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1.
MAbs ; 16(1): 2383013, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39051531

RESUMEN

Targeting antigens with antibodies exhibiting pH/Ca2+-dependent binding against an antigen is an attractive strategy to mitigate target-mediated disposition and antigen buffering. Studies have reported improved serum exposure of antibodies exhibiting pH/Ca2+-binding against membrane-bound receptors. Asialoglycoprotein receptor 1 (ASGR1) is a membrane-bound receptor primarily localized in hepatocytes. With a high expression level of approximately one million receptors per cell, high turnover, and rapid recycling, targeting this receptor with a conventional antibody is a challenge. In this study, we identified an antibody exhibiting pH/Ca2+-dependent binding to ASGR1 and generated antibody variants with increased binding to neonatal crystallizable fragment receptor (FcRn). Serum exposures of the generated anti-ASGR1 antibodies were analyzed in transgenic mice expressing human FcRn. Contrary to published reports of increased serum exposure of pH/Ca2+-dependent antibodies, the pH/Ca2+-dependent anti-ASGR1 antibody had rapid serum clearance in comparison to a conventional anti-ASGR1 antibody. We conducted sub-cellular trafficking studies of the anti-ASGR1 antibodies along with receptor quantification analysis for mechanistic understanding of the rapid serum clearance of pH/Ca2+-dependent anti-ASGR1 antibody. The findings from our study provide valuable insights in identifying the antigens, especially membrane bound, that may benefit from targeting with pH/Ca2+-dependent antibodies to obtain increased serum exposure.


Asunto(s)
Receptor de Asialoglicoproteína , Antígenos de Histocompatibilidad Clase I , Ratones Transgénicos , Receptores Fc , Animales , Humanos , Receptor de Asialoglicoproteína/inmunología , Receptor de Asialoglicoproteína/metabolismo , Ratones , Receptores Fc/inmunología , Receptores Fc/genética , Receptores Fc/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Concentración de Iones de Hidrógeno , Anticuerpos Monoclonales/inmunología , Calcio/metabolismo
2.
MAbs ; 13(1): 1870058, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33397191

RESUMEN

Bispecific antibodies, engineered to recognize two targets simultaneously, demonstrate exceptional clinical potential for the therapeutic intervention of complex diseases. However, these molecules are often composed of multiple polypeptide chains of differing sequences. To meet industrial scale productivity, enforcing the correct quaternary assembly of these chains is critical. Here, we describe Chain Selectivity Assessment (CSA), a high-throughput method to rationally select parental monoclonal antibodies (mAbs) to make bispecific antibodies requiring correct heavy/light chain pairing. By deploying CSA, we have successfully identified mAbs that exhibit a native preference toward cognate chain pairing that enables the production of hetero-IgGs without additional engineering. Furthermore, CSA also identified rare light chains (LCs) that permit positive binding of the non-cognate arm in the common LC hetero-IgGs, also without engineering. This rational selection of parental mAbs with favorable developability characteristics is critical to the successful development of bispecific molecules with optimal manufacturability properties.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Afinidad de Anticuerpos/inmunología , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía Liquida/métodos , Electroforesis Capilar/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Ingeniería de Proteínas/métodos
3.
J Orthop Res ; 36(4): 1238-1247, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-28971529

RESUMEN

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238-1247, 2018.


Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Artropatías/tratamiento farmacológico , Proteínas/administración & dosificación , Animales , Colágeno Tipo II/metabolismo , Femenino , Humanos , Inyecciones Intraarticulares , Masculino , Dominios Proteicos , Ingeniería de Proteínas , Ratas Endogámicas Lew , Ratas Sprague-Dawley
4.
Science ; 305(5681): 222-7, 2004 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-15178751

RESUMEN

Condensins are conserved proteins containing SMC (structural maintenance of chromosomes) moieties that organize and compact chromosomes in an unknown mechanism essential for faithful chromosome partitioning. We show that MukBEF, the condensin in Escherichia coli, cooperatively compacts a single DNA molecule into a filament with an ordered, repetitive structure in an adenosine triphosphate (ATP) binding-dependent manner. When stretched to a tension of approximately 17 piconewtons, the filament extended in a series of repetitive transitions in a broad distribution centered on 45 nanometers. A filament so extended and held at a lower force recondensed in steps of 35 nanometers or its multiples; this cycle was repeatable even in the absence of ATP and free MukBEF. Remarkably, the pattern of transitions displayed by a given filament during the initial extension was identical in every subsequent extension. Hence, after being deformed micrometers in length, each filament returned to its original compact structure without the addition of energy. Incubation with topoisomerase I increased the rate of recondensation and allowed the structure to extend and reform almost reversibly, indicating that supercoiled DNA is trapped in the condensed structure. We suggest a new model for how MukBEF organizes the bacterial chromosome in vivo.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Proteínas Represoras/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Fenómenos Químicos , Química Física , Proteínas Cromosómicas no Histona/química , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Dimerización , Escherichia coli/genética , Proteínas de Escherichia coli/química , Rayos Láser , Microesferas , Modelos Químicos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Subunidades de Proteína , Proteínas Represoras/química
5.
Science ; 307(5714): 1409, 2005 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-15746408
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