Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

The membrane effects of melittin on gastric and colorectal cancer.

The cytotoxic effects of melittin, a bee-venom peptide, have been widely studied towards cancer cells. Typically, these studies have examined the effect of melittin over extended-time courses (6-24 hours), meaning that immediate cellular interactions have been overlooked. In this work, we demonstrate the rapid effects of melittin on both gastric and colorectal cancer, specifically AGS, COLO205 and HCT-15 cell lines, over a period of 15 minutes. Melittin exhibited a dose dependent effect at 4 hours of treatment, with complete cellular death occurring at the highest dose of 20 µg/mL. Interestingly, when observed at shorter time points, melittin induced cellular changes within seconds; membrane damage was observed as swelling, breakage or blebbing. High-resolution imaging revealed treated cells to be compromised, showing clear change in cellular morphology. After 1 minute of melittin treatment, membrane changes were observed, and intracellular material could be seen expelled from the cells. Overall, these results enhance our understanding of the fast acting anti-cancer effects of melittin.

Osteoclastogenesis-related cytokines and peri-prosthetic osteolysis in revision metal-on-metal total hip replacements.

PURPOSE: Peri-prosthetic osteolysis is a major cause for revision hip arthroplasty; various cytokines including those in the osteoclastogenesis pathway have been identified as potentially key in the osteolysis process. Adverse reactions to metal debris in metal-on-metal total hip replacements have led to an increase in revision procedures. This study examines the levels of osteoclastogenesis-related cytokines in serum and synovial fluid samples obtained from patients at the time of revision metal-on-metal total hip replacement and compares between patients with and without radiographic evidence of peri-prosthetic osteolysis. METHODS: Sandwich ELISA techniques were used to detect IL-6, IL-18, M-CSF, sRANKL and OPG in the samples. Results were analysed with linear regression, Fisher's tests and t-tests; p<0.05 considered significant. Samples from 36 patients (18 with osteolysis, 18 without osteolysis) were analysed. RESULTS: There was wide variation in the detectable levels of cytokines. No significant differences were found between patients with and without osteolysis in mean synovial fluid levels of IL-6 (p = 0.863), IL-18 (p = 0.324), M-CSF (p = 0.508), sRANKL (p = 0.884), OPG (p = 0.776) or mean serum levels of OPG (p = 0.993) or sRANKL (p = 0.565) (insufficient detection of IL-6, IL-18 or M-CSF in serum samples). A correlation was found between synovial fluid levels of IL-6 and OPG in patients without osteolysis (r2 = 0.618, p<0.001) but not with osteolysis (r2 = 0.0004). CONCLUSIONS: These results indicate that the process of peri-prosthetic osteolysis is complex and multifactorial; there may also be an influence of metallosis. Further research is needed to increase understanding of peri-prosthetic osteolysis and influence clinical practice.