Development of bispecific antibody mimetics

Chronic neovascular retinal diseases can be treated by antibody-based medicines which are administered by intravitreal injection Ongoing efforts are focused on improving treatment outcomes for patients taking these medicines. One strategy to improve treatments is the development of bispecific antibodies which are capable of binding to 2 intraocular therapeutic targets simultaneously. Bispecific antibodies are suggested as an alternative to the use of combination therapies within the eye. Many different bispecific antibody motifs have been prepared including motifs derived from antibody fragments such as Fabs and single chain variable fragments. Previously our research group developed a monospecific antibody motif called a Fab-PEG-Fab (FpF) in which 2 identical Fabs are conjugated to either end of a PEG di bis-sulfone protein dimerisation reagent 5 mimicking an IgG antibody. It was thought to try and develop the FpF platform further by preparing bispecific FpFs (BsFpF). The aim of this PhD thesis was to synthesise BsFpFs capable of binding to two therapeutic targets within the eye. To prepare BsFpFs, it is required to digest IgGs using the proteolytic enzyme papain to obtain Fabs. Previously an immobilised form of papain was used to digest IgGs with the methodology being laborious and slow. A new method using a soluble form of papain was developed, which allows the digestion of up to 100 mg of IgG within 30 minutes and with a yield in excess of 50%. Purification of the digestion mixtures to obtain highly purified Fabs was achieved using Protein L chromatography. The developed digestion method using soluble papain was published in literature during this PhD – Collins M, Khalili H. Soluble Papain to Digest Monoclonal Antibodies; Time and Cost-Effective Method to Obtain Fab Fragment. Bioengineering (Basel). 2022 May 12;9(5):209. doi: 10.3390/bioengineering9050209. PMID: 35621487; PMCID: PMC9137653. To synthesise BsFpFs, different chemistry methods were used. A conjugation-ligation strategy in which two different Fabs are conjugated to di-functional reagents was explored. Two di-functional reagents PEG bis-sulfone transcyclooctene (TCO) and PEG bis-sulfone tetrazine (Tz) were prepared by our research group. The bis-sulfone group enabled conjugation to Fabs to proceed, forming TCO and Tz functionalised intermediates. The free TCO and Tz moieties ligate, allowing the TCO and Tz functionalised intermediates to combine into a single molecule, a BsFpF. Key to the preparation of BsFpFs via conjugation-ligation was the removal of any free PEG bis-sulfone TCO and PEG bis-sulfone Tz reagents prior to ligation of the TCO and Tz functionalised intermediates. 14 different BsFpFs were prepared via conjugation-ligation. It was possible to obtain highly purified BsFpFs using ion exchange (IEX) and size exclusion chromatographies (SEC) with purity being confirmed via silver staining. Isolated yields of BsFpFs were between 10-15% with yield being limited by the purity of reagents 26 and 27. BsFpFs were stored at their intended storage temperature of 5°C for 6 months with no evidence of deconjugation or aggregation being observed. Ligand binding studies performed using enzyme linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) were able to confirm that BsFpFs were capable of binding to both of their intended targets, with SPR confirming binding in a concentration dependent manner. ELISA demonstrated that an anti-VEGF/IL-6R BsFpF had a similar affinity towards vascular endothelial growth factor (VEGF) and the interleukin-6 receptor (IL-6R) as anti-VEGF and anti-IL-6R TCO and Tz functionalised Fab conjugates. This indicated that the VEGF binding arm of the anti-VEGF/IL-6R BsFpF did not interfere during binding of the BsFpF to IL-6R and vice versa. During SPR binding experiments the same finding was found during VEGF binding assays, however the same was not found when binding to IL-6R. The data presented in this thesis demonstrates that it is possible to reproducibly prepare and purify BsFpFs targeting two intraocular targets simultaneously. Future work would focus on the assessment of the functional activity of BsFpFs using in-vitro cell-based assays. Preliminary work showed that human umbilical vascular endothelial cells (HUVECs) may be a suitable choice of cell model for these assays.