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INTRODUCTION: Hemophilia B is an inherited X chromosome-linked disorder characterized by impaired blood clotting owing to the absence of functional coagulation factor IX. Due to the relatively short half-life of factor IX, patients with hemophilia B require frequent factor IX infusions to maintain prophylaxis. We have developed a recombinant factor IX (rFIX) fused to the Fc region of IgG (rFIXFc) with an extended half-life in animals and humans. MATERIALS AND METHODS: Procoagulant properties of rFIXFc and rFIX (BENEFIX®) were compared to determine the effect of the Fc region on rFIXFc hemostatic function. Specifically, we assessed rFIXFc activation, intermolecular interactions within the Xase complex, inactivation by antithrombin III (AT) and thrombin generation potential compared with rFIX. We also assessed the acute and prophylactic efficacy profiles of rFIXFc and rFIX in vivo in hemophilia B mouse bleeding models. RESULTS AND CONCLUSIONS: The activation by factor XIa or factor VIIa/tissue factor, inhibition by AT, interaction profiles with phospholipids, affinities for factor VIIIa within the context of the Xase complex, and thrombin generation profiles were similar for rFIXFc and rFIX. Xase complexes formed with either molecule exhibited similar kinetic profiles for factor Xa generation. In acute efficacy models, mice infused with rFIXFc or rFIX were equally protected from bleeding. However, in prophylactic efficacy models, protection from bleeding was maintained approximately three times longer in rFIXFc-dosed mice than in those given rFIX; this prolonged efficacy correlates with the previously observed half-life extension. We conclude that rFIXFc retains critical FIX procoagulant attributes and that the extension in rFIXFc half-life translates into prolonged efficacy in hemophilia B mice.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/farmacologia , Fator IX/farmacologia , Hemofilia B/tratamento farmacológico , Hemorragia/tratamento farmacológico , Fragmentos Fc das Imunoglobulinas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antitrombina III/farmacologia , Testes de Coagulação Sanguínea , Modelos Animais de Doenças , Ativação Enzimática/fisiologia , Fator IX/genética , Fator VIIa/farmacologia , Fator XIa/farmacologia , Meia-Vida , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Trombina/biossínteseRESUMO
The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach - depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases.
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Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an â¼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.
Assuntos
Fator VIII/farmacocinética , Hemofilia A/metabolismo , Antígenos de Histocompatibilidade Classe I/farmacologia , Proteínas Recombinantes de Fusão/farmacocinética , Animais , Coagulantes/farmacocinética , Coagulantes/uso terapêutico , Modelos Animais de Doenças , Doenças do Cão/tratamento farmacológico , Doenças do Cão/metabolismo , Cães , Fator VIII/química , Fator VIII/genética , Fator VIII/uso terapêutico , Células HEK293 , Meia-Vida , Hemofilia A/tratamento farmacológico , Hemofilia A/patologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Fc/química , Receptores Fc/metabolismo , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Tempo de Coagulação do Sangue TotalRESUMO
Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short in vivo half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semisynthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied among 2, 11, or 16 amino acids. In addition, either one ("monomeric") or two ("dimeric") ANP peptides were linked to Fc to study whether this modification had an effect on in vitro activity and/or in vivo half-life. The various constructs were studied for in vitro activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16- and â¼375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc's were found to be approximately 2 orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8-5.5 h), but a trend was observed where the C(max) of the monomeric constructs was approximately 3-fold higher than that of the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases.
Assuntos
Fator Natriurético Atrial/farmacocinética , Fragmentos Fc das Imunoglobulinas/química , Animais , Fator Natriurético Atrial/química , Linhagem Celular , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Feminino , Meia-Vida , Humanos , Técnicas In Vitro , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Current factor IX (FIX) products display a half-life (t(1/2)) of â¼ 18 hours, requiring frequent intravenous infusions for prophylaxis and treatment in patients with hemophilia B. This open-label, dose-escalation trial in previously treated adult subjects with hemophilia B examined the safety and pharmacokinetics of rFIXFc. rFIXFc is a recombinant fusion protein composed of FIX and the Fc domain of human IgG(1), to extend circulating time. Fourteen subjects received a single dose of rFIXFc; 1 subject each received 1, 5, 12.5, or 25 IU/kg, and 5 subjects each received 50 or 100 IU/kg. rFIXFc was well tolerated, and most adverse events were mild or moderate in intensity. No inhibitors were detected in any subject. Dose-proportional increases in rFIXFc activity and Ag exposure were observed. With baseline subtraction, mean activity terminal t(1/2) and mean residence time for rFIXFc were 56.7 and 71.8 hours, respectively. This is â¼ 3-fold longer than that reported for current rFIX products. The incremental recovery of rFIXFc was 0.93 IU/dL per IU/kg, similar to plasma-derived FIX. These results show that rFIXFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia B. The trial was registered at www.clinicaltrials.gov as NCT00716716.
Assuntos
Fator IX/metabolismo , Hemofilia B/metabolismo , Hemofilia B/terapia , Proteínas Recombinantes de Fusão/uso terapêutico , Adolescente , Adulto , Idoso , Feminino , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/farmacocinética , Segurança , Adulto JovemRESUMO
Treatment of hemophilia B requires frequent infusions of factor IX (FIX) to prophylax against bleeding episodes. Hemophilia B management would benefit from a FIX protein with an extended half-life. A recombinant fusion protein (rFIXFc) containing a single FIX molecule attached to the Fc region of immunoglobulin G was administered intravenously and found to have an extended half-life, compared with recombinant FIX (rFIX) in normal mice, rats, monkeys, and FIX-deficient mice and dogs. Recombinant FIXFc protein concentration was determined in all species, and rFIXFc activity was measured in FIX-deficient animals. The half-life of rFIXFc was approximately 3- to 4-fold longer than that of rFIX in all species. In contrast, in mice in which the neonatal Fc receptor (FcRn) was deleted, the half-life of rFIXFc was similar to rFIX, confirming the increased circulatory time was due to protection of the rFIXFc via the Fc/FcRn interaction. Whole blood clotting time in FIX-deficient mice was corrected through 144 hours for rFIXFc, compared with 72 hours for rFIX; similar results were observed in FIX-deficient dogs. Taken together, these studies show the enhanced pharmacodynamic and pharmacokinetic properties of the rFIXFc fusion protein and provide the basis for evaluating rFIXFc in patients with hemophilia B.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fator IX/farmacocinética , Fragmentos Fc das Imunoglobulinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Animais , Tempo de Sangramento , Coagulação Sanguínea/genética , Células Cultivadas , Doenças do Cão/sangue , Doenças do Cão/tratamento farmacológico , Cães , Avaliação Pré-Clínica de Medicamentos , Fator IX/genética , Fator IX/metabolismo , Fator IX/fisiologia , Fator IX/uso terapêutico , Feminino , Hemofilia B/sangue , Hemofilia B/tratamento farmacológico , Hemofilia B/veterinária , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Multimerização Proteica , Ratos , Proteínas Recombinantes de Fusão/uso terapêutico , Fatores de TempoRESUMO
The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG-hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Macaca fascicularis/imunologia , Peptídeos/imunologia , Receptores Fc/antagonistas & inibidores , Receptores Fc/imunologia , Albuminas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Imunoglobulina A/imunologia , Imunoglobulina M/imunologia , Macaca fascicularis/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Solubilidade , Ressonância de Plasmônio de SuperfícieRESUMO
We have applied a "physiologic" approach to the pulmonary delivery of therapeutic proteins, utilizing an immunoglobulin (antibody) transport pathway recently shown to be present predominantly in the conducting airways of the human respiratory tract. Therapeutic proteins are fused to the Fc-domain of an IgG1, allowing them to bind with high affinity to the antibody transport receptor, FcRn. Liquid aerosols are administered into the lung using normal breathing maneuvers and efficient delivery of several different Fc-fusion proteins has been achieved with retention of biological activity and an increase in circulating half-life. A new paradigm for the pulmonary delivery of therapeutic proteins and a fundamental advance in the construction of Fc-fusion proteins for this purpose will be described.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Pulmão/metabolismo , Preparações Farmacêuticas/administração & dosagem , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Administração por Inalação , Animais , HumanosRESUMO
The delivery of therapeutic proteins by noninvasive routes of administration has been a challenging goal, hence current modes of delivery generally require injections. However, we have recently shown that a naturally occurring receptor, the neonatal Fc receptor (FcRn) can be utilized to carry aerosolized therapeutic proteins conjugated to a portion of its respective ligand (Fc domain of immunoglobulin G) across epithelial cells of the lung to effectively deliver biologically active molecules to the bloodstream. First-generation dimeric Fc fusion molecules were successfully transported by the pulmonary route and biologic activity was demonstrated in both non-human primates and human volunteers. Continuing efforts to improve transport efficiency have led to the development of an alternate configuration of Fc fusion proteins with improved characteristics. These second generation Fc fusion molecules are monomeric with respect to the therapeutic protein and dimeric with respect to the Fc region, and have been termed Fc fusion 'monomers'. Several different Fc fusion monomers have demonstrated improved transport efficiency, achieving high bioavailabilities for pulmonary delivery in non-human primates. While the traditional dimeric Fc fusion molecule generally increases the half-life compared with the unconjugated effector molecule, the monomer configuration has been shown to result in an even greater extension of the circulating half-life, which improves pharmacokinetic parameters for protein therapeutics, whether administered by pulmonary delivery or injection. Finally, many of the Fc monomer fusions have enhanced biologic activity compared with the dimeric configuration. Because of these many advantages, the monomer configuration promises to be an enabling advance to achieve clinically relevant, noninvasive delivery with potentially less frequent administration regimens for a broad range of protein therapeutics. In addition, molecules that are comprised of heterodimeric subunits or multi-subunit complexes can also be constructed as Fc fusions that result in a molecule with enhanced pharmacokinetics and greater bioactivity. Several examples of novel Fc fusion proteins, both monomer and heterodimer are described herein.
Assuntos
Eritropoetina/metabolismo , Fator IX/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Pulmão/metabolismo , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Animais , Sistemas de Liberação de Medicamentos , Humanos , Ligação Proteica , Transporte ProteicoRESUMO
A novel drug delivery platform has been developed that utilizes a naturally occurring receptor known as the neonatal Fc receptor (FcRn). The receptor is specific for the Fc fragment of IgG and is expressed in epithelial cells where it functions to transport immunoglobulins across these cell barriers. It has been shown that FcRn is expressed in both the upper and central airways in non-human primates as well as in humans. Pulmonary delivery of an erythropoietin- Fc fusion molecule (EpoFc) was previously demonstrated in non-human primates using this FcRn pathway. We have now conducted a phase I clinical study to test whether the FcRn pathway functioned similarly in man using human erythropoietin (Epo) fused to the Fc portion of human IgG1. The design was a three leg, non-randomized study conducted in healthy male volunteers with rising doses (3, 10, and 30 microg/kg) of the fusion protein targeted to the central lung regions. Using a target range of 10-30% vital capacity and 15 breaths per minute, approximately 70% of the lung-deposited dose of aerosolized EpoFc was delivered safely and effectively to the central lung regions. We showed dose-dependent concentrations of the fusion protein in the serum and an increase in circulating reticulocytes was evident in the highest dose group, thus demonstrating that large therapeutic molecules can be delivered to humans via the lung, with retention of biological activity, using the FcRn-mediated transport pathway.
Assuntos
Sistemas de Liberação de Medicamentos , Eritropoetina/administração & dosagem , Antígenos de Histocompatibilidade Classe I/administração & dosagem , Receptores Fc/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Administração por Inalação , Adolescente , Adulto , Aerossóis , Relação Dose-Resposta a Droga , Eritropoetina/sangue , Eritropoetina/farmacocinética , Antígenos de Histocompatibilidade Classe I/sangue , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Humanos , Pulmão/diagnóstico por imagem , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Nebulizadores e Vaporizadores , Cintilografia , Compostos Radiofarmacêuticos , Receptores Fc/sangue , Receptores Fc/metabolismo , Receptores Fc/fisiologia , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/farmacocinética , Reticulócitos/metabolismo , Pentetato de Tecnécio Tc 99m , Capacidade VitalRESUMO
Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRn-mediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of IgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant "monomeric-Epo" Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.