Genome‐wide interaction screen for Mycobacterium tuberculosis ClpCP protease reveals toxin–antitoxin systems as a major substrate class

In Mycobacterium tuberculosis (Mtb), the Clp protease degradation pathway, mediated by the modular ClpCP and ClpXP protease complexes, is essential for growth and presents an attractive drug target. Employing a bacterial adenylate cyclase two‐hybrid (BACTH) screening approach that we adapted to screen the proteome of an Mtb ORF library, we identify protein interaction partners of the ClpC1 chaperone on a genome‐wide level. Our results demonstrate that bipartite type II toxin–antitoxin (TA) systems represent a major substrate class. Out of the 67 type II TA systems known in Mtb, 25 appear as ClpC1 interaction partners in the BACTH screen, including members of the VapBC, MazEF, and ParDE families, as well as a RelBE member that was identified biochemically. We show that antitoxins of the Vap and Rel families are degraded by ClpCP in vitro. We also demonstrate that ClpCP is responsible for mediating the N‐end rule pathway, since the adaptor protein ClpS supports ClpC‐dependent degradation of an N‐end rule model substrate in vitro.ISSN:1742-464XISSN:1742-465

Antibody-based delivery of interleukin-9 to neovascular structures: Therapeutic evaluation in cancer and arthritis

Interleukin-9 is a cytokine with multiple functions, including the ability to activate group 2 innate lymphoid cells, which has been postulated to be therapeutically active in mouse models of arthritis. Similarly, interleukin-9 has been suggested to play an important role in tumor immunity. Here, we describe the cloning, expression, and characterization of three fusion proteins based on murine interleukin-9 and the F8 antibody, specific to the alternatively spliced EDA domain of fibronectin. EDA is strongly expressed in cancer and in various arthritic conditions, while being undetectable in the majority of healthy organs. Interleukin-9-based fusion proteins with an irrelevant antibody specific to hen egg lysozyme served as negative control in our study. The fusion proteins were characterized by quantitative biodistribution analysis in tumor-bearing mice using radioiodinated protein preparations. The highest tumor uptake and best tumor:organ ratios were observed for a format, in which the interleukin-9 moiety was flanked by two units of the F8 antibody in single-chain Fv format. Biological activity of interleukin-9 was retained when the payload was fused to antibodies. However, the targeted delivery of interleukin-9 to the disease site resulted in a modest anti-tumor activity in three different murine models of cancer (K1735M2, CT26, and F9), while no therapeutic benefit was observed in a collagen induced model of arthritis. Collectively, these results confirm the possibility to deliver interleukin-9 to the site of disease but cast doubts about the alleged therapeutic activity of this cytokine in cancer and arthritis, which has been postulated in previous publications

Therapeutic Evaluation of Antibody-Based Targeted Delivery of Interleukin 9 in Experimental Pulmonary Hypertension

Background and Aims: Pulmonary hypertension (PH) is a heterogeneous disorder associated with poor prognosis. For the majority of patients, only limited therapeutic options are available. Thus, there is great interest to develop novel treatment strategies focusing on pulmonary vascular and right ventricular remodeling. Interleukin 9 (IL9) is a pleiotropic cytokine with pro- and anti-inflammatory functions. The aim of this study was to evaluate the therapeutic activity of F8IL9F8 consisting of IL9 fused to the F8 antibody, specific to the alternatively-spliced EDA domain of fibronectin, which is abundantly expressed in pulmonary vasculature and right ventricular myocardium in PH. Methods: The efficacy of F8IL9F8 in attenuating PH progression in the monocrotaline mouse model was evaluated in comparison to an endothelin receptor antagonist (ERA) or an IL9 based immunocytokine with irrelevant antibody specificity (KSFIL9KSF). Treatment effects were assessed by right heart catheterization, echocardiography as well as histological and immunohistochemical tissue analyses. Results: Compared to controls, systolic right ventricular pressure (RVPsys) was significantly elevated and a variety of right ventricular echocardiographic parameters were significantly impaired in all MCT-induced PH groups except for the F8IL9F8 group. Both, F8IL9F8 and ERA treatments lead to a significant reduction in RVPsys and an improvement of echocardiographic parameters when compared to the MCT group not observable for the KSFIL9KSF group. Only F8IL9F8 significantly reduced lung tissue damage and displayed a significant decrease of leukocyte and macrophage accumulation in the lungs and right ventricles. Conclusions: Our study provides first pre-clinical evidence for the use of F8IL9F8 as a new therapeutic agent for PH in terms of a disease-modifying concept addressing cardiovascular remodeling