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
PURPOSE: To determine the feasibility of administration, safety, toxicity, immunogenicity, pharmacokinetics, maximum tolerated dose, and biodistribution of ING-1, a high-affinity, Human-Engineered monoclonal antibody (heMAb) to the Mr 40,000 epithelial cell adhesion molecule Ep-CAM, in patients with advanced adenocarcinomas. EXPERIMENTAL DESIGN: ING-1 was initially administered to patients as a 1-hour intravenous infusion every 3 weeks. Toxicity and pharmacokinetic data led to the evaluation of a weekly schedule. The distribution of iodine-131 (131I)-labeled ING-1 was studied. RESULTS: Twenty-five patients received 82 courses of ING-1. Minimal toxicity was initially observed at the 0.03-, 0.10-, and 0.30-mg/kg dose levels. A patient dosed at 1.0 mg/kg developed acute pancreatitis with severe abdominal pain, nausea, and vomiting. A patient dosed at 0.3 mg/kg had an asymptomatic amylase and lipase elevation to 502 units/L and 1,627 units/L, respectively. Both patients made uncomplicated recoveries. No other dose-limiting toxicities were observed. Regardless of dose, the volume of distribution (mean +/- SEM) was 46.6 +/- 1.6 mL/kg. ING-1 clearance decreased with increasing dose. To minimize toxicity and increase dose intensity, we then administered ING-1 weekly. No significant toxicity was observed in 7 patients dosed at 0.1 mg/kg. Studies of 131I-labeled ING-1 biodistribution showed radiolocalization to colorectal and prostate cancers. A patient with colorectal cancer had an 80% decrement in the levels of carcinoembryonic antigen. CONCLUSION: The recommended dose for ING-1 is 0.10 mg/kg by intravenous infusion weekly. The absence of severe toxicity at this dose, low immunogenicity, and preliminary evidence of ING-1 tumor localization and antitumor efficacy support the further clinical development of this antibody to treat Ep-CAM-positive malignant diseases.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Neoplasias/tratamento farmacológico , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Adulto , Idoso , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias , Antineoplásicos/farmacologia , Área Sob a Curva , Peso Corporal , Moléculas de Adesão Celular , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Even after neutrophil counts return to near normal levels, patients undergoing myeloablative chemotherapy and bone marrow transplantation (BMT) are at risk for invasive bacterial infections, raising the possibility that their neutrophil function might be impaired. To assess potential qualitative defects in neutrophil function in patients undergoing BMT, we measured neutrophil content of the antimicrobial (poly)peptides BPI and defensins. METHODS: Neutrophil extracts were analyzed for content of BPI by Western blotting and ELISA and for defensin peptides by acid-urea polyacrylamide gel electrophoresis (PAGE). Antibacterial activity of neutrophil extracts was measured against Escherichia coli K1/r, a clinical isolate sensitive to synergistic killing by BPI and defensins. RESULTS: Neutrophil extract BPI content on post-BMT days +20, +30, and +100 (169+/-35, 232+/-57, and 160+/-55 ng per 106 neutrophils, respectively) was similar to the neutrophil BPI content of normal controls (163+/-35 ng per 106 neutrophils). Neutrophil defensin content also did not vary during this time-span. Activity of neutrophil extracts against E. coli K1/r did not differ between BMT patients and controls. CONCLUSION: At post-BMT days +20 to +100, neutrophils derived from engrafted marrow contain normal quantities of BPI and defensins. Any deficiencies of neutrophil function during this phase are not due to inadequate expression of these antimicrobial (poly)peptides but could reflect abnormalities in other aspects of neutrophil function.
Assuntos
Proteínas Sanguíneas/metabolismo , Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Defensinas/metabolismo , Proteínas de Membrana , Neutrófilos/fisiologia , Adolescente , Adulto , Peptídeos Catiônicos Antimicrobianos , Extratos Celulares/farmacologia , Criança , Pré-Escolar , Proteínas de Escherichia coli/efeitos dos fármacos , Humanos , Lactente , Neutrófilos/química , Valores de ReferênciaRESUMO
BACKGROUND: Hematopoietic stem cell transplantation (SCT) carries a significant risk of severe therapy-associated complications chief among which is acute graft vs.host disease (aGVHD). Animal models indicate that myeloablative chemotherapy compromises the mucosal barrier, thereby allowing translocation of intestinal flora-derived lipopolysaccharides (or endotoxin) that subsequently trigger aGVHD, but there are no comparable data in humans. Our aim was to gain insight into the potential role of endotoxin and endotoxin-induced acute phase proteins in children undergoing SCT. METHODS: Plasma concentrations of C-reactive protein (CRP) and lipopolysaccharide-binding protein (LBP) were measured in 57 pediatric patients undergoing SCT. In addition plasma endotoxin levels were measured in 25 patients. RESULTS: The previously described rise in CRP was confirmed, and a marked elevation of LBP was observed that peaked at Day 7 (median value, 6.6 microg/ml; P < 0.03 for all pairwise comparisons). CRP and LBP values were significantly correlated (r = 0.77, P < 0.001). A significant but complex relationship was noted between LBP concentrations at Day 0 and severity of subsequent aGVHD (P = 0.02). Of the 25 patients assayed, 11 (44%) had detectable endotoxemia, including 4 who were endotoxin-positive at Day 0. CONCLUSIONS: The detection of endotoxemia coupled with marked elevations in LBP at Day 7 raises the possibility that inflammatory responses early after SCT may be driven in part by the entry of lipopolysaccharide into the bloodstream.
Assuntos
Proteínas de Fase Aguda , Proteína C-Reativa/análise , Proteínas de Transporte/sangue , Endotoxemia/epidemiologia , Transplante de Células-Tronco Hematopoéticas , Glicoproteínas de Membrana , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Estatísticas não ParamétricasRESUMO
Many therapeutic monoclonal antibodies act as antagonists to receptors by targeting and blocking the natural ligand binding site (orthosteric site). In contrast, the use of antibodies to target receptors at allosteric sites (distinct from the orthosteric site) has not been extensively studied. This approach is especially important in metabolic diseases in which endogenous ligand levels are dysregulated. Herein, we review our investigations of 3 categories of human monoclonal antibodies that bind allosterically to the insulin receptor (INSR) and affect its activity: XMetA, XMetS and XMetD. XMetA directly activates the INSR either alone or in combination with insulin. XMetS, in contrast, does not directly activate the INSR but markedly enhances the receptor's ability to bind insulin and potentiate insulin signaling. Both XMetA and XMetS are effective in controlling hyperglycemia in mouse models of diabetes. A third allosteric antibody, XMetD, is an inhibitor of INSR signaling. This antibody reverses insulin-induced hypoglycemia in a mouse model of hyperinsulinemia. These studies indicate, therefore, that allosteric antibodies to INSR can modulate its signaling and correct conditions of glucose dysregulation. These studies also raise the possibility that the use of allosteric antibodies can be expanded to other receptors for the treatment of metabolic disorders.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Hiperglicemia/tratamento farmacológico , Hipoglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Receptor de Insulina/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Insulina/sangue , Insulina/metabolismo , Proteína Oncogênica v-akt/metabolismo , FosforilaçãoRESUMO
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
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.