A CD25-biased interleukin-2 for autoimmune therapy engineered via a semi-synthetic organism.

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.

Prognostic predictive model for PONV in cesarean delivery

Introduction: Apfel simplified risk score for postoperative nausea and vomiting (PONV) has shown to be useful in anesthesia; however, since it has not been calibrated in regional anesthesia or in pregnant patients, its use in cesarean section is limited. Objective: To develop a prognostic predictive model for postoperative nausea and vomiting in pregnant patients undergoing cesarean section under spinal anesthesia. Methods: In a cohort of 703 term pregnant patients scheduled of cesarean section, 15 variables were prospectively assessed, to design a prognostic predictive model for the development of postoperative nausea and vomiting. A logistic regression analysis was used to construct the model and its calibration and discrimination were based on the Hosmer-Lemeshow test, the calibration curves, and C statistic. Additionally, the internal calibration was performed with the Bootstrap resampling method. Results: Postoperative nausea and vomiting were experienced by 27% of the patients during the first six hours after surgery. The model included as prognostic variables the development of intraoperative nausea and vomiting, age under 28 years, a history of PONV, the mother's BMI and the weight of the newborn baby. The model showed an adequate calibration (x2: 4.65 p: 0.5888), though a low discrimination (Statistic C = 0.68). Conclusions: A prognostic predictive model was created for the development of PONV in cesarean section. This model was used to build a prognostic scale for the classification of patients into risk groups.

Introducción: La escala de riesgo simplificada de Apfel para náuseas y vómito posoperatorio (NVPO) ha mostrado utilidad en anestesia; sin embargo, al no haber sido calibrada en anestesia regional o en pacientes embarazadas, su utilidad en cesárea es limitado. Objetivo: Desarrollar un modelo de predicción pronóstica para náuseas y vómito posoperatorios en pacientes embarazadas, llevadas a cesárea bajo anestesia espinal. Métodos: En una cohorte de 703 pacientes con embarazo a término programadas para cesárea, se evaluaron 15 variables de forma prospectiva para construir un modelo de predicción pronóstica para el desarrollo de náuseas y vómito posoperatorio. Se utilizó el análisis de regresión logística para la construcción del modelo y se calculó su calibración y discriminación con la prueba de Hosmer-Lemeshow, las curvas de calibración y el estadístico C. Además, se realizó la calibración interna con el método de remuestreo Bootstrap. Resultados: Las náuseas y vómito posoperatorio se presentaron en el 27% de las pacientes durante las primeras seis horas después de la cirugía. El modelo incluyó como variables pro-nósticas el desarrollo de náuseas y vómito en el intraoperatorio, edad menor de 28 años, antecedentes de NVPO, índice de masa corporal (IMC) de la madre y el peso del recién nacido. El modelo mostró una adecuada calibración (x2: 4,65 p: 0,5888), aunque una baja discriminación (Estadístico C = 0,68). Conclusiones: Se construyó un modelo de predicción pronóstica para el desarrollo de NVPO en cirugía cesárea, y con este se construyó una escala pronóstica que permite clasificar a las pacientes por grupos de riesgo.

An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism.

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.

Pediatric Pulmonology year in review 2018: Asthma, physiology/pulmonary function testing, and respiratory infections.

Pediatric Pulmonology publishes original research, reviews, and case reports related to a wide range of children's respiratory disorders. In our "Year in Review" series, we summarize publications in our major topic areas from 2018, in the context of selected literature in these areas from other journals relevant to our discipline. This review covers selected articles on asthma, physiology/lung function testing, and respiratory infections.

Expression, purification, and characterization of human malonyl-CoA decarboxylase.

The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330-520 microM and a turnover rate kcat of 13-28s(-1). For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4-10); whereas the full-length hMCD appeared to be stable only at pH >/= 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD.