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Abstract 

Background 

Alzheimer’s disease (AD) is the major cause of dementia world-wide, and even though extensive efforts have been made to develop drugs altering disease progression, the only disease modifying FDA approved agent is the Aβ-targeting antibody known as Aducanumab. Development of drugs targeting AD is impeded by the blood brain barrier (BBB) which must be surmounted for the delivery of biopharmaceuticals into the brain parenchyma.

Aim

The current project intended to design, recombinantly express and characterise a BBB penetrating fusion protein construct referred to as DP196 targeting AD. The project also aims to evaluate a parent bispecific antibody R1/DP192, and its linker length variants R1/DP194 and R1/DP195 as a potential drug delivery platform for engineering biologics to enhance uptake into the brain.

Methods

The constructs were recombinantly expressed on EXPI293 cells, followed by purification, in-vitro characterisation involving SDS-PAGE analysis, transferrin receptor (TfR) ELISAs and thermal stability assay. Thioflavin T (ThT) fluorescence assay was used to assess effects of DP196 on Aβ-40 aggregation. Transcytosis of the parent construct R1/DP192 via TfR was evaluated in-vitro using BBB model followed by sandwich ELISA.

Results

All constructs were expressed in adequate yields and purified to acceptable levels of ≥ 80%estimated purity. SDS-PAGE analysis confirmed identity and inflection temperature measurements revealed stable nature of constructs. DP196 was found to completely block 10µM Aβ-40 aggregation at a 1:10 molar ratio of DP196 to Aβ-40. Assessment of transcytosis of R1/DP192 on an in-vitro BBB model using sandwich ELISA showed lower transcytosis compared to the positive control RmAb158-scFv8D3.

Conclusion

DP196 fusion protein was successfully produced and showed promising results as disease modifying agent in terms of inhibiting Aβ-40 aggregation. However, further evaluation is required for its’progress in terms of preclinical and clinical studies. The R1/DP192-linker lengths variants were also successfully produced and characterized; however, the parental antibody performance didn’t show encouraging results as a drug delivery platform.