Production and analysis of novel disulfide stabilised scFv-containing antibody formats

Abstract

Antibody engineering enables the production of antibody-based molecules with modified properties, and is a key tool in the development of modern therapeutics like monoclonal antibodies. Full-length IgG molecules have favourable half-lives and effector functions, however their large size limits tissue penetration. In contrast, single-chain variable fragments (scFv) are smaller molecules formed solely by the antigen-binding domains of the antibody joined by a peptide linker. ScFv molecules have more efficient tissue penetration but tend to be unstable, have short half-lives and altered effector functions. This limitation can be addressed by linking scFv fragments to an Fc region, creating scFv-Fc fusion proteins, which retain similar characteristics to full-length IgGs such as longer half-lives, solubility, bivalent binding, and effector functions. Further engineering and stabilisation of scFv-Fc fusion proteins could yield new therapeutics that combine the favourable properties of full-length IgGs with enhanced tissue penetration and efficacy. Previous research developed ultra-stable scFv fragments (ds-scFv) derived from trastuzumab by introducing a disulfide bridge in a novel position. Trastuzumab is a humanised IgG1 monoclonal antibody targeting the HER2 domain on tumour cells. This is a proof-of-concept study which aims to produce and analyse the properties of two novel ds-scFv-containing trastuzumab antibody formats: (1) a bivalent construct (dsscFv-Fc), and (2) a tetravalent construct (dsscFv-Fc-dsscFv). Both constructs were expressed in mammalian cells, characterised and assessed for stability, binding and efficacy. Results demonstrated increased thermostability in both constructs, compared to their non-disulfide stabilised analogue. Furthermore, showed to be high-affinity binders with a binding profile similar to trastuzumab. Lastly, the cell-based assays confirmed effective inhibition of proliferation in HER2 over-expressing tumour cells. These promising results encourage continued research into disulfide-stabilised scFv-Fc antibody formats for biomedical applications.