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1.
J Orthop Translat ; 29: 134-142, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34249611

RESUMO

BACKGROUND: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characterised by excessive bone formation, is caused by absence of sclerostin, a negative regulator of bone formation that binds LRP5/6 Wnt co-receptors. Current treatment is limited to surgical management of symptoms arising from bone overgrowth. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. METHODS: Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/- mice were treated with 10 mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4 mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (µCT). RESULTS: Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/- mice indicated the presence of hFc increased protein half-life from less than 5 min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively. CONCLUSION: Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/- mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.

2.
Int Immunopharmacol ; 66: 362-365, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30529500

RESUMO

Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by pathogenic immunoglobulin G (IgG) autoantibodies that bind to platelets, causing their phagocytic removal and leading to reductions in platelet number. The neonatal Fc receptor (FcRn) selectively salvages and recycles IgG, including pathogenic IgG, thereby extending the half-life of IgG in plasma. Two anti-mouse FcRn monoclonal antibodies (mAb) (4470 and 4464) were generated to evaluate the effect of inhibiting IgG recycling. Statistically significant reductions in plasma IgG concentration were observed upon administration of 4470 (10, 30 and 100 mg/kg) in wild-type mice. In a passive mouse model of ITP, 4464 alleviated the reduction in platelet number and/or preserved newly produced platelets when dosed prophylactically as well as in a therapeutic dosing regimen once platelet numbers had already been reduced. These results support the investigation of anti-FcRn therapy as a potential treatment for ITP.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Plaquetas/imunologia , Imunoglobulina G/sangue , Imunoglobulinas Intravenosas/uso terapêutico , Imunoterapia/métodos , Anticorpos de Cadeia Única/uso terapêutico , Trombocitopenia/terapia , Animais , Anticorpos Monoclonais/genética , Autoanticorpos/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunidade Humoral , Camundongos , Camundongos Endogâmicos C57BL , Contagem de Plaquetas , Receptores Fc/imunologia , Anticorpos de Cadeia Única/genética , Trombocitopenia/imunologia
3.
MAbs ; 10(7): 1111-1130, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30130439

RESUMO

Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).


Assuntos
Anticorpos Monoclonais Humanizados/química , Antígenos de Histocompatibilidade Classe I/imunologia , Imunossupressores/química , Miastenia Gravis/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptores Fc/imunologia , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/metabolismo , Ensaios Clínicos como Assunto , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoglobulina G/sangue , Imunossupressores/metabolismo , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Ligação Proteica , Receptores Fc/genética , Transgenes/genética
4.
Bone ; 107: 93-103, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29129759

RESUMO

Administration of antibodies to sclerostin (Scl-Ab) has been shown to increase bone mass, bone mineral density (BMD) and bone strength by increasing bone formation and decreasing bone resorption in both animal studies and human clinical trials. In these studies, the magnitude and rate of increase in bone formation markers is attenuated upon repeat dosing with Scl-Ab despite a continuous and progressive increase in BMD. Here, we investigated whether the attenuation in the bone formation response following repeated administration of Scl-Ab was associated with increased expression of secreted antagonists of Wnt signalling and determined how the circulating marker of bone formation, P1NP, responded to single, or multiple doses, of Scl-Ab four days post-dosing. Female Balb/c mice were treated with Scl-Ab and we demonstrated that the large increase in serum P1NP observed following the first dose was reduced following administration of multiple doses of Scl-Ab. This dampening of the P1NP response was not due to a change in the kinetics of the bone formation marker response, or differences in exposure to the drug. The abundance of transcripts encoding several secreted Wnt antagonists was determined in femurs collected from mice following one or six doses of Scl-Ab, or vehicle treatment. Compared with vehicle controls, expression of SOST, SOST-DC1, DKK1, DKK2, SFRP1, SFRP2, FRZB, SFRP4 and WIF1 transcripts was significantly increased (approximately 1.5-4.2 fold) following a single dose of Scl-Ab. With the exception of SFRP1, these changes were maintained or further increased following six doses of Scl-Ab and the abundance of SFRP5 was also increased. Up-regulation of these Wnt antagonists may exert a negative feedback to increased Wnt signalling induced by repeated administration of Scl-Ab and could contribute to self-regulation of the bone formation response over time. After an antibody-free period of four weeks or more, the P1NP response was comparable to the naïve response, and a second phase of treatment with Scl-Ab following an antibody-free period elicited additional gains in BMD. Together, these data demonstrate that the rapid dampening of the bone formation response in the immediate post-dose period which occurs after repeat dosing of Scl-Ab is associated with increased expression of Wnt antagonists, and a treatment-free period can restore the full bone formation response to Scl-Ab.


Assuntos
Glicoproteínas/antagonistas & inibidores , Osteogênese/efeitos dos fármacos , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos/farmacologia , Feminino , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos BALB C , Regulação para Cima
5.
J Immunol ; 198(7): 2876-2885, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28242648

RESUMO

We hypothesized that exudates collected at the beginning of the resolution phase of inflammation might be enriched for tissue protective molecules; thus an integrated cellular and molecular approach was applied to identify novel chondroprotective bioactions. Exudates were collected 6 h (inflammatory) and 24 h (resolving) following carrageenan-induced pleurisy in rats. The resolving exudate was subjected to gel filtration chromatography followed by proteomics, identifying 61 proteins. Fractions were added to C28/I2 chondrocytes, grown in micromasses, ions with or without IL-1ß or osteoarthritic synovial fluids for 48 h. Three proteins were selected from the proteomic analysis, α1-antitrypsin (AAT), hemopexin (HX), and gelsolin (GSN), and tested against catabolic stimulation for their effects on glycosaminoglycan deposition as assessed by Alcian blue staining, and gene expression of key anabolic proteins by real-time PCR. In an in vivo model of inflammatory arthritis, cartilage integrity was determined histologically 48 h after intra-articular injection of AAT or GSN. The resolving exudate displayed protective activities on chondrocytes, using multiple readouts: these effects were retained in low m.w. fractions of the exudate (46.7% increase in glycosaminoglycan deposition; ∼20% upregulation of COL2A1 and aggrecan mRNA expression), which reversed the effect of IL-1ß. Exogenous administration of HX, GSN, or AAT abrogated the effects of IL-1ß and osteoarthritic synovial fluids on anabolic gene expression and increased glycosaminoglycan deposition. Intra-articular injection of AAT or GSN protected cartilage integrity in mice with inflammatory arthritis. In summary, the strategy for identification of novel chondroprotective activities in resolving exudates identified HX, GSN and AAT as potential leads for new drug discovery programs.


Assuntos
Artrite Experimental/patologia , Condrócitos/efeitos dos fármacos , Exsudatos e Transudatos/química , Pleurisia/imunologia , Animais , Modelos Animais de Doenças , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite do Joelho/patologia , Proteômica , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
6.
J Biol Chem ; 284(16): 10890-900, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19208630

RESUMO

The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.


Assuntos
Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/metabolismo , Osteogênese/fisiologia , Conformação Proteica , Proteínas Wnt/metabolismo , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/genética , Marcadores Genéticos/genética , Heparina/química , Heparina/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , beta Catenina/metabolismo
7.
Protein Eng Des Sel ; 20(5): 227-34, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17452434

RESUMO

Antigen-binding fragments (Fab') of antibodies can be site specifically PEGylated at thiols using cysteine reactive PEG-maleimide conjugates. For therapeutic Fab'-PEG, conjugation with 40 kDa of PEG at a single hinge cysteine has been found to confer appropriate pharmacokinetic properties to enable infrequent dosing. Previous methods have activated the hinge cysteine using mildly reducing conditions in order to retain an intact interchain disulphide. We demonstrate that the final Fab-PEG product does not need to retain the interchain disulphide and also therefore that strongly reducing conditions can be used. This alternative approach results in PEGylation efficiencies of 88 and 94% for human and murine Fab, respectively. It also enables accurate and efficient site-specific multi-PEGylation. The use of the non-thiol reductant tris(2-carboxyethyl) phosphine combined with protein engineering enables us to demonstrate the mono-, di- and tri-PEGylation of Fab fragments with a range of PEG size. We present evidence that PEGylated and unPEGylated Fab' molecules that lack an interchain disulphide bond retain very high levels of chemical and thermal stability and normal performance in PK and efficacy models.


Assuntos
Dissulfetos/química , Fragmentos Fab das Imunoglobulinas/química , Polietilenoglicóis/química , Engenharia de Proteínas/métodos , Substâncias Redutoras/química , Sequência de Aminoácidos , Animais , Cisteína/química , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Camundongos , Dados de Sequência Molecular , Oxirredução , Fosfinas/química , Ratos , Ratos Sprague-Dawley
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