RESUMO
Human epidermal growth factor receptor 2 (HER2) overexpression has been demonstrated in a variety of cancers. Targeted therapy with anti-HER2 monoclonal antibodies (mAbs) has been approved as a therapeutic modality. Despite the efficacy of mAbs in tumor treatment, many patients do not benefit from this therapeutic platform. Fragment crystallizable (Fc) engineering is a common approach to improve the efficacy of therapeutic mAbs. Five Fc-engineered mAbs have so far been approved by FDA. We have recently developed an anti-HER2 bispecific mAb, BiHT, constructed from variable domains of trastuzumab, and our novel humanized anti-HER2 mAb, hersintuzumab. BiHT displayed promising antitumor activity as potently as the combination of the parental mAbs. Here, we aimed to modify the Fc of BiHT to improve its therapeutic efficacy. The Fc-engineered BiHT (MBiHT) bound to recombinant HER2 and its subdomains with an affinity similar to BiHT. It also recognized native HER2 on different cell lines, inhibited their proliferation, downregulated HER2 expression, and suppressed downstream signaling pathways similar to BiHT. Compared with BiHT, MBiHT displayed enhanced antibody-dependent cellular cytotoxicity activity against various tumor cell lines. It also inhibited the growth of ovarian xenograft tumors in nude mice more potently than BiHT. Our findings suggest that MBiHT could be a potent therapeutic candidate for the treatment of HER2-overexpressing cancer types.
Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Camundongos , Animais , Humanos , Camundongos Nus , Trastuzumab , Anticorpos Monoclonais/metabolismo , Receptor ErbB-2 , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. Methods: Methods: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most critical quality attributes (CQAs) and effects of process parameters on these quality attributes. Results: Results: The solubilization of IBs was optimized by design of experiment on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in anion exchange chromatography that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate®, as the reference product. Conclusions: Conclusion: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.
Assuntos
Escherichia coli , Corpos de Inclusão , Proteínas Recombinantes de Fusão , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Anti-TNF inhibitors exert their therapeutic effect by inhibition of the excessive amounts of TNF-α within the body. Recombinant TNF-α should be produced in a soluble refolded form to investigate the effectiveness and efficiency of anti-TNF-α compounds. In this research, the designed cassette was subcloned in the pET28a expression vector and expressed in E. coli BL21 (DE3). The identity of the protein was confirmed through SDS-PAGE and Western blotting. After optimizing expression conditions, protein purification was performed using native Ni-NTA affinity chromatography. The biological activity of the soluble recombinant TNF-α was investigated using MTT assay. Also, the affinity of an anti-TNF-α agent, Altebrel, was investigated against the expressed protein through ELISA. Optimization of TNF-α expression conditions represented that the highest expression could be achieved at 37 °C using 0.5 mM IPTG 6 h post-induction. The recombinant protein represented an inhibitory effect on the L929 murine fibroblast cell line and was successfully detected by Altebrel in ELISA. Binding kinetics were also studied using Cimzia as an anti-TNF-α molecule and 7.2 E-13M was calculated as the equilibrium dissociation constant value (KD). The significant expression level of the recombinant protein in the soluble form, its high purity, and assessment of its biological activity showed that the expressed protein could be used in tests of ELISA and MTT to assess the activity of anti-TNF-α agents.
Assuntos
Escherichia coli/genética , Proteínas Recombinantes de Fusão , Fator de Necrose Tumoral alfa , Animais , Linhagem Celular , Cromatografia de Afinidade , Meios de Cultura/metabolismo , Humanos , Camundongos , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Certolizumab pegol is a Fab' antibody fragment for treatment of rheumatoid arthritis and Crohn's disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab' molecule. It was observed that PASylation influenced the properties of the Fab' molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab' antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab' control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.
Assuntos
Certolizumab Pegol , Polietilenoglicóis , Animais , Artrite Reumatoide/tratamento farmacológico , Linhagem Celular , Certolizumab Pegol/química , Certolizumab Pegol/farmacocinética , Certolizumab Pegol/farmacologia , Doença de Crohn/tratamento farmacológico , Feminino , Meia-Vida , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologiaRESUMO
OBJECTIVE: Heart dysfunctions are the major complications of trastuzumab in patients with Human Epidermal growth factor Receptor-2 (HER2)-positive breast cancers. METHODS: In this study, the cytotoxicity of trastuzumab on H9c2 cardiomyoblasts was demonstrated, and the proteome changes of cells were investigated by a tandem mass tagging quantitative approach. The Differentially Abundant Proteins (DAPs) were identified and functionally enriched. RESULTS: We determined that carvedilol, a non-selective beta-blocker, could effectively inhibit trastuzumab toxicity when administrated in a proper dose and at the same time. The proteomics analysis of carvedilol co-treated cardiomyoblasts showed complete or partial reversion in expressional levels of trastuzumab-induced DAPs. CONCLUSION: Downregulation of proteins involved in the translation biological process is one of the most important changes induced by trastuzumab and reversed by carvedilol. These findings provide novel insights to develop new strategies for the cardiotoxicity of trastuzumab.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Antineoplásicos Imunológicos/toxicidade , Carvedilol/farmacologia , Mioblastos Cardíacos/efeitos dos fármacos , Proteoma/metabolismo , Trastuzumab/toxicidade , Antagonistas Adrenérgicos beta/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Cardiomiopatias/prevenção & controle , Carvedilol/uso terapêutico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Regulação para Baixo , Feminino , Humanos , Mioblastos Cardíacos/metabolismo , Mioblastos Cardíacos/patologia , Proteômica , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêuticoRESUMO
Cardiac dysfunction is a major side effect of trastuzumab therapy for patients with HER2-positive breast cancer. Beta blockers, such as carvedilol, have been used for protection of trastuzumab cardiotoxicity but there is no definitive conclusive clinical report on their efficacy. In the present study, the preservability effects of carvedilol on trastuzumab-induced left ventricular (LV) dysfunction and the reversibility of trastuzumab-induced cardiotoxicity were evaluated in Wistar rats by echocardiography method. We showed that trastuzumab treatment of rats could induce the LV dysfunction through increasing the LV internal systolic diameter (LVIDs), increasing the end-systolic volume (ESV), decreasing the ejection fraction (EF), and decreasing the fractional shortening (FS). These parameters were not reversed after 14 days of stopping trastuzumab administration. Interestingly, carvedilol improved LVIDs, ESV, EF, and FS. Collectively, the results of this study have verified clinical observations which simultaneously administration of carvedilol may be considered as a possible therapeutic strategy to prevent trastuzumab-mediated LV dysfunction.
Assuntos
Antagonistas Adrenérgicos beta/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Carvedilol/administração & dosagem , Trastuzumab/efeitos adversos , Disfunção Ventricular Esquerda/prevenção & controle , Administração Oral , Animais , Cardiotoxicidade/diagnóstico , Cardiotoxicidade/etiologia , Cardiotoxicidade/prevenção & controle , Modelos Animais de Doenças , Ecocardiografia , Feminino , Humanos , Injeções Intraperitoneais , Pessoa de Meia-Idade , Ratos , Volume Sistólico/efeitos dos fármacos , Trastuzumab/administração & dosagem , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/diagnósticoRESUMO
The crucial role of VEGF receptor 2 (VEGFR2) signaling in the angiogenesis and metastasis of solid tumors has prompted the development of inhibitors with minimal bystander effects. Recently, Adnectin C has attracted attention for cancer treatment. To overcome the problematic properties of Adnectin, a novel form of Adnectin C has been designed by its fusion to a biodegradable polymeric peptide containing Pro/Ala/Ser (PAS) repetitive residues. E. coli-expressed recombinant fused and unfused proteins were compared in terms of bioactivity, physicochemical, and pharmacokinetic properties using standard methods. Dynamic light scattering (DLS) analysis of PASylated adnectin C revealed an approximate 2-fold increase in particle size with a slight change in the net charge. Additionally, fusion of the PAS sequence improved its stability against the growth of thermo-induced aggregated forms. The high receptor-binding and improved binding kinetic parameters of PASylated Adnectin C was confirmed by ELISA and surface plasmon resonance assays, respectively. Pharmacokinetic studies showed a noticeable increase in the terminal half-life of Adnectin C-PAS#1(200) by a factor of 4.57 after single dose by intravenous injection into female BALB/c mice. The results suggest that PASylation could offer a superior delivery strategy for developing Adnectin-derived drugs with improved patient compliance.
Assuntos
Fibronectinas/farmacologia , Fibronectinas/farmacocinética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Alanina , Animais , Escherichia coli , Feminino , Fibronectinas/isolamento & purificação , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/farmacologia , Prolina , Domínios Proteicos/fisiologia , Engenharia de Proteínas/métodos , Serina , Ressonância de Plasmônio de Superfície/métodos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Listeria monocytogenes/imunologia , Ativação Linfocitária/imunologia , Fator Tu de Elongação de Peptídeos/imunologia , Proteínas de Bactérias/imunologia , Linhagem Celular , Células Dendríticas/citologia , Citometria de Fluxo , Humanos , Imunoterapia/métodos , Teste de Cultura Mista de Linfócitos , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG4 did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG4 levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG4 in this type of recombinant cells.
RESUMO
BACKGROUND: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. CONCLUSION/SIGNIFICANCE: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.
Assuntos
Cisteína Proteases/imunologia , Leishmania/imunologia , Vacinas contra Leishmaniose/imunologia , Proteínas e Peptídeos Salivares/imunologia , Vacinação/métodos , Animais , Anticorpos Antiprotozoários/sangue , Cisteína Proteases/biossíntese , Cisteína Proteases/genética , Modelos Animais de Doenças , Feminino , Leishmaniose/prevenção & controle , Vacinas contra Leishmaniose/administração & dosagem , Vacinas contra Leishmaniose/genética , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Psychodidae , Proteínas e Peptídeos Salivares/biossíntese , Proteínas e Peptídeos Salivares/genética , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Rabies virus invades the nervous system, induces neuronal dysfunction and causes death of the host. The disruption of the cytoskeletal integrity and synaptic structures of the neurons by rabies virus has been postulated as a possible basis for neuronal dysfunction. In the present study, a two-dimensional electrophoresis/mass spectrometry proteomics analysis of neuroblastoma cells revealed a significant effect of a virulent strain of rabies virus on the host cytoskeleton related proteins which was quite different from that of an attenuated strain. Vimentin, actin cytoplasmic 1 isoform, profilin I, and Rho-GDP dissociation inhibitor were host cell cytoskeletal related proteins changed by the virulent strain. The proteomics data indicated that the virulent strain of rabies virus induces significant expression changes in the vimentin and actin cytoskeleton networks of neurons which could be a strong clue for the relation of cytoskeletal integrity distraction and rabies virus pathogenesis. In addition, the expression alteration of other host proteins, particularly some structural and regulatory proteins may have potential roles in rabies virus pathogenesis.
Assuntos
Proteínas do Citoesqueleto/análise , Expressão Gênica , Interações Hospedeiro-Patógeno , Neurônios/química , Neurônios/virologia , Vírus da Raiva/crescimento & desenvolvimento , Animais , Linhagem Celular , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Camundongos , ProteômicaRESUMO
BACKGROUND: We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. METHODOLOGY/PRINCIPAL FINDINGS: Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b(-CTE), pcDNA-cpa/b(-CTE), cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b(-CTE) showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. CONCLUSIONS/SIGNIFICANCE: This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb(-CTE) cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Cisteína Endopeptidases/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Lipossomos/administração & dosagem , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Animais , Cisteína Endopeptidases/genética , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Feminino , Leishmania major/genética , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Proteínas de Protozoários/genética , Vacinas Protozoárias/genética , Doenças dos Roedores/prevenção & controle , Deleção de Sequência , Células Th1/imunologiaRESUMO
Earlier generations of Leishmania vaccines have reached the third-phase of clinical trials, however none of them have shown adequate efficacy due to lack of an appropriate adjuvant. In this study, cationic solid lipid nanoparticles (cSLNs) were used to formulate three pDNAs encoding L. major cysteine proteinase type I (cpa), II (cpb) and III (cpc). BALB/c mice were immunized twice with a 3-week interval, with SLN-pcDNA-cpa/b/c, pcDNA-cpa/b/c, SLN, SLN-pcDNA and PBS. Footpad assessments, parasite burden, cytokine and antibody responses were evaluated. Mice vaccinated with SLN-pcDNA-cpa/b/c significantly (p<0.05) showed higher protection levels with specific Th1 immune response development compared to other groups. This is the first report demonstrating cSLNs as a nanoscale vehicle boosting immune response quality and quantity; in a designable trend. The nanomedical feature of this novel formulation can be applied for wide-spread use in genetic vaccination against leishmaniasis, which is currently managed only through relatively ineffectual therapeutic regimens.
Assuntos
Cisteína Endopeptidases/genética , Leishmania major/enzimologia , Leishmaniose Cutânea/prevenção & controle , Nanopartículas/química , Vacinas de DNA/administração & dosagem , Vacinas de DNA/uso terapêutico , Animais , Formação de Anticorpos , Feminino , Imunoglobulina G/imunologia , Interferon gama/imunologia , Leishmaniose Cutânea/enzimologia , Lipídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host-parasite interactions at the cellular level.