RESUMEN
The complex immunosuppressive nature of solid tumor microenvironments poses a significant challenge to generating efficacious and durable anticancer responses. Photoimmunotherapy is a cancer treatment strategy by which an antibody is conjugated with a non-toxic light-activatable dye. Following administration of the conjugate and binding to the target tumor, subsequent local laser illumination activates the dye, resulting in highly specific target cell membrane disruption. Here we demonstrate that photoimmunotherapy treatment elicited tumor necrosis, thus inducing immunogenic cell death characterized by the release of damage-associated molecular patterns (DAMPs). Photoimmunotherapy-killed tumor cells activated dendritic cells (DC), leading to the production of proinflammatory cytokines, T cell stimulation, priming antigen-specific T cells, and durable memory T cell responses, which led complete responder mice to effectively reject new tumors upon rechallenge. PD-1 blockade in combination with photoimmunotherapy enhanced overall anticancer efficacy, including against anti-PD-1-resistant tumors. The combination treatment also elicited abscopal anticancer activity, as observed by reduction of distal, non-illuminated tumors, further demonstrating the ability of photoimmunotherapy to harness local and peripheral T cell responses. With this work we therefore delineate the immune mechanisms of action for photoimmunotherapy and demonstrate the potential for cancer-targeted photoimmunotherapy to be combined with other immunotherapy approaches for augmented, durable anticancer efficacy. Moreover, we demonstrate responses utilizing various immunocompetent mouse models, as well as in vitro data from human cells, suggesting broad translational potential.
Asunto(s)
Inmunoterapia , Neoplasias , Humanos , Animales , Ratones , Inmunoterapia/métodos , Fototerapia/métodos , Neoplasias/terapia , Modelos Animales de Enfermedad , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Endocannabinoides/farmacología , Inhibidores Enzimáticos/farmacología , Etanolaminas/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Ácidos Oléicos/farmacología , Ácidos Palmíticos/farmacología , Administración Oral , Amidas , Amidohidrolasas/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Endocannabinoides/administración & dosificación , Endocannabinoides/química , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Etanolaminas/administración & dosificación , Etanolaminas/química , Ratones , Estructura Molecular , Esclerosis Múltiple/metabolismo , Ácidos Oléicos/administración & dosificación , Ácidos Oléicos/química , Ácidos Palmíticos/administración & dosificación , Ácidos Palmíticos/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Photoimmunotherapy (PIT) uses a target-specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IR700, to induce tumor necrosis after irradiation with NIR light to kill cancer cells, such as those that remain after surgery. The purpose of the present study was to sterilize the surgical bed after pancreatic cancer resection with PIT in carcinoembryonic antigen (CEA)-expressing, patient-derived, orthotopic xenograft (PDOX) nude mouse models. METHODS: After confirmation of tumor engraftment, mice were randomized to two groups: bright light surgery (BLS)-only and BLS + PIT. Each treatment arm consisted of seven tumor-bearing mice. BLS was performed under standard bright-field with an MVX10 long-working distance, high-magnification microscope on all mice. For BLS + PIT, anti-CEA antibody conjugated with IR700 (anti-CEA-IR700) (50 µg) was injected intravenously in all mice 24 h before surgery. After the surgery, the resection bed was then irradiated with a red-light-emitting diode at 690 ± 5 nm with a power density of 150 mW/cm(2). RESULTS: Anti-CEA-IR700 labelled and illuminated the pancreatic cancer PDOX. Minimal residual cancer of the PDOX was detected by fluorescence after BLS. The local recurrence rate was 85.7 % for BLS-only and 28.6 % for BLS + PIT-treated mice (p = 0.05). The average recurrent tumor weight was 1149.0 ± 794.6 mg for BLS-only and 210.8 ± 336.9 mg for BLS + PIT-treated mice (p = 0.015). CONCLUSION: Anti-CEA-IR700 was able to label and illuminate a pancreatic cancer PDOX nude mouse model sufficiently for PIT. PIT reduced recurrence by eliminating remaining residual cancer cells after BLS.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígeno Carcinoembrionario/inmunología , Inmunoterapia , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Desnudos , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/cirugía , Neoplasia Residual/tratamiento farmacológico , Neoplasia Residual/inmunología , Neoplasia Residual/cirugía , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/cirugía , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Extensive phase II metabolism of an advanced PKCε inhibitor resulted in sub-optimal pharmacokinetics in rat marked by elevated clearance. Synthesis of the O-glucuronide metabolite as a standard was followed by three distinct strategies to specifically temper phase II metabolic degradation of the parent molecule. In this study, it was determined that the introduction of proximal polarity to the primary alcohol generally curbed O-glucuronidation and improved PK and physical chemical properties while maintaining potency against the target. Utilization of a Jacobsen hydrolytic kinetic resolution to obtain optically enriched final compounds is also discussed.
Asunto(s)
Glucurónidos/farmacología , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Perros , Relación Dosis-Respuesta a Droga , Glucurónidos/química , Glucurónidos/metabolismo , Estructura Molecular , Proteína Quinasa C-epsilon/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Photoimmunotherapy (PIT) of cancer utilizes tumor-specific monoclonal antibodies conjugated to a photosensitizer phthalocyanine dye IR700 which becomes cytotoxic upon irradiation with near infrared light. In this study, we aimed to evaluate the efficacy of PIT on human pancreatic cancer cells in vitro and in vivo in an orthotopic nude mouse model. The binding capacity of anti-CEA antibody to BxPC-3 human pancreatic cancer cells was determined by FACS analysis. An in vitro cytotoxicity assay was used to determine cell death following treatment with PIT. For in vivo determination of PIT efficacy, nude mice were orthotopically implanted with BxPC-3 pancreatic tumors expressing green fluorescent protein (GFP). After tumor engraftment, the mice were divided into two groups: (1) treatment with anti-CEA-IR700 + 690 nm laser and (2) treatment with 690 nm laser only. Anti-CEA-IR700 (100 µg) was administered to group (1) via tail vein injection 24 hours prior to therapy. Tumors were then surgically exposed and treated with phototherapy at an intensity of 150 mW/cm2 for 30 minutes. Whole body imaging was done subsequently for 5 weeks using an OV-100 small animal imaging system. Anti-CEA-IR700 antibody bound to the BxPC3 cells to a high degree as shown by FACS analysis. Anti-CEA-IR700 caused extensive cancer cell killing after light activation compared to control cells in cytotoxicity assays. In the orthotopic models of pancreatic cancer, the anti-CEA-IR700 group had significantly smaller tumors than the control after 5 weeks (p<0.001). There was no significant difference in the body weights of mice in the anti-CEA-IR700 and control groups indicating that PIT was well tolerated by the mice.
Asunto(s)
Antígeno Carcinoembrionario/inmunología , Inmunoterapia , Neoplasias Pancreáticas/terapia , Fototerapia , Animales , Anticuerpos Monoclonales , Línea Celular Tumoral , Humanos , Rayos Infrarrojos , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/patología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP(3)) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP(3), with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP(3) at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP(3) and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP(3) in vesicles. This biochemical assay readily miniaturized to 1.8-microl reaction volumes and was validated in a 3456-well screening format.
Asunto(s)
Proteínas Sanguíneas/química , Membrana Celular/metabolismo , Evaluación Preclínica de Medicamentos , Fosfatidilinositoles/metabolismo , Fosfoproteínas/química , Espectrometría de Fluorescencia/métodos , Agammaglobulinemia Tirosina Quinasa , Animales , Unión Competitiva , Relación Dosis-Respuesta a Droga , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Cinética , Metabolismo de los Lípidos , Proteínas Luminiscentes/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa has two complete acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-LasI and RhlR-RhlI. LasI catalyzes the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12-HSL), and LasR is a transcription factor that requires 3OC12-HSL as a ligand. RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (C4), and RhlR is a transcription factor that responds to C4. LasR and RhlR control the transcription of hundreds of P. aeruginosa genes, many of which are critical virulence determinants, and LasR is required for RhlR function. We developed an ultra-high-throughput cell-based assay to screen a library of approximately 200,000 compounds for inhibitors of LasR-dependent gene expression. Although the library contained a large variety of chemical structures, the two best inhibitors resembled the acyl-homoserine lactone molecule that normally binds to LasR. One compound, a tetrazole with a 12-carbon alkyl tail designated PD12, had a 50% inhibitory concentration (IC50) of 30 nM. The second compound, V-06-018, had an IC50 of 10 microM and is a phenyl ring with a 12-carbon alkyl tail. A microarray analysis showed that both compounds were general inhibitors of quorum sensing, i.e., the expression levels of most LasR-dependent genes were affected. Both compounds also inhibited the production of two quorum-sensing-dependent virulence factors, elastase and pyocyanin. These compounds should be useful for studies of LasR-dependent gene regulation and might serve as scaffolds for the identification of new quorum-sensing modulators.
Asunto(s)
Antibacterianos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , Proteínas Bacterianas/genética , Western Blotting , Medios de Cultivo , Proteínas de Unión al ADN/genética , Evaluación Preclínica de Medicamentos , Electroforesis en Gel de Poliacrilamida , Biblioteca de Genes , Genes Bacterianos/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Pseudomonas aeruginosa/genética , Percepción de Quorum/genética , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/farmacología , Transactivadores/genética , Transcripción Genética , Factores de Virulencia/metabolismoRESUMEN
The pathogenic bacterium Pseudomonas aeruginosa uses acyl-homoserine lactone quorum-sensing signals to coordinate the expression of a battery of virulence genes in a cascade of regulatory events. The quorum-sensing signal that triggers the cascade is N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), which interacts with two signal receptor-transcription factors, LasR and QscR. This signal is base labile, and it is degraded by mammalian PON lactonases. We have identified a structurally unrelated triphenyl mimic of 3OC12-HSL that is base-insensitive and PON-resistant. The triphenyl mimic seems to interact specifically with LasR but not with QscR. In silico analysis suggests that the mimic fits into the 3OC12-HSL-binding site of LasR and makes key contacts with LasR. The triphenyl mimic is an excellent scaffold for developing quorum-sensing inhibitors, and its stability and potency make it ideal for biotechnology uses such as heterologous gene expression.