RESUMO
Secretogranin III (Scg3) is a diabetic retinopathy (DR)-restricted angiogenic factor identified in preclinical studies as a target for DR therapy. Previously, our group generated and characterized ML49.3, an anti-Scg3 monoclonal antibody (mAb) which we then converted into an EBP2 humanized antibody Fab fragment (hFab) with potential for clinical application. We also generated anti-Scg3 mT4 mAb and related EBP3 hFab. In this study, to identify the preferred hFab for DR therapy, we compared all four antibodies for binding, neutralizing and therapeutic activities in vitro and in vivo. Octet binding kinetics analyses revealed that ML49.3 mAb, EBP2 hFab, mT4 mAb and EBP3 hFab have Scg3-binding affinities of 35, 8.7, 0.859 and 0.116 nM, respectively. Both anti-Scg3 EBP2 and EBP3 hFabs significantly inhibited Scg3-induced proliferation and migration of human umbilical vein endothelial cells in vitro, and alleviated DR vascular leakage and choroidal neovascularization with high efficacy. Paired assays in DR mice revealed that intravitreally injected EBP3 hFab is 26.4% and 10.3% more effective than EBP2 hFab and aflibercept, respectively, for ameliorating DR leakage. In conclusion, this study confirms the markedly improved binding affinities of hFabs compared to mAbs and further identifies EBP3 hFab as the preferred antibody to develop for anti-Scg3 therapy.
Assuntos
Inibidores da Angiogênese , Anticorpos Neutralizantes , Retinopatia Diabética , Células Endoteliais da Veia Umbilical Humana , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Retinopatia Diabética/imunologia , Retinopatia Diabética/patologia , Humanos , Animais , Camundongos , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de SinalRESUMO
Tongue squamous cell carcinoma (TSCC) occurs frequently in the oral cavity, and because of its high proliferative and metastatic potential, it is necessary to develop a novel treatment for it. We have reported the importance of the inhibition of the periostin (POSTN) pathological splicing variant, including exon 21 (PN1-2), in various malignancies, but its influence is unclear in tongue cancer. In this study, we investigated the potential of POSTN exon 21-specific neutralizing antibody (PN21-Ab) as a novel treatment for TSCC. Human PN2 was transfected into the human TSCC (HSC-3) and cultured under stress, and PN2 was found to increase cell viability. PN2 induced chemotherapy resistance in HSC-3 via the phosphorylation of the cell survival signal Akt. In tissues from human TSCC and primary tumors of an HSC-3 xenograft model, PN1-2 was expressed in the tumor stroma, mainly from fibroblasts. The intensity of PN1-2 mRNA expression was positively correlated with malignancy. In the HSC-3 xenograft model, CDDP and PN21-Ab promoted CDPP's inhibition of tumor growth. These results suggest that POSTN exon 21 may be a biomarker for tongue cancer and that PN21-Ab may be a novel treatment for chemotherapy-resistant tongue cancer. The treatment points towards important innovations for TSCC, but many more studies are needed to extrapolate the results.
Assuntos
Moléculas de Adesão Celular , Resistencia a Medicamentos Antineoplásicos , Éxons , Neoplasias da Língua , Humanos , Neoplasias da Língua/patologia , Neoplasias da Língua/genética , Neoplasias da Língua/tratamento farmacológico , Neoplasias da Língua/metabolismo , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Éxons/genética , Linhagem Celular Tumoral , Camundongos , Masculino , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Feminino , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/efeitos dos fármacos , Pessoa de Meia-Idade , Camundongos Endogâmicos BALB C , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , PeriostinaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic disease. Recent studies have highlighted the persistence of an intermediate state of alveolar stem cells in IPF lungs. In this study, we discovered a close correlation between the distribution pattern of intermediate alveolar stem cells and the progression of fibrotic changes. We showed that amphiregulin (AREG) expression is significantly elevated in intermediate alveolar stem cells of mouse fibrotic lungs and IPF patients. High levels of serum AREG correlate significantly with profound deteriorations in lung function in IPF patients. We demonstrated that AREG in alveolar stem cells is both required and sufficient for activating EGFR in fibroblasts, thereby driving lung fibrosis. Moreover, pharmacological inhibition of AREG using a neutralizing antibody effectively blocked the initiation and progression of lung fibrosis in mice. Our study underscores the therapeutic potential of anti-AREG antibodies in attenuating IPF progression, offering a promising strategy for treating fibrotic diseases.
Assuntos
Anfirregulina , Progressão da Doença , Fibrose Pulmonar Idiopática , Anfirregulina/metabolismo , Animais , Humanos , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/metabolismo , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Masculino , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/metabolismo , Anticorpos Neutralizantes/farmacologia , FemininoRESUMO
Type 2 diabetes (T2D) is on the rise worldwide and is associated with various complications in the oral cavity. Using an adult-onset diabetes preclinical model, we demonstrated profound periodontal alterations in T2D mice, including inflamed gingiva, disintegrated periodontal ligaments (PDLs), marked alveolar bone loss, and unbalanced bone remodeling due to decreased formation and increased resorption. Notably, we observed elevated levels of the Wnt signaling inhibitor sclerostin in the alveolar bone of T2D mice. Motivated by these findings, we investigated whether a sclerostin-neutralizing antibody (Scl-Ab) could rescue the compromised periodontium in T2D mice. Administering Scl-Ab subcutaneously once a week for 4 weeks, starting 4 weeks after T2D induction, led to substantial increases in bone mass. This effect was attributed to the inhibition of osteoclasts and promotion of osteoblasts in both control and T2D mice, effectively reversing the bone loss caused by T2D. Furthermore, Scl-Ab stimulated PDL cell proliferation, partially restored the PDL fibers, and mitigated inflammation in the periodontium. Our study thus established a T2D-induced periodontitis mouse model characterized by inflammation and tissue degeneration. Scl-Ab emerged as a promising intervention to counteract the detrimental effects of T2D on the periodontium, exhibiting limited side effects on other craniofacial hard tissues.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Perda do Osso Alveolar , Diabetes Mellitus Tipo 2 , Animais , Camundongos , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/complicações , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Perda do Osso Alveolar/prevenção & controle , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Masculino , Doenças Periodontais/imunologia , Anticorpos Neutralizantes/farmacologia , Ligamento Periodontal/patologia , Ligamento Periodontal/efeitos dos fármacos , Modelos Animais de Doenças , Diabetes Mellitus Experimental/imunologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Camundongos Endogâmicos C57BL , Periodontite/imunologia , Periodontite/patologia , Periodontite/tratamento farmacológico , Remodelação Óssea/efeitos dos fármacosRESUMO
Rift Valley fever (RVF) is an emerging arboviral disease affecting both humans and livestock. In humans, RVF displays a spectrum of clinical manifestations, including encephalitis. To date, there are no FDA-approved vaccines or therapeutics for human use, although several are in preclinical development. Few small-animal models of RVF encephalitis exist, further complicating countermeasure assessment. Human mAbs RVFV-140, RVFV-268, and RVFV-379 are recombinant potently neutralizing antibodies that prevent infection by binding the RVFV surface glycoproteins. Previous studies showed that both RVFV-268 and RVFV-140 improve survival in a lethal mouse model of disease, and RVFV-268 has prevented vertical transmission in a pregnant rat model of infection. Despite these successes, evaluation of mAbs in the context of brain disease has been limited. This is the first study to our knowledge to assess neutralizing antibodies for prevention of RVF neurologic disease using a rat model. Administration of RVFV-140, RVFV-268, or RVFV-379 24 hours prior to aerosol exposure to the virulent ZH501 strain of RVFV resulted in substantially enhanced survival and lack of neurological signs of disease. These results using a stringent and highly lethal aerosol infection model support the potential use of human mAbs to prevent the development of RVF encephalitis.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Modelos Animais de Doenças , Febre do Vale de Rift , Vírus da Febre do Vale do Rift , Animais , Febre do Vale de Rift/imunologia , Febre do Vale de Rift/prevenção & controle , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Ratos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/farmacologia , Humanos , Vírus da Febre do Vale do Rift/imunologia , Anticorpos Antivirais/imunologia , Feminino , CamundongosRESUMO
BACKGROUND: The interleukin (IL)-1-family receptor antagonist IL-38 has emerged as a negative regulator of auto-inflammation. Given the intricate interplay between antitumor immunity and auto-inflammation, we hypothesized that blocking IL-38 may enhance tumor immune control. METHODS: Our hypothesis was tested in the transgenic polyoma virus middle T oncoprotein mammary carcinoma model that is suitable for identifying strong immunomodulators. To investigate the effect of acute IL-38 blockade, we used a neutralizing antibody, alone or in combination with chemotherapy. Immune cell composition and location in tumors were determined by flow cytometry and immunohistochemistry, respectively. The role of γδ T cells was studied using an antibody blocking γδ T-cell receptor signaling. Whole transcriptome RNA sequencing and RNA expression analysis were employed to determine mechanisms downstream of IL-38 neutralization. Additionally, in vitro assays with γδ T cells, CD8+ T cells and cDC1, followed by in vivo CD8+ T cell depletion, were performed to study the underlying mechanistic pathways. RESULTS: Both, genetic ablation of IL-38 and neutralization with the antibody, reduced tumorigenesis, and IL-38 blockade improved chemotherapy efficacy. This was accompanied by an augmented lymphocyte infiltrate dominated by γδ T cells and CD8+ T cells, and signaling through the γδ-T-cell receptor was required for CD8+ T cell infiltration. Rather than directly interacting with CD8+ T cells, γδ T cells recruited conventional dendritic cells (cDC1) into tumors via the chemokine Xcl1. cDC1 in turn activated CD8+ T cells via the Notch pathway. Moreover, IL-38 negatively correlated with cDC1, XCL1-producing γδ T cells, T-cell infiltrates and survival in patients with mammary carcinoma. CONCLUSIONS: These data suggest that interfering with IL-38 improves antitumor immunity even in immunologically cold tumors.
Assuntos
Anticorpos Neutralizantes , Animais , Feminino , Humanos , Camundongos , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Interleucina-1/metabolismo , Interleucina-1/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T gama-delta/metabolismoRESUMO
Like TNFα, IL-6 is upregulated in Crohn's disease (CD) especially in patients associated with Mycobacterium avium paratuberculosis (MAP) infection, and both cytokines have been targeted as a therapeutic option for the treatment of the disease despite the accepted partial response in some patients. Limited response to anti-IL-6 receptor-neutralizing antibodies therapy may be related to the homeostatic dual role of IL-6. In this study, we investigated the effects and the signaling mechanism of IL-6 involved in intestinal epithelial integrity and function during MAP infection using an in vitro model that consists of THP-1, HT-29 and Caco-2 cell lines. Clinically, we determined that plasma samples from MAP-infected CD patients have higher IL-6 levels compared to controls (P-value < 0.001). In CD-like macrophages, MAP infection has significantly upregulated the secretion of IL-6 and the shedding of (IL-6R) from THP-1 macrophages, P-value < 0.05. Intestinal cell lines (Caco-2 and HT-29) were treated with the supernatant of MAP-infected THP-1 macrophages with or without a neutralizing anti-IL-6R antibody. Treating intestinal Caco-2 cells with supernatant of MAP-infected macrophages resulted in significant upregulation of intestinal damage markers including claudin-2 and SERPINE1/PAI-1. Interestingly, blocking IL-6 signaling exacerbated that damage and further increased the levels of the damage markers. In HT-29 cells, MAP infection upregulated MUC2 expression, a protective response that was reversed when IL-6R was neutralized. More importantly, blocking IL-6 signaling during MAP infection rescued damaged Caco-2 cells from MAP-induced apoptosis. The data clearly supports a protective role of IL-6 in intestinal epithelia integrity and function especially in CD patients associated with MAP infection. The findings may explain the ineffective response to anti-IL6 based therapy and strongly support a therapeutic option that restores the physiologic level of IL-6 in patient's plasma. A new treatment strategy based on attenuation of IL-6 expression and secretion in inflammatory diseases should be considered.
Assuntos
Interleucina-6 , Mucosa Intestinal , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Receptores de Interleucina-6 , Humanos , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Células CACO-2 , Interleucina-6/metabolismo , Interleucina-6/imunologia , Células HT29 , Mucosa Intestinal/microbiologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Paratuberculose/imunologia , Paratuberculose/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Doença de Crohn/imunologia , Doença de Crohn/microbiologia , Células THP-1 , Masculino , Anticorpos Neutralizantes/farmacologia , Feminino , Adulto , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Pessoa de Meia-Idade , Transdução de SinaisRESUMO
Despite advancements in antiretroviral therapy (ART) suppressing HIV-1 replication, existing antiviral drugs pose limitations, including lifelong medication, frequent administration, side effects and viral resistance, necessitating novel HIV-1 treatment approaches. CD4, pivotal for HIV-1 entry, poses challenges for drug development due to neutralization and cytotoxicity concerns. Nevertheless, Ibalizumab, the sole approved CD4-specific antibody for HIV-1 treatment, reignites interest in exploring alternative anti-HIV targets, emphasizing CD4's potential value for effective drug development. Here, we explore anti-CD4 nanobodies, particularly Nb457 from a CD4-immunized alpaca. Nb457 displays high potency and broad-spectrum activity against HIV-1, surpassing Ibalizumab's efficacy. Strikingly, engineered trimeric Nb457 nanobodies achieve complete inhibition against live HIV-1, outperforming Ibalizumab and parental Nb457. Structural analysis unveils Nb457-induced CD4 conformational changes impeding viral entry. Notably, Nb457 demonstrates therapeutic efficacy in humanized female mouse models. Our findings highlight anti-CD4 nanobodies as promising HIV-1 therapeutics, with potential implications for advancing clinical treatment against this global health challenge.
Assuntos
Antígenos CD4 , Camelídeos Americanos , Anticorpos Anti-HIV , Infecções por HIV , HIV-1 , Anticorpos de Domínio Único , HIV-1/imunologia , HIV-1/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/imunologia , Animais , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Humanos , Infecções por HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Camelídeos Americanos/imunologia , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/farmacologia , Camundongos , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Conformação Proteica , Feminino , Internalização do Vírus/efeitos dos fármacos , Células HEK293 , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Anticorpos MonoclonaisRESUMO
Purpose: To explore the effects of the tight junction protein zonula occludens 1 (ZO-1) on experimental corneal neovascularization (CNV). Methods: CNV models were established in the left eyes of BALB/c mice using NaOH. Anti-ZO-1 neutralizing antibody was topically applied to the burnt corneas after modeling thrice a day for 1 week. CD31 expression was analyzed to calculate the ratio of CNV number to area using a corneal whole-mount fluorescent immunohistochemical assay. Messenger ribonucleic acid (mRNA) and protein expression levels of ZO-1, vascular endothelial growth factor (VEGF), interleukin (IL)-1ß, IL-6, IL-8, IL-18, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), phosphorylated protein kinase C (pPKC), and clusterin in burned corneas were detected by reverse transcriptase polymerase chain reaction (PCR) and western blot analyses. Infiltration of neutrophils, macrophages, and progenitor cells was examined by flow cytometry. Results: CNV was obviously greater in 45 s than in 15 s alkali injury group. In another experiment, CNV was obviously greater in the ZO-1 antibody group than in the vehicle-treated group. Corneal mRNA and protein expression levels of VEGF, IL-1ß, IL-6, IL-8, IL-18, and MCP-1 were significantly higher in the ZO-1 antibody group than in the control group. Infiltration of neutrophils, macrophages, and progenitor cells was significantly greater in the ZO-1 antibody group than in the control group. TNF-α expression was much higher in 45 s than in 15 s alkali injury group. However, protein expression of pPKC and clusterin was much lower in 45 s than in 15 s alkali injury group. Conclusions: Anti-ZO-1 neutralizing antibody-treated mice exhibited enhanced alkali-induced CNV through enhanced intracorneal infiltration of progenitor and inflammatory cells.
Assuntos
Neovascularização da Córnea , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Proteína da Zônula de Oclusão-1 , Animais , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Proteína da Zônula de Oclusão-1/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Hidróxido de Sódio , Anticorpos Neutralizantes/farmacologia , Masculino , Junções Íntimas/metabolismo , Citocinas/metabolismoRESUMO
OBJECTIVES: We aimed to characterize and investigate the safety and efficacy of Plonmarlimab, a novel anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF) neutralizing antibody, on the treatment of macrophage activation syndrome (MAS), a life-threatening systemic inflammatory disease, in pre-clinical models. METHODS: The binding affinity was evaluated using Biacore. The neutralizing activity was measured through the blockade of ligand-receptor interaction, inhibition of STAT5 phosphorylation and suppression of TF-1 cell proliferation. The efficacy of Plonmarlimab was evaluated in a humanized MAS model, which was established by engrafting human umbilical cord blood (UCB) cells into NOG-EXL mice. Additionally, the safety profile of Plonmarlimab was investigated in cynomolgus monkeys. RESULTS: At the molecular level, Plonmarlimab showed sub-nanomolar binding affinity with human GM-CSF and effectively blocked the binding of GM-CSF to its receptor. At the cellular level, Plonmarlimab dose-dependently inhibited intracellular STAT5 phosphorylation and suppressed GM-CSF-induced TF-1 proliferation. In the UCB-engrafted NOG-EXL MAS mouse model, Plonmarlimab treatment significantly ameliorated disease progression, demonstrated by the improvements in body weight loss, anaemia and some histopathological features. Furthermore, Plonmarlimab was well tolerated up to 150 mg/kg weekly in monkeys with no reported adverse effects. CONCLUSIONS: Plonmarlimab is a highly potent GM-CSF blocking antibody and has demonstrated promising efficacy in a pre-clinical MAS model with a favourable safety profile, supporting its clinical development.
Assuntos
Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Macaca fascicularis , Síndrome de Ativação Macrofágica , Animais , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Camundongos , Síndrome de Ativação Macrofágica/tratamento farmacológico , Síndrome de Ativação Macrofágica/imunologia , Progressão da Doença , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT5/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Fosforilação , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismoRESUMO
Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by low bone mass and frequent fractures. Children with OI are commonly treated with bisphosphonates to reduce fracture rate, but treatment options for adults are limited. In the Phase 2b ASTEROID trial, setrusumab (a sclerostin neutralizing antibody, SclAb) improved bone density and strength in adults with type I, III, and IV OI. Here, we investigate bone matrix material properties in tetracycline-labeled trans iliac biopsies from 3 groups: (1) control: individuals with no metabolic bone disease, (2) OI: individuals with OI, (3) SclAb-OI: individuals with OI after 6 mo of setrusumab treatment (as part of the ASTEROID trial). In addition to bone histomorphometry, bone mineral and matrix properties were evaluated with nanoindentation, Raman spectroscopy, second harmonic generation imaging, quantitative backscatter electron imaging, and small-angle X-ray scattering. Spatial locations of fluorochrome labels were identified to differentiate inter-label bone of the same tissue age and intra-cortical bone. No difference in collagen orientation was found between the groups. The bone mineral density distribution and analysis of Raman spectra indicate that OI groups have greater mean mineralization, greater relative mineral content, and lower crystallinity than the control group, which was not altered by SclAb treatment. Finally, a lower modulus and hardness were measured in the inter-label bone of the OI-SclAb group compared to the OI group. Previous studies suggest that even though bone from OI has a higher mineral content, the extracellular matrix (ECM) has comparable mechanical properties. Therefore, fragility in OI may stem from contributions from other yet unexplored aspects of bone organization at higher length scales. We conclude that SclAb treatment leads to increased bone mass while not adversely affecting bone matrix properties in individuals with OI.
Individuals with OI, also known as "brittle bone disease," have low bone mass and frequent fractures. Low bone mass occurs due to an imbalance between cells that remove bone and cells that form bone. Pharmaceutical treatments that block removal of bone lead to reduced fracture rates in children with OI. Effective treatment options for adults are limited. Setrusumab is a drug that leads to increased bone mass and strength in adults with OI. Here, we investigate whether setrusumab alters the bone material in addition to improving bone mass. Three groups are compared: individuals with OI treated with setrusumab, individuals with OI not treated with setrusumab, and individuals without OI. A lower modulus and hardness were measured with nanoindentation in the setrusumab-treated group. However, we did not find any changes in the bone's multi-scale structure. Fragility in OI may stem from other yet unexplored aspects of bone organization. We conclude that setrusumab treatment leads to increased bone mass while not adversely affecting bone material properties in individuals with OI.
Assuntos
Matriz Óssea , Osteogênese Imperfeita , Humanos , Osteogênese Imperfeita/tratamento farmacológico , Osteogênese Imperfeita/patologia , Osteogênese Imperfeita/diagnóstico por imagem , Adulto , Masculino , Feminino , Matriz Óssea/efeitos dos fármacos , Matriz Óssea/patologia , Matriz Óssea/metabolismo , Anticorpos Neutralizantes/farmacologia , Densidade Óssea/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Pessoa de Meia-IdadeRESUMO
Substantial evidence supports that Fc-mediated effector functions of anti-spike Abs contribute to anti-SARS-Cov-2 protection. We have previously shown that two non-neutralizing but opsonic mAbs targeting the receptor-binding domain and N-terminal domain (NTD), Ab81 and Ab94, respectively, are protective against lethal Wuhan SARS-CoV-2 infection in K18-hACE2 mice. In this article, we investigated whether these protective non-neutralizing Abs maintain Fc-mediated function and Ag binding against mutated SARS-CoV-2 variants. Ab81 and Ab94 retained their nanomolar affinity and Fc-mediated function toward Omicron and its subvariants, such as BA.2, BA.4, BA.5, XBB, XBB1.5, and BQ1.1. However, when encountering the more heavily mutated BA.2.86, Ab81 lost its function, whereas the 10 new mutations in the NTD did not affect Ab94. In vivo experiments with Ab94 in K18-hACE2 mice inoculated with a stringent dose of 100,000 PFU of the JN.1 variant revealed unexpected results. Surprisingly, this variant exhibited low disease manifestation in this animal model with no weight loss or death in the control group. Still, assessment of mice using a clinical scoring system showed better protection for Ab94-treated mice, indicating that Fc-mediated functions are still beneficial. Our work shows that a protective anti-receptor-binding domain non-neutralizing mAb lost reactivity when BA.2.86 emerged, whereas the anti-NTD mAb was still functional. Finally, this work adds new insight into the evolution of the SARS-CoV-2 virus by reporting that JN.1 is substantially less virulent in vivo than previous strains.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , COVID-19 , Fragmentos Fc das Imunoglobulinas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Camundongos , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Humanos , Virulência/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Feminino , Camundongos Endogâmicos BALB CRESUMO
Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed in tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancers. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms. SIGNIFICANCE: A first-in-class anti-Notch4 agent, E7011, demonstrates strong antitumor effects in murine tumor models including breast carcinoma. Endothelial Notch4 blockade reduces perfusion and vessel area.
Assuntos
Anticorpos Neutralizantes , Neovascularização Patológica , Receptor Notch4 , Animais , Receptor Notch4/metabolismo , Camundongos , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Neovascularização Patológica/metabolismo , Feminino , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismoRESUMO
Purpose: Over the past three years, extensive research has been dedicated to understanding and combating COVID-19. Targeting the interaction between the SARS-CoV-2 Spike protein and the ACE2 receptor has emerged as a promising therapeutic strategy against SARS-CoV-2. This study aimed to develop ACE2-coated virus-like particles (ACE2-VLPs), which can be utilized to prevent viral entry into host cells and efficiently neutralize the virus. Methods: Virus-like particles were generated through the utilization of a packaging plasmid in conjunction with a plasmid containing the ACE2 envelope sequence. Subsequently, ACE2-VLPs and ACE2-EVs were purified via ultracentrifugation. The quantification of VLPs was validated through multiple methods, including Nanosight 3000, TEM imaging, and Western blot analysis. Various packaging systems were explored to optimize the ACE2-VLP configuration for enhanced neutralization capabilities. The evaluation of neutralization effectiveness was conducted using pseudoviruses bearing different spike protein variants. Furthermore, the study assessed the neutralization potential against the Omicron BA.1 variant in Vero E6 cells. Results: ACE2-VLPs showed a high neutralization capacity even at low doses and demonstrated superior efficacy in in vitro pseudoviral assays compared to extracellular vesicles carrying ACE2. ACE2-VLPs remained stable under various environmental temperatures and effectively blocked all tested variants of concern in vitro. Notably, they exhibited significant neutralization against Omicron BA.1 variant in Vero E6 cells. Given their superior efficacy compared to extracellular vesicles and proven success against live virus, ACE2-VLPs stand out as crucial candidates for treating SARS-CoV-2 infections. Conclusion: This novel therapeutic approach of coating VLPs with receptor particles provides a proof-of-concept for designing effective neutralization strategies for other viral diseases in the future.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Células Vero , Chlorocebus aethiops , Humanos , COVID-19/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Neutralizantes/farmacologia , Células HEK293 , Internalização do Vírus/efeitos dos fármacosRESUMO
Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % ß-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.
Assuntos
Anticorpos de Cadeia Única , Animais , Camundongos , Coelhos , Sequência de Aminoácidos , Animais Peçonhentos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/farmacologia , Expressão Gênica , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/química , Neurotoxinas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/química , Venenos de Escorpião/genética , Escorpiões , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos de Cadeia Única/farmacologiaRESUMO
Prostate cancer (PC) is the most common cancer diagnosed in men worldwide. Currently, castration-resistant prostate cancer (CRPC), which is resistant to androgen deprivation therapy, has a poor prognosis and is a therapeutic problem. We investigated the antitumor effects on PC of an antibody neutralizing secreted disintegrin and metalloproteinase domain-containing protein 9 (sADAM9), which is a blood-soluble form. We performed proliferation assays, wound healing assays, invasion assays, Western blot (WB), and an in vivo study in which a sADAM9 neutralizing antibody was administered intratumorally to PC-bearing mice. In invasion assays, the sADAM9 neutralizing antibody significantly inhibited invasion in all cell lines (TRAMP-C2: p = 0.00776, LNCaP: p = 0.000914, PC-3: p = 0.0327, and DU145: p = 0.0254). We examined epithelial-mesenchymal transition (EMT) markers, one of the metastatic mechanisms, in WB and showed downregulation of Slug in TRAMP-C2, LNCaP, and DU145 and upregulation of E-cadherin in TRAMP-C2 and PC-3 by sADAM9 neutralization. In mouse experiments, the sADAM9 neutralizing antibody significantly suppressed tumor growth compared to controls (1.68-fold in TRAMP-C2, 1.89-fold in LNCaP, and 2.67-fold in PC-3). These results suggested that the sADAM9 neutralizing antibody inhibits invasion, migration, and tumor growth in PC. Previous studies examined the anti-tumor effect of knockdown of total ADAM9 or sADAM9, but this study used the new technology of neutralizing antibodies for sADAM9. This may be novel because there was no animal study using a neutralizing antibody for sADAM9 to see the relationship between ADAM9 expression and prostate cancer.
Assuntos
Proteínas ADAM , Movimento Celular , Transição Epitelial-Mesenquimal , Neoplasias da Próstata , Masculino , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Animais , Humanos , Movimento Celular/efeitos dos fármacos , Proteínas ADAM/metabolismo , Camundongos , Linhagem Celular Tumoral , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Anticorpos Neutralizantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human cytomegalovirus (HCMV) is a ß-herpesvirus that poses severe disease risk for immunocompromised patients who experience primary infection or reactivation. Development and optimization of safe and effective anti-HCMV therapeutics is of urgent necessity for the prevention and treatment of HCMV-associated diseases in diverse populations. The use of neutralizing monoclonal antibodies (mAbs) to limit HCMV infection poses a promising therapeutic strategy, as anti-HCMV mAbs largely inhibit infection by targeting virion glycoprotein complexes. In contrast, the small-molecule compounds currently approved for patients (e.g., ganciclovir, letermovir, and maribavir) target later stages of the HCMV life cycle. Here, we present a broadly neutralizing human mAb, designated 1C10, elicited from a VelocImmune mouse immunized with infectious HCMV particles. Clone 1C10 neutralizes infection after virion binding to cells by targeting gH/gL envelope complexes and potently reduces infection of diverse HCMV strains in fibroblast, trophoblast, and epithelial cells. Antibody competition assays found that 1C10 recognizes a region of gH associated with broad neutralization and binds to soluble pentamer in the low nanomolar range. Importantly, 1C10 treatment significantly reduced virus proliferation in both fibroblast and epithelial cells. Further, the combination treatment of mAb 1C10 with ganciclovir reduced HCMV infection and proliferation in a synergistic manner. This work characterizes a neutralizing human mAb for potential use as a HCMV treatment, as well as a possible therapeutic strategy utilizing combination-based treatments targeting disparate steps of the viral life cycle. Collectively, the findings support an antibody-based therapy to effectively treat patients at risk for HCMV-associated diseases. IMPORTANCE: Human cytomegalovirus is a herpesvirus that infects a large proportion of the population and can cause significant disease in diverse patient populations whose immune systems are suppressed or compromised. The development and optimization of safe anti-HCMV therapeutics, especially those that have viral targets and inhibition mechanisms different from current HCMV treatments, are of urgent necessity to better public health. Human monoclonal antibodies (mAbs) that prevent HCMV entry of cells were identified by immunizing transgenic mice and screened for broad and effective neutralization capability. Here, we describe one such mAb, which was found to target gH/gL envelope complexes and effectively limit HCMV infection and dissemination. Further, administration of the antibody in combination with the antiviral drug ganciclovir inhibited HCMV in a synergistic manner, highlighting this approach and the use of anti-HCMV mAbs more broadly, as a potential therapeutic strategy for the treatment of diverse patient populations.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Infecções por Citomegalovirus , Citomegalovirus , Camundongos Transgênicos , Proteínas do Envelope Viral , Animais , Humanos , Citomegalovirus/imunologia , Citomegalovirus/efeitos dos fármacos , Camundongos , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/virologia , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Vírion/imunologia , Fibroblastos/virologia , Replicação Viral/efeitos dos fármacos , Anticorpos Amplamente Neutralizantes/imunologia , Antivirais/farmacologia , ImunizaçãoRESUMO
SARS-CoV-2 infection is initiated by Spike glycoprotein binding to the human angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain. Blocking this interaction has been proven to be an effective approach to inhibit virus infection. Here we report the discovery of a neutralizing nanobody named VHH60, which was directly produced from an engineering nanobody library based on a commercialized nanobody within a very short period. VHH60 competes with human ACE2 to bind the receptor binding domain of the Spike protein at S351, S470-471and S493-494 as determined by structural analysis, with an affinity of 2.56 nM. It inhibits infections of both ancestral SARS-CoV-2 strain and pseudotyped viruses harboring SARS-CoV-2 wildtype, key mutations or variants at the nanomolar level. Furthermore, VHH60 suppressed SARS-CoV-2 infection and propagation 50-fold better and protected mice from death for twice as long as the control group after SARS-CoV-2 nasal infections in vivo. Therefore, VHH60 is not only a powerful nanobody with a promising profile for disease control but also provides evidence for a highly effective and rapid approach to generating therapeutic nanobodies.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , COVID-19 , SARS-CoV-2 , Anticorpos de Domínio Único , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/imunologia , SARS-CoV-2/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/imunologia , Humanos , Animais , COVID-19/imunologia , COVID-19/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Camundongos , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Tratamento Farmacológico da COVID-19 , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Células HEK293 , Camundongos Endogâmicos BALB C , Ligação Proteica , FemininoRESUMO
Allergic rhinitis is a common non-infectious inflammatory disease that affects approximately 15â¯% of people worldwide and has a complex and unclear aetiology. In recent years, pyroptosis has been found to play a role in the development of allergic rhinitis. IL-9, pyroptosis, serum and glucocorticoid-induced protein kinase 1 (SGK1), NOD-like receptor 3 (NLRP3), and nuclear factor kappa B (NF-κB) have been shown to influence each other. Herein, we aimed to explore the role of IL-9 neutralising antibody in pyroptosis involving IL-9, SGK1, NF-κB, and NLRP3 in allergic rhinitis. We observed a decrease in cytokines involved in pyroptosis and gasdermin D (GSDMD) compared with those in mice with allergic rhinitis. Further, phosphorylation of NF-κB/p65 decreased compared with that in mice with allergic rhinitis; NLRP3 and ASC also decreased, although the levels were higher than those in controls. SGK1 levels decreased compared with that in mice with allergic rhinitis and increased after using IL-9 neutralising antibodies, thus demonstrating its negative regulatory effects. The IL-9 neutralising antibody reduced the inflammatory and pyroptosis responses via SGK1 and NF-κB/NLRP3/GSDMD pathway. Our research results indicate that IL-9 regulates allergic rhinitis via the influence of SGK1 and NF-κB/NLRP3/GSDMD signalling pathway, providing new insights for developing novel drugs to treat allergic rhinitis.
Assuntos
Anticorpos Neutralizantes , Proteínas Imediatamente Precoces , Interleucina-9 , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Serina-Treonina Quinases , Piroptose , Rinite Alérgica , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piroptose/efeitos dos fármacos , NF-kappa B/metabolismo , Rinite Alérgica/tratamento farmacológico , Rinite Alérgica/imunologia , Camundongos , Proteínas Imediatamente Precoces/metabolismo , Interleucina-9/metabolismo , Anticorpos Neutralizantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação a Fosfato/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Feminino , Citocinas/metabolismoRESUMO
BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.