A specific CD4 epitope bound by tregalizumab mediates activation of regulatory T cells by a unique signaling pathway.

CD4(+)CD25(+) regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

Tetrahydrochromenoimidazoles as potassium-competitive acid blockers (P-CABs): structure-activity relationship of their antisecretory properties and their affinity toward the hERG channel.

Potassium-competitive acid blockers (P-CABs) constitute a new therapeutic option for the treatment of acid-related diseases that are widespread and constitute a significant economical burden. Enantiomerically pure tetrahydrochromenoimidazoles were prepared using the readily available candidate 4 (BYK 405879) as starting material or the Noyori asymmetric reduction of ketones as key reaction. A comprehensive SAR regarding the influence of the 5-carboxamide and the 8-aryl residue on in vitro activity, acid-suppression in the Ghosh Schild rat, and affinity toward the hERG channel was established. In addition, efficacy and duration of the antisecretory action was examined for the most promising target compounds by 24 h pH-metry in the fistula dog and a significantly different SAR was observed as compared to the Ghosh Schild rat. Several tetrahydrochromenoimidazoles were identified that possessed a comparable profile as the candidate 4.

Recombinant protein expression by targeting pre-selected chromosomal loci.

BACKGROUND: Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. RESULTS: We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context. CONCLUSION: RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.

Synthesis and evaluation of 7H-8,9-dihydropyrano[2,3-c]imidazo[1,2-a]pyridines as potassium-competitive acid blockers.

7H-8,9-Dihydropyrano[2,3-c]imidazo[1,2-a]pyridines with excellent physicochemical and pharmacological properties were identified that represent interesting candidates for further development as potassium-competitive acid blockers (P-CABs). The title compounds were prepared following synthetic pathways that relied either on a Claisen rearrangement/cross-metathesis reaction or on the (asymmetric) reduction of prochiral ketones. The influence of the character of the substituents R3, R6, and Ar on the biological activity and the physicochemical properties of the target compounds was examined. In contrast to the parent system (R6 = H), compounds in which R6 represents a carboxamide residue generally show improved in vivo activity and favorable pKa/log D values. Whereas variation of R3 is useful to obtain target compounds with modified basicity and lipophilicity, strong inhibition of the H+/K+-ATPase and potent in vivo activity is observed for R3 = methyl only. Small modifications of the aryl group, e.g., replacement of hydrogen versus a fluoro atom or a methyl group, are allowed. The (9S)-enantiomers are responsible for the gastric acid secretion inhibiting action, whereas the (9R)-enantiomers are virtually inactive.

AggA is required for aggregation and increased biofilm formation of a hyper-aggregating mutant of Shewanella oneidensis MR-1.

Shewanella oneidensis COAG, a hyper-aggregating mutant of MR-1, was isolated from a rifampicin-challenged culture. Compared to the wild-type, COAG exhibited increased biofilm formation on glass carrier material. The role of surface-located proteins in the process of COAG auto-aggregation was confirmed by different proteolytic treatments of the aggregates. All of the tested proteolytic enzymes resulted in deflocculation within 3 h of incubation. In order to examine the altered expression of outer-membrane proteins in COAG, membrane-enriched cell preparations were analysed by proteomics and the protein pattern was compared to that of MR-1. From the proteomics results, it was hypothesized that the agglutination protein AggA, associated with the secretion of a putative RTX protein, was involved in the hyper-aggregating phenotype. These results were confirmed with a DNA microarray study of COAG versus MR-1. An insertional mutation in the S. oneidensis COAG aggA locus resulted in loss of the hyper-aggregating properties and the increased biofilm-forming capability. The insertional mutation resulted in strongly decreased attachment during the initial stage of biofilm formation. By complementing this mutation with the vector pCM62, expressing the aggA gene, this effect could be nullified and biofilm formation was restored to at least the level of the MR-1 wild-type.