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Surface-exposed calreticulin (CRT) serves as a crucial cell damage-associated molecular pattern for immunogenic apoptosis, by generating an "eat me" signal to macrophages. Aiming at precision immunotherapies we intended to artificially label tumoral cells in vivo with a recombinant CRT, in a targeted way. For that, we have constructed a CRT fusion protein intended to surface attach CXCR4+ cancer cells, to stimulate their immunological destruction. As a targeting ligand of the CRT construct and to drive its specific cell adhesion, we used the peptide V1, a derivative of the vMIP-II cytokine and an antagonist of CXCR4. The modular protein tends to self-assemble as regular 16 nm nanoparticles, assisted by ionic Zn. Through both in vivo and in vitro experiments, we have determined that CRT itself confers cell targeting capabilities to the construct overcoming those of V1, that are only moderate. In particular, CRT binds HeLa cells in absence of further internalization, by a route fully independent of CXCR4. Furthermore, by cytometry in THP-1 cells, we observed that the binding of the protein is preferential for dead cells over live cells, a fact that cannot be associated to a mere artefactual adsorption. These data are discussed in the context of the oligomerizing properties of CRT and the potential clinical applicability of proteins and protein materials functionalized with this novel cell surface ligand.
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Calreticulina , Nanopartículas , Receptores CXCR4 , Humanos , Calreticulina/metabolismo , Nanopartículas/química , Células HeLa , Receptores CXCR4/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Células THP-1 , Animales , Apoptosis/efectos de los fármacos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Línea Celular Tumoral , Adhesión Celular/efectos de los fármacos , RatonesRESUMEN
Poor long-term survival in localized high-risk soft tissue sarcomas (STSs) of the extremities and trunk highlights the need to identify new prognostic factors. CXCR4 is a chemokine receptor involved in tumor progression, angiogenesis, and metastasis. The aim of this study was to evaluate the association between CXCR4 expression in tumor tissue and survival in STSs patients treated with neoadjuvant therapy. CXCR4 expression was retrospectively determined by immunohistochemical analysis in serial specimens including initial biopsies, tumors post-neoadjuvant treatment, and tumors after relapse. We found that a positive cytoplasmatic expression of CXCR4 in tumors after neoadjuvant treatment was a predictor of poor recurrence-free survival (RFS) (p = 0.003) and overall survival (p = 0.019) in synovial sarcomas. We also found that positive nuclear CXCR4 expression in the initial biopsies was associated with poor RFS (p = 0.022) in undifferentiated pleomorphic sarcomas. In conclusion, our study adds to the evidence that CXCR4 expression in tumor tissue is a promising prognostic factor for STSs.
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Developing time-sustained drug delivery systems is a main goal in innovative medicines. Inspired by the architecture of secretory granules from the mammalian endocrine system it has generated non-toxic microscale amyloid materials through the coordination between divalent metals and poly-histidine stretches. Like their natural counterparts that keep the functionalities of the assembled protein, those synthetic structures release biologically active proteins during a slow self-disintegration process occurring in vitro and upon in vivo administration. Being these granules formed by a single pure protein species and therefore, chemically homogenous, they act as highly promising time-sustained drug delivery systems. Despite their enormous clinical potential, the nature of the clustering process and the quality of the released protein have been so far neglected issues. By using diverse polypeptide species and their protein-only oligomeric nanoscale versions as convenient models, a conformational rearrangement and a stabilization of the building blocks during their transit through the secretory granules, being the released material structurally distinguishable from the original source is proved here. This fact indicates a dynamic nature of secretory amyloids that act as conformational arrangers rather than as plain, inert protein-recruiting/protein-releasing granular depots.
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Amiloide , Amiloide/metabolismo , Amiloide/química , Humanos , Vesículas Secretoras/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Conformación ProteicaRESUMEN
The need for more effective and precision medicines for cancer has pushed the exploration of new materials appropriate for drug delivery and imaging, and alternative receptors for targeting. Among the most promising strategies, finding suitable cell surface receptors and targeting agents for cancer-associated platelet derived growth factor receptor ß (PDGFR-ß)+ stromal fibroblasts is highly appealing. As a neglected target, this cell type mechanically and biologically supports the growth, progression, and infiltration of solid tumors in non-small cell lung, breast, pancreatic, and colorectal cancers. We have developed a family of PDGFR-ß-targeted nanoparticles based on biofabricated, self-assembling proteins, upon hierarchical and iterative selective processes starting from four initial candidates. The modular protein PDGFD-GFP-H6 is well produced in recombinant bacteria, resulting in structurally robust oligomeric particles that selectively penetrates into PDGFR-ß+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-ß ligand PDGFD. Upon in vivo administration, these GFP-carrying protein nanoparticles precisely accumulate in tumor tissues and enlighten them for IVIS observation. When GFP is replaced by a microbial toxin, selective tumor tissue destruction is observed associated with a significant reduction in tumor volume growth. The presented data validate the PDGFR-ß/PDGFD pair as a promising toolbox for targeted drug delivery in the tumor microenvironment and oligomeric protein nanoparticles as a powerful instrument to mediate highly selective biosafe targeting in cancer through non-cancer cells. STATEMENT OF SIGNIFICANCE: We have developed a transversal platform for nanoparticle-based drug delivery into cancer-associated fibroblasts. This is based on the engineered modular protein PDGFD-GFP-H6 that spontaneously self-assemble and selectively penetrates into PDGFR-ß+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-ß ligand PDGFD. In vivo, these protein nanoparticles accumulate in tumor and when incorporating a microbial toxin, they destroy tumor tissues with a significant reduction in tumor volume, in absence of side toxicities. The data presented here validate the PDGFR-ß/PDGFD pair as a fully versatile toolbox for targeted drug delivery in the tumor microenvironment intended as a synergistic treatment.
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Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to â¼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.
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COVID-19 , Contención de Riesgos Biológicos , Animales , Humanos , Preparaciones de Acción Retardada/farmacología , SARS-CoV-2 , Prótesis e Implantes , Modelos Animales de EnfermedadRESUMEN
Two human proteins involved in the inflammatory cell death, namely Gasdermin D (GSDMD) and the Mixed Lineage Kinase Domain-Like (MLKL) protein have been engineered to accommodate an efficient ligand of the tumoral cell marker CXCR4, and a set of additional peptide agents that allow their spontaneous self-assembling. Upon production in bacterial cells and further purification, both proteins organized as stable nanoparticles of 46 and 54 nm respectively, that show, in this form, a moderate but dose-dependent cytotoxicity in cell culture. In vivo, and when administered in mouse models of colorectal cancer through repeated doses, the nanoscale forms of tumor-targeted GSDMD and, at a lesser extent, of MLKL promoted CD8+ and CD20+ lymphocyte infiltration in the tumor and an important reduction of tumor size, in absence of systemic toxicity. The potential of these novel pharmacological agents as anticancer drugs is discussed in the context of synergistic approaches to more effective cancer treatments.
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Antineoplásicos , Nanopartículas , Animales , Ratones , Humanos , Péptidos y Proteínas de Señalización Intracelular , Piroptosis , Antineoplásicos/farmacología , LinfocitosRESUMEN
Despite advances in the development of targeted therapies for acute myeloid leukemia (AML), most patients relapse. For that reason, it is still necessary to develop novel therapies that improve treatment effectiveness and overcome drug resistance. We developed T22-PE24-H6, a protein nanoparticle that contains the exotoxin A from the bacterium Pseudomonas aeruginosa and is able to specifically deliver this cytotoxic domain to CXCR4+ leukemic cells. Next, we evaluated the selective delivery and antitumor activity of T22-PE24-H6 in CXCR4+ AML cell lines and BM samples from AML patients. Moreover, we assessed the in vivo antitumor effect of this nanotoxin in a disseminated mouse model generated from CXCR4+ AML cells. T22-PE24-H6 showed a potent, CXCR4-dependent antineoplastic effect in vitro in the MONO-MAC-6 AML cell line. In addition, mice treated with nanotoxins in daily doses reduced the dissemination of CXCR4+ AML cells compared to buffer-treated mice, as shown by the significant decrease in BLI signaling. Furthermore, we did not observe any sign of toxicity or changes in mouse body weight, biochemical parameters, or histopathology in normal tissues. Finally, T22-PE24-H6 exhibited a significant inhibition of cell viability in CXCR4high AML patient samples but showed no activity in CXCR4low samples. These data strongly support the use of T22-PE24-H6 therapy to benefit high-CXCR4-expressing AML patients.
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Fundamental clinical areas such as drug delivery and regenerative medicine require biocompatible materials as mechanically stable scaffolds or as nanoscale drug carriers. Among the wide set of emerging biomaterials, polypeptides offer enticing properties over alternative polymers, including full biocompatibility, biodegradability, precise interactivity, structural stability and conformational and functional versatility, all of them tunable by conventional protein engineering. However, proteins from non-human sources elicit immunotoxicities that might bottleneck further development and narrow their clinical applicability. In this context, selecting human proteins or developing humanized protein versions as building blocks is a strict demand to design non-immunogenic protein materials. We review here the expanding catalogue of human or humanized proteins tailored to execute different levels of scaffolding functions and how they can be engineered as self-assembling materials in form of oligomers, polymers or complex networks. In particular, we emphasize those that are under clinical development, revising their fields of applicability and how they have been adapted to offer, apart from mere mechanical support, highly refined functions and precise molecular interactions.
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Materiales Biocompatibles , Proteínas , Humanos , Materiales Biocompatibles/química , Medicina Regenerativa , Polímeros/química , Sistemas de Liberación de Medicamentos , Ingeniería de TejidosRESUMEN
Advanced endometrial cancer (EC) lacks therapy, thus, there is a need for novel treatment targets. CXCR4 overexpression is associated with a poor prognosis in several cancers, whereas its inhibition prevents metastases. We assessed CXCR4 expression in EC in women by using IHC. Orthotopic models were generated with transendometrial implantation of CXCR4-transduced EC cells. After in vitro evaluation of the CXCR4-targeted T22-GFP-H6 nanocarrier, subcutaneous EC models were used to study its uptake in tumor and normal organs. Of the women, 91% overexpressed CXCR4, making them candidates for CXCR4-targeted therapies. Thus, we developed CXCR4+ EC mouse models to improve metastagenesis compared to current models and to use them to develop novel CXCR4-targeted therapies for unresponsive EC. It showed enhanced dissemination, especially in the lungs and liver, and displayed 100% metastasis penetrance at all clinically relevant sites with anti-hVimentin IHC, improving detection sensitivity. Regarding the CXCR4-targeted nanocarrier, 60% accumulated in the SC tumor; therefore, selectively targeting CXCR4+ cancer cells, without toxicity in non-tumor organs. Our CXCR4+ EC models will allow testing of novel CXCR4-targeted drugs and development of nanomedicines derived from T22-GFP-H6 to deliver drugs to CXCR4+ cells in advanced EC. This novel approach provides a therapeutic option for women with metastatic, high risk or recurrent EC that have a dismal prognosis and lack effective therapies.
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Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma (HNSCC) mortality. The overexpression of chemokine receptor 4 (CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4+ tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4+ HNSCC cells, achieving a high accumulation in CXCR4+ tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4+ cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted protein-only nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.
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Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, metastases are the main cause of death. CRC treatment remains limited by systemic toxicity and chemotherapy resistance. Therefore, nanoparticle-mediated delivery of cytotoxic agents selectively to cancer cells represents an efficient strategy to increase the therapeutic index and overcome drug resistance. We have developed the T22-PE24-H6 therapeutic protein-only nanoparticle that incorporates the exotoxin A from Pseudomonas aeruginosa to selectively target CRC cells because of its multivalent ligand display that triggers a high selectivity interaction with the CXCR4 receptor overexpressed on the surface of CRC stem cells. We here observed a CXCR4-dependent cytotoxic effect for T22-PE24-H6, which was not mediated by apoptosis, but instead capable of inducing a time-dependent and sequential activation of pyroptotic markers in CRC cells in vitro. Next, we demonstrated that repeated doses of T22-PE24-H6 inhibit tumor growth in a subcutaneous CXCR4+ CRC model, also through pyroptotic activation. Most importantly, this nanoparticle also blocked the development of lymphatic and hematogenous metastases, in a highly aggressive CXCR4+ SW1417 orthotopic CRC model, in the absence of systemic toxicity. This targeted drug delivery approach supports for the first time the clinical relevance of inducing GSDMD-dependent pyroptosis, a cell death mechanism alternative to apoptosis, in CRC models, leading to the selective elimination of CXCR4+ cancer stem cells, which are associated with resistance, metastases and anti-apoptotic upregulation.
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Antineoplásicos , Neoplasias Colorrectales , Proteínas de Unión a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Piroptosis , Receptores CXCR4 , Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Humanos , Metástasis de la Neoplasia/prevención & control , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR4/uso terapéutico , Transducción de SeñalRESUMEN
Loco-regional recurrences and metastasis represent the leading causes of death in head and neck squamous cell carcinoma (HNSCC) patients, highlighting the need for novel therapies. Chemokine receptor 4 (CXCR4) has been related to loco-regional and distant recurrence and worse patient prognosis. In this regard, we developed a novel protein nanoparticle, T22-DITOX-H6, aiming to selectively deliver the diphtheria toxin cytotoxic domain to CXCR4+ HNSCC cells. The antimetastatic effect of T22-DITOX-H6 was evaluated in vivo in an orthotopic mouse model. IVIS imaging system was utilized to assess the metastatic dissemination in the mouse model. Immunohistochemistry and histopathological analyses were used to study the CXCR4 expression in the cancer cells, to evaluate the effect of the nanotoxin treatment, and its potential off-target toxicity. In this study, we report that CXCR4+ cancer cells were present in the invasive tumor front in an orthotopic mouse model. Upon repeated T22-DITOX-H6 administration, the number of CXCR4+ cancer cells was significantly reduced. Similarly, nanotoxin treatment effectively blocked regional and distant metastatic dissemination in the absence of systemic toxicity in the metastatic HNSCC mouse model. The repeated administration of T22-DITOX-H6 clearly abrogates tumor invasiveness and metastatic dissemination without inducing any off-target toxicity. Thus, T22-DITOX-H6 holds great promise for the treatment of CXCR4+ HNSCC patients presenting worse prognosis.
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High rates of relapsed and refractory diffuse large B-cell lymphoma (DLBCL) patients and life-threatening side effects associated with immunochemotherapy make an urgent need to develop new therapies for DLBCL patients. Immunotoxins seem very potent anticancer therapies but their use is limited because of their high toxicity. Accordingly, the self-assembling polypeptidic nanoparticle, T22-DITOX-H6, incorporating the diphtheria toxin and targeted to CXCR4 receptor, which is overexpressed in DLBCL cells, could offer a new strategy to selectively eliminate CXCR4+ DLBCL cells without adverse effects. In these terms, our work demonstrated that T22-DITOX-H6 showed high specific cytotoxicity towards CXCR4+ DLBCL cells at the low nanomolar range, which was dependent on caspase-3 cleavage, PARP activation and an increase of cells in early/late apoptosis. Repeated nanoparticle administration induced antineoplastic effect, in vivo and ex vivo, in a disseminated immunocompromised mouse model generated by intravenous injection of human luminescent CXCR4+ DLBCL cells. Moreover, T22-DITOX-H6 inhibited tumor growth in a subcutaneous immunocompetent mouse model bearing mouse CXCR4+ lymphoma cells in the absence of alterations in the hemogram, liver or kidney injury markers or on-target or off-target organ histology. Thus, T22-DITOX-H6 demonstrates a selective cytotoxicity towards CXCR4+ DLBCL cells without the induction of toxicity in non-lymphoma infiltrated organs nor hematologic toxicity.
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Antineoplásicos , Linfoma de Células B Grandes Difuso , Nanopartículas , Receptores CXCR4 , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Toxina Diftérica/farmacología , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Inmunocompetencia , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Receptores CXCR4/metabolismoRESUMEN
BACKGROUND: Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC. METHODS: T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4+ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining. RESULTS: First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4+ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy. CONCLUSIONS: Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.
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Sistemas de Liberación de Medicamentos/métodos , Nanotecnología/métodos , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptosis/genética , Receptores CXCR4/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Animales , Femenino , Humanos , Ratones , Ratones DesnudosRESUMEN
Self-assembling non-immunoglobulin scaffold proteins are a promising class of nanoscale carriers for drug delivery and interesting alternatives to antibody-based carriers that are not sufficiently efficient in systemic administration. To exploit their potentialities in clinics, protein scaffolds need to be further tailored to confer appropriate targeting and to overcome their potential immunogenicity, short half-life in plasma and proteolytic degradation. We have here engineered three human scaffold proteins as drug carrier nanoparticles to target the cytokine receptor CXCR4, a tumoral cell surface marker of high clinical relevance. The capability of these scaffolds for the selective delivery of Monomethyl auristatin E has been comparatively evaluated in a disseminated mouse model of human, CXCR4+ acute myeloid leukemia. Monomethyl auristatin E is an ultra-potent anti-mitotic drug used against a range of hematological neoplasias, which because of its high toxicity is not currently administered as a free drug but as payload in antibody-drug conjugates. The protein nanoconjugates generated here offer a collective strength of simple manufacturing process, high proteolytic and structural stability and multivalent ligand receptor interactions that result in a highly efficient and selective delivery of the payload drug and in a potent anticancer effect. The approach shown here stresses this class of human scaffold proteins as promising alternatives to antibodies for targeted drug delivery in the rapidly evolving drug development landscape.
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Antineoplásicos , Inmunoconjugados , Animales , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Humanos , Inmunoconjugados/química , Ratones , Nanoconjugados , ProteínasRESUMEN
The sustained release of small, tumor-targeted cytotoxic drugs is an unmet need in cancer therapies, which usually rely on punctual administration regimens of non-targeted drugs. Here, we have developed a novel concept of protein-drug nanoconjugates, which are packaged as slow-releasing chemically hybrid depots and sustain a prolonged secretion of the therapeutic agent. For this, we covalently attached hydrophobic molecules (including the antitumoral drug Monomethyl Auristatin E) to a protein targeting a tumoral cell surface marker abundant in several human neoplasias, namely the cytokine receptor CXCR4. By this, a controlled aggregation of the complex is achieved, resulting in mechanically stable protein-drug microparticles. These materials, which are mimetics of bacterial inclusion bodies and of mammalian secretory granules, allow the slow leakage of fully functional conjugates at the nanoscale, both in vitro and in vivo. Upon subcutaneous administration in a mouse model of human CXCR4+ lymphoma, the protein-drug depots release nanoconjugates for at least 10 days, which accumulate in the tumor with a potent antitumoral effect. The modification of scaffold cell-targeted proteins by hydrophobic drug conjugation is then shown as a novel transversal platform for the design of slow releasing protein-drug depots, with potential application in a broad spectrum of clinical settings.
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Current therapy in acute myeloid leukemia (AML) is based on chemotherapeutic drugs administered at high doses, lacking targeting selectivity and displaying poor therapeutic index because of severe adverse effects. Here, we develop a novel nanoconjugate that combines a self-assembled, multivalent protein nanoparticle, targeting the CXCR4 receptor, with an Oligo-Ara-C prodrug, a pentameric form of Ara-C, to highly increase the delivered payload to target cells. This 13.4 nm T22-GFP-H6-Ara-C nanoconjugate selectively eliminates CXCR4+ AML cells, which are protected by its anchoring to the bone marrow (BM) niche, being involved in AML progression and chemotherapy resistance. This nanoconjugate shows CXCR4-dependent internalization and antineoplastic activity in CXCR4+ AML cells in vitro. Moreover, repeated T22-GFP-H6-Ara-C administration selectively eliminates CXCR4+ leukemic cells in BM, spleen and liver. The leukemic dissemination blockage induced by T22-GFP-H6-Ara-C is significantly more potent than buffer or Oligo-Ara-C-treated mice, showing no associated on-target or off-target toxicity and, therefore, reaching a highly therapeutic window. In conclusion, T22-GFP-H6-Ara-C exploits its 11 ligands-multivalency to enhance target selectivity, while the Oligo-Ara-C prodrug multimeric form increases 5-fold its payload. This feature combination offers an alternative nanomedicine with higher activity and greater tolerability than current intensive or non-intensive chemotherapy for AML patients.
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Antineoplásicos , Leucemia Mieloide Aguda , Profármacos , Animales , Antineoplásicos/farmacología , Citarabina/uso terapéutico , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Nanoconjugados/uso terapéutico , Profármacos/uso terapéuticoRESUMEN
Patients with advanced endometrial cancer (EC) show poor outcomes. Thus, the development of new therapeutic approaches to prevent metastasis development in high-risk patients is an unmet need. CXCR4 is overexpressed in EC tumor tissue, epitomizing an unexploited therapeutic target for this malignancy. The in vitro antitumor activity of two CXCR4-targeted nanoparticles, including either the C. diphtheriae (T22-DITOX-H6) or P. aeruginosa (T22-PE24-H6) toxin, was evaluated using viability assays. Apoptotic activation was assessed by DAPI and caspase-3 and PARP cleavage in cell blocks. Both nanotoxins were repeatedly administrated to a subcutaneous EC mouse model, whereas T22-DITOX-H6 was also used in a highly metastatic EC orthotopic model. Tumor burden was assessed through bioluminescence, while metastatic foci and toxicity were studied using histological or immunohistochemical analysis. We found that both nanotoxins exerted a potent antitumor effect both in vitro and in vivo via apoptosis and extended the survival of nanotoxin-treated mice without inducing any off-target toxicity. Repeated T22-DITOX-H6 administration in the metastatic model induced a dramatic reduction in tumor burden while significantly blocking peritoneal, lung and liver metastasis without systemic toxicity. Both nanotoxins, but especially T22-DITOX-H6, represent a promising therapeutic alternative for EC patients that have a dismal prognosis and lack effective therapies.
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CXCR4 is a cytokine receptor used by HIV during cell attachment and infection. Overexpressed in the cancer stem cells of more than 20 human neoplasias, CXCR4 is a convenient antitumoral drug target. T22 is a polyphemusin-derived peptide and an effective CXCR4 ligand. Its highly selective CXCR4 binding can be exploited as an agent for the cell-targeted delivery and internalization of associated antitumor drugs. Sharing chemical and structural traits with antimicrobial peptides (AMPs), the capability of T22 as an antibacterial agent remains unexplored. Here, we have detected T22-associated antimicrobial activity and biofilm formation inhibition over Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, in a spectrum broader than the reference AMP GWH1. In contrast to GWH1, T22 shows neither cytotoxicity over mammalian cells nor hemolytic activity and is active when displayed on protein-only nanoparticles through genetic fusion. Under the pushing need for novel antimicrobial agents, the discovery of T22 as an AMP is particularly appealing, not only as its mere addition to the expanding catalogue of antibacterial drugs. The recognized clinical uses of T22 might allow its combined and multivalent application in complex clinical conditions, such as colorectal cancer, that might benefit from the synchronous destruction of cancer stem cells and local bacterial biofilms.
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Through the controlled addition of divalent cations, polyhistidine-tagged proteins can be clustered in form of chemically pure and mechanically stable micron-scale particles. Under physiological conditions, these materials act as self-disintegrating protein depots for the progressive release of the forming polypeptide, with potential applications in protein drug delivery, diagnosis, or theragnosis. Here we have explored the in vivo disintegration pattern of a set of such depots, upon subcutaneous administration in mice. These microparticles were fabricated with cationic forms of either Zn, Ca, Mg, or Mn, which abound in the mammalian body. By using a CXCR4-targeted fluorescent protein as a reporter building block we categorized those cations regarding their ability to persist in the administration site and to sustain a slow release of functional protein. Ca2+ and specially Zn2+ have been observed as particularly good promoters of time-prolonged protein leakage. The released polypeptides result is available for selective molecular interactions, such as specific fluorescent labeling of tumor tissues, in which the protein reaches nearly steady levels.