Chua, Jia Xin
(2017)
The generation and characterisation of anti-glycan monoclonal antibodies.
PhD thesis, University of Nottingham.
Abstract
Glycomic profiling of tumour tissues consistently shows alterations in N- and O-glycosylation profiles of glycoproteins and glycolipids compared to healthy tissues, with important functional implications for cancer cell biology. Despite the attractiveness of the targets, there are very few mAbs recognising glycans as they induce low affinity IgM responses.
The aim of this study was to develop a novel immunisation protocol, with the objective of increasing the immunogenicity of tumour-associated carbohydrate or glycolipid antigens, leading to the generation of novel anti-glycan monoclonal antibodies (mAbs) with potent cytotoxicity against cancers.
Mice were immunised with, tumour cell plasma membrane glycolipid extracts incorporated into liposomes in the presence or absence of alpha-galactosylceramide (α-GalCer) and/or anti-CD40 mAb and/or with engineered cells, which express specific glycolipids. Three IgG3 murine mAbs (FG88.2, FG88.7 and FG2811.72) with subnanomolar potency were generated, suggesting that the immunisation protocols succeeded in increasing the immunogenicity of glycolipid antigens which promoted antibody class switching and affinity maturation. FG88.2 and FG88.7 mAbs recognised the novel Lewis a/c/x (Lea/c/x) glycans expressed on glycolipids and glycoproteins whereas FG2811.72 is an ultraspecific mAb targeting SSEA-4 glycolipid.
Immunohistochemical analysis showed that FG88.2 glyco-epitopes were overexpressed on a broad range of tumour types with limited cross reactivity with normal human tissues. Characterisation of effector functions of FG88 and FG2811.72 mAbs demonstrated that they were capable of inducing potent antibody dependent cellular cytotoxicity (ADCC; EC50 FG88 mAbs: 10-9 M; EC50 FG2811.72 mAb: 10-10 M) and complement dependent cytotoxicity (CDC; EC50 FG88 mAbs: 10-9 M; EC50 FG2811.72 mAb: 10-9 M). In addition, FG88 mAbs were capable of inducing direct tumour cell death via oncosis which induces pore formation and lysis of tumour cells. Preliminary results showed that FG2811.72 mAb inhibited tumour cell growth in the absence of immune cells and complement proteins, but the mechanism of tumour cell growth inhibition remains unexplored. The excellent in vitro cytotoxicity of FG88 mAbs translated into potent anti-tumour efficacy and significant survival improvement in a colorectal hepatic metastasis xenograft model in which mAb treatment was initiated 10 days after tumour initiation and development of liver metastasis. No other studies, to date, have shown tumour eradication of 10-day established tumours and liver metastases.
Traditional cancer chemotherapy is often accompanied with severe side effects to cancer patients. Antibody-drug conjugates (ADCs) are composed of a therapeutic mAb chemically linked to a potent cytotoxic drug. By linking mAbs to cytotoxic drugs, ADCs selectively target tumour cells, thus improving the therapeutic index of chemotherapeutic agents. ADCs work via the internalisation of antibody-antigen complexes into cancer cells, followed by the release of the cytotoxic drugs in the lysosomes which then causes cancer cell death. FG88 and FG2811.72 mAbs deliver Zaporin conjugates into tumour cells with subnanomolar potency, suggesting they have the potential to be used as ADCs.
Murine mAbs have limited clinical utility due to their short half-life in serum, their suboptimal in stimulation of Fc effector functions and their high immunogenicity in humans. Therefore chimeric FG88.2 (CH88.2; IgG1) mAb was generated. Although CH88.2 mAb maintained antigen specificity and affinity, it had reduced CDC activity and importantly, it lost its ability to induce direct tumour cell death. In this study, the E345R mutation [a single amino acid substitution at position 345 (Glu345→ Arg)] was introduced into CH88.2 to generate a mutant version of CH88.2 named ECH88.2 mAb. The ECH88.2 mAb showed restored direct cell killing ability and the CDC activity was improved. In addition, ECH88.2 demonstrated excellent ADCC activity.
Lewis a/c/x (Lea/c/x) glycans were expressed by both glycolipids and glycoproteins. Studies on the role of glycolipids/glycoproteins in inducing direct tumour cell death suggested that it was primarily mediated by glycolipids. In contrast, both glycolipids and glycoproteins were involved in ADCC, CDC and mAb internalisation into tumour cells.
In summary, the aim of this study was achieved by generating three novel anti-glycan mAbs which bind to tumour associated carbohydrate antigens with high affinity. Their strong tumour specificity and limited cross reactivity to normal tissues, as well as their potent cytotoxicity effects, suggest that they should have great potential as therapeutic anti-cancer mAbs. Furthermore, their ability to internalise into tumour cells suggests that they may have potential as drug carriers. Importantly, this study demonstrates a novel approach: by introducing the E345R mutation in the Fc region of the chimeric mAbs it promotes non-covalent interaction in the presence of antigen leading to increased cell surface binding, improved CDC activity and restoration of the direct killing ability of the mouse mAbs.
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