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1.
Oncoimmunology ; 11(1): 2141007, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36352891

RESUMEN

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.


Asunto(s)
Neoplasias , Linfocitos T Reguladores , Ratones , Animales , Humanos , Linfocitos T Reguladores/metabolismo , Receptor de Muerte Celular Programada 1 , Inmunoterapia/métodos , Neoplasias/terapia , Microambiente Tumoral , Fragmentos Fc de Inmunoglobulinas/metabolismo , Receptores CCR8/metabolismo
2.
MAbs ; 11(7): 1289-1299, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31199179

RESUMEN

IgG4, a common type of therapeutic antibody, is less stable during manufacturing processes compared with IgG1. Aggregation and fragmentation are the two main challenges. Here, we report instability of the heavy chain (HC) C-terminal region under acidic conditions, which leads to cleavage and aggregation. Leu445, at the C-terminal region of the HC in IgG4, plays a critical role in its acid-induced fragmentation and subsequent aggregation. We found that mutating HC C-terminal Leu445 to Pro (the corresponding residue in IgG1) in IgG4_CDR-X significantly reduces fragmentation and aggregation, while mutating Pro445 to Leu in IgG1_CDR-X promotes fragmentation and aggregation. HC C-terminal Gly446 cleavage was observed in low pH citrate buffer and resulted in further fragmentation and aggregation, whereas, glycine buffer can completely inhibit the cleavage and aggregation. It is proposed that cleavages occur through acid-induced hydrolysis under acidic conditions and glycine stabilizes IgG4 via two main mechanisms: 1) product feedback inhibition of the hydrolysis reaction, and 2) stabilization of protein conformation by direct interaction with the peptide backbone and charged side chains. Experiments using IgG4 molecules IgG4_CDR-Y and IgG4_CDR-Z with the same CH domains as IgG4_CDR-X, but different complementarity-determining regions (CDRs), indicate that the stability of the HC C-terminal region is also closely related to the sequence of the CDRs. The stability of IgG4_CDR-X is significantly improved when binding to its target. Both observations suggest that there are potential interactions between Fab and CH2-CH3 domains, which could be the key factor affecting the stability of IgG antibodies.


Asunto(s)
Regiones Determinantes de Complementariedad/química , Glicina/química , Inmunoglobulina G/química , Cadenas Pesadas de Inmunoglobulina/química , Fragmentos de Péptidos/química , Regiones Determinantes de Complementariedad/genética , Glicina/genética , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Mutación/genética , Fragmentos de Péptidos/genética , Agregado de Proteínas , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estabilidad Proteica , Proteolisis
3.
BMC Biotechnol ; 14: 79, 2014 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-25148894

RESUMEN

BACKGROUND: Switchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated. RESULTS: A novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used. CONCLUSION: The results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.


Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Ingeniería Genética/métodos , Panicum/genética , Polen/genética , Transgenes , Regulación de la Expresión Génica de las Plantas , Flujo Génico , Vectores Genéticos , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas , Transformación Genética
4.
Antonie Van Leeuwenhoek ; 85(3): 229-35, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15028871

RESUMEN

The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a P nosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK(2).


Asunto(s)
Proteínas Bacterianas/genética , Bradyrhizobium/enzimología , Bradyrhizobium/genética , Genes Bacterianos , Proteínas de la Membrana/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Óxido Nitroso/metabolismo , Oxidorreductasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
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