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1.
Sci Rep ; 11(1): 15430, 2021 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-34326410

RESUMEN

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody-drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen.


Asunto(s)
Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Inmunoconjugados/administración & dosificación , Terapia Molecular Dirigida/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Línea Celular Tumoral , Regiones Determinantes de Complementariedad/inmunología , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/inmunología , Xenoinjertos , Humanos , Inmunoconjugados/inmunología , Inmunoglobulina G/inmunología , Inyecciones Intravenosas , Masculino , Mesotelina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/patología , Ingeniería de Proteínas/métodos , Neoplasias Gástricas/patología , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos
2.
PLoS One ; 15(9): e0239813, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32986768

RESUMEN

Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/metabolismo , Inmunoconjugados/farmacología , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Ado-Trastuzumab Emtansina/química , Ado-Trastuzumab Emtansina/farmacología , Anticuerpos Monoclonales Humanizados/química , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Inmunoconjugados/química , Concentración 50 Inhibidora , Oligopéptidos/química , Oligopéptidos/farmacología
3.
Sci Rep ; 10(1): 13318, 2020 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-32770098

RESUMEN

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10-15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Influenza A/inmunología , Biblioteca de Péptidos , Proteínas del Núcleo Viral/inmunología , Animales , Perros , Ensayo de Inmunoadsorción Enzimática , Humanos , Gripe Humana/diagnóstico , Gripe Humana/inmunología , Células de Riñón Canino Madin Darby , Ratones
4.
MAbs ; 11(2): 373-387, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30526270

RESUMEN

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.


Asunto(s)
Aprendizaje Automático , Ingeniería de Proteínas/métodos , Anticuerpos de Cadena Única/química , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Humanos , Biblioteca de Péptidos , Relación Estructura-Actividad
5.
MAbs ; 11(1): 153-165, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30365359

RESUMEN

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.


Asunto(s)
Aminobenzoatos/farmacología , Inmunoconjugados/farmacología , Terapia Molecular Dirigida/métodos , Oligopéptidos/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Neoplasias Gástricas/patología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Sci Rep ; 7(1): 14455, 2017 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-29089574

RESUMEN

Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural antibody repertoires. With continuing advancement in designing and constructing synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Orthomyxoviridae/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/inmunología , Bacteriófagos/inmunología , Brotes de Enfermedades , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Influenza A/inmunología , Gripe Humana/virología , Pandemias , Anticuerpos de Cadena Única/inmunología
7.
PLoS Negl Trop Dis ; 9(4): e0003692, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25855976

RESUMEN

Enterovirus 71 (EV71) and coxsackieviruses (CV) are the major causative agents of hand, foot and mouth disease (HFMD). There is not currently a vaccine available against HFMD, even though a newly developed formalin-inactivated EV71 (FI-EV71) vaccine has been tested in clinical trial and has shown efficacy against EV71. We have designed and genetically engineered a recombinant adenovirus Ad-EVVLP with the EV71 P1 and 3CD genes inserted into the E1/E3-deleted adenoviral genome. Ad-EVVLP were produced in HEK-293A cells. In addition to Ad-EVVLP particles, virus-like particles (VLPs) formed from the physical association of EV71 capsid proteins, VP0, VP1, and VP3 expressed from P1 gene products. They were digested by 3CD protease and confirmed to be produced by Ad-EVVLP-producing cells, as determined using transmission electron microscopy and western blotting. Mouse immunogenicity studies showed that Ad-EVVLP-immunized antisera neutralized the EV71 B4 and C2 genotypes. Activation of VLP-specific CD4+ and CD8+/IFN-γ T cells associated with Th1/Th2-balanced IFN-ɣ, IL-17, IL-4, and IL-13 was induced; in contrast, FI-EV71 induced only Th2-mediated neutralizing antibody against EV71 and low VLP-specific CD4+ and CD8+ T cell responses. The antiviral immunity against EV71 was clearly demonstrated in mice vaccinated with Ad-EVVLP in a hSCARB2 transgenic (hSCARB2-Tg) mouse challenge model. Ad-EVVLP-vaccinated mice were 100% protected and demonstrated reduced viral load in both the CNS and muscle tissues. Ad-EVVLP successfully induced anti-CVA16 immunities. Although antisera had no neutralizing activity against CVA16, the 3C-specific CD4+ and CD8+/IFN-γ T cells were identified, which could mediate protection against CVA16 challenge. FI-EV71 did not induce 3C-mediated immunity and had no efficacy against the CVA16 challenge. These results suggest that Ad-EVVLP can enhance neutralizing antibody and protective cellular immune responses to prevent EV71 infection and cellular immune responses against CV infection.


Asunto(s)
Proteínas de la Cápside/inmunología , Enterovirus Humano A/inmunología , Enfermedad de Boca, Mano y Pie/prevención & control , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Femenino , Ingeniería Genética , Enfermedad de Boca, Mano y Pie/virología , Sueros Inmunes/inmunología , Interleucina-13/inmunología , Interleucina-17 , Interleucina-4/inmunología , Ratones , Ratones Transgénicos , Vacunas de Productos Inactivados/inmunología , Vacunas Sintéticas/inmunología
8.
PLoS One ; 8(10): e77133, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24098578

RESUMEN

Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90ß siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90ß resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90ß and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. In vivo studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.


Asunto(s)
Benzoquinonas/farmacología , Enterovirus Humano A/efectos de los fármacos , Infecciones por Enterovirus/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Terapia Molecular Dirigida , Virión/efectos de los fármacos , Animales , Anticuerpos/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/genética , Infecciones por Enterovirus/virología , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Transgénicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Células Vero , Virión/genética , Virión/patogenicidad , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos
9.
PLoS One ; 8(2): e57591, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23451246

RESUMEN

Enterovirus 71 (EV71) and coxsackievirus (CVA) are the most common causative factors for hand, foot, and mouth disease (HFMD) and neurological disorders in children. Lack of a reliable animal model is an issue in investigating EV71-induced disease manifestation in humans, and the current clinical therapies are symptomatic. We generated a novel EV71-infectious model with hSCARB2-transgenic mice expressing the discovered receptor human SCARB2 (hSCARB2). The challenge of hSCARB2-transgenic mice with clinical isolates of EV71 and CVA16 resulted in HFMD-like and neurological syndromes caused by E59 (B4) and N2838 (B5) strains, and lethal paralysis caused by 5746 (C2), N3340 (C4), and CVA16. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases. Transgenic mice pre-immunized with live E59 or the FI-E59 vaccine was able to resist the subsequent lethal challenge with EV71. These results indicate that hSCARB2-transgenic mice are a useful model for assessing anti-EV71 medications and for studying the pathogenesis induced by EV71.


Asunto(s)
Enterovirus Humano A/genética , Enfermedad de Boca, Mano y Pie/genética , Proteínas de Membrana de los Lisosomas/genética , Receptores Depuradores/genética , Animales , Células Cultivadas , Chlorocebus aethiops , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/inmunología , Modelos Animales de Enfermedad , Enterovirus/genética , Enterovirus/inmunología , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/genética , Infecciones por Enterovirus/inmunología , Genotipo , Enfermedad de Boca, Mano y Pie/inmunología , Humanos , Inflamación/inmunología , Proteínas de Membrana de los Lisosomas/inmunología , Ratones , Ratones Transgénicos , Receptores Depuradores/inmunología , Linfocitos T/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Células Vero
10.
PLoS One ; 8(12): e84978, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24386439

RESUMEN

Regulated RNA translation is critical to provide proteins needed to maintain persistent modification of synaptic strength, which underlies the molecular basis of long-term memory (LTM). Cytoplasmic polyadenylation element-binding proteins (CPEBs) are sequence-specific RNA-binding proteins and regulate translation in various tissues. All four CPEBs in vertebrates are expressed in the brain, including the hippocampal neurons, suggesting their potential roles in translation-dependent plasticity and memory. Although CPEB1 and CPEB3 have been shown to control specific kinds of hippocampus-related LTM, the role of CPEB2 and CPEB4 in learning and memory remains elusive. Thus, we generated CPEB4 knockout (KO) mice and analyzed them using several behavioral tests. No difference was found in the anxiety level, motor coordination, hippocampus-dependent learning and memory between the KO mice and their wild-type (WT) littermates. Electrophysiological recordings of multiple forms of synaptic plasticity in the Schaffer collateral pathway-CA1 neurons also showed normal responses in the KO hippocampal slices. Morphological analyses revealed that the CPEB4-lacking pyramidal neurons possessed slightly elongated dendritic spines. Unlike its related family members, CPEB1 and CPEB3, CPEB4 seems to be dispensable for hippocampus-dependent plasticity, learning and memory.


Asunto(s)
Conducta Animal/fisiología , Región CA1 Hipocampal , Memoria/fisiología , Plasticidad Neuronal/genética , Proteínas de Unión al ARN/genética , Animales , Aprendizaje/fisiología , Ratones , Ratones Noqueados , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo
11.
PLoS One ; 7(1): e30507, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22272359

RESUMEN

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.


Asunto(s)
Endocitosis , Enterovirus Humano A/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Receptores Depuradores/metabolismo , Internalización del Virus , Animales , Western Blotting , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Chlorocebus aethiops , Colesterol/metabolismo , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endosomas/química , Enterovirus Humano A/crecimiento & desarrollo , Infecciones por Enterovirus/virología , Humanos , Concentración de Iones de Hidrógeno , Inmunoprecipitación , Proteínas de Membrana de los Lisosomas/genética , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Unión Proteica , Interferencia de ARN , Receptores Depuradores/genética , Transfección , Células Vero
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