Engineering novel antibody-based molecules to probe the role of the immunoglobulin hinge and generate a potential cancer vaccine

  • Tein Foong Leong

Student thesis: Doctoral ThesisDoctor of Philosophy


In different immunoglobulin (Ig) isotypes, the hinge region, which separates the antigen-binding Fab region from the Fc region, varies considerably in length. The hinge region is believed to provide flexibility for the Ig to conduct better positioning for antigen binding via the Fab arms or better receptor association via the Fc region. However, the reason for the difference in hinge length among different Igs is not fully understood. In this project it is hypothesised that the longer the upper hinge length of an IgG the further the distance that the antigen binding sites at the tips of Fab arms are able to reach between neighbouring antigens.

In order to investigate the relation between upper hinge region of IgG and the distance between antigen binding sites, human IgG1 mutants specific for the hapten NIP which featured different upper hinge lengths were generated. Rigid bivalent ligands or “molecular rulers” (dsNIP-DNA) comprising defined lengths of double stranded DNA with NIP covalently attached to both ends were produced. ELISA tests demonstrated that anti-NIP human IgG1 bound to dsNIP-DNA. Through employment of molecular rulers of different lengths, surface plasmon resonance experiments indicated that the maximum distance between antigen binding sites is influenced by the length of the upper part of the IgG hinge. In addition, the role of upper hinge length on the ability of IgG to bridge between antigen on a target cell surface and Fc receptors on the surface of a phagocyte was investigated. Rosette formation between antibody-coated erythrocytes and Fc receptor-positive effector cells was utilised as an assay for this bridging. When anti-NIP human IgG1 hinge mutants were compared to wildtype IgG1, differences in rosette formation efficiency were observed indicating that the upper hinge region has a role in facilitating the bridging between an antigenic target cell and effector cell.

In a second IgG engineering approach, potential cancer vaccines that feature the Fc region of IgG fused to the tumour associated cancer-testis antigen, MAGE-A, were generated and tested. Such vaccines are designed to promote formation of immune complexes, which are preferentially taken up by dendritic cells via their FcRn receptors, with the aim of stimulating a strong anti-tumour immune response. Human MAGE-A2 fused to either human IgG1 Fc or mouse IgG2a Fc had been successfully produced and purified using Protein-G Sepharose. Results from Coomassie staining and western blotting analyses suggested that the Fc fusion proteins were suffering from protein aggregations and possibly protein degradations. To test if MAGE-A2 Fc fusion protein can be a potential cancer vaccine, an 8 amino acid peptide sequence, SIINFEKL, was incorporated into MAGE-A2 protein of the Fc fusion protein containing mouse IgG2a Fc. IgG-immune complexes of SIINFEKL incorporated Fc fusion protein were incubated with bone marrow derived dendritic cells (BMDC) and corresponding cytotoxic T cells, B3Z, to investigate the induction of antigen cross presentation. Unfortunately, SIINFEKL incorporated Fc fusion protein was unable to activate B3Z T cells. Further investigation showed that BMDC seems to have a low expression of FcRn receptor, which is crucial for inducing an effective antigen cross presentation, and might be the reason for the unsuccessful antigen cross presentation assay. As a result, further investigations are required to determine if MAGE-A2 Fc fusion protein can be a potential cancer vaccine. More studies are also required to understand the characteristics of MAGE-A2 Fc fusion protein to minimise protein aggregations or protein degradations.
Date of Award2017
Original languageEnglish
SupervisorJennifer Woof (Supervisor) & David Meek (Supervisor)

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