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1.
Life Sci ; 265: 118866, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33301810

RESUMO

AIMS: GnRH-DFF40 (gonadotropin releasing hormone-DNA fragmentation factor 40) humanized recombinant immunotoxin serves as a prospective candidate for targeted therapy of malignancies with over-expressed gonadotropin releasing hormone receptor (GnRHR). In this study, we attempted to generate a GnRH-based chimeric protein composed of human DFF40 fused with GnRH which encodes an apoptotic nuclease and specifically targets cancer cells displaying GnRH receptor overexpression. MATERIALS AND METHODS: A codon optimized, synthetic GnRH-DFF40 fusion gene and its single counterpart (DFF40) were constructed in pET28a expression vector. Cytotoxicity of these expressed proteins were evaluated on three breast cancer cell lines (MCF7, MDA-MB231, and SKBR3). The stability and biological activity of the recombinant proteins were investigated in the treated cell line and cell-free system. Also, the ability of this fusion and its single form in inducing apoptosis, and inhibiting metastasis and migration were evaluated by flow cytometry, migration assay and wound healing analysis, respectively. In silico analyses were also done to understand the specific interactions between GnRH and its receptor. KEY FINDINGS: GnRH-DFF40 fusion protein and DFF40 were successfully expressed. The purified chimeric protein showed dose-dependent cytotoxicity against all three cell lines. The recombinant fusion protein was biologically active with nucleolytic functionality and apoptosis induction ability. Moreover, the fusion could inhibit the invasion property of MDA-MB-231 cells. In silico analysis also showed that four residues from GnRH domain and 11 GnRHR residues had the most interaction sites for specific targeted delivery of the immunotoxin in cancer cells. SIGNIFICANCE: Fusion construct could be a prospective candidate for targeted therapy of cancers upregulating GnRH receptor.


Assuntos
Neoplasias da Mama/terapia , Desoxirribonucleases/genética , Imunotoxinas/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores LHRH/genética , Proteínas Recombinantes de Fusão/farmacologia , Apoptose/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sistema Livre de Células , Simulação por Computador , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Humanos , Imunotoxinas/administração & dosagem , Células MCF-7 , Terapia de Alvo Molecular , Proteínas Recombinantes de Fusão/administração & dosagem
2.
Toxicol Lett ; 335: 71-81, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33122006

RESUMO

Destruction of liver sinusoidal endothelial cells (LSECs) is an initial event in sinusoidal obstruction syndrome (SOS) that leads to accumulation of platelets in the liver. Herein, we explored the role of platelets during progression of experimental SOS induced by monocrotaline (MCT) in mice. Depletion of platelets using an anti-CD41 antibody or anti-thrombocyte serum exacerbated MCT-induced liver injury in C57BL/6 mice, as indicated by an increase in the alanine transaminase (ALT) level, which was associated with hemorrhagic necrosis. Thrombocytosis induced by thrombopoietin (TPO) or the TPO receptor agonist romiplostim (ROM) attenuated MCT-induced liver injury, as evidenced by lower levels of ALT and mRNA encoding matrix metalloproteinase (MMP) 9, and higher levels of mRNA encoding vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3. The level of activated hepatic platelets was higher in TPO- and ROM-treated mice than in saline-treated mice. Co-culture with a high number of platelets increased the viability of LSECs and their mRNA levels of CD31, VEGFR2, and VEGFR3, and decreased their mRNA level of MMP9. The level of VEGF-A was increased in the culture medium of LSECs co-cultured with platelets. These results indicate that platelets attenuate MCT-induced liver injury by minimizing damage to LSECs.


Assuntos
Plaquetas/efeitos dos fármacos , Doença Hepática Crônica Induzida por Substâncias e Drogas/sangue , Hepatopatia Veno-Oclusiva/sangue , Hepatopatia Veno-Oclusiva/induzido quimicamente , Monocrotalina/toxicidade , Trombocitose/sangue , Animais , Plaquetas/citologia , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Crônica Induzida por Substâncias e Drogas/prevenção & controle , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Hepatopatia Veno-Oclusiva/prevenção & controle , Testes de Função Hepática , Masculino , Camundongos Endogâmicos C57BL , Contagem de Plaquetas , Receptores Fc , Receptores de Trombopoetina/agonistas , Proteínas Recombinantes de Fusão/farmacologia , Trombocitose/induzido quimicamente , Trombopoetina/farmacologia
3.
PLoS One ; 15(10): e0241230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33095843

RESUMO

Optical coherence tomography angiography (OCTA) is a novel, non-invasive imaging tool used to detect vascular flow. The absence of a flow signal in OCTA in polyps revealed by indocyanine green angiography (ICGA) in patients with polypoidal choroidal vasculopathy (PCV) may indicate slow or compromised filling of blood flow from choroidal vessels. Naïve patients with PCV treated with intravitreal injections of aflibercept (IVI-A) were enrolled in this study to validate the hypothesis that baseline flow may affect the outcome of polyp regression in ICGA. The flow signal of polyps in OCTA was detected by manual segmentation in the corresponding location by ICGA. Polyps were defined as high-flow if both OCTA and ICGA showed positive findings, and low-flow if OCTA showed a negative flow signal in 3 consecutive horizontal scans at the polyp area shown in ICGA. A total of 24 polyps were identified in 13 PCV patients at baseline. Of these 24 polyps, 22 (91.7%) were high-flow and 2 (8.3%) were low-flow. After 3 monthly IVI-A, all low-flow polyps had complete regression in ICGA. Among 17 (77%) high-flow polyps at baseline that had regression after treatment, 10 (58.8%) became low-flow, while 5 (22.7%) persistent polyps remained high-flow. Flow signal of polyps as detected by OCTA could be a predictive factor for treatment response in patients with PCV. Monitoring changes in flow signal after treatment is clinically relevant.


Assuntos
Pólipos/tratamento farmacológico , Fluxo Sanguíneo Regional , Processamento de Sinais Assistido por Computador , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Neovascularização de Coroide/diagnóstico por imagem , Neovascularização de Coroide/tratamento farmacológico , Feminino , Angiofluoresceinografia , Humanos , Verde de Indocianina , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Pólipos/diagnóstico por imagem , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fluxo Sanguíneo Regional/efeitos dos fármacos , Tomografia de Coerência Óptica , Resultado do Tratamento , Fatores de Crescimento do Endotélio Vascular/metabolismo
4.
Int J Biol Macromol ; 165(Pt B): 1626-1633, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33080267

RESUMO

Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for SARS-CoV-2, and recombinant ACE2 decoys are being evaluated as new antiviral therapies. We designed and tested an antibody-like ACE2-Fc fusion protein, which has the benefit of long pharmacological half-life and the potential to facilitate immune clearance of the virus. Out of a concern that the intrinsic catalytic activity of ACE2 may unintentionally alter the balance of its hormonal substrates and cause adverse cardiovascular effects in treatment, we performed a mutagenesis screening for inactivating the enzyme. Three mutants, R273A, H378A and E402A, completely lost their enzymatic activity for either surrogate or physiological substrates. All of them remained capable of binding SARS-CoV-2 and could suppress the transduction of a pseudotyped virus in cell culture. This study established new ACE2-Fc candidates as antiviral treatment for SARS-CoV-2 without potentially harmful side effects from ACE2's catalytic actions toward its vasoactive substrates.


Assuntos
Fragmentos Fc das Imunoglobulinas , Proteínas Recombinantes de Fusão , /metabolismo , Substituição de Aminoácidos , /genética , Animais , /metabolismo , Linhagem Celular , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação de Sentido Incorreto , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia
5.
Minerva Med ; 111(5): 467-477, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32955827

RESUMO

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy, characterized by poor prognosis if treated with conventional therapy. Allogenic hematologic stem cell transplant can improve survival and can be curative, but it is available in a small percentage of patients given that the median age at diagnosis is 70 years. In this scenario it is assumed that only the development of precision medicine-driven therapy will change BPDCN patient prognosis. CD123 (the α-subunit of interleukin (IL)-3 receptor) is over-expressed on BPDCN cells surface and seems to be the ideal marker to develop antibody-based therapies. Tagraxofusp (Elzonris®), a recombinant immunotoxin consisting of human interleukin-3 fused to a truncated diphtheria toxin, has been approved by FDA in December 2018 for the treatment of BPDCN in adult and pediatric patients. tagraxofusp has shown promising clinical activity, with a high overall response rate and quite manageable safety profile even in elderly patients. It seems to improve overall survival too, but comparative trials are necessary to confirm this. Adverse events are commonly reported and the most important are transaminitis, thrombocytopenia and capillary leak syndrome (CLS). Therefore, to prevent the onset of severe CLS is recommended to reserve tagraxofusp for patients with preserved hepatic and cardiac functions, and to strictly observe serum albumin level. Further studies are required to resolve many several unanswered questions about tagraxofusp. In this review, we will resume and discuss pharmacological characteristic of tagraxofusp, results of clinical trials leading to its approval by FDA in 2018 and future perspectives about its use in BPDCN and other hematological malignancies.


Assuntos
Células Dendríticas , Neoplasias Hematológicas/tratamento farmacológico , Subunidade alfa de Receptor de Interleucina-3/antagonistas & inibidores , Medicina de Precisão , Proteínas Recombinantes de Fusão/farmacologia , Idoso , Síndrome de Vazamento Capilar/induzido quimicamente , Síndrome de Vazamento Capilar/prevenção & controle , Criança , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Rotulagem de Medicamentos , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/patologia , Humanos , Prognóstico , Proteínas Recombinantes de Fusão/efeitos adversos , Trombocitopenia/induzido quimicamente , Transaminases/sangue
6.
Int J Hematol ; 112(6): 787-794, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32876852

RESUMO

Eltrombopag (EPAG) and romiplostim (ROM), thrombopoietin receptor-agonists with demonstrated efficacy against aplastic anemia (AA) in prospective controlled studies, were authorized in Japan for use in adults with aplastic anemia in 2017 and 2019, respectively. So far, no data are available on the potential contribution of switching from ROM to EPAG or vice versa in terms of efficacy or tolerance. Efficacies and tolerance profiles of ten patients, who failed to respond to the maximum dose of EPAG and then switched to ROM, were evaluated. All ten patients received a maximum dose of ROM (20 µg/kg/week). At a median follow-up of twelve months, seven of ten patients (70%) had achieved either neutrophil, erythroid, or platelet response, including one complete response. No patients showed platelet count fluctuations that were reported during ROM treatment for immune thrombocytopenia. In univariate analysis of the relationship between efficacy and demographics, the response had a correlation with neither factors. None of the patients stopped the ROM treatment because of adverse events. Although a larger number of patients and a longer follow-up period are needed to confirm our findings, our results show the efficacy of ROM in patients with EPAG-refractory AA.


Assuntos
Anemia Aplástica/tratamento farmacológico , Anemia Refratária/tratamento farmacológico , Benzoatos , Tolerância a Medicamentos , Hidrazinas , Pirazóis , Receptores Fc/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Trombopoetina/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Aplástica/sangue , Substituição de Medicamentos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Estudos Prospectivos , Receptores de Trombopoetina/agonistas , Proteínas Recombinantes de Fusão/farmacologia , Estudos Retrospectivos , Trombopoetina/farmacologia
7.
Biosci Rep ; 40(9)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32808659

RESUMO

Endotoxemia-induced acute kidney injury (AKI) is a common clinical condition that lacks effective treatments. Elabela (ELA) is a recently discovered kidney peptide hormone, encoded by the gene apela, and has been reported to improve cardio-renal outcomes in sepsis. However, ELA is a small peptide and is largely unsuitable for clinical use because of its short in vivo half-life. In the present study, we evaluated the potential renoprotective effects of a long-acting constant fragment (Fc)-ELA fusion protein in liposaccharide (LPS)-induced AKI in mice. LPS administration in mice for 5 days greatly lowered the gene expression of apela and impaired kidney function, as evidenced by elevated serum creatinine and the ratio of urine protein to creatinine. In addition, renal inflammation and macrophage infiltration were apparent in LPS-challenged mice. Treatment with the Fc-ELA fusion protein partially restored apela expression and attenuated the kidney inflammation. Moreover, LPS treatment induced reactive oxygen species (ROS) production and apoptosis in kidney HK-2 cells as well as in the mouse kidney, which were mitigated by ELA or Fc-ELA treatment. Finally, we found that ELA promoted the survival of HK-2 cells treated with LPS, and this action was abolished by LY204002, a PI3K/Akt inhibitor. Collectively, we have demonstrated that the Fc-ELA fusion protein has significant renoprotective activities against LPS-induced AKI in mice.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Fragmentos Fc das Imunoglobulinas/genética , Lipopolissacarídeos/toxicidade , Hormônios Peptídicos/genética , Proteínas Recombinantes de Fusão/farmacologia , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Camundongos Endogâmicos C57BL , Hormônios Peptídicos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética
8.
J Pharmacol Exp Ther ; 375(1): 69-75, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32669367

RESUMO

Therapeutic fragmented antibodies show a poor pharmacokinetic profile that leads to frequent high-dose administration. In the current study, for the first time, a novel proline, alanine, serine (PAS) repeat sequence called PAS#208 was designed to extend the plasma half-life of a nanosized anti-vascular endothelial growth factor-A single-domain antibody. Polyacrylamide gel electrophoresis, circular dichroism, dynamic light scattering, and electrophoretic light scattering were used to assess the physicochemical properties of the newly designed PAS sequence. The effect of PAS#208 on the biologic activity of a single-domain antibody was studied using an in vitro proliferation assay. The pharmacokinetic parameters, including terminal half-life, the volume of distribution, elimination rate constant, and clearance, were determined in mice model and compared with the native protein and PAS#1(200) sequence. The novel PAS repeat sequence showed comparable physicochemical, biologic, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The PAS#208 increased the hydrodynamic radius and decreased significantly the electrophoretic mobility of the native protein without any change in zeta potential. Surprisingly, the fusion of PAS#208 to the single-domain antibody increased the binding potency. In addition, it did not alter the biologic activity and did not show any cytotoxicity on the normal cells. The PAS#208 sequence improved the terminal half-life (14-fold) as well as other pharmacokinetic parameters significantly. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of therapeutic fragmented antibodies. SIGNIFICANCE STATEMENT: In the current study, a new proline, alanine, serine (PAS) sequence was developed that showed comparable physicochemical, biological, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of recombinant small proteins.


Assuntos
Alanina/genética , Prolina/genética , Serina/genética , Anticorpos de Domínio Único/sangue , Fator A de Crescimento do Endotélio Vascular/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Células HEK293 , Meia-Vida , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/farmacologia , Distribuição Tecidual
9.
Sci Rep ; 10(1): 12164, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699335

RESUMO

Cryptdins are disulfide-rich cationic antimicrobial peptides secreted by mouse Paneth cells and are known to exhibit potent antimicrobial activity against various deadly pathogens. Keeping in view the extremely low yield obtained from mouse Paneth cells and high cost of synthetic peptide(s), herein, we have attempted to produce cryptdin-2 in Escherichia coli using recombinant technology. To avoid lethal effects of peptide on the host cells, cryptdin-2 was expressed as a fusion protein with thioredoxin as fusion partner which yielded 40 mg/L protein in the soluble fraction. Subsequently, mature cryptdin-2 was cleaved from the fusion partner and purified by cation exchange chromatography. Since conjugation of poly(ethylene) glycol (PEG) has been known to improve the biological properties of biomolecules, therefore, we further attempted to prepare PEG-conjugated variant of cryptdin-2 using thiol specific PEGylation. Though the antimicrobial activity of PEGylated cryptdin-2 was compromised to some extent, but it was found to have enhanced serum stability for longer duration as compared to its un-modified forms. Also, it was found to exhibit reduced toxicity to the host cells. Further, its synergism with gentamicin suggests that PEGylated cryptdin-2 can be used with conventional antibiotics, thereby indicating its possibility to be used as an adjunct therapy.


Assuntos
Defensinas/farmacologia , Polietilenoglicóis/química , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Defensinas/química , Defensinas/genética , Defensinas/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Células RAW 264.7 , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia
10.
Exp Hematol ; 87: 33-41.e4, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32619459

RESUMO

Multiple myeloma remains a hard-to-treat cancer as all patients eventually progress because of drug resistance. Thus, there is a need for novel and non-cross-resistant treatment options, and we aimed to address this issue by introducing a new immuno-oncology drug (APO010) in multiple myeloma treatment. APO010 is a hexameric Fas-ligand that mimics cytotoxic T-lymphocyte signaling through the Fas-receptor to induce apoptosis. APO010 is currently in clinical trials with multiple myeloma patients. Thus, an understanding of the mechanisms contributing to resistance to APO010 will be essential for future clinical studies with APO010, and it might be possible to develop strategies to circumvent this resistance. We developed APO010-resistant variants of human multiple myeloma cell lines (LP1, MOLP-8, and KMS-12-BM) and a human Burkitt's lymphoma cell line (Raji) by exposing the cells to gradually increasing concentrations of APO010 over a period of 6-12 months. The resistant cell lines were characterized on the basis of immunocytochemistry, Fas-receptor protein expression, mRNA expression analysis, and pathway analysis. APO010-resistant cell lines exhibited a 4- to 520-fold increase in resistance to APO010 and still remained sensitive to other chemotherapeutics. Downregulation of the Fas-receptor protein expression was observed in all resistant cell lines. mRNA expression analysis of the resistant versus parental cell lines confirmed a significant alteration in FAS expression between sensitive and resistant cell lines (p = 0.03), while pathway analysis revealed alterations in mRNA signaling pathways of Fas. On the basis of the pre-clinical data obtained, it can be concluded that downregulation of Fas-receptor can mediate resistance to APO010.


Assuntos
Linfoma de Burkitt/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/metabolismo , Receptor fas/metabolismo
11.
Sci Rep ; 10(1): 12695, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728160

RESUMO

Intravitreal anti-vascular endothelial growth factor (VEGF) agents have revolutionized the treatment of retinopathy of prematurity (ROP); however, there are concerns regarding the potential systemic complications caused by those treatments. This study aimed to determine the serum concentrations of cytokines in infants with ROP and to evaluate the changes in serum VEGF concentrations after intravitreal conbercept (IVC). Sixty infants with ROP treated with IVC 0.25 mg were included. Blood samples were collected before treatment as well as 1 week and 4 weeks after treatment. Serum levels of 45 types of cytokines were measured by a multiplex bead assay. We observed that IVC 0.25 mg in ROP patients suppressed the circulating levels of VEGF-A and VEGF-D as of 1 week after injection, and these growth factor levels returned to baseline at 4 weeks. No significant differences were observed in the serum levels of the other cytokines between baseline and 1 or 4 weeks after IVC.


Assuntos
Citocinas/sangue , Proteínas Recombinantes de Fusão/administração & dosagem , Retinopatia da Prematuridade/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/sangue , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lactente , Injeções Intravítreas , Masculino , Proteínas Recombinantes de Fusão/farmacologia , Retinopatia da Prematuridade/sangue , Resultado do Tratamento
12.
Int J Mol Sci ; 21(11)2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503354

RESUMO

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a "lectibody". Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.


Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Lectinas/metabolismo , Trans-Splicing , Brevibacillus/imunologia , Clorófitas/metabolismo , HIV/fisiologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Lectinas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Vírus da SARS/fisiologia , Internalização do Vírus/efeitos dos fármacos
13.
Cancer Immunol Immunother ; 69(12): 2561-2569, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32583154

RESUMO

Many cancer cells express CD47 as a 'don't eat me' signal to mask their presences from immune recognition and destruction. Such a signal is transmitted when CD47 binds to the signal regulatory protein-α (SIRPα) on macrophages to cut the phagocytic reaction. Most recent studies have focused on developing CD47 blocking agents with different affinities and avidities in order to optimize the therapeutic window between efficacy and toxicities involving normal cells expressing CD47. We described in this study a new design to fuse one CD47 binding domain of SIRPα with a pharmacokinetics modifying domain F8. The resulted single valent long-acting CD47 antagonist SIRPα-F8 was able to bind to CD47 and disrupt CD47-SIRPα axis. However, by itself it cannot trigger endocytosis and has no effect on tumor growth. Only when used in combination with the anti-CD20 mAbs, there were greatly improved phagocytic activities towards CD20 positive cancer cells. In vivo the combination also resulted in better tumor growth inhibition comparing to the vehicle control group. In addition, we showed that the F8 fusion bound to hFcRn only inside endosomes at pH 6.0, enabled hFcRn mediated recycling and thus greatly extended the circulation half-life in hFcRn knock-in mice. Taken together, the SIRPα-F8 design may suggest a new option to improve the therapeutic index of antibody treatment in clinical use towards tumors.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antígeno CD47/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fagocitose/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/farmacologia , Antígenos de Diferenciação/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Antígeno CD47/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Técnicas de Introdução de Genes , Células HEK293 , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Injeções Intralesionais , Macrófagos , Camundongos Transgênicos , Monócitos , Neoplasias/imunologia , Neoplasias/patologia , Fagocitose/imunologia , Receptores Fc/genética , Receptores Imunológicos/genética , Receptores Imunológicos/uso terapêutico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Cancer Immunol Immunother ; 69(11): 2291-2303, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32504247

RESUMO

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-ß, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/imunologia , Molécula de Adesão da Célula Epitelial/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
15.
J Biol Chem ; 295(30): 10446-10455, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32518163

RESUMO

Transthyretin (TTR) is an abundant homotetrameric serum protein and was selected here for engineering higher-valency molecules because of its compact size, simple structure, and natural propensity to tetramerize. To demonstrate this utility, we fused TTR to the C terminus of conatumumab, an antibody that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2, as heavy chains to form antibody dimers and Fab heavy chains to form Fab tetramers. Moreover, we used constant heavy domain 3 heterodimerization substitutions to create TTR-mediated conatumumab tetramers. The conatumumab-TTR fusions displayed substantially enhanced potency in cell-based assays, as well as in murine tumor xenograft models. We conclude that antibody-TTR fusions may provide a powerful platform for multimerizing antibody and Fab fragments to enhance the capabilities of human therapeutics that benefit from target clustering and higher-order antigen-binding valency.


Assuntos
Anticorpos Monoclonais , Antineoplásicos Imunológicos , Fragmentos Fab das Imunoglobulinas , Neoplasias Experimentais , Pré-Albumina , Multimerização Proteica , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/farmacologia , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Pré-Albumina/genética , Pré-Albumina/farmacocinética , Pré-Albumina/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Sci Rep ; 10(1): 8439, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439930

RESUMO

Non-response to intravitreal ranibizumab represents a frequent problem in pachychoroid neovasculopathy (PNV). To investigate the effectivity of switching to aflibercept, the database of the Ludwig Maximilians University, Munich, was screened for patients fulfilling the following inclusion criteria: (i) diagnosis of PNV; (ii) inadequate response to ≥ 3 ranibizumab injections, in spite of monthly dosing, defined as persistence of subretinal-fluid four weeks after the last ranibizumab injection; (iii) resulting switch to aflibercept administered as three monthly injections. Primary outcome measure was percentage of eyes with a dry macula four weeks after the third aflibercept injection. Secondary outcome measures included changes in maximum subretinal fluid (SRF), central subfield thickness (CST) and subfoveal choroidal thickness (SFCT). In total, 14 eyes of 14 patients were included. Mean age was 64.1 ± 7.5 (range: 51-78) years. Switching to aflibercept was performed after mean 8.4 ± 4.1 (3-15) ranibizumab injections. While no eye (0%) achieved a dry macula status during ranibizumab treatment, switching to aflibercept achieved a dry macula status in eight eyes (57.1%) after three injections. While both ranibizumab and aflibercept showed an effect on CST (p = 0.027, p = 0.003), only aflibercept showed a significant effect on SRF (p = 0.0009) and SFCT (p = 0.044). In cases of PNV not responding to intravitreal ranibizumab, switching treatment to aflibercept induces a favorable short-term response resolving persistent fluid and achieving a dry macula. Further studies with longer follow-up are warranted.


Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Substituição de Medicamentos/métodos , Macula Lutea/efeitos dos fármacos , Ranibizumab/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Idoso , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Feminino , Seguimentos , Humanos , Macula Lutea/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores de Fatores de Crescimento do Endotélio Vascular , Estudos Retrospectivos
17.
Artif Cells Nanomed Biotechnol ; 48(1): 854-866, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32468873

RESUMO

In the present study, a novel single domain antibody (sdAb) fusion protein, named everestmab, composing of a mutated GLP-1(A8G) fused to the tandem bispecific humanized GLP-1R-targeting and albumin-binding nanobodies was designed and characterized for the therapies for type 2 diabetes mellitus (T2DM). Surface plasmon resonance (SPR) measurements demonstrated everestmab associates with serum albumins of rat and monkey species with high affinity, and tends to be cross-reactive with rat and monkey species. In vitro GLP-1R binding and activation assays revealed that everestmab can specifically activate the GLP-1R, and the antagonist exendin-4 (9-39) did not inhibit the activation yet. In vivo multiple oral glucose tolerance tests (OGTTs) and hypoglycaemic efficacy tests proved that a single injection of everestmab reduced the blood glucose for at least 144 h in Goto-Kakizaki (GK) rats. The plasma half-lives of 4.1 and 7.8 days were observed after a single s.c. administration of everestmab in SD rats and cynomolgus monkeys, respectively. Chronic treatment of everestmab to GK and diet induced obese (DIO) rats achieved beneficial effects on weight reducing, HbA1c lowering, glucose tolerance, liver and pancreas islet function impairment. In summary, everestmab is a unique G-protein-coupled receptor-targeted nanobody fusion protein and exerts potential as a therapeutic treatment for T2DM.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/imunologia , Hipoglicemiantes/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Domínio Único/farmacologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Macaca fascicularis , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Domínio Único/uso terapêutico , Distribuição Tecidual
18.
Exp Hematol ; 85: 33-46.e6, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32417303

RESUMO

Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F_Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação de Sentido Incorreto , Receptores de Trombopoetina/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Trombopoese/efeitos dos fármacos , Trombopoetina/farmacologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Receptores Fc , Receptores de Trombopoetina/genética , Trombopoese/genética
19.
Protein Expr Purif ; 174: 105658, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32360598

RESUMO

The recombinant multi-epitope vaccine called VBP3 is designed to suppress tumor growth and angiogenesis through targeting both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA). We are aiming to produce VBP3 vaccine in a large scale and provide sufficient protein for pre-clinical study. High cost and potential toxicity are severe limitations of IPTG and we investigated whether lactose can mediate VBP3 induction. Firstly, we identified the biological characteristics and established a culture bank of VBP3 strains. The best-performing strains were selected and the fermentation mode of medium, bacterial growth and protein expression were optimized in shake flasks. We scaled up the VBP3 production in 10 L bioreactor using lactose as inducer and the protein yield was comparable with IPTG induction. Next, the target protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, with a SDS-PAGE purity over 90%. Further, the purified VBP3 vaccine was subcutaneously injected in BALB/c mice and elicited high-titer anti-bFGF (1:32,000) and anti-VEGFA (1:4000) antibodies. Take together, lactose was an applicable inducer for VBP3 production and the eligible product of VBP3 was harvested in the large-scale fermentation, supporting the industrial production and pre-clinical study in the future. The VBP3 vaccine with superior immunogenicity might be used as a potential therapeutic vaccine for tumor treatment.


Assuntos
Vacinas Anticâncer , Escherichia coli/crescimento & desenvolvimento , Fator 2 de Crescimento de Fibroblastos , Proteínas Recombinantes de Fusão , Fator A de Crescimento do Endotélio Vascular , Animais , Vacinas Anticâncer/biossíntese , Vacinas Anticâncer/genética , Vacinas Anticâncer/isolamento & purificação , Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/farmacologia
20.
J Biol Chem ; 295(28): 9379-9391, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32398258

RESUMO

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t 1/2 in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.


Assuntos
Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/farmacologia , Mutação de Sentido Incorreto , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Proteínas Recombinantes de Fusão/genética , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/genética
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