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1.
Biopharm Drug Dispos ; 41(3): 111-125, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32080869

RESUMO

The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de-immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de-immunization strategies reduce the risk of anti-drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human-cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation-prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de-immunized), compared with only 336 hours (adalimumab) or 336-672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes.


Assuntos
Adalimumab/imunologia , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Animais , Feminino , Humanos , Imunização , Macaca fascicularis , Masculino , Fator de Necrose Tumoral alfa/imunologia
2.
Br J Clin Pharmacol ; 85(5): 935-948, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30677788

RESUMO

AIMS: Erythropoiesis-stimulating agents used to treat anaemia in patients with chronic kidney disease (CKD) have been associated with cardiovascular adverse events. Hepcidin production, controlled by bone morphogenic protein 6 (BMP6), regulates iron homeostasis via interactions with the iron transporter, ferroportin. High hepcidin levels are thought to contribute to increased iron sequestration and subsequent anaemia in CKD patients. To investigate alternative therapies to erythropoiesis-stimulating agents for CKD patients, monoclonal antibodies, LY3113593 and LY2928057, targeting BMP6 and ferroportin respectively, were tested in CKD patients. METHODS: Preclinical in vitro/vivo data and clinical data in healthy subjects and CKD patients were used to illustrate the translation of pharmacological properties of LY3113593 and LY2928057, highlighting the novelty of targeting these nodes within the hepcidin-ferroportin pathway. RESULTS: LY2928057 bound ferroportin and blocked interactions with hepcidin, allowing iron efflux, leading to increased serum iron and transferrin saturation levels and increased hepcidin in monkeys and humans. In CKD patients, LY2928057 led to slower haemoglobin decline and reduction in ferritin (compared to placebo). Serum iron increase was (mean [90% confidence interval]) 1.98 [1.46-2.68] and 1.36 [1.22-1.51] fold-relative to baseline following LY2928057 600 mg and LY311593 150 mg respectively in CKD patients. LY3113593 specifically blocked BMP6 binding to its receptor and produced increases in iron and transferrin saturation and decreases in hepcidin preclinically and clinically. In CKD patients, LY3113593 produced an increase in haemoglobin and reduction in ferritin (compared to placebo). CONCLUSION: LY3113593 and LY2928057 pharmacological effects (serum iron and ferritin) were translated from preclinical-to-clinical development. Such interventions may lead to new CKD anaemia treatments.


Assuntos
Anemia/tratamento farmacológico , Fármacos Hematológicos/farmacologia , Hepcidinas/metabolismo , Insuficiência Renal Crônica/complicações , Transdução de Sinais/efeitos dos fármacos , Adulto , Anemia/sangue , Anemia/etiologia , Anemia/metabolismo , Animais , Proteína Morfogenética Óssea 6/antagonistas & inibidores , Proteína Morfogenética Óssea 6/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Ferritinas/sangue , Ferritinas/metabolismo , Voluntários Saudáveis , Fármacos Hematológicos/uso terapêutico , Hemoglobinas/análise , Humanos , Ferro/sangue , Ferro/metabolismo , Macaca fascicularis , Masculino , Camundongos , Pessoa de Meia-Idade , Ratos , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/metabolismo , Resultado do Tratamento , Adulto Jovem
3.
Biotechnol Bioeng ; 115(3): 705-718, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29150961

RESUMO

Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG CH 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function.


Assuntos
Cricetulus/metabolismo , Imunoglobulina G/biossíntese , Engenharia de Proteínas , Rituximab/biossíntese , Animais , Cricetulus/genética , Glicosilação , Imunoglobulina G/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Rituximab/genética
4.
Am J Physiol Renal Physiol ; 312(6): F951-F962, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28249836

RESUMO

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.


Assuntos
Nefropatias Diabéticas/metabolismo , Rim/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Idoso , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Pressão Sanguínea , Células Cultivadas , Dependovirus/genética , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Receptores ErbB/metabolismo , Matriz Extracelular/metabolismo , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Taxa de Filtração Glomerular , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/fisiopatologia , Rim/cirurgia , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Pessoa de Meia-Idade , Nefrectomia , Fosforilação , Sistema Renina-Angiotensina , Transdução de Sinais , Fatores de Tempo , Fator de Crescimento Transformador alfa/antagonistas & inibidores , Fator de Crescimento Transformador alfa/deficiência , Fator de Crescimento Transformador alfa/genética
5.
Gut ; 63(12): 1951-9, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24598129

RESUMO

OBJECTIVE: Hypoxia affects body iron homeostasis; however, the underlying mechanisms are incompletely understood. DESIGN: Using a standardised hypoxia chamber, 23 healthy volunteers were subjected to hypoxic conditions, equivalent to an altitude of 5600 m, for 6 h. Subsequent experiments were performed in C57BL/6 mice, CREB-H knockout mice, primary hepatocytes and HepG2 cells. RESULTS: Exposure of subjects to hypoxia resulted in a significant decrease of serum levels of the master regulator of iron homeostasis hepcidin and elevated concentrations of platelet derived growth factor (PDGF)-BB. Using correlation analysis, we identified PDGF-BB to be associated with hypoxia mediated hepcidin repression in humans. We then exposed mice to hypoxia using a standardised chamber and observed downregulation of hepatic hepcidin mRNA expression that was paralleled by elevated serum PDGF-BB protein concentrations and higher serum iron levels as compared with mice housed under normoxic conditions. PDGF-BB treatment in vitro and in vivo resulted in suppression of both steady state and BMP6 inducible hepcidin expression. Mechanistically, PDGF-BB inhibits hepcidin transcription by downregulating the protein expression of the transcription factors CREB and CREB-H, and pharmacological blockade or genetic ablation of these pathways abrogated the effects of PDGF-BB toward hepcidin expression. CONCLUSIONS: Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis.


Assuntos
Hepcidinas/metabolismo , Hipóxia/metabolismo , Ferro/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Adulto , Animais , Becaplermina , Modelos Animais de Doenças , Regulação para Baixo , Eritropoese/fisiologia , Feminino , Voluntários Saudáveis , Fármacos Hematológicos/farmacologia , Células Hep G2 , Humanos , Hipóxia/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
6.
J Pharmacol Exp Ther ; 349(2): 330-43, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24518034

RESUMO

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Epirregulina , Humanos , Imunoglobulina G/imunologia , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Ligação Proteica , Fator de Crescimento Transformador alfa/genética
7.
Haematologica ; 99(9): 1516-24, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24895335

RESUMO

Anemia of chronic disease is a multifactorial disorder, resulting mainly from inflammation-driven reticuloendothelial iron retention, impaired erythropoiesis, and reduced biological activity of erythropoietin. Erythropoiesis-stimulating agents have been used for the treatment of anemia of chronic disease, although with varying response rates and potential adverse effects. Serum concentrations of hepcidin, a key regulator of iron homeostasis, are increased in patients with anemia of chronic disease and linked to the pathogenesis of this disease, because hepcidin blocks cellular iron egress, thus limiting availability of iron for erythropoiesis. We tested whether serum hepcidin levels can predict and affect the therapeutic efficacy of erythropoiesis-stimulating agent treatment using a well-established rat model of anemia of chronic disease. We found that high pre-treatment hepcidin levels correlated with an impaired hematologic response to an erythropoiesis-stimulating agent in rats with anemia of chronic disease. Combined treatment with an erythropoiesis-stimulating agent and an inhibitor of hepcidin expression, LDN-193189, significantly reduced serum hepcidin levels, mobilized iron from tissue stores, increased serum iron levels and improved hemoglobin levels more effectively than did the erythropoiesis-stimulating agent or LDN-193189 monotherapy. In parallel, both the erythropoiesis-stimulating agent and erythropoiesis-stimulating agent/LDN-193189 combined reduced the expression of cytokines known to inhibit erythropoiesis. We conclude that serum hepcidin levels can predict the hematologic responsiveness to erythropoiesis-stimulating agent therapy in anemia of chronic disease. Pharmacological inhibition of hepcidin formation improves the erythropoiesis-stimulating agent's therapeutic efficacy, which may favor a reduction of erythropoiesis-stimulating agent dosages, costs and side effects.


Assuntos
Anemia/tratamento farmacológico , Eritropoetina/farmacologia , Hematínicos/farmacologia , Hepcidinas/genética , Ferro/sangue , RNA Mensageiro/genética , Anemia/sangue , Anemia/induzido quimicamente , Anemia/patologia , Animais , Biomarcadores/sangue , Doença Crônica , Combinação de Medicamentos , Sinergismo Farmacológico , Eritropoese/efeitos dos fármacos , Feminino , Expressão Gênica , Hepcidinas/antagonistas & inibidores , Hepcidinas/sangue , Humanos , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Ferro/agonistas , Polissacarídeos Bacterianos , Prognóstico , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/sangue , Ratos , Ratos Endogâmicos Lew , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese
8.
Hum Mol Genet ; 20(6): 1232-40, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21208937

RESUMO

The genetic determinants of variation in iron status are actively sought, but remain incompletely understood. Meta-analysis of two genome-wide association (GWA) studies and replication in three independent cohorts was performed to identify genetic loci associated in the general population with serum levels of iron and markers of iron status, including transferrin, ferritin, soluble transferrin receptor (sTfR) and sTfR-ferritin index. We identified and replicated a novel association of a common variant in the type-2 transferrin receptor (TFR2) gene with iron levels, with effect sizes highly consistent across samples. In addition, we identified and replicated an association between the HFE locus and ferritin and confirmed previously reported associations with the TF, TMPRSS6 and HFE genes. The five replicated variants were tested for association with expression levels of the corresponding genes in a publicly available data set of human liver samples, and nominally statistically significant expression differences by genotype were observed for all genes, although only rs3811647 in the TF gene survived the Bonferroni correction for multiple testing. In addition, we measured for the first time the effects of the common variant in TMPRSS6, rs4820268, on hepcidin mRNA in peripheral blood (n = 83 individuals) and on hepcidin levels in urine (n = 529) and observed an association in the same direction, though only borderline significant. These functional findings require confirmation in further studies with larger sample sizes, but they suggest that common variants in TMPRSS6 could modify the hepcidin-iron feedback loop in clinically unaffected individuals, thus making them more susceptible to imbalances of iron homeostasis.


Assuntos
Variação Genética , Ferro/sangue , Receptores da Transferrina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Receptores da Transferrina/metabolismo , Adulto Jovem
9.
JCI Insight ; 8(13)2023 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-37427592

RESUMO

B cells contribute to multiple aspects of autoimmune disorders, and B cell-targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti-human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell-dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell-targeting therapies in treatment of autoimmune conditions without causing B cell depletion.


Assuntos
Doenças Autoimunes , Linfócitos B , Camundongos , Animais , Antígenos CD19 , Doenças Autoimunes/tratamento farmacológico
10.
Pathogens ; 12(12)2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38133292

RESUMO

As part of the non-clinical safety package characterizing bamlanivimab (SARS-CoV-2 neutralizing monoclonal antibody), the risk profile for antibody-dependent enhancement of infection (ADE) was evaluated in vitro and in an African green monkey (AGM) model of COVID-19. In vitro ADE assays in primary human macrophage, Raji, or THP-1 cells were used to evaluate enhancement of viral infection. Bamlanivimab binding to C1q, FcR, and cell-based effector activity was also assessed. In AGMs, the impact of bamlanivimab pretreatment on viral loads and clinical and histological pathology was assessed to evaluate enhanced SARS-CoV-2 replication or pathology. Bamlanivimab did not increase viral replication in vitro, despite a demonstrated effector function. In vivo, no significant differences were found among the AGM groups for weight, temperature, or food intake. Treatment with bamlanivimab reduced viral loads in nasal and oral swabs and BAL fluid relative to control groups. Viral antigen was not detected in lung tissue from animals treated with the highest dose of bamlanivimab. Bamlanivimab did not induce ADE of SARS-CoV-2 infection in vitro or in an AGM model of infection at any dose evaluated. The findings suggest that high-affinity monoclonal antibodies pose a low risk of mediating ADE in patients and support their safety profile as a treatment of COVID-19 disease.

11.
Thorax ; 67(11): 985-92, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22735687

RESUMO

BACKGROUND: Therapeutic strategies to modulate the host response to bacterial pneumonia are needed to improve outcomes during community-acquired pneumonia. This study used mice with impaired Fas signalling to examine susceptibility to pneumococcal pneumonia and decoy receptor 3 analogue (DcR3-a) to correct factors associated with increased susceptibility. METHODS: Wild-type mice and those with varying degrees of impairment of Fas (lpr) or Fas ligand signalling (gld) were challenged with Streptococcus pneumoniae and microbiological and immunological outcomes measured in the presence or absence of DcR3-a. RESULTS: During established pneumonia, neutrophils became the predominant cell in the airway and gld mice were less able to clear bacteria from the lungs, demonstrating localised impairment of pulmonary neutrophil function in comparison to lpr or wild-type mice. T-cells from gld mice had enhanced activation and reduced apoptosis in comparison to wild-type and lpr mice during established pneumonia. Treatment with DcR3-a reduced T-cell activation and corrected the defect in pulmonary bacterial clearance in gld mice. CONCLUSIONS: The results suggest that imbalance in tumour necrosis factor superfamily signalling and excessive T-cell activation can impair bacterial clearance in the lung but that DcR3-a treatment can reduce T-cell activation, restore optimal pulmonary neutrophil function and enhance bacterial clearance during S pneumoniae infection.


Assuntos
Proteína Ligante Fas/metabolismo , Neutrófilos/imunologia , Fagócitos/imunologia , Pneumonia Pneumocócica/imunologia , Membro 6b de Receptores do Fator de Necrose Tumoral/farmacologia , Animais , Modelos Animais de Doenças , Proteína Ligante Fas/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neutrófilos/efeitos dos fármacos , Fagócitos/efeitos dos fármacos , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/terapia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Streptococcus pneumoniae/imunologia
12.
Drug Metab Dispos ; 40(8): 1545-55, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22584253

RESUMO

The pH-dependent binding of IgGs to the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. Enhancing interactions between Fc and FcRn via protein engineering has been successfully used as an approach for improving the pharmacokinetics of monoclonal antibodies (mAbs). Although the quantitative translatability of the in vitro FcRn affinity enhancement to an in vivo pharmacokinetic benefit has been supported by several studies, there are also published reports indicating a disconnect in this relation. The body of literature suggests there are likely additional biochemical and biophysical properties of the mAbs along with their FcRn affinity that influence the in vivo pharmacokinetics. Herein, we more broadly evaluate the in vitro Fc-FcRn interactions and biochemical properties of five humanized IgG4 antibodies each with two Fc variant sequences (T250Q/M428L and V308P) and their corresponding pharmacokinetics in cynomolgus monkeys. Our findings indicate that the FcRn affinity-pharmacokinetic relationship does not show a direct correlation either across different IgGs or between the two variant sequences within a platform. Other parameters that have been suggested to contribute to mAb pharmacokinetic properties, such as the pH-dependent dissociation of the FcRn-IgG complexes, mAb biophysical properties, and nonspecific/charge binding characteristics of the mAbs, also did not independently explain the differing pharmacokinetic behaviors. Our results suggest that there is likely not a single in vitro parameter that readily predicts in vivo pharmacokinetics, but that the relative contribution and interplay of several factors along with the FcRn binding affinity are important determinants of mAb pharmacokinetic properties.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animais , Linhagem Celular , Humanos , Imunoglobulina G/química , Técnicas In Vitro , Macaca fascicularis , Ligação Proteica
13.
Blood ; 113(21): 5277-86, 2009 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-19293425

RESUMO

The anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and the master regulator hepcidin. Hepcidin controls cellular iron efflux on binding to the iron export protein ferroportin. Many patients, however, present with both ACD and iron deficiency anemia (ACD/IDA), the latter resulting from chronic blood loss. We used a rat model of ACD resulting from chronic arthritis and mimicked ACD/IDA by additional phlebotomy to define differing iron-regulatory pathways. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In rats and humans with ACD, serum hepcidin concentrations are elevated, which is paralleled by reduced duodenal and macrophage expression of ferroportin. Individuals with ACD/IDA have significantly lower hepcidin levels than ACD subjects, and ACD/IDA persons, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Circulating hepcidin levels affect iron traffic in ACD and ACD/IDA and are more responsive to the erythropoietic demands for iron than to inflammation. Hepcidin determination may aid to differentiate between ACD and ACD/IDA and in selecting appropriate therapy for these patients.


Assuntos
Anemia Ferropriva/patologia , Anemia/patologia , Homeostase , Ferro/metabolismo , Anemia/sangue , Anemia/metabolismo , Anemia Ferropriva/sangue , Anemia Ferropriva/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/sangue , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/análise , Doença Crônica , Modelos Animais de Doenças , Duodeno/metabolismo , Hepcidinas , Humanos , Inflamação , Macrófagos/metabolismo , Macrófagos/patologia , Ratos , Ratos Endogâmicos Lew , Baço/metabolismo , Baço/patologia
14.
Proc Natl Acad Sci U S A ; 105(17): 6320-5, 2008 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-18443287

RESUMO

The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.


Assuntos
Aciltransferases/metabolismo , Grelina/metabolismo , Acilação , Aciltransferases/genética , Animais , Caprilatos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Sequência Conservada , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Grelina/sangue , Grelina/genética , Humanos , Dados de Sequência Molecular , Pâncreas/enzimologia , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/metabolismo , Estômago/enzimologia
15.
Clin Chem ; 56(11): 1725-32, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20847326

RESUMO

BACKGROUND: Hepcidin, a key regulator of iron metabolism, binds to the iron transporter ferroportin to cause its degradation. In humans, hepcidin deficiency has been linked to hemochromatosis and iron overload, whereas increased concentrations have been reported in anemia of cancer and chronic disease. There is currently an unmet clinical need for a specific immunoassay with a low limit of quantification to measure serum concentrations of hepcidin-25, the active form of the protein. METHODS: We generated 2 antihepcidin-25 monoclonal antibodies and used them to build a sandwich ELISA. We correlated ELISA results to hepcidin-25 measurements by LC-MS and used ELISA to measure serum hepcidin-25 concentrations in normal individuals, cancer patients, and patients with rheumatoid arthritis. RESULTS: The sandwich ELISA was highly specific for hepcidin-25, having a limit of quantification of 0.01 µg/L (10 pg/mL). Serum concentrations of hepcidin-25 measured by ELISA correlated with hepcidin-25 concentrations measured by using an independent LC-MS assay (r = 0.98, P < 0.001). Hepcidin-25 concentrations were increased in patients with cancer (median 54.8 µg/L, 25%-75% range 23.2-93.5 µg/L, n = 34) and rheumatoid arthritis (median 10.6 µg/L, 25%-75% range 5.9-18.4 µg/L, n = 76) compared with healthy individuals (median 1.20 µg/L, 25%-75% range 0.42-3.07 µg/L, n = 100). CONCLUSIONS: The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of the active form of hepcidin. This ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism.


Assuntos
Anticorpos Monoclonais , Peptídeos Catiônicos Antimicrobianos/sangue , Adolescente , Adulto , Peptídeos Catiônicos Antimicrobianos/imunologia , Artrite Reumatoide/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Hepcidinas , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Grupos Raciais , Valores de Referência , Fatores Sexuais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem
16.
MAbs ; 12(1): 1770028, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32486889

RESUMO

Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) administration. As a means to improve mAb drug-ability and the patient experience, subcutaneous (SC) administration is an increasingly important delivery route for mAbs. Unlike IV administration, bioavailability limitations for antibodies have been reported following SC injection and can dictate whether a mAb is administered via this parenteral route. The SC bioavailability of antibodies has been difficult to predict, and it can be variable and partial, with values ranging from ~50% to 100%. The mechanisms leading to the incomplete bioavailability of some mAbs relative to others are not well understood. There are some limited data that suggest the physiochemical properties inherent to a mAb can contribute to its SC absorption, bioavailability, and in vivo fate. In this study, we evaluated the integrated influence of multiple mAb physiochemical factors on the SC absorption and bioavailability of six humanized mAbs in both rats and cynomolgus monkeys. We demonstrate the physiochemical properties of mAbs are critical to their rate and extent of SC absorption. The combination of high positive charge and hydrophobic interaction significantly reduced the rate of the evaluated mAb's SC absorption and bioavailability. Reduction or balancing of both these attributes via re-engineering the mAbs restored desirable properties of the molecules assessed. This included reduced association with SC tissue, improvements in mAb absorption from the SC space and overall SC bioavailability. Our findings point to the importance of evaluating the relative balance between various physiochemical factors, including charge, hydrophobicity, and stability, to improve the SC drug-ability of mAbs for selecting or engineering mAbs with enhanced in vivo absorption and bioavailability following SC administration.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Físico-Química/métodos , Animais , Anticorpos Monoclonais Humanizados/química , Bioengenharia , Disponibilidade Biológica , Desenvolvimento de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Injeções Subcutâneas , Macaca fascicularis , Ligação Proteica , Estabilidade Proteica , Ratos , Absorção Subcutânea
17.
MAbs ; 12(1): 1831880, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33183151

RESUMO

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Afinidade de Anticorpos , Quimiotaxia de Leucócito/imunologia , Humanos , Macaca fascicularis , Camundongos , Neutrófilos/imunologia , Ratos
18.
Br J Haematol ; 144(5): 789-93, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19120353

RESUMO

Iron absorption is inadequately increased in patients with chronic haemolytic anaemia, which is commonly complicated by iron overload. Growth differentiation factor 15 (GDF15) has been identified as a bone marrow-derived factor that abrogates hepcidin-mediated protection from iron overload under conditions of increased erythropoiesis. Increased concentrations of GDF15 have been reported in beta-thalassaemia patients and GDF15 has been found to suppress hepcidin expression in vitro. To further study the interdependencies of iron metabolism and erythropoiesis in vivo, the concentrations of hepcidin and GDF15 were determined in sera from 22 patients with pyruvate kinase deficiency (PKD) and 21 healthy control subjects. In PKD patients, serum hepcidin levels were 13-fold lower than in controls (2.0 ng/ml vs. 26.2 ng/ml) and GDF15 was significantly higher (859 pg/ml vs. 528 pg/ml). Serum hepcidin concentrations correlated positively with haemoglobin and negatively with serum GDF15. These results suggest that GDF15 contributes to low hepcidin expression and iron loading in PKD.


Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Fator 15 de Diferenciação de Crescimento/sangue , Sobrecarga de Ferro/sangue , Ferro/metabolismo , Piruvato Quinase/deficiência , Adolescente , Adulto , Idoso , Peptídeos Catiônicos Antimicrobianos/metabolismo , Estudos de Casos e Controles , Eritropoese/fisiologia , Feminino , Fator 15 de Diferenciação de Crescimento/metabolismo , Hemoglobinas/análise , Hepcidinas , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Reticulócitos , Estatísticas não Paramétricas , Adulto Jovem
19.
Biotechnol J ; 14(3): e1800007, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29802766

RESUMO

There is a rapidly growing reinvigoration of the investigation of small proteins, cyclic peptides, and mAb derived domains as biotherapies. The drugability of these structures are challenged by fast peripheral clearance properties that can reduce their potential to be realized as medicines. Engineering strategies have been of limited value because mechanistically the half-life benefit is manifested by increasing the molecular weight and/or the hydrodyanimc radius which slows the molecule's renal elimination, but can result in the inherent loss of activity and target accessibility. The present work evaluated an alternative approach using smaller peptide sequences which bind to the neonatal Fc receptor (FcRn). Results revealed, small linear and cyclic FcRn binding peptides (FcRnBPs) fused to a combination of the N- and C-termini of a Fab can significantly improve the pharmacokinetics of the protein in cynomolgus monkeys relative to the parental Fab. The linear and cyclic conformations, as well as, the number of FcRnBPs fused to the Fab both influence the clearance and the extent of pharmacokinetic benefit. FcRnBP fusion protein kinetics were also affected by a combination of post-translation modifications and non-specific binding properties. The results in this report lay some foundation in fostering the advent of newer technologies toward successfully improving the pharmacokinetics of proteins, peptides, and mAb-derived domains. Additional work in the integration of a variety of factors including the intended site of action, tissue disposition, metabolism, toxicity and pharmacokinetic, and pharmacodynamics relationship of the intended therapeutic modality are key areas for advancement of these approaches.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Peptídeos/metabolismo , Receptores Fc/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Meia-Vida , Humanos , Cinética , Macaca fascicularis , Masculino , Ligação Proteica/fisiologia
20.
J Clin Endocrinol Metab ; 93(5): 1980-7, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18349056

RESUMO

CONTEXT: Ghrelin, an acylated peptide hormone secreted from the gut, regulates appetite and metabolism. Elucidating its pattern of secretion in the fed and fasted states is important in the face of the obesity epidemic. OBJECTIVE: Our objective was to examine changes in circulating ghrelin and des-acyl ghrelin in response to meals and fasting using newly developed two-site sandwich assays and sample preservation protocols to allow specific detection of full-length forms. DESIGN: Ten-minute sampling was done for 26.5 h during a fed admission with standardized meals and on a separate admission during the final 24 h of a 61.5-h fast and continuing for 2.5 h after terminating the fast. SETTING: The study was conducted at the University Hospital General Clinical Research Center. PARTICIPANTS: Eight male volunteers participated, mean +/- sd age 24.5 +/- 3.7 yr and body mass index 24 +/- 2.1 kg/m(2). MAIN OUTCOME MEASURES: Ten-minute sampling profiles were assessed for ghrelin and des-acyl ghrelin, fed and fasting. RESULTS: In the fed state, ghrelin and des-acyl ghrelin showed similar dynamics; both were sharply inhibited by meals and increased at night. During fasting, ghrelin decreased to nadir levels seen postprandially, and des-acyl ghrelin remained near peak levels seen preprandially. Total full-length ghrelin (acyl plus des-acyl) levels remained unchanged. CONCLUSIONS: Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.


Assuntos
Grelina/sangue , Acilação , Adulto , Butirilcolinesterase/sangue , Ensaio de Imunoadsorção Enzimática , Jejum/sangue , Humanos , Soros Imunes/imunologia , Masculino , Sensibilidade e Especificidade
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