Lectin-anticancer peptide fusion demonstrates a significant cancer-cell-selective cytotoxic effect and inspires the production of "clickable" anticancer peptide in Escherichia coli.

Targeted killing of tumor cells while protecting healthy cells is the pressing priority in cancer treatment. Lectins that target a specific glycan marker abundant in cancer cells can be valuable new tools for selective cancer cell killing. The lectin Shiga-like toxin 1 B subunit (Stx1B) is an example that specifically binds globotriaosylceramide (CD77 or Gb3), which is overexpressed in certain cancers. In this study, a human lactoferricin-derived synthetic retro di-peptide R-DIM-P-LF11-215 with antitumor efficacy was fused to the lectin Stx1B to selectively target and kill Gb3+ cancer cells. We produced lectin-peptide fusion proteins in Escherichia coli, isolated them by Gb3-affinity chromatography, and assessed their ability to selectively kill Gb3+ cancer cells in a Calcein AM assay. Furthermore, to expand the applications of R-DIM-P-LF11-215 in developing therapeutic bioconjugates, we labeled R-DIM-P-LF11-215 with the unique reactive non-canonical amino acid Nε -((2-azidoethoxy)carbonyl)-L-lysine (AzK) at a selected position by amber stop codon suppression. The R-DIM-P-LF11-215 20AzK and the unlabeled R-DIM-P-LF11-215 parent peptide were produced as GST-fusion proteins for soluble expression in E. coli for the first time. We purified both variants by size-exclusion chromatography and analyzed their peptide masses. Finally, a cyanin 3 fluorophore was covalently conjugated to R-DIM-P-LF11-215 20AzK by strain-promoted alkyne-azide cycloaddition. Our results showed that the recombinant lectin-peptide fusion R-DIM-P-LF11-215-Stx1B killed >99% Gb3+ HeLa cells while Gb3-negative cells were unaffected. The peptides R-DIM-P-LF11-215 and R-DIM-P-LF11-215 20AzK were produced recombinantly in E. coli in satisfactory amounts and were tested functional by cytotoxicity and cell-binding assays, respectively.

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.

Oriental freshwater mussels arose in East Gondwana and arrived to Asia on the Indian Plate and Burma Terrane.

Freshwater mussels cannot spread through oceanic barriers and represent a suitable model to test the continental drift patterns. Here, we reconstruct the diversification of Oriental freshwater mussels (Unionidae) and revise their taxonomy. We show that the Indian Subcontinent harbors a rather taxonomically poor fauna, containing 25 freshwater mussel species from one subfamily (Parreysiinae). This subfamily most likely originated in East Gondwana in the Jurassic and its representatives arrived to Asia on two Gondwanan fragments (Indian Plate and Burma Terrane). We propose that the Burma Terrane was connected with the Indian Plate through the Greater India up to the terminal Cretaceous. Later on, during the entire Paleogene epoch, these blocks have served as isolated evolutionary hotspots for freshwater mussels. The Burma Terrane collided with mainland Asia in the Late Eocene, leading to the origin of the Mekong's Indochinellini radiation. Our findings indicate that the Burma Terrane had played a major role as a Gondwanan "biotic ferry" alongside with the Indian Plate.

Recombinant spider silk coatings functionalized with enzymes targeting bacteria and biofilms.

Bacteria forming biofilms on surgical implants is a problem that might be alleviated by the use of antibacterial coatings. In this article, recombinant spider silk was functionalized with the peptidoglycan degrading endolysin SAL-1 from the staphylococcal bacteriophage SAP-1 and the biofilm-matrix-degrading enzyme Dispersin B from Aggregatibacter actinomycetemcomitans using direct genetic fusion and/or covalent protein-protein fusion catalyzed by Sortase A. Spider silk assembly and enzyme immobilization was monitored using quartz crystal microbalance analysis. Enzyme activity was investigated both with a biochemical assay using cleavage of fluorescent substrate analogues and bacterial assays for biofilm degradation and turbidity reduction. Spider silk coatings functionalized with SAL-1 and Disperin B were found to exhibit bacteriolytic effect and inhibit biofilm formation, respectively. The strategy to immobilize antibacterial enzymes to spider silk presented herein show potential to be used as surface coatings of surgical implants and other medical equipment to avoid bacterial colonization.