RESUMEN
During immune surveillance, CD8 T cells scan the surface of antigen-presenting cells using dynamic microvillar palpation and movements as well as by having their receptors preconcentrated into patches. Here, we use real-time lattice light-sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independent of the TCR. An exception to this is the CD8 coreceptor which largely comigrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is random prior to antigen detection and that such random variation may play into the generation of many individually composed receptor patch compositions at a single synapse.
Asunto(s)
Células Presentadoras de Antígenos , Linfocitos T CD8-positivos , Microvellosidades , Receptores de Antígenos de Linfocitos T , Células Presentadoras de Antígenos/citología , Linfocitos T CD8-positivos/citología , Membrana Celular/metabolismo , Humanos , Vigilancia Inmunológica , Sinapsis Inmunológicas , Microvellosidades/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell-mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor-ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti-PD-1 and anti-PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Inmunoterapia/métodos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/inmunología , Antígeno B7-H1/inmunología , Células CHO , Cricetulus , Femenino , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas de Unión a Retinoblastoma/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas de Unión a Retinoblastoma/genética , Transducción de Señal , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521-31. ©2017 AACR.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Cetuximab/uso terapéutico , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Línea Celular Tumoral , Cetuximab/farmacología , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , HumanosRESUMEN
Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Anticuerpos Neutralizantes/farmacología , Receptores de Somatomedina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Desnudos , Microscopía Confocal , Neoplasias Experimentales/tratamiento farmacológico , Estabilidad Proteica , Receptor IGF Tipo 1 , Resonancia por Plasmón de Superficie , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
HECT (homologous to the E6AP C terminus) ubiquitin ligases have diverse functions in eukaryotic cells. In screens for proteins that bind to the HECT ubiquitin ligase WWP1, we identified Spartin, which is also known as SPG20. This protein is truncated in a neurological disease, Troyer syndrome. In this study, we show that SPG20 associates with the surface of lipid droplets (LDs) and can regulate their size and number. SPG20 binds to another LD protein, TIP47, and both proteins compete with an additional LD protein, adipophilin/adipocyte differentiation-related protein, for occupancy of LDs. The mutant SPG20 present in Troyer syndrome does not possess these activities. Depletion of SPG20 using RNA interference increases the number and size of LDs when cells are fed with oleic acid. Binding of WWP1 to SPG20 and the consequent ubiquitin transfer remove SPG20 from LDs and reduce the levels of coexpressed SPG20. These experiments suggest functions for ubiquitin ligases and SPG20 in the regulation of LD turnover and potential pathological mechanisms in Troyer syndrome.
Asunto(s)
Cuerpos de Inclusión/metabolismo , Metabolismo de los Lípidos , Neuronas/metabolismo , Proteínas/metabolismo , Paraplejía Espástica Hereditaria/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Ácido Oléico/metabolismo , Perilipina-2 , Perilipina-3 , Proteínas Gestacionales/metabolismo , Proteínas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Paraplejía Espástica Hereditaria/genética , Síndrome , Transfección , Ubiquitina/metabolismo , Proteínas de Transporte VesicularRESUMEN
Foamy viruses (FVs) assemble using pathways distinct from those of orthoretroviruses. FV capsid assembly takes place near the host microtubule-organizing center (MTOC). Assembled capsids then migrate by an unknown mechanism to the trans-Golgi network to colocalize with the FV glycoprotein, Env. Interaction with Env is required for FV capsid egress from cells; the amino terminus of FV Gag contains a cytoplasmic targeting/retention signal that is responsible for targeting assembly to the MTOC. A mutant Gag was constructed by addition of a myristylation (M) signal in an attempt to target assembly to the plasma membrane and potentially overcome the dependence upon Env for budding (S. W. Eastman and M. L. Linial, J. Virol. 75:6857-6864, 2001). Using this and additional mutants, we now show that assembly is not redirected to the plasma membrane. Addition of an M signal leads to gross morphological defects. The aberrant particles still assemble near the MTOC but do not produce infectious virus. Although extracellular Gag can be detected in a pelletable form in the absence of Env, the mutant particles contain very little genomic RNA and are less dense. Our analyses indicate that the amino terminus of Gag contains an Env interaction domain that is critical for bona fide egress of assembled capsids.
Asunto(s)
Productos del Gen env/metabolismo , Productos del Gen gag/genética , Spumavirus/genética , Ensamble de Virus/genética , Secuencia de Aminoácidos , Western Blotting , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , Productos del Gen gag/metabolismo , Humanos , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutagénesis , Mutación/genética , Spumavirus/ultraestructuraRESUMEN
Foamy viruses (FV) are unusual retroviruses that differ in many aspects of their life cycle from the orthoretroviruses such as human immunodeficiency virus. Similar to Mason-Pfizer monkey virus (MPMV), FV assemble into capsids intracellularly. The capsids are then transported to a cellular membrane for acquisition of envelope (Env) glycoproteins and budding. However, unlike MPMV, budding of FV is dependent upon the presence of Env. Previous work suggested that FV Env proteins are localized to the endoplasmic reticulum (ER) where budding takes place. However, very little was known about the details of FV assembly. We have used immunofluorescence and electron microscopy to visualize the intracellular location of FV assembly and budding. We have found that, as in the case of MPMV, FV capsids assemble at a pericentriolar site in the cytoplasm. Surprisingly, FV Env is mostly absent from this site and, contrary to expectations, FV capsid structural protein (Gag) is absent from the ER. Gag and Env only co-localize at the trans-Golgi network, suggesting that Env-Gag interactions that are required for viral egress from the cell, occurs at this site. Finally, inhibitor studies suggest an important role of microtubule networks for foamy viral assembly and budding.
Asunto(s)
Cápside/fisiología , Centriolos , Citoplasma/metabolismo , Productos del Gen gag/metabolismo , Transducción de Señal , Spumavirus/fisiología , Línea Celular , Retículo Endoplásmico/metabolismo , Productos del Gen env/metabolismo , Aparato de Golgi/metabolismo , Humanos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Transporte de ProteínasRESUMEN
The human immunodeficiency virus (HIV) type-1 viral protein U (Vpu) protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV) Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP) were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes.
Asunto(s)
Membrana Celular/fisiología , Endocitosis/fisiología , Endosomas/virología , Retroviridae/fisiología , Proteínas Reguladoras y Accesorias Virales/fisiología , Virión/fisiología , Animales , Línea Celular , Endosomas/metabolismo , Endosomas/fisiología , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/farmacocinética , VIH-1/fisiología , Células HeLa/virología , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Virus de la Leucemia Murina/metabolismo , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes de Fusión/farmacocinética , Subtilisina/farmacologíaRESUMEN
Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif-dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.
Asunto(s)
Secuencias de Aminoácidos , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/metabolismo , Vacuolas/metabolismo , Replicación Viral , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/genética , ATPasas de Translocación de Protón Vacuolares , Proteínas de Transporte Vesicular , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/metabolismoRESUMEN
Endosomal sorting complex required for transport-I (ESCRT-I) is one of three defined protein complexes in the class E vacuolar protein sorting (VPS) pathway required for the sorting of ubiquitinated transmembrane proteins into internal vesicles of multivesicular bodies. In yeast, ESCRT-I is composed of three proteins, VSP23, VPS28, and VPS37, whereas in mammals only Tsg101(VPS23) and VPS28 were originally identified as ESCRT-I components. Using yeast two-hybrid screens, we identified one of a family of human proteins (VPS37C) as a Tsg101-binding protein. VPS37C can form a ternary complex with Tsg101 and VPS28 by binding to a domain situated toward the carboxyl terminus of Tsg101 and binds to another class E VPS factor, namely Hrs. In addition, VPS37C is recruited to aberrant endosomes induced by overexpression of Tsg101, Hrs, or dominant negative form of the class E VPS ATPase, VPS4. Enveloped viruses that encode PTAP motifs to facilitate budding exploit ESCRT-I as an interface with the class E VPS pathway, and accordingly, VPS37C is recruited to the plasma membrane along with Tsg101 by human immunodeficiency virus, type 1 (HIV-1) Gag. Moreover, direct fusion of VPS37C to HIV-1 Gag obviates the requirement for a PTAP motif to induce virion release. Depletion of VPS37C from cells does not inhibit murine leukemia virus budding, which is not mediated by ESCRT-I, however, if murine leukemia virus budding is engineered to be ESCRT-I-dependent, then it is inhibited by VPS37C depletion, and this inhibition is accentuated if VPS37B is simultaneously depleted. Thus, this study identifies VPS37C as a functional component of mammalian ESCRT-I.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Línea Celular , Vesículas Citoplasmáticas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Productos del Gen gag/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Virus de la Leucemia Murina/fisiología , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Replicación ViralRESUMEN
Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.