RESUMEN
BACKGROUND: Natural cytokines are poorly suited as therapeutics for systemic administration due to suboptimal pharmacological and pharmacokinetic (PK) properties. Recombinant human interleukin-2 (rhIL-2) has shown promise for treatment of autoimmune (AI) disorders yet exhibits short systemic half-life and opposing immune responses that negate an appropriate therapeutic index. METHODS: A semi-synthetic microbial technology platform was used to engineer a site-specifically pegylated form of rhIL-2 with enhanced PK, specificity for induction of immune-suppressive regulatory CD4 + T cells (Tregs), and reduced stimulation of off-target effector T and NK cells. A library of rhIL-2 molecules was constructed with single site-specific, biorthogonal chemistry-compatible non-canonical amino acids installed near the interface where IL-2 engages its cognate receptor ßγ (IL-2Rßγ) signaling complex. Biorthogonal site-specific pegylation and functional screening identified variants that retained engagement of the IL-2Rα chain with attenuated potency at the IL-2Rßγ complex. RESULTS: Phenotypic screening in mouse identifies SAR444336 (SAR'336; formerly known as THOR-809), rhIL-2 pegylated at H16, as a potential development candidate that specifically expands peripheral CD4+ Tregs with upregulation of markers that correlate with their suppressive function including FoxP3, ICOS and Helios, yet minimally expands CD8 + T or NK cells. In non-human primate, administration of SAR'336 also induces dose-dependent expansion of Tregs and upregulated suppressive markers without significant expansion of CD8 + T or NK cells. SAR'336 administration reduces inflammation in a delayed-type hypersensitivity mouse model, potently suppressing CD4+ and CD8 + T cell proliferation. CONCLUSION: SAR'336 is a specific Treg activator, supporting its further development for the treatment of AI diseases.
Interleukin-2 (IL-2) is a protein that functions as a master regulator of immune responses. A key function of IL-2 is the stimulation of immune-regulatory cells that suppress autoimmune disease, which occurs when the body's immune system mistakenly attacks healthy tissues. However, therapeutic use of IL-2 is limited by its short duration of action and incomplete selectivity for immune-suppressive cells over off-target immune-stimulatory cells. We employ a platform that we have previously developed, which is a bacterial organism with an expanded DNA code, to identify a new version of IL-2, SAR444336 (SAR'336), with an extended duration of activity and increased selectivity for immune-suppressive cells. In mice and monkeys, SAR'336 was a specific activator of immune suppression, with minimal effect on immune cells that stimulate autoimmunity. Our results support further development of SAR'336 for treatment of autoimmune disorders.
RESUMEN
Cancer therapy directed at specific, frequently occurring molecular alterations in signaling pathways of cancer cells has been validated through the clinical development and regulatory approval of agents such as Herceptin for the treatment of advanced breast cancer and Gleevec for chronic myelogenous leukemia and gastrointestinal stromal tumors. While most novel, target-directed cancer drugs have pregenomic origins, one can anticipate a postgenomic wave of sophisticated "smart drugs" to fundamentally change the treatment of all cancers. With these prospects, interest in this new class of therapeutics extends from basic research scientists to practicing oncologists and their patients. An extension of the initial successes in molecular oncology will occur more quickly and successfully through an appreciation of lessons learned with the first group of agents in their progress through clinical development.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Proteínas de Fusión bcr-abl , Humanos , Neoplasias/irrigación sanguínea , Neovascularización Patológica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial VascularRESUMEN
The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug accumulation in the tumor tissue, stimulates tumor-infiltrating CD8+ T and NK cells, and leads to a dose-dependent reduction of tumor growth. These results support further characterization of the immune modulatory, anti-tumor properties of THOR-707 and represent a fundamental advance in the application of synthetic biology to medicine, leveraging engineered semi-synthetic organisms as cellular factories to facilitate discovery and production of differentiated classes of chemically modified biologics.
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Antineoplásicos/uso terapéutico , Interleucina-2/química , Interleucina-2/metabolismo , Interleucina-2/farmacología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Descubrimiento de Drogas , Ingeniería Genética , Humanos , Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Linfocitos/efectos de los fármacos , Ratones , Biología SintéticaRESUMEN
SU101 or leflunomide, has been studied extensively because of its anti-cancer and immunomodulating properties. The parent isoxazole compound is converted in vitro and metabolized in vivo to an open ring isomeric form, SU0020. Several pharmacological activities have been reported for the parent and metabolite compounds including inhibition of platelet-derived growth factor (PDGF)-mediated signaling for the parent compound and inhibition of de novo pyrimidine biosynthesis for the metabolite. The inhibition of PDGF-mediated signaling and the anti-tumor properties have been ascribed to the parent compound. In spite of its short plasma half-life of the parent molecule, SU101 can be administered intermittently in animal tumor models and retain efficacy. Therefore, the relationship between plasma levels of SU101 and its efficacy in tumor-implanted immuno-compromised mice is not well established. This study was conducted to assess the concentration of SU101 in 3T3/PDGFr alpha and beta cells (NIH3T3 mouse fibroblasts engineered to overexpress human PDGFr alpha or beta) to better understand the cellular levels of SU101 and SU0020. Two strains of 3T3/PDGFr cells (alpha and beta) were incubated with 1, 25, and 100 microM concentrations of SU101 for 1, 6, 24, and 48 hours. Quantitation of SU101 and SU0020 in these cell lines was achieved by a specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Interestingly, in both alpha and beta cell lysates SU101 was much more concentrated than SU0020. The greater concentration of SU101 versus SU0020 that was observed may be due to the preferential partitioning of SU101 into the cells and this shows that significant levels of the parent drug can reach the pharmacological site of action for inhibition of PDGF receptors. The data suggest that the conversion of SU101 to SU0020 is much slower in these cells than in the incubation media.
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Compuestos de Anilina/análisis , Compuestos de Anilina/metabolismo , Antineoplásicos/metabolismo , Isoxazoles/metabolismo , Nitrilos/análisis , Nitrilos/metabolismo , Células 3T3 , Algoritmos , Animales , Antineoplásicos/análisis , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Isoxazoles/análisis , Leflunamida , Espectrometría de Masas , Ratones , Receptores del Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Estándares de Referencia , Espectrofotometría UltravioletaRESUMEN
The molecular characteristics of recurrent ovarian cancers following chemotherapy treatment have been poorly characterized. Such knowledge could impact salvage therapy selection. Since 2008, we have profiled 168 patients' ovarian cancers to determine the expression of proteins that may predict chemotherapy response or are targets for drugs that are in clinical trials for ovarian cancer treatment. Expression of epidermal growth factor receptor (EGFR), HER2, VEGF, ER, c-Met, IGF1R, Ki67, COX2, PGP/MDR1, BCRP, MRP1, excision repair complementation group 1 (ERCC1), MGMT, TS, RRM1, TOPO1, TOP2A, and SPARC was measured by immunohistochemical analyses at Clinical Laboratory Improvement Amendments-certified laboratories. Our univariate analysis of 56 primary and 50 recurrent tumors from patients with advanced stage ovarian serous carcinoma revealed that PGP and ERCC1 were significantly upregulated in recurrent lesions (P < 0.05). To determine whether these or any of the other markers were differentially expressed in specimens obtained from the same individual at diagnosis and at recurrence, we analyzed 43 matched tumor specimens from 19 advanced stage ovarian carcinoma patients. We confirmed the expression differences in PGP and ERCC1 that were observed in the cohort analysis but discovered that the expression levels of BCRP, RRM1, and COX2 were also discordant in more than 40% of the matched tumor specimens. These results may have implications both for the use of biomarkers in therapy selection as well as for their discovery and validation. Expression of these and other candidate response biomarkers must be evaluated in much larger studies and, if confirmed, support the need for profiling of recurrent tumor specimens in future clinical trials.