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1.
Front Immunol ; 13: 1016179, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36569945

RESUMO

The optimal use of many biotherapeutics is restricted by Anti-drug antibodies (ADAs) and hypersensitivity responses which can affect potency and ability to administer a treatment. Here we demonstrate that Re-surfacing can be utilized as a generalizable approach to engineer proteins with extensive surface residue modifications in order to avoid binding by pre-existing ADAs. This technique was applied to E. coli Asparaginase (ASN) to produce functional mutants with up to 58 substitutions resulting in direct modification of 35% of surface residues. Re-surfaced ASNs exhibited significantly reduced binding to murine, rabbit and human polyclonal ADAs, with a negative correlation observed between binding and mutational distance from the native protein. Reductions in ADA binding correlated with diminished hypersensitivity responses in an in vivo mouse model. By using computational design approaches to traverse extended distances in mutational space while maintaining function, protein Re-surfacing may provide a means to generate novel or second line therapies for life-saving drugs with limited therapeutic alternatives.


Assuntos
Asparaginase , Escherichia coli , Humanos , Animais , Camundongos , Coelhos , Asparaginase/genética , Asparaginase/uso terapêutico , Escherichia coli/genética , Anticorpos , Proteínas de Membrana
2.
Mol Ther ; 29(7): 2227-2238, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-33677092

RESUMO

mRNA vaccines induce potent immune responses in preclinical models and clinical studies. Adjuvants are used to stimulate specific components of the immune system to increase immunogenicity of vaccines. We utilized a constitutively active mutation (V155M) of the stimulator of interferon (IFN) genes (STING), which had been described in a patient with STING-associated vasculopathy with onset in infancy (SAVI), to act as a genetic adjuvant for use with our lipid nanoparticle (LNP)-encapsulated mRNA vaccines. mRNA-encoded constitutively active STINGV155M was most effective at maximizing CD8+ T cell responses at an antigen/adjuvant mass ratio of 5:1. STINGV155M appears to enhance development of antigen-specific T cells by activating type I IFN responses via the nuclear factor κB (NF-κB) and IFN-stimulated response element (ISRE) pathways. mRNA-encoded STINGV155M increased the efficacy of mRNA vaccines encoding the E6 and E7 oncoproteins of human papillomavirus (HPV), leading to reduced HPV+ TC-1 tumor growth and prolonged survival in vaccinated mice. This proof-of-concept study demonstrated the utility of an mRNA-encoded genetic adjuvant.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Neoplasias Pulmonares/terapia , Proteínas de Membrana/imunologia , Proteínas E7 de Papillomavirus/imunologia , RNA Mensageiro/imunologia , Vacinas de mRNA/imunologia , Adjuvantes Imunológicos , Animais , Apoptose , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Lipossomos/química , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , RNA Mensageiro/genética , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Vacinas de mRNA/administração & dosagem , Vacinas de mRNA/genética
3.
J Clin Invest ; 130(11): 5976-5988, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33016924

RESUMO

BACKGROUNDTherapeutic vaccinations against cancer have mainly targeted differentiation antigens, cancer-testis antigens, and overexpressed antigens and have thus far resulted in little clinical benefit. Studies conducted by multiple groups have demonstrated that T cells recognizing neoantigens are present in most cancers and offer a specific and highly immunogenic target for personalized vaccination.METHODSWe recently developed a process using tumor-infiltrating lymphocytes to identify the specific immunogenic mutations expressed in patients' tumors. Here, validated, defined neoantigens, predicted neoepitopes, and mutations of driver genes were concatenated into a single mRNA construct to vaccinate patients with metastatic gastrointestinal cancer.RESULTSThe vaccine was safe and elicited mutation-specific T cell responses against predicted neoepitopes not detected before vaccination. Furthermore, we were able to isolate and verify T cell receptors targeting KRASG12D mutation. We observed no objective clinical responses in the 4 patients treated in this trial.CONCLUSIONThis vaccine was safe, and potential future combination of such vaccines with checkpoint inhibitors or adoptive T cell therapy should be evaluated for possible clinical benefit in patients with common epithelial cancers.TRIAL REGISTRATIONPhase I/II protocol (NCT03480152) was approved by the IRB committee of the NIH and the FDA.FUNDINGCenter for Clinical Research, NCI, NIH.


Assuntos
Antígenos de Neoplasias , Vacinas Anticâncer , Neoplasias Gastrointestinais , Imunidade Celular , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro , Linfócitos T/imunologia , Substituição de Aminoácidos , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/terapia , Humanos , Masculino , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/imunologia
4.
J Cereb Blood Flow Metab ; 37(5): 1829-1840, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27432225

RESUMO

Spontaneous spreading depolarizations are frequent after various forms of human brain injury such as ischemic or hemorrhagic stroke and trauma, and worsen the outcome. We have recently shown that supply-demand mismatch transients trigger spreading depolarizations in ischemic stroke. Here, we examined the mechanisms triggering recurrent spreading depolarization events for many days after subarachnoid hemorrhage. Despite large volumes of subarachnoid hemorrhage induced by cisternal injection of fresh arterial blood in rodents, electrophysiological recordings did not detect a single spreading depolarization for up to 72 h after subarachnoid hemorrhage. Cortical susceptibility to spreading depolarization, measured by direct electrical stimulation or topical KCl application, was suppressed after subarachnoid hemorrhage. Focal cerebral ischemia experimentally induced after subarachnoid hemorrhage revealed a biphasic change in the propensity to develop peri-infarct spreading depolarizations. Frequency of peri-infarct spreading depolarizations decreased at 12 h, increased at 72 h and normalized at 7 days after subarachnoid hemorrhage compared with sham controls. However, ischemic tissue and neurological outcomes were significantly worse after subarachnoid hemorrhage even when peri-infarct spreading depolarization frequency was reduced. Laser speckle flowmetry implicated cerebrovascular hemodynamic mechanisms worsening the outcome. Altogether, our data suggest that cerebral ischemia is required for spreading depolarizations to be triggered after subarachnoid hemorrhage, which then creates a vicious cycle leading to the delayed cerebral ischemia syndrome.


Assuntos
Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular/fisiologia , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Hemorragia Subaracnóidea/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Isquemia Encefálica/complicações , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/complicações
5.
Am J Physiol Cell Physiol ; 300(4): C919-26, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21270296

RESUMO

Sphingosine-1-phosphate (S1P) has been shown to modulate intracellular Ca(2+) through both G protein-coupled receptors and intracellular second messenger pathways. The precise mechanism by which S1P activates store-operated calcium entry (SOCE) in vascular smooth muscle cells (VSMCs) has not been fully characterized. Because sphingolipids and Ca(2+) modulate proliferation and constriction in VSMCs, characterizing the connection between S1P and SOCE may provide novel therapeutic targets for vascular diseases. We found that S1P triggered STIM1 puncta formation and SOCE in VSMCs. S1P-activated SOCE was inhibited by 2-aminoethoxydiphenyl borate (2-APB), diethylstilbestrol (DES), and gadolinium (Gd(3+)). SOCE was observed in VSMCs lacking either S1P(2) or S1P(3) receptors, suggesting that S1P acts via multiple signaling pathways. Indeed, both extracellular and intracellular S1P application increased the total internal reflection fluorescence signal in VSMCs cells transfected with STIM1-yellow fluorescent protein in a 2-APB-sensitive manner. These data, and the fact that 2-APB, DES, and Gd(3+) all inhibited S1P-induced cerebral artery constriction, suggest that SOCE modulates S1P-induced vasoconstriction in vivo. Finally, S1P-induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P(1), and S1P(3) receptor mRNA. These data demonstrate that S1P can act through both receptors and a novel intracellular pathway to activate SOCE. Because S1P-induced SOCE contributes to vessel constriction and is increased in proliferative VSMCs, it is likely that S1P/SOCE signaling in proliferative VSMCs may play a role in vascular dysfunction such as atherosclerosis and diabetes.


Assuntos
Cálcio/metabolismo , Lisofosfolipídeos/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais/fisiologia , Esfingosina/análogos & derivados , Animais , Compostos de Boro/metabolismo , Canais de Cálcio , Células Cultivadas , Expressão Gênica , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Molécula 1 de Interação Estromal , Vasoconstrição/fisiologia
6.
Pharmacol Res ; 62(6): 465-74, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20850539

RESUMO

Sphingosine-1-phosphate (S1P) constricts cerebral arteries through S1P(3) receptor stimulation. Because the activity of the key S1P-synthesizing enzyme, sphingosine kinase (SPK), can be stimulated by agonists of various G protein-coupled receptors, it is likely that S1P also acts as a second messenger for other vasoconstrictors. We investigated the effect of SPK inhibitors and SPK gene deletion on the contractile responses of isolated vessels to vasoactive agonists and KCl-induced depolarization. Basilar and femoral arteries of rat, mounted in a wire myograph, were incubated with dimethylsphingosine (DMS), 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (Compound 2) or FTY720, and exposed to KCl, 5-hydroxytryptamine (5-HT), S1P or phenylephrine (PE). Vasomotor responses in basilar artery were decreased by DMS, Compound 2 and FTY720, while they were not affected in femoral artery. Basilar arteries from SPK1(-/-) mice exhibited weaker vasoconstriction to both KCl and agonists (S1P and the prostanoid U46619) when compared to either wild type (WT) or SPK2(-/-). In contrast, in mesenteric resistance arteries, neither the contraction to KCl nor the maximum contraction to PE and S1P significantly differed among WT, SPK1(-/-) and SPK2(-/-). Quantitative analysis of SPK mRNA (reverse transcription and real time polymerase chain reaction) in mouse arteries showed 40-80-fold higher SPK1 expression in cerebral arteries than in aorta or mesenteric arteries. SPK1 critically modulates the reactivity of cerebral vasculature to vasoconstrictors. S1P plays a specific role as modulator of cerebral blood flow, potentially acting either directly outside vascular smooth muscle cells on S1P(3) receptors, or indirectly after being generated inside the cell in response to vasoconstrictors.


Assuntos
Artérias/efeitos dos fármacos , Artérias/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Vasoconstrição , Vasoconstritores/farmacologia , Animais , Artérias Cerebrais/fisiologia , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Cloreto de Potássio/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley
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