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1.
Structure ; 32(9): 1404-1418.e7, 2024 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-39146931

RESUMEN

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.


Asunto(s)
Antígenos de Histocompatibilidad Clase I , Inmunoglobulina G , Receptores Fc , Concentración de Iones de Hidrógeno , Inmunoglobulina G/metabolismo , Inmunoglobulina G/química , Humanos , Receptores Fc/metabolismo , Receptores Fc/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Ingeniería de Proteínas/métodos , Unión Proteica , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Antígenos/metabolismo , Antígenos/química , Animales , Modelos Moleculares
2.
Nat Commun ; 15(1): 2007, 2024 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-38453922

RESUMEN

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions.


Asunto(s)
Neoplasias , Receptores Fc , Ratones , Animales , Humanos , Inmunoglobulina G , Semivida , Antibacterianos/uso terapéutico , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Ratones Transgénicos , Anticuerpos Monoclonales , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias/terapia , Neoplasias/tratamiento farmacológico
3.
Science ; 379(6639): 1336-1341, 2023 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-36996217

RESUMEN

Aggregates of the protein tau are proposed to drive pathogenesis in neurodegenerative diseases. Tau can be targeted by using passively transferred antibodies (Abs), but the mechanisms of Ab protection are incompletely understood. In this work, we used a variety of cell and animal model systems and showed that the cytosolic Ab receptor and E3 ligase TRIM21 (T21) could play a role in Ab protection against tau pathology. Tau-Ab complexes were internalized to the cytosol of neurons, which enabled T21 engagement and protection against seeded aggregation. Ab-mediated protection against tau pathology was lost in mice that lacked T21. Thus, the cytosolic compartment provides a site of immunotherapeutic protection, which may help in the design of Ab-based therapies in neurodegenerative disease.


Asunto(s)
Anticuerpos Monoclonales , Inmunización Pasiva , Ribonucleoproteínas , Tauopatías , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Proteínas tau , Animales , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Citosol/metabolismo , Modelos Animales de Enfermedad , Receptores Fc , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas tau/inmunología , Tauopatías/terapia , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
4.
Bioconjug Chem ; 33(8): 1494-1504, 2022 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-35875886

RESUMEN

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.


Asunto(s)
Elapidae , Anticuerpos de Dominio Único , Animales , Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Elapidae/metabolismo , Neurotoxinas/química , Neurotoxinas/metabolismo , Anticuerpos de Dominio Único/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
5.
Biomolecules ; 10(4)2020 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-32344647

RESUMEN

The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. The crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure-function studies of both full-length AnxA11 and the disease variants being identified in ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Anexinas/química , Anexinas/genética , Mutación/genética , Secuencia de Aminoácidos , Animales , Modelos Moleculares , Proteínas Mutantes/química , Multimerización de Proteína , Estabilidad Proteica , Ratas , Dispersión del Ángulo Pequeño , Solubilidad , Soluciones , Relación Estructura-Actividad , Temperatura , Difracción de Rayos X
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