to 74.2% (p <0.05), which proves the body’s immune response. The frequency of cells with an unstable genome, as an indicator of the genetic safety of the AL-vaccine, was rare both in expe-rimental and control animals, and did not exceed 1.5%. Conclusion. Thus, the new pneumococcal vaccine Irivax and its components have mutagenic and toxic safety.

A Genetically Engineered Biofilm Material for SARS-CoV-2 Capturing and Isolation.

The novel severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is continuously infecting people all around the world since its outbreak in 2019. Studies for numerous infection detection strategies are continuing. The sensitivity of detection methods is crucial to separate people with mild infections from people who are asymptomatic. In this sense, a strategy that would help to capture and isolate the SARS‐CoV‐2 virus prior to tests can be effective and beneficial. To this extent, genetically engineered biomaterials grounding from the biofilm protein of Escherichia coli are beneficial due to their robustness and adaptability to various application areas. Through functionalizing the E. coli biofilm protein, diverse properties can be attained such as enzyme display, nanoparticle production, and medical implant structures. Here, E. coli species are employed to express major curli protein CsgA and Griffithsin (GRFT) as fusion proteins, through a complex formation using SpyTag and SpyCatcher domains. In this study, a complex system with a CsgA scaffold harboring the affinity of GRFT against Spike protein to capture and isolate SARS‐CoV‐2 virus is successfully developed. It is shown that the hybrid recombinant protein can dramatically increase the sensitivity of currently available lateral flow assays for Sars‐CoV‐2 diagnostics.

Study of immunogenicity and safety of a candidate rotavirus vaccine based on a recombinant hybrid protein

BACKGROUND: Rotaviruses are the main cause of acute gastroenteritis in children in both developed and developing countries. Vaccination is the only way to prevent severe and fatal course of this disease. Live attenuated viruses-based vaccines currently available can have a number of side effects. A candidate rotavirus vaccine reported is based on a hybrid recombinant protein FliCVP6VP8, which includes a VP6 protein fragment, a rotavirus A VP8 protein fragment, and S. typhimurium FliC flagellin components. AIM: The aim was to evaluate the immunogenicity and safety of а preparation Rotavirus vaccine, recombinant in preclinical studies. MATERIALS AND METHODS: The immunogenicity of vaccine (blood antibody titers, antigen-specific proliferative response of spleen cells) was evaluated in BALB/c mice. The acute and subchronic toxicity, the possible irritating effect, pyrogenicity and the anaphylactic effect and delayed type hypersensitivity were evaluated in laboratory mice, rats, Guinea pigs, and rabbits. RESULTS: Double immunization of mice with the candidate vaccine demonstrated a significant increase in antibody titers in mouse sera compared to that in control mice. Evaluation of antigen-specific proliferative response after double immunization with a candidate vaccine demonstrated a significant increase in the values of stimulated proliferation. Evaluation of safety through acute and chronic toxicity studies demonstrated no toxicity. The immunostimulatory effect of vaccine was demonstrated when evaluating the number of antibody-producing cells with sheep red blood cells as antigens. The number of white blood cells was demonstrated to increase after the prolonged vaccine administration. CONCLUSIONS: The preclinical studies have demonstrated safety of the candidate rotavirus vaccine and its capability to produce the immune response.

Cross-reactive immunogenicity of group A streptococcal vaccines designed using a recurrent neural network to identify conserved M protein linear epitopes

The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal sequence of the protein defines the more than 200 M types of Strep A and also contains epitopes that elicit opsonic antibodies, some of which cross-react with heterologous M types. Current efforts to develop broadly protective M protein-based vaccines are directed at identifying potential cross-protective epitopes located in the N-terminal regions of cluster-related M proteins for use as vaccine antigens. In this study, we have used a comprehensive approach using the recurrent neural network ABCpred and IEDB epitope conservancy analysis tools to predict 16 residue linear B-cell epitopes from 117 clinically relevant M types of Strep A (~88% of global Strep A infections). To examine the immunogenicity of these epitope-based vaccines, nine peptides that together shared ≥60% sequence identity with 37 heterologous M proteins were incorporated into two recombinant hybrid protein vaccines, in which the epitopes were repeated 2 or 3 times, respectively. The combined immune responses of immunized rabbits showed that the vaccines elicited significant levels of antibodies against all nine vaccine epitopes present in homologous N-terminal 1–50 amino acid synthetic M peptides, as well as cross-reactive antibodies against 16 of 37 heterologous M peptides predicted to contain similar epitopes. The epitope-specificity of the cross-reactive antibodies was confirmed by ELISA inhibition assays and functional opsonic activity was assayed in HL-60-based bactericidal assays. The results provide important information for the future design of broadly protective M protein-based Strep A vaccines.

Identification of intermolecular bonds between human factor B and Cobra Venom Factor important for C3 convertase stability

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF, Bb is an enzyme that cleaves C3 and C5. However, CVF, Bb exhibits significantly different functional properties from C3b,Bb. Whereas both, CVF, Bb and C3b, Bb exhibit spontaneous decay-dissociation into the respective subunits, thereby eliminating the enzymatic activity, the CVF, Bb convertase is physico-chemically far more stable, decaying with a half-life that is more than two orders of magnitude slower than that of C3b,Bb. In addition, CVF, Bb is completely resistant to inactivation by Factors H and I. These two properties of CVF, Bb allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the physico-chemical stability of CVF,Bb, we have created recombinant hybrid proteins of CVF and human C3, based on structural differences between CVF and human C3b in the C-terminal C345C domain. Here we describe three human C3/CVF hybrid proteins which differ in only one, two, or five amino acid residues from earlier described hybrid proteins. In all three cases, the hybrid proteins containing CVF residues form more stable convertases, and exhibit stronger complement-depletion activity than hybrid proteins with human C3 residues. Three bonds between CVF residues and Factor Bb residues could be identified by crystallographic modeling that contribute to the greater stability of the convertases.

An efficient production of hybrid recombinant protein comprising non-structural proteins (NS 1 & NS 3) of bluetongue virus in prokaryotic expression system

Strategic design and suitable purification techniques are of paramount importance in the production of recombinant proteins, if intended for use in a diagnostic assay. However, there is no single protocol that can be universally adopted for obtaining proteins in requisite quality and quantity across various platforms. In this study, we have targeted proteins of bluetongue virus (BTV), which is the causative agent of an arthropod-borne infectious disease in ruminants. Traditionally, serological diagnosis of the disease has rested upon either virus neutralization test or on an ELISA test that employed a recombinant structural (VP1, VP7) protein. Among the non-structural (NS) proteins of BTV, NS1 and NS3, are preferred candidate antigens in development of immuno-diagnostics as these provide the option for identifying recent/ongoing infection. However, the difficulty in production/purification of recombinant full length NS proteins of BTV in sufficient quantity and quality in various expression systems, due to inherent structural complexities, have restricted their wider applicability as immunodiagnostic reagents. To circumvent the difficulties associated with production/purification, we developed a novel NS1 and NS3 fusion gene (∼1302 bp) encoding for NS1 N-terminus (1M to G252 aa) and NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains along with intervening variable central domain (118A to A182 aa) of bluetongue virus 23. This construct was cloned, over-expressed and efficiently purified by single step affinity chromatography under unique denaturing/renaturing condition. The purified fusion protein was found suitable for detection of antibodies against BTV in an indirect ELISA (iELISA).

Membrane-permeable Rab27A is a regulator of the acrosome reaction: Role of geranylgeranylation and guanine nucleotides

The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized — but not in non-permeabilized — cells when geranylgeranylated and active. When GDP-β-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-β-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.

Leptospira borgpetersenii hybrid leucine-rich repeat protein: Cloning and expression, immunogenic identification and molecular docking evaluation

Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein. The fusion DNA was cloned into pET160/GW/D-TOPO® to form the pET160_hKU_R21_2271 plasmid. The plasmid was used to express the rhKU_Sej_LRR_2271 protein in Escherichia coli BL21 Star™ (DE3). The expressed protein was immunologically detected by Western blotting and immunoreactivity detection with hyperimmune sera, T cell epitope prediction by HLA allele and epitope peptide binding affinity, and potential T cell reactivity analysis. The immunogenic epitopes of the protein were evaluated and verified by HLA allele and epitope peptide complex structure molecular docking. Among fourteen best allele epitopes of this protein, binding affinity values of 12 allele epitopes remained unchanged compared to LBJ_2271. Two epitopes for alleles HLA-A0202 and -A0301 had higher IC50 values, while T cell reactivity values of these peptides were better than values from LBJ_2271 epitopes. Eight of twelve epitope peptides had positive T-cell reactivity scores. Although the molecular docking of two epitopes, 3FPLLKEFLV11/47FPLLKEFLV55 and 50KLSTVPEGV58, into an HLA-A0202 model revealed a good fit in the docked structures, 50KLSTVPEGV58 and 94KLSTVPEEV102 are still considered as the proteins' best epitopes for allele HLA-A0202. The results of this study showed that rhKU_Sej_LRR_2271 protein contained natural immunological properties that should be further examined with respect to antigenic immune stimulation for vaccine development to prevent prevalent leptospiral serovar infection in Thailand.

PRODUCTION OF HYBRID RECOMBINANT PROTEIN Flu-Chim, CONTAINING INFLUENZA VIRUSES A AND B MAJOR EPITOPES

The influenza virus is highly contagious diseases of people, birds and mammals. Approximately 250 000– 500 000 deaths are caused by influenza epidemics worldwide yearly, and the death number may be up to millions in a possible influenza pandemic. Vaccination is the most cost-effective way to reduce the considerable disease burden of seasonal influenza. Although seasonal influenza vaccines are effective, their performance in the elderly and immunocompromised individuals would benefit from improvement. Major problems related to the development and production of pandemic influenza vaccines are response time and production capacity as well as vaccine efficacy and safety. Reverse genetics techniques can speed up the generation of seed viruses and new mathematical modelling methods improve vaccine strain selection. Using vaccines based on recombinant proteins, we avoid the risks associated with the introduction of the virus into the body, even inactivated. In this paper, we have got a highly purified recombinant fusion protein composed of fragments of the hemagglutinin of influenza viruses A and B. As adjuvant we used components of flagellin. We used the most immunogenic and conserved areas of hemagglutinin H1, H3, H5 and B, which cause the formation of specific antibodies which can cross-react with homologous epitopes among the various strains of influenza A and B. Vaccine efficacy is increased by using multiple epitopes of various proteins. The aim of this study was to clone and express the hybrid recombinant protein Flu-Chim, containing immunogenic epitopes of influenza A/H1N1, A/H3N2, A/H5N1 and B fused with fragments of flagellin in Escherichia coli expression system and its subsequent purification. During the study was created high-yield E. coli strain, which produces the recombinant protein Flu-Chim, selected the optimal protocol of induction of the gene encoding the protein. The protein was purified using metal affinity chromatography. The purity of the final preparation reached 98%. In the future, we are going to study the immunogenic properties of the protein and use it as a component of the candidate vaccine against influenza.

VACCINATION AGAINST MALARIA: REALITY AND PERSPECTIVES

Malaria continues to be a major international public health problem. However a significant reduction in the morbidity rate has been achieved over the past decade. The effective vaccine against malaria, caused by P. falciparun, could contribute significantly to the prevention and control of the disease, as tropical malaria is most widely distributed in the world. Nowadays there is the only RTS,S/AS01 vaccine had passed the accomplished phase 3 of clinical trial and received endorsement of certain bodies of World Health Organization and European Medicines Agency. RTS,S/AS01 is a pre-erythrocytic hybrid recombinant protein vaccine. Both immunogenicity and effectiveness of this vaccine particularly in children aged of 5-12 months were demonstrated by the trial at the time for first vaccination. Vaccine should be administered 3 times as the initial series of inoculation with 4 weeks interval and then the 4th dose should be given 15-18 months later. Since there is still a number of issues required certain clarifications, the decision has been taken to undertake another relatively large pilot project in African countries, prior to final recommendations on the vaccine use could be developed and proposed to countries. The implementation of this new trial will definitely take appropriate time. The contribution of vaccine to malaria control might be significant only if used simultaneously with other proved malaria control measures, such as the use of insecticide-treated nets, detection of malaria cases with the use of rapid diagnostic tests and subsequent its treatment, chemoprevention when appropriate and, of course vector control. The consideration of the vaccine use as the mechanism, which should allow achieve the eradication of the disease is not appropriate in principle.

308P - The effectiveness of neoadjuvant chemotherapy with recombinant tumor necrosis factor-thymosin-&agr;1in locally advanced breast cancer and effect on tumor angiogenesis

The little is known about the influence of recombinant hybrid protein of tumor necrosis factor-alpha-thymosin-alpha1 (TNF-T) on angiogenesis in breast cancer (ВС). The aim of the study was to investigate markers of angiogenesis and vascular proliferation in the tumor and to evaluate the efficacy of TNF-T in the neoadjuvant treatment of locally advanced breast cancer (LABC). Eligibility criteria included LABC of IIB-IIIB stages, ECOG ≤ 1, adequate kidney, liver and bone marrow function, no brain metastases. TNF-T 200000 IU was used peritumorally (injected around the tumor) on D1-5 (30 min before cytostatics injection), combined with standard FAC or PA regimens. The factors of angiogenesis CD31 and CD34 were studied in trephine-biopsy specimens before treatment and in postsurgical biopsies of the tumor after TNF-T courses. 82 women were recruited between April 2012 – October 2013 (mean age 53.3 ± 1.1 years). Group A (30 pts) received TNF-T combined to PA (17) and FAC (13) up to 6 courses. Group B (52pts) received standard PA (31) and FAC (21). Tumor response (TR) in Group A was 80% and in Group B 71.9% (p < 0.05), including CR 15% and 6.25%, correspondingly (p < 0.05). The number of vessels of the microcirculation in sight was for CD31: 5,9 ± 0,34 before treatment and 2,24 ± 0,35 after treatment (p < 0.05); CD34: 6.88 ± 1.07 and 2,75 ± 0,72, respectively (p < 0.05). The VEGF level in the main group decreased from 0,62 ± 0,11 to 0,11 ± 0,07(p < 0.05). The level of EGFR in the main group decreased from 0,87 ± 0,13 to 0,18 ± 0,05 (p < 0.05). All patients in Group A were operated successfully, no postoperative complications connected with TNF-T use were observed. By the time of abstract submission OS in Group A was 35.6 ± 1.2mos, in Group B was 34.1 ± 1.1mos; EFS in Group A was 33.5 ± 1.1mos, in Group B was 28.7 ± 1.2mos (p < 0.05). Three-year EFS in group A is 20% higher than in group B (83% and 63% respectively, log-rank test p = 0,04778). TNF-T injected peritumorally allows to increase the antitumor activity of cytostatics (CR) which is of great importance for survival in LABC. The data acquired in the biopsies show that the recombinant protein of TNF-T has the suppressive effect on angiogenesis.

Tetrameric RGD induces clustering of integrin αvβ3 on the melanoma cell surface and decreases cell viability

The recombinant hybrid protein SR15 composed of streptavidin fused with RGD peptide is obtained. The ability of this protein to recognize human melanoma cells (MeWo line) is demonstrated. Types of expressed RGD-binding integrins in the cells of this line are identified. It is found that the recombinant hybrid protein SR15 binds to integrin αvβ3 on the surface of human melanoma cells. The binding to the protein SR15 results in clustering of αvβ3 receptors on the surface of MeWo cells, internalization of the recombinant protein, and an almost twofold decrease in cell viability.

Abstract P1-14-19: Clinical and morphological results of neoadjuvant treatment with tumor necrosis factor-alpha-thymosin-alpha1 (TNF-T) of triple-negative locally advanced breast cancer patients

Abstract Background. There is a few data on tumor necrosis factor drugs used in neoadjuvant treatment in locally advanced breast cancer (LABC) patients but there is a clinical need to increase the efficiency of standard therapy and to gain complete regression which is of great importance for survival. The aim of the study was to evaluate the efficacy of recombinant hybrid protein of tumor necrosis factor-alpha-thymosin-alpha1 (TNF-T) in neoadjuvant treatment of triple-negative LABC (TN LABC). Methods. Eligibility criteria included TN LABC of IIB-IIIB stages, ECOG≤1, adequate liver, kidney and bone marrow function, no brain metastases. Recombinant hybrid protein of TNF-T 200000 IU was used peritumorally (injected around the tumor) on D1-5 (30 min before cytostatics injection), combined with standard FAC or PA regimens. Results. 52 women were recruited between April 2012 – October 2013 (mean age 53.3±1.1 years). Group A (20 pts) received recombinant hybrid protein of TNF-T combined to PA (11) and FAC (9) up to 6 courses. Group B (32pts) received standard PA (18) and FAC (14). Tumor response (TR) in Group A was 80% and in Group B 71.9% (p&amp;lt;0.05), including CR 15% and 6.25% correspondingly (p&amp;lt;0.05). After neoadjuvant chemotherapy quantity of CD3+CD8+ cells in group A (PA) was significantly higher than in group B (PA) (31.5±2.8% and 21.7±2.25% correspondingly, p&amp;lt;0.05). Content of B-lymphocytes (CD20) decreased during the treatment in group B (from 15.5±0.53% to 13.7±0.55% (FAC) and from 16.7±0.97% to 12.7±1.0% (PA) correspondingly, p&amp;lt;0.05). CD20 level in group A after the treatment PA+TNF-T was 15,0±1,0%, after FAC+TNF-T was 15,4±1,4% which were different significantly from those in group B (p&amp;lt;0.05). Common toxicities in Group A were: neutropenia Gr1 – 15.9% courses, nausea and vomiting Gr2– 27.2%, polyneuropathy Gr1/2 – 9%. TNF-T-specified toxicities were: hyperthermia Gr1 – 45.4%; local reaction (aula, pain) Gr1 lasted for 24-48 hrs – 90.9%. Group B: neutropenia Gr 1/2 – 21.2%, nausea and vomiting Gr2– 32.5%, polyneuropathy Gr1/2 – 10,6%, diarrhea Gr1 – 4,5. All patients in Group A were operated successfully, no postoperative complications connected with recombinant hybrid protein of TNF-T use were observed. By the time of abstract submission OS in Group A was 28.4±1.1mos, in Group B was 26.1±1.4mos; EFS in Group A was 26.9±1.3mos, in Group B was 22.9±1.2mos (p&amp;lt;0.05). Conclusions. TNF-T injected peritumorally is well-tolerated and allows to enhance an antitumor effect of cytostatics (pCR) which is of great importance for survival in TN LABC. The proposed method of the locally application of recombinant hybrid protein of TNF-T has mainly immunostimulant effect on CD3+CD8+ in patients with PA regimen. Recombinant TNF-T with PA and FAC has protected B-cell component of the immune system from immunosuppressive effect as well. Citation Format: Vladimirova LY, Podzorova NA, Zlatnik EY, Nepomnyaschaya EM, Zakharova NP. Clinical and morphological results of neoadjuvant treatment with tumor necrosis factor-alpha-thymosin-alpha1 (TNF-T) of triple-negative locally advanced breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-14-19.

THE POSSIBILITY OF USING TUMOR NECROSIS FACTOR-THYMOSIN-α1 IN NEOADJUVANT CHEMOTHERAPY OF BREAST CANCER

The article covers the results of clinical application of a domestic drug based on recombinant tumor necrosis factor-alphathymosin-alpha1 (TNF-T) combined with chemotherapy FAC and РА during neoadjuvant treatment of patients with breast cancer IIB-IIIB stage. In contrast to the standard subcutaneous application method, the authors proposed peritumoral administration of TNF-T to maximize the drug’s action directly on the tumor, peritumoral tissue. TNF-T 200000 IU peritumorally on days 1–5 of each treatment course during chemotherapy showed significant increase in objective effect rate, including increase in frequency of complete regressions, and decrease in frequency and intensity of chemotherapy complications. Morphological examination revealed an increase rate of medical pathomorphosis grade III-IV, decreased number of microvessels in tumor in comparison with primary tumors. Study of immune status of patients in dynamics shows that the proposed method of peritumoral application of recombinant hybrid protein of tumor necrosis factor-alpha- thymosin-alpha1 has an immunocorrective effect because of its effect on T- and B-cell component of the immune system.

Recombinant hybrid protein of tumor necrosis factor in neoadjuvant treatment of patients with locally advanced breast cancer.

e12020 Background: There is a clinical need to increase the efficiency of standard neoadjuvant treatment in breast cancer patients. But there is a few data on tumor necrosis factor drugs used in ne...

Evaluation of the immune response induced by a mite derived fusion protein in BALB/c mice

Background The mite Dermatophagoides pteronyssinus is an important source of allergens and a major risk factor for allergic rhinitis and asthma. A more effective and safe way for allergen specific immunotherapy could be using recombinant allergens or their derivatives, such as hybrid molecules. This study was aimed to evaluate the immune response induced in mice by a hybrid recombinant protein, harboring several segments of major allergens of D. pteronyssinus.

Influence of Tumor Necrosis Factor-Alpha-Thymosin-Alpha1 on the Humoral and Cellular Immunity of Patients with Locally Advanced Breast Cancer

ABSTRACT Background The purpose of the study was to assess the effect of recombinant hybrid protein of tumor necrosis factor-alpha-thymosin-alpha1 (TNF-T) on B-cell component of the immune system of patients with locally advanced breast cancer (LABC). Methods Eligibility criteria included LABC of IIB-IIIB stages, ECOG ≤ 1, adequate liver, kidney and bone marrow function, no brain metastases. TNF-T 200000 IU was injected locally around the tumor on D1-5 (30 min before cytostatics injection), combined with standard FAC or PA regimens. Results 56 women were recruited between April 2012 and October 2013 (mean age 53.3 ± 1.1 years). Group A (25 pts) received TNF-T combined with PA (17) and FAC (8) up to 4 courses. Group B (31pts) received standard PA (21) and FAC (10) up to 6 courses. Immune status in both groups of patients was assessed before the treatment and after neoadjuvant chemotherapy (CT). The study showed that levels of T-lymphocytes and CD3 + CD4+ cells did not differ significantly neither in comparison of groups A and B nor in the course of treatment. Quantity of CD3 + CD8+ cells in group A (PA) was significantly higher than in group B (PA) (31.5 ± 2.8% and 21.7 ± 2.25%, respectively, p Conclusions The proposed method of the local application of recombinant hybrid protein of TNF-T has a mainly immunostimulant effect on B-cell component of the immune system. Disclosure All authors have declared no conflicts of interest.