A Shiga Toxin B-Subunit-Based Lectibody Boosts T Cell Cytotoxicity towards Gb3-Positive Cancer Cells.

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.

Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy.

In recent years, receptor-mediated drug delivery has gained major attention in the treatment of cancer. The pathogen-derived Shiga Toxin B subunit (STxB) can be used as a carrier that detects the tumor-associated glycosphingolipid globotriaosylceramide (Gb3) receptors. While drug conjugation via lysine or cysteine offers random drug attachment to carriers, click chemistry has the potential to improve the engineering of delivery systems as the site specificity can eliminate interference with the active binding site of tumor ligands. We present the production of recombinant STxB in its wild-type (STxBwt) version or incorporating the noncanonical amino acid azido lysine (STxBAzK). The STxBwt and STxBAzK were manufactured using a growth-decoupled Escherichia coli (E. coli)-based expression strain and analyzed via flow cytometry for Gb3 receptor recognition and specificity on two human colorectal adenocarcinoma cell lines-HT-29 and LS-174-characterized by high and low Gb3 abundance, respectively. Furthermore, STxBAzK was clicked to the antineoplastic agent monomethyl auristatin E (MMAE) and evaluated in cell-killing assays for its ability to deliver the drug to Gb3-expressing tumor cells. The STxBAzK-MMAE conjugate induced uptake and release of the MMAE drug in Gb3-positive tumor cells, reaching 94% of HT-29 cell elimination at 72 h post-treatment and low nanomolar doses while sparing LS-174 cells. STxBAzK is therefore presented as a well-functioning drug carrier, with a possible application in cancer therapy. This research demonstrates the feasibility of lectin carriers used in delivering drugs to tumor cells, with prospects for improved cancer therapy in terms of straightforward drug attachment and effective cancer cell elimination.

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.

Neutralizing the Impact of the Virulence Factor LecA from Pseudomonas aeruginosa on Human Cells with New Glycomimetic Inhibitors.

Bacterial adhesion, biofilm formation and host cell invasion of the ESKAPE pathogen Pseudomonas aeruginosa require the tetravalent lectins LecA and LecB, which are therefore drug targets to fight these infections. Recently, we have reported highly potent divalent galactosides as specific LecA inhibitors. However, they suffered from very low solubility and an intrinsic chemical instability due to two acylhydrazone motifs, which precluded further biological evaluation. Here, we isosterically substituted the acylhydrazones and systematically varied linker identity and length between the two galactosides necessary for LecA binding. The optimized divalent LecA ligands showed improved stability and were up to 1000-fold more soluble. Importantly, these properties now enabled their biological characterization. The lead compound L2 potently inhibited LecA binding to lung epithelial cells, restored wound closure in a scratch assay and reduced the invasiveness of P. aeruginosa into host cells.

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.

Extending Janus lectins architecture: Characterization and application to protocells.

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being exploited for producing tools with precise topology and specificity. We developed the concept of engineered chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the ß-propeller domain from Ralstonia solanacearum and the ß-trefoil domain from fungus Marasmius oreades, is specific for fucose and α-galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The engineered protein recognizes and binds H1299 human lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering.

The choanoflagellate pore-forming lectin SaroL-1 punches holes in cancer cells by targeting the tumor-related glycosphingolipid Gb3.

Choanoflagellates are primitive protozoa used as models for animal evolution. They express a large variety of multi-domain proteins contributing to adhesion and cell communication, thereby providing a rich repertoire of molecules for biotechnology. Adhesion often involves proteins adopting a ß-trefoil fold with carbohydrate-binding properties therefore classified as lectins. Sequence database screening with a dedicated method resulted in TrefLec, a database of 44714 ß-trefoil candidate lectins across 4497 species. TrefLec was searched for original domain combinations, which led to single out SaroL-1 in the choanoflagellate Salpingoeca rosetta, that contains both ß-trefoil and aerolysin-like pore-forming domains. Recombinant SaroL-1 is shown to bind galactose and derivatives, with a stronger affinity for cancer-related α-galactosylated epitopes such as the glycosphingolipid Gb3, when embedded in giant unilamellar vesicles or cell membranes. Crystal structures of complexes with Gb3 trisaccharide and GalNAc provided the basis for building a model of the oligomeric pore. Finally, recognition of the αGal epitope on glycolipids required for hemolysis of rabbit erythrocytes suggests that toxicity on cancer cells is achieved through carbohydrate-dependent pore-formation.

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli.

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell.

Microbial carbohydrate-binding toxins - From etiology to biotechnological application.

Glycan-recognizing toxins play a significant role in the etiology of many diseases afflicting humanity. The carbohydrate recognition domains of these toxins play essential roles in the virulence of many microbial organisms with multiple modes of action, from promoting pore formation to facilitating the entry of toxic enzymatic subunits into the host cell. Carbohydrate-binding domains with an affinity for specific glycan-based receptors can also be exploited for various applications, including detecting glycobiomarkers, as drug delivery systems, and new generation biopharmaceutical products and devices (e.g. glycoselective capture of tumor-derived exosomes). Therefore, understanding how to efficiently express and purify recombinant toxins and their carbohydrate-binding domains can enable opportunities for the formulation of innovative biopharmaceuticals that can improve human health. Here, we provide an overview of carbohydrate-binding toxins in the context of biotechnological innovation. We review 1) structural characteristics concerning the toxins' mode of action; 2) applications and therapeutic design with a particular emphasis on exploiting carbohydrate-binding toxins for production of anti-tumor biopharmaceuticals; discuss 3) possible ways to manufacture those molecules at a bioreactor scale using microbial expression systems, and 4) their purification using their affinity for glycans.

The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake.

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation-a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions.

Structure-activity relationships of agonists for the orphan G protein-coupled receptor GPR27.

GPR27 belongs, with GPR85 and GPR173, to a small subfamily of three receptors called "Super-Conserved Receptors Expressed in the Brain" (SREB). It has been postulated to participate in key physiological processes such as neuronal plasticity, energy metabolism, and pancreatic ß-cell insulin secretion and regulation. Recently, we reported the first selective GPR27 agonist, 2,4-dichloro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (I, pEC50 6.34, Emax 100%). Here, we describe the synthesis and structure-activity relationships of a series of new derivatives and analogs of I. All products were evaluated for their ability to activate GPR27 in an arrestin recruitment assay. As a result, agonists were identified with a broad range of efficacies including partial and full agonists, showing higher efficacies than the lead compound I. The most potent agonist was 4-chloro-2,5-difluoro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7y, pEC50 6.85, Emax 37%), and the agonists with higher efficacies were 4-chloro-2-methyl-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7p, pEC50 6.04, Emax 123%), and 2-bromo-4-chloro-N-(4-(N-phenylsulfamoyl)phenyl)benzamide (7r, pEC50 5.99, Emax 123%). Docking studies predicted the putative binding site and interactions of agonist 7p with GPR27. Selected potent agonists were found to be soluble and devoid of cellular toxicity within the range of their pharmacological activity. Therefore, they represent important new tools to further characterize the (patho)physiological roles of GPR27.

Metabolic Syndrome, Anxiety and Depression in a Sample of Italian Primary Care Patients.

This cross-sectional study aimed at measuring the correlation and association of anxiety, depression and comorbid anxiety-depression symptoms with metabolic syndrome (MetS) in a sample of Italian primary care patients who attended their General Practitioner clinics over a 1-month period in 2013. The Hospital Anxiety and Depression Scale (HADS) was used to assess anxiety and depressive symptoms. The sample was made up of 129 patients (57% women; mean age, 61 ± 12 years). The prevalence of MetS varied from 40% (Adult Treatment Panel III-Revised criteria) to 48% (International Diabetes Federation criteria). The prevalence of symptoms of anxiety, depression and comorbid anxiety and depression was, respectively, 26%, 2%, and 15%. MetS (defined according to Adult Treatment Panel III-Revised criteria) was associated with comorbid anxiety-depressive symptoms (odds ratio [OR] = 3.84, 95% confidence interval [CI] = 1.26-11.71), but not with anxiety or depressive symptoms only. Out of the individual components of MetS, enlarged waist circumference was associated with anxiety symptoms (OR = 4.22, 95% CI = 1.56-11.44).

Effects of lanreotide Autogel on growth hormone, insulinlike growth factor 1, and tumor size in acromegaly: a 1-year prospective multicenter study.

OBJECTIVE: To evaluate the safety and effectiveness of lanreotide Autogel on growth hormone and insulinlike growth factor 1 (IGF-1) concentrations and tumor size in patients with acromegaly. METHODS: Between September 2004 and March 2006, patients with active acromegaly who had not previously been treated with somatostatin analogues or received irradiation were enrolled in a 1-year, prospective, open, multicenter study. Lanreotide Autogel was injected subcutaneously starting with 90 mg every 4 weeks for 2 cycles and then individually titrated, aiming for safe growth hormone concentrations (<2.5 ng/mL) and normal age-matched IGF-1 concentrations. Tumor shrinkage, clinical score, pituitary function, and safety parameters were evaluated. RESULTS: Twenty-seven patients (15 women, 12 men) were enrolled. One patient withdrew because of treatment intolerance, and 5 proceeded to neurosurgery 6 months into the study. Lanreotide Autogel was the primary treatment in 19 patients (4 with microadenoma, 15 with macroadenoma) and the adjuvant treatment in 8 patients in whom it followed a previous unsuccessful neurosurgery. In the 26 patients, safe growth hormone values were achieved in 11 (42%), normal IGF-1 values in 14 (54%), and both targets were achieved in 10 (38%). Tumors shrank in 16 of the 22 patients (73%) in whom tumor shrinkage could be evaluated. The maximal vertical diameter of the tumor decreased by a mean of 24% (range, 0% to 50%), from 14.4 +/- 8.4 mm to 10.4 +/- 7 mm, and tumor volume decreased by a mean of 44% (range, 0% to 76%), from 2536 mm3 (range, 115-7737 mm(3)) to 1461 mm(3) (range, 63-6217 mm(3)) (both P<.015). Symptom scores and lipid levels significantly improved. In the 26 patients, glucose metabolism deteriorated in 3 (12%) and improved in 4 (15%). New biliary alterations appeared in 26%. Pituitary function and safety parameters did not change. CONCLUSIONS: Lanreotide Autogel treatment, titrated for optimal hormonal control, effectively controls IGF-1 and growth hormone levels, shrinks tumors, reduces acromegalic symptoms, and is well tolerated.

First-line octreotide-LAR therapy induces tumour shrinkage and controls hormone excess in patients with acromegaly: results from an open, prospective, multicentre trial.

BACKGROUND: The majority of patients with acromegaly have large tumours and the outcome of conventional management remains poor. OBJECTIVE: To investigate the clinical application of octreotide-LAR as primary treatment in newly diagnosed patients with GH-secreting pituitary tumours. DESIGN: Open, prospective, multicentre, 24-week follow-up study. PATIENTS: Thirty-four patients were enrolled (20 men, 14 women; mean age, 50 years); 13 had microadenoma [median tumour volume 327 mm(3) (range 31-629 mm(3))], 21 had macroadenoma [median tumour volume 1,325 mm(3) (range 503-11,583 mm(3))]. Interventions Octreotide-LAR at the dosage of 20 mg every 28 days for the first 12 weeks increased to 30 mg every 28 days to control GH and/or IGF-I excess in 20 patients (64.7%). MAIN OUTCOME MEASURES: Primary endpoints were control of GH (fasting < 2.5 microg/l) and IGF-I secretion (gender- and age-normalized) and presence and entity of tumour mass shrinkage. Secondary endpoint was improvement of symptoms score. RESULTS: In patients with micro- and macroadenomas GH levels decreased to < 2.5 microg/l in 84.6% and 45%, serum IGF-I levels normalized for age and gender in 61.5% and 35% of cases. Failure in achieving either GH < 2.5 microg/l or normal IGF-I levels was found in none of the patients with micro- and in 45% of patients with macroadenoma. Median tumour volume was reduced by 54% (range: -90% to +350%) in micro- and by 49% (range -94% to -14%) in macroadenomas. Headache, perspiration and osteo-arthralgias disappeared in 21%, paresthesias in 38%, fatigue in 26% and carpal tunnel syndrome in 15%. The treatment was well tolerated: more frequent adverse events were gastrointestinal (in 44%). CONCLUSIONS: In both patients with micro- or macroadenoma, primary octreotide-LAR treatment controls hormone excess, induces tumour shrinkage and improves symptoms of acromegaly with limited side effects and can be therefore successfully employed in patients with contraindications for surgery or in those who refuse surgery.