Structural recognition of carbohydrates by antibody candidates for immunotherapy of cancer and infection
Creators: name: SOLIMAN, Caroline
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310112
subject: Structural biology (incl. macromolecular modelling)
classificationCode: 320402
subject: Applied immunology (incl. antibody engineering, xenotransplantation and t-cell therapies)
classificationCode: 320409
subject: Tumour immunology

description: Monoclonal antibody therapies are increasingly being developed for the treatment of a range of diseases including cancer, and as therapies for infectious disease. While most of the approved therapeutic antibodies target protein or glycoprotein antigens, it has been known for many years that carbohydrate-binding antibodies are capable of specifically recognizing altered self-cells (e.g. cancer cells) and being effective in providing protective responses to pathogens. However, progress has been slow when delivering carbohydrate-binding monoclonal antibodies (mAbs) as therapeutics for cancer and for antibiotic resistant bacterial infections. 

The overall goal of this thesis is to determine the structural basis for recognition of carbohydrates by antibodies with potential uses in the immunotherapy of several cancers and a broad range of infections. This was done using a range of integrated computational and experimental techniques. Chapter 2 examines the three-dimensional structural basis for the effective targeting of a broadly expressed microbial polysaccharide, known as poly-N-acetyl glucosamine (PNAG). Crystal structures of the human anti-PNAG mAb F598 showed a large deep binding groove that accommodates at least 5 PNAG residues via an anchored binding mechanism, recognising the core GlcNAc residue in a conserved manner. This study revealed the mechanism for broad-spectrum antibody-based targeting of microbial pathogens by a fully human antibody. 

Chapters 3 and 5 identify two other carbohydrate-binding antibodies that are being developed as cancer therapeutics. Importantly, understanding the molecular details of glycan recognition may help advance a new class of cancer-targeting immunotherapy. In Chapter 3, the structural basis for the effective targeting of stage-specific embryonic antigen-4 (SSEA-4) is examined using crystallography and molecular dynamics. SSEA-4 is a glycan that is overexpressed on some cancer cells including breast and ovarian. The chimeric antibody ch28/11 was shown to be highly specific for SSEA-4, with X-ray structures revealing carbohydrate binding also occurred in a large deep binding groove, similar to F598-PNAG. SSEA-4 bound in a horseshoe-like conformation with the highly specific nature of the antibody explained by the dominant binding of the terminal sialic acid, as further supported by molecular dynamics simulations. 

Chapter 5 examines the structural basis for antibody binding to a group of Lewis glycans, which are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary. The chimeric antibody ch88.2 showed reactivity to colorectal cancer cells and significantly reduced cell viability. Glycan recognition of the Lea-Lex hexasaccharide was predicted to occur in an extended but shallow groove by X-ray crystallography and computational techniques, in contrast to the binding observed for F598 and ch28/11. Interestingly, ch88.2 bound to both glycans, with one site for Lea and a second adjacent site for Lex. These results provide structural insights into the cross-reactivity of the ch88.2 mAb for various Lewis-a and Lewis-x containing glycans and its high selectivity for Lewis-positive tumours.

Finally, to identify nanoscale imaging techniques that are appropriate for visualizing cancer cell membrane damage, a model peptide was employed in Chapter 4. Melittin, a bee-venom peptide with wide reaching cytotoxic effects, has the potential to be used as a targeted immunotherapy. Melittin was shown to cause rapid changes to colorectal and gastric cancer cells. High-resolution microscopy, membrane staining, AFM and SEM techniques all revealed extensive changes to cancer cells within seconds of melittin treatment in the form of cell swelling, membrane breakage and blebbing. These techniques were further employed in Chapter 5 to image antibody-induced nanoscale transformations of target cell membranes, where ch88.2 was observed to cause cancer cell death via pore formation. 

Overall, this thesis provides a detailed understanding of the 3D structural basis for recognition of carbohydrates by antibodies in cancer and bacterial infection, which may have future applications for the development of therapeutic antibodies.