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1.
J Thromb Haemost ; 22(2): 430-440, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37940048

RESUMO

BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.


Assuntos
Anticorpos Biespecíficos , Hemofilia A , Hemostáticos , Humanos , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Trombina/metabolismo , Hemostasia , Coagulação Sanguínea , Fator VIII
2.
MAbs ; 7(1): 120-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25524207

RESUMO

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG4 antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non-antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG4-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non-antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.


Assuntos
Anticorpos Biespecíficos/química , Materiais Biomiméticos/química , Fator IXa/química , Fator VIII , Fator X/química , Anticorpos Biespecíficos/genética , Humanos
3.
J Alzheimers Dis ; 38(1): 185-200, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23948938

RESUMO

Cerebral clearance of amyloid-ß peptide (Aß), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aß1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aß1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAß1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAß1-40, suggesting that hAß1-40 and hANP share a common elimination process. Internalization of [125I]hAß1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAß1-40, suggesting that there is no direct interaction between Npr-C and hAß1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAß1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAß1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.


Assuntos
Fator Natriurético Atrial/metabolismo , Encéfalo/citologia , Células Endoteliais/efeitos dos fármacos , Insulina/metabolismo , Insulisina/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Fator Natriurético Atrial/genética , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Insulisina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , RNA Interferente Pequeno/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Fatores de Tempo
4.
PLoS One ; 8(2): e57479, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23468998

RESUMO

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.


Assuntos
Anticorpos Biespecíficos/imunologia , Fator VIII/fisiologia , Imunoglobulina G/imunologia , Epitopos/imunologia , Fator VIII/imunologia , Fator VIII/farmacocinética , Humanos , Ponto Isoelétrico , Solubilidade , Linfócitos T/imunologia
5.
Nat Med ; 18(10): 1570-4, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23023498

RESUMO

Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.


Assuntos
Anticorpos Biespecíficos , Fator IXa/imunologia , Fator VIII/fisiologia , Fator X/imunologia , Hemofilia A/terapia , Hemostasia , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Hemofilia A/imunologia , Macaca fascicularis
6.
J Cereb Blood Flow Metab ; 31(2): 457-66, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20628403

RESUMO

Cerebral atrial natriuretic peptide (ANP), which is generated in the brain, has functions in the regulation of brain water and electrolyte balance, blood pressure and local cerebral blood flow, as well as in neuroendocrine functions. However, cerebral ANP clearance is still poorly understood. The purpose of this study was to clarify the mechanism of blood-brain barrier (BBB) efflux transport of ANP in mouse. Western blot analysis showed expression of natriuretic peptide receptor (Npr)-A and Npr-C in mouse brain capillaries. The brain efflux index (BEI) method confirmed elimination of [(125)I]human ANP (hANP) from mouse brain across the BBB. Inhibition studies suggested the involvement of Npr-C in vivo. Furthermore, rapid internalization of [(125)I]hANP by TM-BBB4 cells (an in vitro BBB model) was significantly inhibited by Npr-C inhibitors and by two different Npr-C-targeted short interfering RNAs (siRNAs). Finally, treatment with 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) significantly increased Npr-C expression in TM-BBB4 cells, as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted absolute proteomics. Our results indicate that Npr-C mediates brain-to-blood efflux transport of ANP at the mouse BBB as a pathway of cerebral ANP clearance. It seems likely that levels of natriuretic peptides in the brain are modulated by 1,25(OH)(2)D(3) through upregulation of Npr-C expression at the BBB.


Assuntos
Fator Natriurético Atrial/metabolismo , Barreira Hematoencefálica/fisiologia , Química Encefálica/efeitos dos fármacos , Peptídeo Natriurético Tipo C/biossíntese , Receptores do Fator Natriurético Atrial/fisiologia , Animais , Western Blotting , Calcitriol/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Capilares/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Células Endoteliais/metabolismo , Humanos , Radioisótopos do Iodo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno/genética , Receptores do Fator Natriurético Atrial/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Transfecção
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