RESUMEN
BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.
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Movimiento Celular , Proliferación Celular , Receptores de Interleucina-6 , Humanos , Proliferación Celular/efectos de los fármacos , Receptores de Interleucina-6/metabolismo , Movimiento Celular/efectos de los fármacos , Células HEK293 , Línea Celular Tumoral , Unión Proteica/efectos de los fármacosRESUMEN
Immunoglobulin A (IgA) proteinase from Clostridium ramosum is the enzyme which cleaves IgA of both subclasses; in contrast, the other bacterial proteinases cleave only IgA1 proteins. Previous reports characterized the activity of proteinase naturally secreted by C. ramosum specific for the normal human serum IgA of IgA1 and IgA2m(1) subclasses and also for secretory IgA (SIgA). Its amino acid sequence was determined, and the recombinant proteinase which cleaved IgA of both subclasses was prepared. Here we report the optimized expression, purification, storage conditions and activity testing against purified human milk SIgA. The recombinant C. ramosum IgA proteinase isolated in the high degree of purity exhibited almost complete cleavage of SIgA of both subclasses. The proteinase remained active upon storage for more than 10 month at -20 °C without substantial loss of enzymatic activity. Purified SIgA fragments are suitable for studies of all antigen-binding and Fc-dependent functions of SIgA involved in the protection against infections with mucosal pathogens.
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Proteínas Bacterianas/química , Firmicutes/enzimología , Inmunoglobulina A Secretora/química , Fragmentos Fab de Inmunoglobulinas , Fragmentos Fc de Inmunoglobulinas , Péptido Hidrolasas/química , Proteínas Bacterianas/genética , Firmicutes/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Péptido Hidrolasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells' proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2-oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down-regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9-based loss-of-function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1-FOXO3A pathway and that chelation- and 2-oxoglutarate competition-mediated down-regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron- and 2-oxoglutarate-dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2-oxoglutarate-dependent dioxygenase inhibitors as a novel therapy of MCL.
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Ciclina D1/metabolismo , Dioxigenasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Quelantes del Hierro/farmacología , Ácidos Cetoglutáricos/farmacología , Linfoma de Células del Manto/enzimología , Aminoácidos Dicarboxílicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN , Deferoxamina/farmacología , Dioxigenasas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Humanos , Hidroxilación , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Deficiencias de Hierro , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Non-small cell lung cancer (NSCLC) is largely promoted by a multistep tumorigenesis process involving various genetic and epigenetic alterations, which essentially contribute to the high incidence of mortality among patients with NSCLC. Clinical observations revealed that NSCLC also co-opts a multifaceted immune checkpoint dysregulation as an important driving factor in NSCLC progression and development. For example, a deregulated PI3K/AKT/mTOR pathway has been noticed in 50-70% of NSCLC cases, primarily modulated by mutations in key oncogenes such as ALK, EGFR, KRAS, and others. Additionally, genetic association studies containing patient-specific factors and local reimbursement criteria expose/reveal mutations in EGFR/ALK/ROS/BRAF/KRAS/PD-L1 proteins to determine the suitability of available immunotherapy or tyrosine kinase inhibitor therapy. Thus, the expression of such checkpoints on tumors and immune cells is pivotal in understanding the therapeutic efficacy and has been extensively studied for NSCLC treatments. Therefore, this review summarizes current knowledge in NSCLC tumorigenesis, focusing on its genetic and epigenetic intricacies, immune checkpoint dysregulation, and the evolving landscape of targeted therapies. In the context of current and future therapies, we emphasize the significance of antibodies targeting PD-1/PD-L1 and CTLA-4 interactions as the primary therapeutic strategy for immune system reactivation in NSCLC. Other approaches involving the promising potential of nanobodies, probodies, affibodies, and DARPINs targeting immune checkpoints are also described; these are under active research or clinical trials to mediate immune regulation and reduce cancer progression. This comprehensive review underscores the multifaceted nature, current state and future directions of NSCLC research and treatment.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Antígeno B7-H1/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Transformación Celular Neoplásica , Carcinogénesis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores ErbB/metabolismoRESUMEN
IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN.
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Comunicación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glomerulonefritis por IGA/inmunología , Inmunoglobulina A/metabolismo , Sistema de Señalización de MAP Quinasas , Células Mesangiales/inmunología , Podocitos/inmunología , Adulto , Anciano , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Complejo Antígeno-Anticuerpo , Antígenos CD/metabolismo , Biopsia , Presión Sanguínea , Calcio/metabolismo , Comunicación Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Activación Enzimática , Femenino , Glomerulonefritis por IGA/enzimología , Glomerulonefritis por IGA/patología , Glomerulonefritis por IGA/fisiopatología , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Células Mesangiales/efectos de los fármacos , Células Mesangiales/enzimología , Células Mesangiales/patología , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Podocitos/efectos de los fármacos , Podocitos/enzimología , Podocitos/patología , Proteinuria/enzimología , Proteinuria/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Transferrina/metabolismo , Sistema Renina-Angiotensina , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
Introduction: Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified. Methods: In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes. Results: In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro. Discussion: Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.
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Anticuerpos Anti-VIH , VIH-1 , Animales , Ratones , Epítopos , Anticuerpos ampliamente neutralizantes , Anticuerpos Neutralizantes , Proteína gp120 de Envoltorio del VIH , PolisacáridosRESUMEN
BACKGROUND: Circulating immune complexes (CIC) containing galactose (Gal)-deficient IgA1 from adults with IgA nephropathy (IgAN) induce proliferation of cultured mesangial cells, but activities of CIC from pediatric patients with the disease have not been studied. METHODS: CIC of different sizes were isolated from sera of pediatric and adult IgAN patients and their effects on cultured human mesangial cells (MC) were assessed by measuring cellular proliferation, expression of IL-6 and IL-8 and laminin and phosphotyrosine signaling. RESULTS: Large CIC from pediatric IgAN patients (>800 kDa) containing Gal-deficient IgA1 stimulated cellular proliferation, whereas in some patients, smaller CIC were inhibitory. Addition of stimulatory and inhibitory CIC to MC differentially altered phosphorylation patterns of three major tyrosine-phosphorylated proteins of molecular mass 37, 60 and 115 kDa. The stimulatory CIC transiently increased tyrosine-phosphorylation of the 37-kDa protein and decreased phosphorylation of the other two proteins, whereas the inhibitory CIC increased phosphorylation of all three proteins. Furthermore, we investigated the influence of IgA1-containing CIC from sera of children with IgAN with clinically active disease (i.e., abnormal urinalysis and/or serum creatinine concentration) or inactive disease (i.e., normal urinalysis and serum creatinine concentration) on the expression of IL-6 and IL-8 genes by mesangial cells. Real-time reverse transcription-polymerase chain reaction results showed that the CIC from a patient with active disease stimulated MC to express the two cytokine genes at higher levels than did the CIC from a patient with inactive disease. Moreover, stimulatory CIC increased production of the extracellular matrix protein laminin. CONCLUSION: These data indicate that sera of pediatric IgAN patients contain biologically active CIC with Gal-deficient IgA1.
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Complejo Antígeno-Anticuerpo/farmacología , Proliferación Celular , Mesangio Glomerular/citología , Glomerulonefritis por IGA/fisiopatología , Inmunoglobulina A/metabolismo , Células Mesangiales/efectos de los fármacos , Adolescente , Adulto , Complejo Antígeno-Anticuerpo/sangre , Western Blotting , Células Cultivadas , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Galactosa/deficiencia , Tasa de Filtración Glomerular , Mesangio Glomerular/inmunología , Mesangio Glomerular/metabolismo , Glomerulonefritis por IGA/etiología , Glicosilación , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina A/inmunología , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Células Mesangiales/citología , Células Mesangiales/inmunología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this "non-cognate ligand" concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.
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Infecciones por VIH , VIH-1 , Animales , Anticuerpos Neutralizantes , Anticuerpos ampliamente neutralizantes , Epítopos , Anticuerpos Anti-VIH , Infecciones por VIH/prevención & control , VIH-1/genética , Ratones , Pseudotipado Viral , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Mucosal surfaces are colonized by highly diverse commensal microbiota. Coating with secretory IgA (SIgA) promotes the survival of commensal bacteria while it inhibits the invasion by pathogens. Bacterial coating could be mediated by antigen-specific SIgA recognition, polyreactivity, and/or by the SIgA-associated glycans. In contrast to many in vitro studies, only a few reported the effect of SIgA glycans in vivo. Here, we used a germ-free antibody-free newborn piglets model to compare the protective effect of SIgA, SIgA with enzymatically removed N-glycans, Fab, and Fc containing the secretory component (Fc-SC) during oral necrotoxigenic E. coli O55 challenge. SIgA, Fab, and Fc-SC were protective, whereas removal of N-glycans from SIgA reduced SIgA-mediated protection as demonstrated by piglets' intestinal histology, clinical status, and survival. In vitro analyses indicated that deglycosylation of SIgA did not reduce agglutination of E. coli O55. These findings highlight the role of SIgA-associated N-glycans in protection. Further structural studies of SIgA-associated glycans would lead to the identification of those involved in the species-specific inhibition of attachment to corresponding epithelial cells.
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Infecciones por Escherichia coli/inmunología , Escherichia coli/fisiología , Inmunoglobulina A Secretora/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Polisacáridos/metabolismo , Anticuerpos de Cadena Única/metabolismo , Aglutinación , Animales , Animales Recién Nacidos , Resistencia a la Enfermedad , Femenino , Vida Libre de Gérmenes , Glicosilación , Embarazo , PorcinosRESUMEN
BACKGROUND: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies. METHODS: We employed a highly complex combinatorial protein library derived from a 5â¯kDa albumin-binding domain scaffold, fused with support protein of total 38â¯kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses. FINDINGS: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface. INTERPRETATION: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. FUND: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic.