MAGPIX and FLEXMAP 3D Luminex platforms for direct detection of miR-122-5p through dynamic chemical labelling.

MicroRNAs (miRs) have emerged as promising biomarkers for diagnosing and predicting the prognosis of liver injury. This study aimed to compare the performance of two Luminex platforms, MAGPIX and FLEXMAP 3D, utilizing the innovative Dynamic Chemical Labelling (DCL) technology for direct detection and analysis of miR-122-5p in serum samples from patients with liver injury. Serum samples were collected from four patients with liver injury and four healthy controls. The levels of miR-122-5p were measured using the DCL method on both MAGPIX and FLEXMAP 3D platforms. The performance evaluation included the limit of detection (LOD), intra-assay and inter-assay precision, as well as accuracy. The results demonstrated that both platforms exhibited high sensitivity and specificity in detecting miR-122-5p in serum samples from patients with liver injury. However, FLEXMAP 3D indicated a lower LOD compared to MAGPIX. The precision of miR-122-5p detection was similar between the two platforms. In conclusion, both MAGPIX and FLEXMAP 3D Luminex platforms, in conjunction with DCL reagents, proved to be reliable and sensitive tools for detecting miR-122-5p in serum samples from patients with liver injury. Although both platforms were effective, FLEXMAP 3D exhibited slightly better performance, suggesting its preference for miR detection in clinical settings. These findings offer valuable insights for selecting the appropriate Luminex platform for miR detection in patients with liver injury and beyond.

Genetic code expansion in E. coli enables production of a functional 'ready-to-click' T cell receptor-specific scFv.

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition ('click chemistry'). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a 'ready-to-click' scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death.

BACKGROUND: Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called "lectibody", a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. METHODS: The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. RESULTS: This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis-up to 93%-of Gb3+ tumor cells in vitro. CONCLUSIONS: This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.

Molecular cloning and functional characterization of a two highly stereoselective borneol dehydrogenases from Salvia officinalis L.

Enzymes for selective terpene functionalization are of particular importance for industrial applications. Pure enantiomers of borneol and isoborneol are fragrant constituents of several essential oils and find frequent application in cosmetics and therapy. Racemic borneol can be easily obtained from racemic camphor, which in turn is readily available from industrial side-streams. Enantioselective biocatalysts for the selective conversion of borneol and isoborneol stereoisomers would be therefore highly desirable for their catalytic separation under mild reaction conditions. Although several borneol dehydrogenases from plants and bacteria have been reported, none show sufficient stereoselectivity. Despite Croteau et al. describing sage leaves to specifically oxidize one borneol enantiomer in the late 70s, no specific enzymes have been characterized. We expected that one or several alcohol dehydrogenases encoded in the recently elucidated genome of Salvia officinalis L. would, therefore, be stereoselective. This study thus reports the recombinant expression in E. coli and characterization of two enantiospecific enzymes from the Salvia officinalis L. genome, SoBDH1 and SoBDH2, and their comparison to other known ADHs. Both enzymes produce preferentially (+)-camphor from racemic borneol, but (-)-camphor from racemic isoborneol.