RESUMEN
The chemokine (C-X-C) motif ligand 9 (CXCL9) is one of the lymphocyte-traffic-involved chemokines. Despite the immunotherapeutic potential of CXCL9 for recruiting effector T cells (cluster of differentiation 4+ (CD4+) and CD8+ T cells) and natural killer cells (NK cells) around the tumors, practical applications of CXCL9 have been limited because of its immune toxicity and lack of stability in vivo. To overcome these limitations, we designed and synthesized Pt-Te nanorods (PtTeNRs), which exhibited excellent photothermal conversion efficiency with stable CXCL9 payload characteristics under the physiological conditions of in vivo environments. We developed a CXCL9-based immunotherapy strategy by utilizing the unique physicochemical properties of developed PtTeNRs. The investigation revealed that the PtTeNR-loaded CXCL9 was effectively accumulated in the tumor, subsequently released in a sustained manner, and successfully recruited effector T cells for immunotherapy of the designated tumor tissue. In addition, a synergistic effect was observed between the photothermal (PT) therapy and antiprogrammed cell death protein 1 (aPD-1) antibody. In this study, we demonstrated that PtTeNR-based CXCL9, PT, and aPD-1 antibody trimodal therapy delivers an outstanding tumor suppression effect in all stages of cancer, including phases 1-4 and tumor recurrence.
Asunto(s)
Inmunidad Adaptativa , Inmunidad Innata , Inmunoterapia , Nanotubos , Animales , Ratones , Inmunidad Innata/efectos de los fármacos , Nanotubos/química , Inmunidad Adaptativa/efectos de los fármacos , Humanos , Terapia Fototérmica , Quimiocina CXCL9/química , Platino (Metal)/química , Platino (Metal)/farmacología , Línea Celular Tumoral , Neoplasias/terapia , Neoplasias/inmunología , Ratones Endogámicos BALB C , FemeninoRESUMEN
Renal rejection is a major incidence in patients after kidney transplantation and associated with allograft scarring and function loss, especially in antibody-mediated rejection. Regular clinical monitoring of kidney-transplanted patients is thus necessary, but measuring donor-specific antibodies is not always predictive, and graft biopsies are time-consuming and costly and may come up with a histological result unsuspicious for rejection. Therefore, a noninvasive diagnostic approach to estimate an increased probability of kidney graft rejection by measuring specific biomarkers is highly desired. The chemokine CXCL9 is described as an early indicator of rejection. In this work, we identified clickmers and an aptamer by split-combine click-SELEX (systematic evolution of ligands by exponential enrichment) that bind CXLC9 with high affinity. The aptamers recognize native CXCL9 and maintain binding properties under urine conditions. These features render the molecules as potential binding and detector probes for developing point-of-care devices, e.g., lateral flow assays, enabling the noninvasive monitoring of CXCL9 in renal allograft patients.
Asunto(s)
Quimiocina CXCL9/química , Química Clic , Rechazo de Injerto/metabolismo , Biomarcadores/metabolismo , Humanos , Ligandos , Unión ProteicaRESUMEN
Vitiligo is a hypopigmentary skin pathology resulting from the death of melanocytes due to the activity of CD8+ cytotoxic lymphocytes and overexpression of chemokines. These include CXCL9, CXCL10, and CXCL11 and its receptor CXCR3, both in peripheral cells of the immune system and in the skin of patients diagnosed with vitiligo. The three-dimensional structure of CXCR3 and CXCL9 has not been reported experimentally; thus, homology modeling and molecular dynamics could be useful for the study of this chemotaxis-promoter axis. In this work, a homology model of CXCR3 and CXCL9 and the structure of the CXCR3/Gαi/0ßγ complex with post-translational modifications of CXCR3 are reported for the study of the interaction of chemokines with CXCR3 through all-atom (AA-MD) and coarse-grained molecular dynamics (CG-MD) simulations. AA-MD and CG-MD simulations showed the first activation step of the CXCR3 receptor with all chemokines and the second activation step in the CXCR3-CXCL10 complex through a decrease in the distance between the chemokine and the transmembrane region of CXCR3 and the separation of the ßγ complex from the α subunit in the G-protein. Additionally, a general protein-ligand interaction model was calculated, based on known antagonists binding to CXCR3. These results contribute to understanding the activation mechanism of CXCR3 and the design of new molecules that inhibit chemokine binding or antagonize the receptor, provoking a decrease of chemotaxis caused by the CXCR3/chemokines axis.
Asunto(s)
Quimiocina CXCL10/química , Quimiocina CXCL11/química , Quimiocina CXCL9/química , Diseño de Fármacos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Receptores CXCR3 , Vitíligo/tratamiento farmacológico , Humanos , Receptores CXCR3/agonistas , Receptores CXCR3/antagonistas & inhibidores , Receptores CXCR3/química , Vitíligo/metabolismoRESUMEN
In the past decade, immune-based therapies such as monoclonal antibodies against tumor epitopes or immune checkpoint inhibitors have become an integral part of contemporary cancer treatment in many entities. However, a fundamental prerequisite for the success of such therapies is a sufficient trafficking of tumor-infiltrating lymphocytes into the tumor microenvironment. This infiltration is facilitated by chemokines, a group of about 50 small proteins capable of chemotactically guiding leukocytes. Proteolytic inactivation of chemokines leading to an impaired infiltration of immune effector cells appears to be an efficient immune escape mechanism of solid cancers.The CXCR3 and CX3CR1 chemokine receptor ligands CXCL9-11 and CX3CL1, respectively, are mainly responsible for the tumor-suppressive lymphocytic infiltration into the tumor micromilieu. Their structure explains the biochemical basis of their proteolytic cleavage, while in vivo data from mouse models and patient samples shed light on the corresponding processes in cancer. The emerging roles of proteases, e.g., matrix metalloproteinases, cathepsins, and dipeptidyl peptidase 4, in chemokine inactivation define new resistance mechanisms against immunotherapies and identify attractive new targets to enhance immune intervention in cancer.
Asunto(s)
Quimiocinas/inmunología , Quimiocinas/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Receptor 1 de Quimiocinas CX3C/inmunología , Receptor 1 de Quimiocinas CX3C/metabolismo , Quimiocina CXCL10/química , Quimiocina CXCL10/inmunología , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/química , Quimiocina CXCL11/inmunología , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/química , Quimiocina CXCL9/inmunología , Quimiocina CXCL9/metabolismo , Quimiocinas/química , Humanos , Linfocitos Infiltrantes de Tumor/enzimología , Linfocitos Infiltrantes de Tumor/patología , Modelos Moleculares , Neoplasias/enzimología , Neoplasias/patología , Péptido Hidrolasas/metabolismo , Proteolisis , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismoRESUMEN
Zbasic-ΔI-CM is a novel intein-based self-cleavable tag we developed to accelerate the soluble expression of recombinant proteins in Escherichia coli (E. coli). Previously we found that intein activity could be interfered by its flanking exteins, and thus reducing the production efficiency and final yield. In this work, we used CXC-chemokine 9 (CXCL9) as a model C-extein, which fusion with Zbasic-ΔI-CM showed high intein activity. When the fusion protein got soluble expression, CXCL9 was released immediately and purified directly from cell lysis supernatant. The results demonstrated that Zbasic-ΔI-CM tag had successfully mediated the efficient production of high-quality CXCL9 with reduced time and resources consumption in comparison with inclusion bodies expression. Molecular dynamics simulations suggested that the improved cleavage activity of Zbasic-ΔI-CM upon fusion with CXCL9 may be due to the higher dynamics of the first half loop and stabilization of the second half loop of intein. Our results proved that the self-cleavable Zbasic-ΔI-CM mediated soluble expression could be a feasible process for cytokines like CXCL9, thus of attractive potentials for production of therapeutic proteins using E. coli expression system.
Asunto(s)
Quimiocina CXCL9/genética , Escherichia coli/genética , Inteínas , Proteínas Recombinantes de Fusión/genética , Quimiocina CXCL9/química , Escherichia coli/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas Recombinantes de Fusión/química , SolubilidadRESUMEN
BACKGROUND: To recruit leucocytes to an inflammatory site, chemokine binding to glycosaminoglycans (GAGs) is critical. Therefore, strategies to interfere with this interaction, aiming at the production of anti-inflammatory agents, were developed. These include production of modified chemokines without affinity for G protein-coupled receptors but with enhanced affinity for GAGs. Such modified chemokines compete with functional chemokines for GAG binding, prevent chemokine immobilization and presentation, and inhibit leucocyte migration. In addition to modified chemokines, a GAG-binding peptide consisting of the 30 COOH-terminal residues of CXCL9, that is CXCL9(74-103), inhibited CXCL8- and monosodium urate crystal-induced neutrophil migration. OBJECTIVE: We wanted to explore whether interference with chemokine-GAG interactions by CXCL9(74-103) reduces inflammation in neutrophil-dependent dinitrofluorobenzene-induced contact hypersensitivity. METHODS: For this study, we evaluated several inflammatory parameters, including ear swelling and the levels of chemokines, cytokines, proteases and neutrophils in the ears of dinitrofluorobenzene-induced mice treated with CXCL9(74-103) or buffer. RESULTS: One intravenous injection of CXCL9(74-103), just before painting with dinitrofluorobenzene on the ear, did not affect protein levels of the major murine neutrophil attractant, that is CXCL6, in this contact hypersensitivity model. However, IL-6, CXCL1, CCL2 and matrix metalloproteinase-9 (MMP-9) protein concentrations and peroxidase activity in challenged ears were reduced. In addition, intravenous injection of the CXCL9-derived peptide led to a reduced ear swelling response, indicating that the locally produced chemokines were hindered to attract leucocytes. The inhibiting potential of CXCL9(74-103) was explained by its competition for GAG binding with CXCL1, CXCL6 and CCL3 and inhibition of transendothelial migration of neutrophils to CXCL6. CONCLUSIONS AND CLINICAL RELEVANCE: The CXCL9(74-103) peptide inhibited dinitrofluorobenzene-induced infiltration of neutrophils and neutrophil-dependent inflammation in ears. Therefore, CXCL9(74-103) may be a lead molecule for the development of therapeutic peptides or peptide derivatives that compete with functional chemokines for GAG binding.
Asunto(s)
Antiinflamatorios/farmacología , Quimiocina CXCL9/química , Dermatitis por Contacto/etiología , Dermatitis por Contacto/metabolismo , Dinitrofluorobenceno/efectos adversos , Glicosaminoglicanos/metabolismo , Péptidos/farmacología , Animales , Citocinas/metabolismo , Dermatitis por Contacto/tratamiento farmacológico , Femenino , Leucocitos/inmunología , Leucocitos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Unión Proteica , Piel/inmunología , Piel/metabolismo , Piel/patología , Migración Transendotelial y TransepitelialRESUMEN
The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.
Asunto(s)
Antiinflamatorios/uso terapéutico , Quimiocina CXCL9/uso terapéutico , Gota/tratamiento farmacológico , Interleucina-8/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Péptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Antiinflamatorios/química , Inhibición de Migración Celular/efectos de los fármacos , Quimiocina CXCL9/química , Quimiotaxis de Leucocito/efectos de los fármacos , Glicosaminoglicanos/inmunología , Gota/inducido químicamente , Gota/inmunología , Humanos , Interleucina-8/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neutrófilos/citología , Neutrófilos/inmunología , Péptidos/química , Ácido ÚricoRESUMEN
Monokine induced by IFN-γ (Mig) is a member of CXC-chemokines and recruits T-lymphocytes to activate the immune response. In recent years, it has raised much interest in the areas of autoimmune disease and allograft rejection, as the production of recombinant human Mig (rHuMig) would be of considerable significance for both research and potential clinical use. Here we report the expression, preparation and characterization of non-tagged recombinant human Mig (rHuMig) using a prokaryotic expression system. Following expression in Escherichia coli (E. coli) BL21, the 103 amino acid residue of rHuMig was purified from bacteria inclusion bodies with a one-step S-Sepharose cation exchange chromatography. The product was immunologically characterized via Western blot and its purity was determined via SDS-PAGE and silver staining to be above 99%, with an endotoxin level <0.5EU/µg via a chemotaxis assay, rHuMig demonstrated chemotactic activity on mouse spleen lymphocytes with an ED50 of 15 ng/mL. Additionally, using a proliferation assay, rHuMig significantly inhibited proliferation of the human bladder cell line T24. In vivo experiments revealed that rHuMig could inhibit mouse bone marrow mononuclear cells cycling into the S-phase and reduced intestinal cell proliferation. Our results demonstrate that rHuMig is fully functional in the mouse model.
Asunto(s)
Quimiocina CXCL9/genética , Quimiocina CXCL9/inmunología , Escherichia coli/genética , Animales , Secuencia de Bases , Proliferación Celular , Quimiocina CXCL9/química , Quimiocina CXCL9/aislamiento & purificación , Quimiotaxis de Leucocito , Cromatografía por Intercambio Iónico , Expresión Génica , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Linfocitos T/citologíaRESUMEN
Dual-specific antibodies are characterized by an antigen-combining site mediating specific interactions with two different antigens. We have generated five dual-specific single chain variable fragments (scFv) that neutralize the activity of the two chemokines, CXCL9 and CXCL10, to bind to their receptor CXCR3. To better understand how these dual-specific scFvs bind these two chemokines that only share a 37% sequence identity, we mapped their epitopes on human CXCL9 and CXCL10 and identified serine 13 (Ser(13)) as a critical residue. It is conserved between the two chemokines but not in the third ligand for CXCR3, CXCL11. Furthermore, Ser(13) is exposed in the tetrameric structure of CXCL10, which is consistent with our finding that the scFvs are able to bind to CXCL9 and CXCL10 immobilized on glycosaminoglycans. Overall, the data indicate that these dual-specific scFvs bind to a conserved surface involved in CXCR3 receptor interaction for CXCL10 and CXCL9. Thus, structural mimicry between the two targets is likely to be responsible for the observed dual specificity of these antibody fragments.
Asunto(s)
Especificidad de Anticuerpos , Quimiocina CXCL10/química , Quimiocina CXCL9/química , Imitación Molecular , Anticuerpos de Cadena Única/química , Animales , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Quimiocina CXCL11/química , Quimiocina CXCL11/genética , Quimiocina CXCL11/inmunología , Quimiocina CXCL9/genética , Quimiocina CXCL9/inmunología , Humanos , Macaca fascicularis , Macaca mulatta , Ratones , Conejos , Receptores CXCR3/química , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
The chemokines play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes. These comprise over 50 proteins grouped into four classes, in basis to the arrangement of conserved cysteine residues within the sequence. CXCL9, CXCL10 and CXCL11 are the members of the family of ELR-CXC chemokines and bind the same CXCR3 receptor. During the past few years, several studies have demonstrated a pathogenetic role of CXCR3 and its ligands in many human inflammatory diseases. The blockade of CXCR3 interactions with its ligands has been suggested as a possible therapeutic target for the treatment of these diseases. Therefore, we modelled the three-dimensional structure of CXCL9 and CXCR3, and, successively, of the CXCL9/CXCR3 complex in comparison to CXCL10/CXCR3 and CXCL11/CXCR3 complexes. We have then shown the structural determinants of these interactions and their physico-chemical features. Finally, the interaction residues involved in the formation of the complexes have been highlighted and analyzed in order to be used for drug design.
Asunto(s)
Membrana Celular/química , Quimiocina CXCL10/química , Quimiocina CXCL11/química , Quimiocina CXCL9/química , Diseño de Fármacos , Modelos Moleculares , Receptores CXCR3/química , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Rodopsina/química , Alineación de Secuencia , Propiedades de Superficie , TermodinámicaRESUMEN
Electrochemical detection of protein binding at physiological salt concentration by planar field effect transistor platforms has yet to be documented convincingly. Here we report detection of streptavidin and clinically relevant levels of biotinylated monokine induced by interferon gamma (MIG) at physiological salt concentrations with AlGaN heterojunction field effect transistors (HFETs). The AlGaN HFETs are functionalized with a silane linker and analyte-specific affinity elements. Polarity of sensor responses is as expected from n-type HFETs to negatively and positively charged analytes. Sensitivity of the HFET sensors increases when salt concentration decreases, and the devices also exhibit dose-dependent responses to analyte. Detection of clinically relevant MIG concentrations at physiological salt levels demonstrates the potential for AlGaN devices to be used in development of in vivo biosensors.