Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

High molecular mass radioimmunoconjugates are promising for intraperitoneal α-emitter immunotherapy due to prolonged retention in the peritoneum

Abstract Aim. Therapeutic efficacy of intraperitoneal radioimmunotherapy is dependent on the time of retention of the radioimmunoconjugates within the peritoneal cavity. Therefore, the aim of this study was to investigate intraperitoneal retention of Fab, IgG and IgM radioimmunoconjugates. Methods. Female Balb/c mice were injected with 213Bi- or 111In-labeled IgM, IgG and recombinant Fab conjugates intraperitoneally or intravenously. At different time points after injection, whole body distribution of radionuclides was imaged using a gamma camera. Distribution of radionuclides in selected organs was determined via γ-counting after sacrifice. Biological half-lives of the conjugates were calculated from whole body activities. Results. After i.p. injection 213Bi-Fab rapidly accumulated in the kidneys indicative of glomerular filtration and reabsorption. Accumulation of 213Bi-IgG in the kidneys was significantly lower. 213Bi-IgM showed a striking accumulation in the liver 180 min after i.p. injection. 111In-IgG persisted in the circulation up to 72 h both after i.p. and i.v. injection. 111In-IgM showed a continuous accumulation in the liver. Moreover, 111In-IgM was significantly higher 24 h after i.v. injection than i.p. injection both in liver and spleen. These differences could be confirmed via scintigraphy. After injection of 111In-IgG differences in scintigraphic images between i.v. and i.p. were clearly visible only at 3 h. Biological half lives were 24 h, 45 h and 165 h for 111In-IgM, 111In-Fab and 111In-IgG, respectively. Conclusion. Retention of radioimmunoconjugates in the peritoneal cavity positively correlates with the molecular mass of the antibody. Therefore, IgM radioimmunoconjugates should be preferably used in intraperitoneal radioimmunotherapy.JRC.E.5-Nuclear chemistr

Regeneration after radiation- and immune-mediated tissue injury is not enhanced by type III interferon signaling.

Purpose: Type I interferon (IFN-I) and interleukin (IL)-22 modulate regeneration of the thymus and intestinal epithelial cells (IECs) after cytotoxic stress such as irradiation. Radiation-induced damage to thymic tissues and IECs is a crucial aspect during the pathogenesis of inadequate immune reconstitution and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with myeloablative total body irradiation (TBI), respectively. IL-22 and IFN-I reduce the severity of acute GVHD after allo-HSCT with myeloablative TBI. However, the role of biologically related type III interferon (IFN-III), also known as interferon lambda (IFN-lambda) or IL-28, in this context is unclear. We therefore studied the role of the IFN-III pathway in thymic regeneration and GVHD after TBI and allo-HSCT.Methods and Materials: Cohoused wild-type (WT) and IFN-III receptor-deficient (IL-28 receptor alpha subunit-deficient/IL-28Ra(-/-)) mice were analyzed in models of TBI-induced thymus damage and a model of GVHD after allo-HSCT with myeloablative TBI. PASylated IFN-III (PASylated IL-28A, XL-protein GmbH) was generated to prolong the plasma half-life of IFN-III. Pharmacologic activity and the effects of PASylated IL-28A on radiation-induced thymus damage and the course of GVHD after allo-HSCT with myeloablative TBI were tested.Results: The course and severity of GVHD after myeloablative TBI and allo-HSCT in IL-28Ra(-/-) mice was comparable to those in WT mice. Activation of the IFN-III pathway by PASylated IL-28A did not significantly modulate GVHD after allo-HSCT with TBI. Furthermore, IL28Ra(-/-) mice and WT mice showed similar thymus regeneration after radiation, which could also not be significantly modulated by IFN-III receptor engagement using PASylated IL-28A.Conclusions: We analyzed the role of IFN-III signaling during radiation-mediated acute tissue injury. Despite molecular and biologic homologies with IFN-I and IL-22, IFN-III signaling did not improve thymus regeneration after radiation or the course of GVHD after myeloablative TBI and allo-HSCT. (C) 2018 Elsevier Inc. All rights reserved

Reprogramming the body weight set point by a reciprocal interaction of hypothalamic leptin sensitivity and Pomc gene expression reverts extreme obesity

Objective: A major challenge for obesity treatment is the maintenance of reduced body weight. Diet-induced obese mice are resistant to achieving normoweight once the obesogenic conditions are reversed, in part because lowered circulating leptin leads to a reduction in metabolic rate and a rebound of hyperphagia that defend the previously elevated body weight set point. Because hypothalamic POMC is a central leptin target, we investigated whether changes in circulating leptin modify Pomc expression to maintain normal energy balance in genetically predisposed obese mice. Methods: Mice with reversible Pomc silencing in the arcuate nucleus (ArcPomc−/−) become morbidly obese eating low-fat chow. We measured body composition, food intake, plasma leptin, and leptin sensitivity in ArcPomc−/− mice weight-matched to littermate controls by calorie restriction, either from weaning or after developing obesity. Pomc was reactivated by tamoxifen-dependent Cre recombinase transgenes. Long acting PASylated leptin was administered to weight-reduced ArcPomc−/− mice to mimic the super-elevated leptin levels of obese mice. Results: ArcPomc−/− mice had increased adiposity and leptin levels shortly after weaning. Despite chronic calorie restriction to achieve normoweight, ArcPomc−/− mice remained moderately hyperleptinemic and resistant to exogenous leptin's effects to reduce weight and food intake. However, subsequent Pomc reactivation in weight-matched ArcPomc−/− mice normalized plasma leptin, leptin sensitivity, adiposity, and food intake. In contrast, extreme hyperleptinemia induced by PASylated leptin blocked the full restoration of hypothalamic Pomc expression in calorie restricted ArcPomc−/− mice, which consequently regained 30% of their lost body weight and attained a metabolic steady state similar to that of tamoxifen treated obese ArcPomc−/− mice. Conclusions: Pomc reactivation in previously obese, calorie-restricted ArcPomc−/− mice normalized energy homeostasis, suggesting that their body weight set point was restored to control levels. In contrast, massively obese and hyperleptinemic ArcPomc−/− mice or those weight-matched and treated with PASylated leptin to maintain extreme hyperleptinemia prior to Pomc reactivation converged to an intermediate set point relative to lean control and obese ArcPomc−/− mice. We conclude that restoration of hypothalamic leptin sensitivity and Pomc expression is necessary for obese ArcPomc−/− mice to achieve and sustain normal metabolic homeostasis; whereas deficits in either parameter set a maladaptive allostatic balance that defends increased adiposity and body weight. Keywords: Body weight set point, Hypothalamus, Leptin, Leptin resistance, Obesity, PASylation, POM

PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.

Interferon α (IFNα) so far is the only therapeutic option for chronic hepatitis B virus (HBV) infection that can lead to virus clearance. Unfortunately, its application is limited by side effects and response rates are low. The aim of this study was to generate a novel long-acting IFNα with the help of PASylation technology that adds a polypeptide comprising Proline, Alanine and Serine (PAS) to increase plasma half-life. Following evaluation of four selected recombinant murine IFNα (mIFNα) subtypes in cell culture, the most active subtype, mIFNα11, was fused with a 600 amino acid PAS chain. The activity of PAS-mIFNα was assessed by interferon bioassay and further evaluated for induction of interferon-stimulated genes (ISG) and antiviral efficacy in cell culture as well as in HBV-transgenic mice. PAS-mIFNα induced expression of ISG comparable to unmodified mIFNα and, likewise, evoked dose-dependent reduction of HBV replication in vitro. In vivo, PAS-mIFNα led to pronounced suppression of HBV replication without detectable liver damage whereas conventional mIFNα treatment only had a modest antiviral effect. Importantly, all PAS-mIFNα treated mice showed an anti-HBs antibody response, lost HBsAg and achieved seroconversion after three weeks. PASylated IFNα showed a profoundly increased antiviral effect in vivo compared to the non-modified version without toxicity, providing proof-of-concept that an improved IFNα can achieve higher rates of HBV antiviral and immune control