RESUMO
Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Interleucina-1beta/fisiologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Bioengenharia , Fibroblastos/citologia , Fibroblastos/fisiologia , Células HeLa/efeitos dos fármacos , Células HeLa/fisiologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Interleucina-1/fisiologia , Interleucina-1beta/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/fisiologia , Cinética , Ligantes , Luciferases/genética , Pulmão/citologia , Pulmão/fisiologia , NF-kappa B/fisiologia , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Receptores de Interleucina-1/efeitos dos fármacos , Receptores de Interleucina-1/fisiologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.
Assuntos
Anticorpos/metabolismo , Fibroblastos/metabolismo , Integrina alfaV/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Anticorpos/imunologia , Bleomicina , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Fibroblastos/citologia , Humanos , Integrina alfaV/imunologia , Masculino , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Propionatos/farmacologia , Ligação Proteica , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controleRESUMO
Purpose: Tumor-associated PD-L1 expression is predictive of clinical response to PD-1-directed immunotherapy. However, PD-L1-negative patients may also respond to PD-1 checkpoint blockade, suggesting that other PD-1 ligands may be relevant to the clinical activity of these therapies. The prevalence of PD-L2, the other known ligand of PD-1, and its relationship to response to anti-PD-1 therapy were evaluated.Experimental Design: PD-L2 expression was assessed in archival tumor tissue from seven indications using a novel immunohistochemical assay. In addition, relationships between clinical response and PD-L2 status were evaluated in tumor tissues from patients with head and neck squamous cell carcinoma (HNSCC) with recurrent or metastatic disease, treated with pembrolizumab.Results: PD-L2 expression was observed in all tumor types and present in stromal, tumor, and endothelial cells. The prevalence and distribution of PD-L2 correlated significantly with PD-L1 (P = 0.0012-<0.0001); however, PD-L2 was detected in the absence of PD-L1 in some tumor types. Both PD-L1 and PD-L2 positivity significantly predicted clinical response to pembrolizumab on combined tumor, stromal and immune cells, with PD-L2 predictive independent of PD-L1. Response was greater in patients positive for both PD-L1 and PD-L2 (27.5%) than those positive only for PD-L1 (11.4%). PD-L2 status was also a significant predictor of progression-free survival (PFS) with pembrolizumab independent of PD-L1 status. Longer median times for PFS and overall survival were observed for PD-L2-positive than PD-L2-negative patients.Conclusions: Clinical response to pembrolizumab in patients with HNSCC may be related partly to blockade of PD-1/PD-L2 interactions. Therapy targeting both PD-1 ligands may provide clinical benefit in these patients. Clin Cancer Res; 23(12); 3158-67. ©2017 AACR.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Imunoterapia , Proteína 2 Ligante de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/administração & dosagem , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/imunologia , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Previously we reported studies of XMetA, an agonist antibody to the insulin receptor (INSR). We have now utilized phage display to identify XMetS, a novel monoclonal antibody to the INSR. Biophysical studies demonstrated that XMetS bound to the human and mouse INSR with picomolar affinity. Unlike monoclonal antibody XMetA, XMetS alone had little or no agonist effect on the INSR. However, XMetS was a strong positive allosteric modulator of the INSR that increased the binding affinity for insulin nearly 20-fold. XMetS potentiated insulin-stimulated INSR signaling â¼15-fold or greater including; autophosphorylation of the INSR, phosphorylation of Akt, a major enzyme in the metabolic pathway, and phosphorylation of Erk, a major enzyme in the growth pathway. The enhanced signaling effects of XMetS were more pronounced with Akt than with Erk. In cultured cells, XMetS also enhanced insulin-stimulated glucose transport. In contrast to its effects on the INSR, XMetS did not potentiate IGF-1 activation of the IGF-1 receptor. We studied the effect of XMetS treatment in two mouse models of insulin resistance and diabetes. The first was the diet induced obesity mouse, a hyperinsulinemic, insulin resistant animal, and the second was the multi-low dose streptozotocin/high-fat diet mouse, an insulinopenic, insulin resistant animal. In both models, XMetS normalized fasting blood glucose levels and glucose tolerance. In concert with its ability to potentiate insulin action at the INSR, XMetS reduced insulin and C-peptide levels in both mouse models. XMetS improved the response to exogenous insulin without causing hypoglycemia. These data indicate that an allosteric monoclonal antibody can be generated that markedly enhances the binding affinity of insulin to the INSR. These data also suggest that an INSR monoclonal antibody with these characteristics may have the potential to both improve glucose metabolism in insulinopenic type 2 diabetes mellitus and correct compensatory hyperinsulinism in insulin resistant conditions.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD/metabolismo , Glucose/metabolismo , Receptor de Insulina/metabolismo , Sítio Alostérico , Animais , Peptídeo C/química , Células CHO , Separação Celular , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Citometria de Fluxo , Humanos , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Insulina/química , Resistência à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Biblioteca de Peptídeos , Fosforilação , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.
Assuntos
Anticorpos Monoclonais/imunologia , Hiperinsulinismo Congênito/imunologia , Receptor de Insulina/antagonistas & inibidores , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/imunologia , Células CHO , Hiperinsulinismo Congênito/tratamento farmacológico , Hiperinsulinismo Congênito/patologia , Cricetinae , Cricetulus , Glucose/imunologia , Resistência à Insulina/imunologia , Camundongos , Ratos , Receptor de Insulina/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
OBJECTIVE: Interleukin-1ß (IL-1ß) has recently been implicated as a major cytokine that is involved in the pancreatic islet inflammation of type 2 diabetes mellitus. This inflammation impairs insulin secretion by inducing beta-cell apoptosis. Recent evidence has suggested that in obesity-induced inflammation, IL-1ß plays a key role in causing insulin resistance in peripheral tissues. DESIGN AND METHODS: To further investigate the pathophysiological role of IL-1ß in causing insulin resistance, the inhibitory effects of IL-1ß on several insulin-dependent metabolic processes in vitro has been neutralized by XOMA 052. The role IL-1ß plays in insulin resistance in adipose tissue was assessed using differentiated 3T3-L1 adipocytes and several parameters involved in insulin signaling and lipid metabolism were examined. RESULTS AND CONCLUSION: IL-1ß inhibited insulin-induced activation of Akt phosphorylation, glucose transport, and fatty acid uptake. IL-1ß also blocked insulin-mediated downregulation of suppressor of cytokine signaling-3 expression. Co-preincubation of IL-1ß with XOMA 052 neutralized nearly all of these inhibitory effects in 3T3-L1 adipocytes. These studies provide evidence, therefore, that IL-1ß is a key proinflammatory cytokine that is involved in inducing insulin resistance. These studies also suggest that the monoclonal antibody XOMA 052 may be a possible therapeutic to effectively neutralize cytokine-mediated insulin resistance in adipose tissue.
Assuntos
Adipócitos/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/farmacologia , Resistência à Insulina , Interleucina-1beta/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/antagonistas & inibidores , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/antagonistas & inibidores , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Metabolismo dos Lipídeos , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Many patients with diabetes mellitus (both type 1 and type 2) require therapy to maintain normal fasting glucose levels. To develop a novel treatment for these individuals, we used phage display technology to target the insulin receptor (INSR) complexed with insulin and identified a high affinity, allosteric, human monoclonal antibody, XMetA, which mimicked the glucoregulatory, but not the mitogenic, actions of insulin. Biophysical studies with cultured cells expressing human INSR demonstrated that XMetA acted allosterically and did not compete with insulin for binding to its receptor. XMetA was found to function as a specific partial agonist of INSR, eliciting tyrosine phosphorylation of INSR but not the IGF-IR. Although this antibody activated metabolic signaling, leading to enhanced glucose uptake, it neither activated Erk nor induced proliferation of cancer cells. In an insulin resistant, insulinopenic model of diabetes, XMetA markedly reduced elevated fasting blood glucose and normalized glucose tolerance. After 6 weeks, significant improvements in HbA(1c), dyslipidemia, and other manifestations of diabetes were observed. It is noteworthy that hypoglycemia and weight gain were not observed during these studies. These studies indicate, therefore, that allosteric monoclonal antibodies have the potential to be novel, ultra-long acting, agents for the regulation of hyperglycemia in diabetes.
Assuntos
Anticorpos Monoclonais/farmacologia , Glicemia/fisiologia , Diabetes Mellitus Experimental/terapia , Receptor de Insulina/agonistas , Animais , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Biomarcadores , Células CHO , Células Cultivadas , Cricetinae , Teste de Tolerância a Glucose , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Organismos Livres de Patógenos EspecíficosRESUMO
Interleukin-1ß (IL-1ß) is a potent mediator of inflammatory responses and plays a role in the differentiation of a number of lymphoid cells. In several inflammatory and autoimmune diseases, serum levels of IL-1ß are elevated and correlate with disease development and severity. The central role of the IL-1 pathway in several diseases has been validated by inhibitors currently in clinical development or approved by the FDA. However, the need to effectively modulate IL-1ß-mediated local inflammation with the systemic delivery of an efficacious, safe and convenient drug still exists. To meet these challenges, we developed XOMA 052 (gevokizumab), a potent anti-IL-1ß neutralizing antibody that was designed in silico and humanized using Human Engineering™ technology. XOMA 052 has a 300 femtomolar binding affinity for human IL-1ß and an in vitro potency in the low picomolar range. XOMA 052 binds to a unique IL-1ß epitope where residues critical for binding have been identified. We have previously reported that XOMA 052 is efficacious in vivo in a diet-induced obesity mouse model thought to be driven by low levels of chronic inflammation. We report here that XOMA 052 also reduces acute inflammation in vivo, neutralizing the effect of exogenously administered human IL-1ß and blocking peritonitis in a mouse model of acute gout. Based on its high potency, novel mechanism of action, long half-life, and high affinity, XOMA 052 provides a new strategy for the treatment of a number of inflammatory, autoimmune and metabolic diseases in which the role of IL-1ß is central to pathogenesis.