RESUMO
Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1ß. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.
Assuntos
Síndrome da Liberação de Citocina , Interleucina-6 , Humanos , Síndrome da Liberação de Citocina/tratamento farmacológico , Interleucina-6/metabolismo , Interleucina-6/antagonistas & inibidores , Cristalografia por Raios X , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/metabolismo , Interleucina-1/metabolismo , Interleucina-1/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/química , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Desenho de Fármacos , Receptor gp130 de Citocina/metabolismo , Receptor gp130 de Citocina/antagonistas & inibidores , Receptor gp130 de Citocina/química , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Receptores Tipo I de Interleucina-1/metabolismoRESUMO
Background: Trametinib is a MEK1 (mitogen-activated extracellular signal-related kinase kinase 1) inhibitor used in the treatment of BRAF (rapid accelerated fibrosarcoma B-type)-mutated metastatic melanoma. Roughly 11% of patients develop cardiomyopathy following long-term trametinib exposure. Although described clinically, the molecular landscape of trametinib cardiotoxicity has not been characterized. Objectives: The aim of this study was to test the hypothesis that trametinib promotes widespread transcriptomic and cellular changes consistent with oxidative stress and impairs cardiac function. Methods: Mice were treated with trametinib (1 mg/kg/d). Echocardiography was performed pre- and post-treatment. Gross, histopathologic, and biochemical assessments were performed to probe for molecular and cellular changes. Human cardiac organoids were used as an in vitro measurement of cardiotoxicity and recovery. Results: Long-term administration of trametinib was associated with significant reductions in survival and left ventricular ejection fraction. Histologic analyses of the heart revealed myocardial vacuolization and calcification in 28% of animals. Bulk RNA sequencing identified 435 differentially expressed genes and 116 differential signaling pathways following trametinib treatment. Upstream gene analysis predicted interleukin-6 as a regulator of 17 relevant differentially expressed genes, suggestive of PI3K/AKT and JAK/STAT activation, which was subsequently validated. Trametinib hearts displayed elevated markers of oxidative stress, myofibrillar degeneration, an 11-fold down-regulation of the apelin receptor, and connexin-43 mislocalization. To confirm the direct cardiotoxic effects of trametinib, human cardiac organoids were treated for 6 days, followed by a 6-day media-only recovery. Trametinib-treated organoids exhibited reductions in diameter and contractility, followed by partial recovery with removal of treatment. Conclusions: These data describe pathologic changes observed in trametinib cardiotoxicity, supporting the exploration of drug holidays and alternative pharmacologic strategies for disease prevention.
RESUMO
Acute cardiac injuries occur in 20%-25% of hospitalized COVID-19 patients. Herein, we demonstrate that human cardiac organoids (hCOs) are a viable platform to model the cardiac injuries caused by COVID-19 hyperinflammation. As IL-1ß is an upstream cytokine and a core COVID-19 signature cytokine, it was used to stimulate hCOs to induce the release of a milieu of proinflammatory cytokines that mirror the profile of COVID-19 cytokine storm. The IL-1ß treated hCOs recapitulated transcriptomic, structural, and functional signatures of COVID-19 hearts. The comparison of IL-1ß treated hCOs with cardiac tissue from COVID-19 autopsies illustrated the critical roles of hyper-inflammation in COVID-19 cardiac insults and indicated the cardioprotective effects of endothelium. The IL-1ß treated hCOs thus provide a defined and robust model to assess the efficacy and potential side effects of immunomodulatory drugs, as well as the reversibility of COVID-19 cardiac injuries at baseline and simulated exercise conditions.
Assuntos
COVID-19 , Síndrome da Liberação de Citocina , Cardiopatias , COVID-19/complicações , Síndrome da Liberação de Citocina/virologia , Citocinas/metabolismo , Cardiopatias/virologia , Humanos , Modelos Biológicos , OrganoidesRESUMO
Acute cardiac injuries occur in 20-25% of hospitalized COVID-19 patients. Despite urgent needs, there is a lack of 3D organotypic models of COVID-19 hearts for mechanistic studies and drug testing. Herein, we demonstrate that human cardiac organoids (hCOs) are a viable platform to model the cardiac injuries caused by COVID-19 hyperinflammation. As IL-1ßis an upstream cytokine and a core COVID-19 signature cytokine, it was used to stimulate hCOs to induce the release of a milieu of proinflammatory cytokines that mirror the profile of COVID-19 cytokine storm. The IL-1 ß treated hCOs recapitulated transcriptomic, structural, and functional signatures of COVID-19 hearts. The comparison of IL-1ß treated hCOs with cardiac tissue from COVID-19 autopsies illustrated the critical roles of hyper-inflammation in COVID-19 cardiac insults and indicated the cardioprotective effects of endothelium. The IL-1ß treated hCOs also provide a viable model to assess the efficacy and potential side effects of immunomodulatory drugs, as well as the reversibility of COVID-19 cardiac injuries at baseline and simulated exercise conditions.
RESUMO
Adoptive T cell transfer therapy induces objective responses in patients with advanced malignancies. Despite these results, some individuals do not respond due to the generation of terminally differentiated T cells during the expansion protocol. As the gamma and delta catalytic subunits in the PI3K pathway are abundant in leukocytes and involved in cell activation, we posited that blocking both subunits ex vivo with the inhibitor IPI-145 would prevent their differentiation, thereby increasing antitumor activity in vivo. However, IPI-145 treatment generated a product with reduced antitumor activity. Instead, T cells inhibited of PI3Kγ (IPI-549) or PI3Kδ (CAL-101 or TGR-1202) alone were more potent in vivo. While T cells coinhibited of PI3Kγ and PI3Kδ were less differentiated, they were functionally impaired, indicated by reduced production of effector cytokines after antigenic re-encounter and decreased persistence in vivo. Human CAR T cells expanded with either a PI3Kγ or PI3Kδ inhibitor possessed a central memory phenotype compared to vehicle cohorts. We also found that PI3Kδ-inhibited CARs lysed human tumors in vitro more effectively than PI3Kγ-expanded or traditionally expanded CAR T cells. Our data imply that sole blockade of PI3Kγ or PI3Kδ generates T cells with remarkable antitumor properties, a discovery that has substantial clinical implications.
Assuntos
Linfócitos T CD8-Positivos/transplante , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Imunoterapia Adotiva/métodos , Animais , Classe I de Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Isoquinolinas/farmacologia , Camundongos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos QuiméricosRESUMO
Adoptive T cell transfer therapy (ACT) using tumor infiltrating lymphocytes or lymphocytes redirected with antigen receptors (CAR or TCR) has revolutionized the field of cancer immunotherapy. Although CAR T cell therapy mediates robust responses in patients with hematological malignancies, this approach has been less effective for treating patients with solid tumors. Additionally, toxicities post T cell infusion highlight the need for safer ACT protocols. Current protocols traditionally expand T lymphocytes isolated from patient tumors or from peripheral blood to large magnitudes in the presence of high dose IL-2 prior to infusion. Unfortunately, this expansion protocol differentiates T cells to a full effector or terminal phenotype in vitro, consequently reducing their long-term survival and antitumor effectiveness in vivo. Post-infusion, T cells face further obstacles limiting their persistence and function within the suppressive tumor microenvironment. Therapeutic manipulation of T cells with common γ chain cytokines, which are critical growth factors for T cells, may be the key to bypass such immunological hurdles. Herein, we discuss the primary functions of the common γ chain cytokines impacting T cell survival and memory and then elaborate on how these distinct cytokines have been used to augment T cell-based cancer immunotherapy.
Assuntos
Citocinas/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Humanos , Imunoterapia/métodos , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologiaRESUMO
Research into long-circulating nanoparticles has in the past focused on reducing their clearance by macrophages. By engineering a hierarchical polyethylene glycol (PEG) structure on nanoparticle surfaces, we revealed an alternative mechanism to enhance nanoparticle blood circulation. The conjugation of a second PEG layer at a density close to but lower than the mushroom-to-brush transition regime on conventional PEGylated nanoparticles dramatically prolongs their blood circulation via reduced nanoparticle uptake by non-Kupffer cells in the liver, especially liver sinusoidal endothelial cells. Our study also disclosed that the dynamic outer PEG layer reduces protein binding affinity to nanoparticles, although not the total number of adsorbed proteins. These effects of the outer PEG layer diminish in the higher density regime. Therefore, our results suggest that the dynamic topographical structure of nanoparticles is an important factor in governing their fate in vivo. Taken together, this study advances our understanding of nanoparticle blood circulation and provides a facile approach for generating long circulating nanoparticles.
Assuntos
Difusão Dinâmica da Luz , Células Endoteliais/metabolismo , Fígado/metabolismo , Nanopartículas/metabolismo , Polietilenoglicóis/metabolismo , Animais , Células Endoteliais/química , Feminino , Fígado/química , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/química , Tamanho da Partícula , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Propriedades de SuperfícieRESUMO
One of the major challenges in applying nanomedicines to cancer therapy is their low interstitial diffusion in solid tumors. Although the modification of nanocarrier surfaces with enzymes that degrade extracellular matrix is a promising strategy to improve nanocarrier diffusion in tumors, it remains challenging to apply this strategy in vivo via systemic administration of nanocarriers due to biological barriers, such as reduced blood circulation time of enzyme-modified nanocarriers, loss of enzyme function in vivo, and life-threatening side effects. Here, we report the conjugation of recombinant human hyaluronidase PH20 (rHuPH20), which degrades hyaluronic acid, on the surfaces of poly(lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG) nanoparticles followed by anchoring a relatively low density layer of PEG, which reduces the exposure of rHuPH20 for circumventing rHuPH20-mediated clearance. Despite the extremely short serum half-life of rHuPH20, our unique design maintains the function of rHuPH20 and avoids its effect on shortening nanocarrier blood circulation. We also show that rHuPH20 conjugated on nanoparticles is more efficient than free rHuPH20 in facilitating nanoparticle diffusion. The facile surface modification quadruples the accumulation of conventional PLGA-PEG nanoparticles in 4T1 syngeneic mouse breast tumors and enable their uniform tumor distribution. The rHuPH20-modified nanoparticles encapsulating doxorubicin efficiently inhibit the growth of aggressive 4T1 tumors under a low drug dose. Thus, our platform technology may be valuable to enhance the clinical efficacy of a broad range of drug nanocarriers. This study also provides a general strategy to modify nanoparticles with enzymes that otherwise may reduce nanoparticle circulation or lose function in the blood.