RESUMO
Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.
Assuntos
Adjuvantes Imunológicos/farmacocinética , Timalfasina/farmacocinética , Acetilação , Acetiltransferases/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Feminino , Meia-Vida , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Neoplasias/tratamento farmacológico , Peptídeos/química , Proteólise , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/ultraestrutura , Proteínas Ribossômicas/metabolismo , Timalfasina/sangue , Timalfasina/química , Timalfasina/genética , Viroses/tratamento farmacológico , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND: Thymosin alpha 1 (Tα1) had been used in the treatment of viral infections as an immune response modifier for many years. However, clinical benefits and the mechanism of Tα1 treatment for COVID-19 patients are still unclear. METHODS: We retrospectively reviewed the clinical outcomes of 76 severe COVID-19 cases admitted to 2 hospitals in Wuhan, China, from December 2019 to March 2020. The thymus output in peripheral blood mononuclear cells from COVID-19 patients was measured by T-cell receptor excision circles (TRECs). The levels of T-cell exhaustion markers programmed death-1 (PD-1) and T-cell immunoglobulin and mucin domain protein 3 (Tim-3) on CD8+ T cells were detected by flow cytometry. RESULTS: Compared with the untreated group, Tα1 treatment significantly reduced the mortality of severe COVID-19 patients (11.11% vs 30.00%, P = .044). Tα1 enhanced blood T-cell numbers in COVID-19 patients with severe lymphocytopenia. Under such conditions, Tα1 also successfully restored CD8+ and CD4+ T-cell numbers in elderly patients. Meanwhile, Tα1 reduced PD-1 and Tim-3 expression on CD8+ T cells from severe COVID-19 patients compared with untreated cases. It is of note that restoration of lymphocytopenia and acute exhaustion of T cells were roughly parallel to the rise of TRECs. CONCLUSIONS: Tα1 treatment significantly reduced mortality of severe COVID-19 patients. COVID-19 patients with counts of CD8+ T cells or CD4+ T cells in circulation less than 400/µL or 650/µL, respectively, gained more benefits from Tα1. Tα1 reversed T-cell exhaustion and recovered immune reconstitution through promoting thymus output during severe acute respiratory syndrome-coronavirus 2 infection.
Assuntos
COVID-19/mortalidade , Linfopenia/metabolismo , SARS-CoV-2/patogenicidade , Timalfasina/metabolismo , Adulto , Idoso , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , COVID-19/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Timalfasina/genética , Timo/metabolismoRESUMO
The use of interferon α-2 in combination with thymosin α-1 shows higher anti-cancer effect in comparison when both are used individually because of their synergistic effects. In this study we produced an important human interferon α-2-thymosin α-1 (IFNα2-Tα1) fusion protein with probable pharmaceutical properties coupled to its high-level expression, characterization, and study of its biological activity. The IFNα2-Tα1 fusion gene was constructed by over-lap extension PCR and expressed in Escherichia coli expression system. The expression of IFNα2-Tα1 fusion protein was optimized to higher level and its maximum expression was obtained in modified terrific broth medium when lactose was used as inducer. The fusion protein was refolded into its native biologically active form with maximum yield of 83.14% followed by purification with â¼98% purity and 69% final yield. A band of purified IFNα2-Tα1 fusion protein equal to â¼23 kDa was observed on 12 % SDS-PAGE gel. The integrity of IFNα2-Tα1 fusion protein was confirmed by western blot analysis and secondary structure was assessed by CD spectroscopy. When IFNα2-Tα1 fusion protein was subjected to its biological activity analysis it was observed that it exhibits both IFNα2 & Tα1 activities as well as significantly higher anticancer activity as compared to IFNα-2 alone.
Assuntos
Interferon-alfa , Proteínas Recombinantes de Fusão , Timalfasina , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interferon-alfa/química , Interferon-alfa/genética , Interferon-alfa/isolamento & purificação , Interferon-alfa/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Timalfasina/química , Timalfasina/genética , Timalfasina/isolamento & purificação , Timalfasina/farmacologiaRESUMO
Thymosin alpha 1 (Tα1) is a biological response modifier that has been introduced into markets for treating several diseases. Given the short serum half-life of Tα1 and the rapid development of Fc fusion proteins, we used genetic engineering method to construct the recombinant plasmid to express Tα1-Fc (Fc domain of human IgG4) fusion protein. A single-factor experiment was performed with different inducers of varying concentrations for different times to get the optimal condition of induced expression. Pure proteins higher than 90.3% were obtained by using 5 mM lactose for 4 h with a final production about 160.4 mg/L. The in vivo serum half-life of Tα1-Fc is 25 h, almost 13 times longer than Tα1 in mice models. Also, the long-acting protein has a stronger activity in repairing immune injury through increasing number of lymphocytes. Tα1-Fc displayed a more effective antitumor activity in the 4T1 and B16F10 tumor xenograft models by upregulating CD86 expression, secreting IFN-γ and IL-2, and increasing the number of tumor-infiltrating CD4+ T and CD8+ T cells. Our study on the novel modified Tα1 with the Fc segment provides valuable information for the development of new immunotherapy in cancer.
Assuntos
Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Fragmentos Fc das Imunoglobulinas/imunologia , Melanoma/etiologia , Melanoma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Timalfasina/metabolismo , Animais , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Meia-Vida , Humanos , Hidrocortisona/sangue , Hidrocortisona/metabolismo , Hospedeiro Imunocomprometido , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunomodulação/efeitos dos fármacos , Masculino , Melanoma/patologia , Melanoma Experimental , Camundongos , Prognóstico , Ratos , Proteínas Recombinantes de Fusão/sangue , Timalfasina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Thymosins have been extracted, characterized, and identified from Thymus. The Thymosins are hormones whose therapeuric applications have seen a recent increase. The action of Thymosin α1 is based on the stimulation of the immune response with a large number of results in a variety of pathologies. The absence of a specific receptor prompted us to investigate the direct interaction with membranes, particularly those exposing phosphatidylserine thus contributing to assess the Thymosin α1's pleiotropy. AREAS COVERED: The interaction with membranes has been studied with a number of models indicating that Thymosin α1 interacts preferentially with negative regions of the membrane (SDS mixed with dodecylphosphocholine) or, better, with vesicles of dipalmitoylphosphatidylcholine with exposed phosphatidylserine. EXPERT OPINION: The study of the role of the membrane in the mechanism of action of Thymosin α1 indicated that probably the first interaction occurs at the membrane level with recognition of negative surface due to the phosphatidylserine exposure. Upon assuming a conformation, with two helices with a disordered tract in between, it diffuses on the membrane surface by lateral diffusion. Then the interaction with membrane receptor(s) causes a membrane complex to be formed, with an activation of a signalling cascade. This can be considered the basis of its pleiotropy. Differences in structuration mechamism of Thymosin ß4 was outlined.
Assuntos
Membrana Celular/metabolismo , Timalfasina/química , Timalfasina/metabolismo , Animais , Humanos , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade por Substrato , Timalfasina/genéticaRESUMO
INTRODUCTION: Cystic fibrosis (CF) is a multisystem illness caused by abnormalities in the CF transmembrane conductance regulator (CFTR) gene and protein. CFTR is an ion channel regulating transport of chloride, bicarbonate, and water, and influencing sodium resorption. It is inherited as an autosomal recessive disorder, and with about 70,000 CF patients worldwide, it is the most common life shortening disease among persons of European descent. CFTR disease-causing mutations have been organized into six classes. : AREAS COVERED: Recently, small molecule targeted therapy for specific classes of CFTR abnormalities have included CFTR correctors that decrease protein degradation and CFTR potentiators that increase channel open probability enhancing chloride transport. EXPERT OPINION: Although there are many novel medications in preclinical and clinical testing, there is need for safe and effective CFTR modulating drugs and immunomodulatory medications to decrease the abundant neutrophilic inflammation response in the airway without unwanted adverse effects. Thymosin alpha 1 treatment of airway cells isolated from phe508del CF patients and from CF knockout mice, decreased inflammation, increased CFTR maturation, and facilitated translocation of CFTR protein to the plasma membrane increasing channel activity. If similar results are seen in humans with CF, thymosin alpha 1 has the unique potential to be a single molecule therapy for treating CF airway disease.
Assuntos
Fibrose Cística/tratamento farmacológico , Descoberta de Drogas , Necessidades e Demandas de Serviços de Saúde , Timalfasina/uso terapêutico , Animais , Fibrose Cística/epidemiologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Descoberta de Drogas/normas , Humanos , Inflamação/genética , Inflamação/prevenção & controle , Camundongos , Camundongos Knockout , Mutação , Timalfasina/genéticaRESUMO
Thymosin α1 (Tα1) and bursin-like peptide (BLP) are both immunopotentiators. In order to investigate adjuvant of thymosin α1-bursin-like peptide (Tα1-BLP), we cloned the gene of Tα1-BLP and provided evidence that the gene of Tα1-BLP in a recombinant prokaryotic expression plasmid was successfully expressed in E. coli BL21. To evaluate the immune adjuvant properties of Tα1-BLP, chickens were immunized with Tα1-BLP combined with H9N2 avian influenza whole-inactivated virus (WIV). The titers of HI antibody, antigen-specific antibodies, AIV-neutralizing antibodies, levels of Th1-type cytokines (IFN-γ) and Th2-type cytokines (IL-4) and lymphocyte proliferation responses were determined. What's more, the viral loads and pathologic changes of lung tissue were observed by virus challenge experiment and HE staining to evaluate the immune protection of chickens. We found that Tα1-BLP enhanced HI antibody and antigen-specific IgG antibodies titers, increased the level of AIV-neutralizing antibodies, induced the secretion of Th1- and Th2-type cytokines, and promoted the proliferation of T and B lymphocyte, Furthermore, virus challenge experiment and HE staining confirmed that Tα1-BLP contributed to inhibition replication of the virus from chicken lungs and protected the lungs from damage. Altogether, this study suggested that Tα1-BLP is a novel adjuvant suitable for H9N2 avian influenza vaccine.