RESUMEN
Innovative strategies for enhancing efficacy and overcoming drug resistance in hematologic cancers, such as using antibody-drug conjugates (ADC), have shifted the paradigm of conventional care by delivering promising outcomes in cancer therapies with a significant reduction in the risk of relapse. Transferrin receptor (TfR1), cluster of differentiation 71 (CD71), is known to be overexpressed in malignant cells and considered a potent antitumor target. Therefore, we developed an anti-CD71 ADC, INA03, a humanized antibody conjugated to monomethyl auristatin E through a 3-arylpropiolonitrile-valine-citrulline linker. In this study, we investigated the potency and safety of INA03, in competition with Transferrin (Tf), the CD71's natural ligand, as a novel strategy to specifically target highly proliferative cells. The high expression of CD71 was confirmed on different leukemic cell lines, allowing INA03 to bind efficiently. Subsequently, INA03 rapidly internalizes into lysosomal compartments, in which its cytotoxic drug is released following cathepsin B cleavage. Downregulation of CD71 expression using shRNA highlighted that INA03-induced cell death was dependent on CD71 density at the cell surface. INA03 intravenous treatment in acute leukemia mouse models significantly reduced tumor burden, increased mouse survival, and showed no residual disease compared with conventional chemotherapies. Because INA03 competes with human Tf, a double knock-in (human CD71/human Tf) competent mouse model was generated to mimic human pharmacokinetics and pharmacodynamics. INA03 administration in human CD71/hTf mice did not reveal any improper toxicities, even at high doses. Hence, these data demonstrate the promising preclinical efficacy and safety of INA03 and support its development as a novel acute leukemia treatment. Significance: The Tf receptor is believed to be undruggable because of its ubiquitous expression. By entering into competition with its cognate ligand, the Tf and INA03 ADC can safely achieve potency.
Asunto(s)
Antígenos CD , Inmunoconjugados , Receptores de Transferrina , Transferrina , Humanos , Animales , Ratones , Receptores de Transferrina/metabolismo , Receptores de Transferrina/antagonistas & inhibidores , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Antígenos CD/metabolismo , Transferrina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Leucemia/tratamiento farmacológico , Leucemia/patología , FemeninoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to have a significant impact on global public health. Multiple mechanisms for SARS-CoV-2 cell entry have been described; however, the role of transferrin receptor 1 (TfR1) in SARS-CoV-2 infection has received little attention. We used ferristatin II to induce the degradation of TfR1 on the surface of Vero cells and to study the consequences of such treatment on the viability of the cells and the replication of SARS-CoV-2. We demonstrated that ferristatin II is non-toxic for Vero cells in concentrations up to 400 µM. According to confocal microscopy data, the distribution of the labeled transferrin and receptor-binding domain (RBD) of Spike protein is significantly affected by the 18h pretreatment with 100 µM ferristatin II in culture medium. The uptake of RBD protein is nearly fully inhibited by ferristatin II treatment, although this protein remains bound on the cell surface. The findings were well confirmed by the significant inhibition of the SARS-CoV-2 infection of Vero cells by ferristatin II with IC50 values of 27 µM (for Wuhan D614G virus) and 40 µM (for Delta virus). A significant reduction in the infectious titer of the Omicron SARS-CoV-2 variant was noted at a ferristatin II concentration as low as 6.25 µM. We hypothesize that ferristatin II blocks the TfR1-mediated SARS-CoV-2 host cell entry; however, further studies are needed to elucidate the full mechanisms of this virus inhibition, including the effect of ferristatin II on other SARS-CoV-2 receptors, such as ACE2, Neuropilin-1 and CD147. The inhibition of viral entry by targeting the receptor on the host cells, rather than the viral mutation-prone protein, is a promising COVID-19 therapeutic strategy.
Asunto(s)
Compuestos de Bifenilo/farmacología , SARS-CoV-2/efectos de los fármacos , Sulfonas/farmacología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Chlorocebus aethiops , Concentración 50 Inhibidora , Unión Proteica , Dominios Proteicos , Receptores de Transferrina/antagonistas & inhibidores , Células VeroRESUMEN
Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.
Asunto(s)
Antígenos CD/metabolismo , Arenaviridae/metabolismo , Fiebre Hemorrágica Americana/metabolismo , Receptores de Transferrina/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Células A549 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Arenaviridae/efectos de los fármacos , Arenaviridae/fisiología , Chlorocebus aethiops , Fiebre Hemorrágica Americana/prevención & control , Fiebre Hemorrágica Americana/virología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Virus Junin/efectos de los fármacos , Virus Junin/fisiología , Ratones Endogámicos C57BL , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Unión Proteica/efectos de los fármacos , Receptores de Transferrina/antagonistas & inhibidores , Receptores de Transferrina/inmunología , Células VeroRESUMEN
Recent advancements in gene delivery systems that specifically target a variety of cancer types have increased demand for tissue-specific gene therapy. The current study describes the synthesis of a copolymer (GPgWSC) composed of a polyethylenimine (PEI)-grafted water-soluble chitosan (WSC) and gambogic acid (GA). It was validated as a ligand capable of enabling targeted attachment to transferrin receptors in HCT116 cancer cell lines. GPgWSC demonstrated superior antitumor activity in vitro in HCT116 compared to LoVo or MCF-7 cell lines, facilitated by the apoptotic activity of psiRNA-hBCL2. Pre-incubation of transferrin significantly inhibited GFP expression in the GPgWSC polyplex, demonstrating that GA is an extremely effective transferrin receptor targeting molecule. Additionally, in the HCT116-bearing mouse model, the tumor mass of PBS-treated mice increased to 2270 mm2 after 22 days, but the injection of GPgWSC polyplex significantly reduced the mass-increasing rate as a mass size of 248 mm2.
Asunto(s)
Antineoplásicos/farmacología , Quitosano/análogos & derivados , Polietileneimina/análogos & derivados , Polímeros/farmacología , Receptores de Transferrina/antagonistas & inhibidores , Xantonas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quitosano/síntesis química , Quitosano/química , Quitosano/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Polietileneimina/síntesis química , Polietileneimina/química , Polietileneimina/farmacología , Polímeros/química , Receptores de Transferrina/genética , Xantonas/químicaRESUMEN
Alterations in sialylation of terminal residues of glycoproteins have been implicated in forming tumor-associated glycans. ST6GALNAC transfers sialyl moiety to N-acetylgalactosamine residue via α2,6 linkage. Although the oncogenic characteristics of ST6GALNACI or II have been demonstrated in various cancer cells, the impact of ST6GALNACIII on tumor progression remains undefined. In this study, we evaluated the effect of ST6GALNACIII knockdown on the growth of A549 non-small cell lung cancer cells. ST6GALNACIII depletion resulted in significant retardation in growth of A549 cells under various culture conditions, including collagen-supported 3D culture and anchorage-independent soft agar culture conditions. Liquid chromatography with tandem mass spectrometry revealed that two glycopeptides of transferrin receptor protein 1 (TFR1) containing N-acetylhexosamine-sialic acid were not detected in ST6GALNACIII-depleted A549 cells compared with control cells. Subsequent lectin binding assay, western blotting, and real-time RT-PCR indicated that TFR1 sialylation was not significantly changed, but TFR1 protein and mRNA expressions were decreased after ST6GALNACIII knockdown. However, cell growth retardation by ST6GALNACIII knockdown was partially rescued by TFR1 overexpression. Additionally, TFR1 mRNA degradation was accelerated following ST6GALNACIII knockdown with concomitant reduction in mRNA levels of iron regulatory protein 1 and 2, the upstream regulators of TFR1 mRNA stability. Therefore, our results indicated an important role of ST6GALNACIII in promoting A549 cell growth through quantitative regulation of TFR1 expression and provided therapeutic implications for ST6GALNACIII targeting in tumor growth suppression in vivo.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/prevención & control , Hierro/metabolismo , Neoplasias Pulmonares/prevención & control , Estabilidad del ARN , Receptores de Transferrina/antagonistas & inhibidores , Sialiltransferasas/deficiencia , Antígenos CD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Receptores de Transferrina/metabolismoRESUMEN
The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/efectos de los fármacos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Péptidos/farmacología , Receptores de Transferrina/antagonistas & inhibidores , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Receptores de Transferrina/metabolismo , TranscitosisRESUMEN
The transferrin receptor 1 (TfR1), also known as cluster of differentiation 71 (CD71), is a type II transmembrane glycoprotein that binds transferrin (Tf) and performs a critical role in cellular iron uptake through the interaction with iron-bound Tf. Iron is required for multiple cellular processes and is essential for DNA synthesis and, thus, cellular proliferation. Due to its central role in cancer cell pathology, malignant cells often overexpress TfR1 and this increased expression can be associated with poor prognosis in different types of cancer. The elevated levels of TfR1 expression on malignant cells, together with its extracellular accessibility, ability to internalize, and central role in cancer cell pathology make this receptor an attractive target for antibody-mediated therapy. The TfR1 can be targeted by antibodies for cancer therapy in two distinct ways: (1) indirectly through the use of antibodies conjugated to anti-cancer agents that are internalized by receptor-mediated endocytosis or (2) directly through the use of antibodies that disrupt the function of the receptor and/or induce Fc effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Although TfR1 has been used extensively as a target for antibody-mediated cancer therapy over the years, interest continues to increase for both targeting the receptor for delivery purposes and for its use as direct anti-cancer agents. This review focuses on the developments in the use of antibodies targeting TfR1 as direct anti-tumor agents.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Receptores de Transferrina/antagonistas & inhibidores , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos CD , Antineoplásicos Inmunológicos/uso terapéutico , Transporte Biológico/efectos de los fármacos , Biomarcadores de Tumor , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Regulación Neoplásica de la Expresión Génica , Humanos , Hierro/metabolismo , Terapia Molecular Dirigida/efectos adversos , Terapia Molecular Dirigida/métodos , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
As many patients ultimately relapse after chimeric antigen receptor (CAR) T-cell therapy, identification of alternative targets is currently being evaluated. Substantial research efforts are underway to develop new targets. The transferrin receptor (TfR) is prevalently expressed on rapidly proliferating tumor cells and holds the potential to be the alternative target. In order to investigate the efficacy and challenges of TfR-targeting on the CAR-based therapy strategy, we generated a TfR-specific CAR and established the TfR-CAR-modified T cells. To take the advantage of TfR being widely shared by multiple tumors, TfR-CAR T cells were assessed against several TfR+ hematological malignant cell lines. Data showed that TfR-CAR T cells were powerfully potent in killing all these types of cells in vitro and in killing T-ALL cells in vivo. These findings suggest that TfR could be a universal target to broaden and improve the therapeutic efficacy of CAR T cells and warrant further efforts to use these cells as an alternative CAR T cell product for the therapy of hematological malignancies.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Receptores Quiméricos de Antígenos/metabolismo , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores Quiméricos de Antígenos/genética , Receptores de Transferrina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In Hunter syndrome (mucopolysaccharidosis II [MPS-II]), systemic accumulation of glycosaminoglycans (GAGs) due to a deficiency of iduronate-2-sulfatase (IDS), caused by mutations in the IDS gene, leads to multiple somatic manifestations and in patients with the severe (neuronopathic) phenotype, also to central nervous system (CNS) involvement. These symptoms cannot be effectively treated with current enzyme-replacement therapies, as they are unable to cross the blood-brain barrier (BBB). Pabinafusp alfa, a novel IDS fused with an anti-human transferrin receptor antibody, was shown to penetrate the BBB and to address neurodegeneration in preclinical studies. Subsequent phase 1/2 and 2/3 clinical studies in Japan have shown marked reduction of GAG accumulation in the cerebrospinal fluid (CSF), along with favorable clinical responses. A 26-week, open-label, randomized, parallel-group phase 2 study was conducted in Brazil to further evaluate the safety and efficacy of intravenously administered pabinafusp alfa at 1.0, 2.0, and 4.0 mg/kg/week in MPS-II patients. The safety profiles in the three dosage groups were similar. Neurodevelopmental evaluation suggested positive neurocognitive signals despite a relatively short study period. The 2.0-mg/kg group, which demonstrated marked reductions in substrate concentrations in the CSF, serum, and urine, was considered to provide the best combination regarding safety and efficacy signals.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Terapia de Reemplazo Enzimático/métodos , Iduronato Sulfatasa/administración & dosificación , Mucopolisacaridosis II/tratamiento farmacológico , Receptores de Transferrina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/administración & dosificación , Adolescente , Adulto , Brasil/epidemiología , Niño , Quimioterapia Combinada , Femenino , Humanos , Masculino , Mucopolisacaridosis II/epidemiología , Mucopolisacaridosis II/genética , Mucopolisacaridosis II/patología , Receptores de Transferrina/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
Mucopolysaccharidosis II (MPS II), a lysosomal storage disease caused by mutations in iduronate-2-sulfatase (IDS), is characterized by a wide variety of somatic and neurologic symptoms. The currently approved intravenous enzyme replacement therapy with recombinant IDS (idursulfase) is ineffective for CNS manifestations due to its inability to cross the blood-brain barrier (BBB). Here, we demonstrate that the clearance of heparan sulfate (HS) deposited in the brain by a BBB-penetrable antibody-enzyme fusion protein prevents neurodegeneration and neurocognitive dysfunctions in MPS II mice. The fusion protein pabinafusp alfa was chronically administered intravenously to MPS II mice. The drug reduced HS and attenuated histopathological changes in the brain, as well as in peripheral tissues. The loss of spatial learning abilities was completely suppressed by pabinafusp alfa, but not by idursulfase, indicating an association between HS deposition in the brain, neurodegeneration, and CNS manifestations in these mice. Furthermore, HS concentrations in the brain and reduction thereof by pabinafusp alpha correlated with those in the cerebrospinal fluid (CSF). Thus, repeated intravenous administration of pabinafusp alfa to MPS II mice decreased HS deposition in the brain, leading to prevention of neurodegeneration and maintenance of neurocognitive function, which may be predicted from HS concentrations in CSF.
Asunto(s)
Encéfalo/metabolismo , Heparitina Sulfato/metabolismo , Mucopolisacaridosis II/tratamiento farmacológico , Trastornos Neurocognitivos/prevención & control , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Administración Intravenosa , Animales , Anticuerpos/genética , Barrera Hematoencefálica , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Glicoproteínas/genética , Heparitina Sulfato/líquido cefalorraquídeo , Humanos , Iduronato Sulfatasa/administración & dosificación , Iduronato Sulfatasa/farmacología , Inmunoglobulina G/química , Inmunoglobulina G/genética , Ratones , Mucopolisacaridosis II/líquido cefalorraquídeo , Mucopolisacaridosis II/psicología , Trastornos Neurocognitivos/etiología , Receptores de Transferrina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Aprendizaje Espacial/efectos de los fármacosRESUMEN
Pabinafusp alfa (JR-141) is a novel enzyme drug that crosses the blood-brain barrier by transcytosis via transferrin receptors. In order to establish its efficacy and safety, a multicenter, single-arm, open-label phase 2/3 clinical trial was conducted in 28 Japanese patients with mucopolysaccharidosis II (MPS-II, Hunter syndrome) by intravenous administrations of 2.0 mg/kg of pabinafusp alfa for 52 weeks. The primary efficacy endpoint was changes in heparan sulfate (HS) concentrations in the cerebrospinal fluid (CSF). Secondary endpoints included assessments of neurocognitive development for central efficacy, and changes in plasma HS and dermatan sulfate (DS) concentrations for peripheral efficacy. HS concentrations in the CSF significantly decreased from baseline to week 52 (p < 0.001), suggesting continuous inhibition of substrate accumulations in the CNS, i.e., hitherto unaddressed progressive neurodegeneration. Evaluations of neurocognitive developments showed positive changes in 21 of the 28 patients. Serum HS and DS concentrations, liver and spleen volumes, and other assessments suggested the peripheral efficacy of pabinafusp alfa was comparable to that of idursulfase. Drug-related adverse events were mild or moderate in severity, transient, and manageable. The results establish delivery across the BBB of pabinafusp alfa as an effective therapeutic for treating both the CNS and peripheral symptoms of patients with MPS-II.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Iduronato Sulfatasa/administración & dosificación , Mucopolisacaridosis II/tratamiento farmacológico , Receptores de Transferrina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Quimioterapia Combinada , Humanos , Mucopolisacaridosis II/diagnóstico , Resultado del TratamientoRESUMEN
Iron uptake via the transferrin receptor (CD71) is a pivotal mechanism for T cell proliferation. Yet, it is incompletely understood if targeting of CD71 also affects the differentiation and functional polarization of primary human T cells. In this study, we demonstrate that inhibition of iron ingestion with blocking mAbs against CD71 induces nonproliferating T cells, which release high amounts of IL-2. Targeting of CD71 with blocking or nonblocking mAbs did not alter major signaling pathways and the activation of the transcription factors NF-κB, NFAT, or AP-1 as analyzed in Jurkat T cells. Growth arrest in iron-deficient (Fe-def) T cells was prevented upon addition of exogenous iron in the form of ferric ammonium citrate but was not reversible by exogenous IL-2. Surprisingly, protein synthesis was found to be intact in Fe-def T cells as demonstrated by comparable levels of CD69 upregulation and cytokine production with iron-sufficient T cells upon stimulation with CD3 plus CD28 mAbs. Indeed, high amounts of IL-2 were detectable in the supernatant of Fe-def T cells, which was accompanied with a reduced cell surface expression of IL-2R. When we used such Fe-def T cells in allogeneic MLRs, we observed that these cells acquired an accessory cell function and stimulated the proliferation of bystander T cells by providing IL-2. Thus, the results of our study demonstrate that iron deprivation causes nonproliferating, altruistic T cells that can help and stimulate other immune cells by providing cytokines such as IL-2.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Proliferación Celular/efectos de los fármacos , Deficiencias de Hierro , Transducción de Señal/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Antígenos CD/inmunología , Donantes de Sangre , Antígenos CD28/antagonistas & inhibidores , Antígenos CD28/inmunología , Complejo CD3/antagonistas & inhibidores , Complejo CD3/inmunología , Femenino , Compuestos Férricos/farmacología , Sangre Fetal/citología , Humanos , Interleucina-2/metabolismo , Células Jurkat , Ratones , Compuestos de Amonio Cuaternario/farmacología , Receptores de Transferrina/antagonistas & inhibidores , Receptores de Transferrina/inmunologíaRESUMEN
The prognosis for breast cancer patients diagnosed with brain metastases is poor, with survival time measured merely in months. This can largely be attributed to the limited treatment options capable of reaching the tumor as a result of the highly restrictive blood-brain barrier (BBB). While methods of overcoming this barrier have been developed and employed with current treatment options, the majority are highly invasive and nonspecific, leading to severe neurotoxic side effects. A novel approach to address these issues is the development of therapeutics targeting receptor-mediated transport mechanisms on the BBB endothelial cell membranes. Using this approach, we intercalated doxorubicin (DOX) into a bifunctional aptamer targeting the transferrin receptor on the BBB and epithelial cell adhesion molecule (EpCAM) on metastatic cancer cells. The ability of the DOX-loaded aptamer to transcytose the BBB and selectively deliver the payload to EpCAM-positive tumors was evaluated in an in vitro model and confirmed for the first time in vivo using the MDA-MB-231 breast cancer metastasis model (MDA-MB-231Br). We show that colocalized aptamer and DOX are clearly detectable within the brain lesions 75 min postadministration. Collectively, results from this study demonstrate that through intercalation of a cytotoxic drug into the bifunctional aptamer, a therapeutic delivery vehicle can be developed for specific targeting of EpCAM-positive brain metastases.
Asunto(s)
Antígenos CD/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacología , Molécula de Adhesión Celular Epitelial/genética , Receptores de Transferrina/genética , Animales , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Molécula de Adhesión Celular Epitelial/antagonistas & inhibidores , Femenino , Humanos , Ratones , Receptores de Transferrina/antagonistas & inhibidoresRESUMEN
Currently, bioengineered apoferritin nanocages with flexible protein shells and functionalized modifications have become an attractive approach for efficient anti-tumor therapy. Here, we modified the N-terminus of H-chain subunits in apoferritin with different amounts of lysine via genetic recombination to obtain a poly(l-lysine) modified H-chain apoferritin (nL-HFn) nanocage for siRNA delivery and gene therapy. To achieve excellent cellular affinity and uptake, the nanocarriers were internalized through transferrin receptor-mediated endocytosis, then escaped from the endosome for cytoplasmic transport. Compared with natural apoferritin, the siRNA-loaded genetic recombination NPs modified with lysine exhibit stronger RNA-interference and antitumor efficiency both in vitro and in 4T1 tumor model mice. Therefore, bioengineered apoferritin nanocages modified with lysine might be a promising platform for nucleic acid drug delivery.
Asunto(s)
Apoferritinas/genética , Neoplasias de la Mama/terapia , Polilisina/genética , Receptores de Transferrina/antagonistas & inhibidores , Animales , Apoferritinas/química , Neoplasias de la Mama/genética , Cápside/química , Línea Celular Tumoral , Endocitosis , Femenino , Terapia Genética , Células HeLa , Humanos , Ratones , Nanopartículas , Tamaño de la Partícula , ARN Interferente Pequeño/genética , Recombinación Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In order to deliver Salvianolic acid B (Sal B) and Baicalin (BA) to the brain tissue to repair neuron damage and improve cerebral ischemia-reperfusion injury (IRI), in our previous study, a nanostructured lipid carrier (NLC) containing BA and Sal B, and modified by the transferrin receptor monoclonal antibody OX26 (OX26-BA/Sal B-NLC) was constructed. The present study is to evaluate its in vitro release behavior, in vitro and in vivo targeting ability, in vitro pharmacodynamics and brain pharmacokinetics. The results showed that the release mechanism of the formulation was in line with the Weibull model release equation. The in-vitro and in-vivo targeting ability study exhibited that OX26 modified formulations was obviously higher than that of non-modified and solution groups. The results of in vitro preliminary study to investigate the protective effect of OX26-BA/Sal B-NLC on oxygen-glucose deprivation/reperfusion injured cells showed that it could decrease the injury. Furthermore, the results of brain microdialysis study showed that the OX26-modified preparation group could significantly increase the content of BA in the brain. In the solution group and the unmodified group, Sal B can only be detected at few time points, while OX26-modified BA/Sal B-NLC could be detected within 4â¯h. These results indicating that OX26-modified NLC can promote the brain delivery of Sal B and BA combination.
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Anticuerpos Monoclonales/química , Benzofuranos/administración & dosificación , Portadores de Fármacos/química , Medicamentos Herbarios Chinos/administración & dosificación , Flavonoides/administración & dosificación , Daño por Reperfusión/tratamiento farmacológico , Administración Intravenosa , Animales , Anticuerpos Monoclonales/farmacología , Benzofuranos/farmacocinética , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Portadores de Fármacos/farmacología , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Liberación de Fármacos , Medicamentos Herbarios Chinos/farmacocinética , Flavonoides/farmacocinética , Humanos , Lípidos/química , Masculino , Ratones , Microdiálisis , Nanopartículas/química , Permeabilidad , Receptores de Transferrina/antagonistas & inhibidores , Daño por Reperfusión/patología , Distribución TisularRESUMEN
Bispecific T-cell engager antibodies (BiTE) have been explored as a means to recruit cytolytic T cells to kill tumor cells. The transferrin receptor (TfR) is highly expressed on the surface of rapidly proliferating tumor cells. Therefore, it holds great potential in T cell redirecting therapies. In this research, we developed a BiTE targeting TfR and CD3 (TfR-BiTE) and studied its therapeutic impact on TfR-positive cancer. TfR-BiTE had the ability to induce the selective lysis of various TfR-positive cancer cells through the activation of T cells, the release of cytokines, and then the coming proliferation of T cells, whereas TfR-negative cells were not affected. In a subcutaneous HepG2 xenograft model, low concentrations of TfR-BiTE inhibited tumor growth. Overall, these results reveal that TfR-BiTE can selectively deplete TfR-positive HepG2 cells; hence, it represents a novel immunotherapeutic approach for the treatment of hepatocellular carcinoma.
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Anticuerpos Biespecíficos/farmacología , Complejo CD3/antagonistas & inhibidores , Inmunoterapia/métodos , Neoplasias Experimentales/inmunología , Receptores de Transferrina/antagonistas & inhibidores , Linfocitos T Citotóxicos/inmunología , Animales , Células Hep G2 , Humanos , Activación de Linfocitos/inmunología , Ratones , Linfocitos T Citotóxicos/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIMS: Although neuroblastoma is a heterogeneous cancer, a substantial portion overexpresses CD71 (transferrin receptor 1) and MYCN. This study provides a mechanistically driven rationale for a combination therapy targeting neuroblastomas that doubly overexpress or have amplified CD71 and MYCN. For this subset, CD71 was targeted by its natural ligand, gambogic acid (GA), and MYCN was targeted with an HDAC inhibitor, vorinostat. A combination of GA and vorinostat was then tested for efficacy in cancer and non-cancer cells. METHODS: Microarray analysis of cohorts of neuroblastoma patients indicated a subset of neuroblastomas overexpressing both CD71 and MYCN. The viability with proliferation changes were measured by MTT and colony formation assays in neuroblastoma cells. Transfection with CD71 or MYCN along with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect expression changes. For pathway analysis, gene ontology (GO) and Protein-protein interaction analyses were performed to evaluate the potential mechanisms of GA and vorinostat in treated cells. RESULTS: For both GA and vorinostat, their pathways were explored for specificity and dependence on their targets for efficacy. For GA-treated cells, the viability/proliferation loss due to GA was dependent on the expression of CD71 and involved activation of caspase-3 and degradation of EGFR. It relied on the JNK-IRE1-mTORC1 pathway. The drug vorinostat also reduced cell viability/proliferation in the treated cells and this was dependent on the presence of MYCN as MYCN siRNA transfection led to a blunting of vorinostat efficacy and conversely, MYCN overexpression improved the vorinostat potency in those cells. Vorinostat inhibition of MYCN led to an increase of the pro-apoptotic miR183 levels and this, in turn, reduced the viability/proliferation of these cells. The combination treatment with GA and vorinostat synergistically reduced cell survival in the MYCN and CD71 overexpressing tumor cells. The same treatment had no effect or minimal effect on HEK293 and HEF cells used as models of non-cancer cells. CONCLUSION: A combination therapy with GA and vorinostat may be suitable for MYCN and CD71 overexpressing neuroblastomas.
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Antígenos CD , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Sistemas de Liberación de Medicamentos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma , Receptores de Transferrina , Antígenos CD/genética , Antígenos CD/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Células HEK293 , Humanos , MicroARNs/biosíntesis , MicroARNs/genética , Proteína Proto-Oncogénica N-Myc/antagonistas & inhibidores , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Receptores de Transferrina/antagonistas & inhibidores , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Vorinostat/farmacología , Xantonas/farmacologíaRESUMEN
Erythropoietin (EPO) is a potential therapeutic for Alzheimer's disease (AD); however, limited blood-brain barrier (BBB) penetration reduces its applicability as a CNS therapeutic. Antibodies against the BBB transferrin receptor (TfRMAbs) act as molecular Trojan horses for brain drug delivery, and a fusion protein of EPO and TfRMAb, designated TfRMAb-EPO, is protective in a mouse model of AD. TfRMAbs have Fc effector function side effects, and removal of the Fc N-linked glycosylation site by substituting Asn with Gly reduces the Fc effector function. However, the effect of such Fc mutations on the pharmacokinetics (PK) of plasma clearance of TfRMAb-based fusion proteins, such as TfRMAb-EPO, is unknown. To examine this, the plasma PK of TfRMAb-EPO (wild-type), which expresses the mouse IgG1 constant heavy chain region and includes the Asn residue at position 292, was compared to the mutant TfRMAb-N292G-EPO, in which the Asn residue at position 292 is mutated to Gly. Plasma PK was compared following IV, IP, and SQ administration for doses between 0.3 and 3 mg/kg in adult male C57 mice. The results show a profound increase in clearance (6- to 8-fold) of the TfRMAb-N292G-EPO compared with the wild-type TfRMAb-EPO following IV administration. The clearance of both the wild-type and mutant TfRMAb-EPO fusion proteins followed nonlinear PK, and a 10-fold increase in dose resulted in a 7- to 11-fold decrease in plasma clearance. Following IP and SQ administration, the Cmax values of the TfRMAb-N292G-EPO mutant were profoundly (37- to 114-fold) reduced compared with the wild-type TfRMAb-EPO, owing to comparable increases in plasma clearance of the mutant fusion protein. The wild-type TfRMAb fusion protein was associated with reticulocyte suppression, and the N292G mutation mitigated this suppression of reticulocytes. Overall, the beneficial suppression of effector function via the N292G mutation may be offset by the deleterious effect this mutation has on the plasma levels of the TfRMAb-EPO fusion protein, especially following SQ administration, which is the preferred route of administration in humans for chronic neurodegenerative diseases including AD.
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Anticuerpos Monoclonales/farmacocinética , Barrera Hematoencefálica/metabolismo , Eritropoyetina/farmacocinética , Inmunoconjugados/farmacocinética , Receptores de Transferrina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacocinética , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular , Células Endoteliales , Eritropoyetina/administración & dosificación , Eritropoyetina/genética , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Regiones Constantes de Inmunoglobulina/administración & dosificación , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inyecciones Intravenosas , Inyecciones Subcutáneas , Masculino , Ratones , Mutación , Receptores de Transferrina/inmunología , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Reticulocitos/efectos de los fármacosRESUMEN
The aim of the present study was to clarify the activation of ferroptosis in different breast cancer cells by sulfasalazine (SAS) and to explore the relationship between the estrogen receptor (ER) and the transferrin receptor (TFRC). MDAMB231 and T47D cells were treated with SAS for 24 h. Changes in cell morphology were observed under a microscope. CCK8 was used to detect the proliferation inhibition rate and determine the IC50 values. Western blotting was used to detect the expression of glutathione peroxidase 4 (GPX4) and xCT. Flow cytometry was used to identify changes in the production of reactive oxygen species (ROS). Mitochondrial morphological changes in T47D were observed using transmission electron microscopy. Changes in the mitochondrial membrane potential (MMP) were observed using confocal fluorescence microscopy. RTPCR was used to detect the mRNA expression levels of TFRC and divalent metal transporter 1 (DMT1). Bioinformatics analysis was performed on TFRC expression in 1,208 breast cancer samples and its relationship with ER. TFRC expression was detected in various breast cancer tissues using immunohistochemistry and in various breast cancer cells using western blotting. Small interfering RNA (siRNA) knocked down ER expression in T47D cells, and changes in the TFRC mRNA and protein levels were observed. RTPCR was used to detect TFRC expression in 87 clinical specimens. The results of the present study revealed that SAS could inhibit breast cancer cell viability, which was accompanied by an abnormal increase in ROS and a depletion of GPX4 and system xc. Liproxstatin1 reversed the SASinduced increase in ROS. The cells treated with SAS had shrunken mitochondria and decreased MMP. SAS upregulated TFRC and DMT1. Knockdown of the ER increased TFRC expression in breast cancer cells. Immunohistochemistry indicated that TFRC expression was lower in ER+ tissues than in ER tissues. After confirmation with RTPCR in 87 clinical specimens, TFRC expression in ER tissue was revealed to be significantly higher than that of ER+ tissue. In conclusion SAS could trigger ferroptosis in breast cancer cells, especially in cells with low ER expression. Therefore, SAS is a potential agent for breast cancer treatment.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Ferroptosis/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Receptores de Transferrina/antagonistas & inhibidores , Sulfasalazina/farmacología , Antiinflamatorios no Esteroideos/farmacología , Antígenos CD , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Persona de Mediana Edad , Pronóstico , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
Enucleation is the process whereby the nucleus is extruded from the erythroblast during late stage mammalian erythropoiesis. However, the specific signaling pathways involved in this process remain unclear. To better understand the mechanisms underlying erythroblast enucleation, we investigated erythroblast enucleation using both the spleens of adult mice with phenylhydrazine-induced anemia and mouse fetal livers. Our results indicated that both iron-bound transferrin (holo-Tf) and the small-molecule iron transporter hinokitiol with iron ions (hinokitiol plus iron) promote hemoglobin synthesis and the enucleation of mouse spleen-derived erythroblasts. Although an antitransferrin receptor 1 (TfR1) monoclonal antibody inhibited both enucleation and hemoglobin synthesis promoted by holo-Tf, it inhibited only enucleation, but not hemoglobin synthesis, promoted by hinokitiol plus iron. Furthermore, siRNA against mouse TfR1 were found to suppress the enucleation of mouse fetal liver-derived erythroblasts, and the endocytosis inhibitor MitMAB inhibited enucleation, hemoglobin synthesis, and the internalization of TfR1 promoted by both types of stimuli. Collectively, our results suggest that TfR1, iron ions, and endocytosis play important roles in mouse erythroblast enucleation.