Recombinant antibodies against putative biomarkers for research and early diagnosis of Alzheimer´s Disease

GRIPPO V; BOZZO JI; RUSSO MJ; OBERHOLZER MV; SEBOLLELA A; ALLEGRI RF; JERUSALINSKY D

Simposio; LATBrain 2022 Estado actual y retos de las investigaciones en Latinoamérica de las enfermedades neurodegenerativas y epilepsia; 2022

LATBrain

Single-domain antibodies (Abs) derived from camelids (Nanobodies or VHH) represent an advantageous tool for new applications with unique characteristics compared to conventional monoclonal Abs: i.e., they are smaller, easier to produce, show higher stability to changes in temperature and chemical environments. In a fraction of camelid Abs the antigen-combining site is composed of a single variable region called VHH. Alzheimer´s disease (AD) is a progressive degenerative disorder of the nervous system, affecting more than 35 million people worldwide. Reproducible differences were found in plasma of AD patients compared to cognitively healthy older people, for proposed biomarkers such as Aβ42/Aβ40 ratio, tau, and NfL and also for RAGE, LRP-1, alpha2-macroglobulin (A2M), α-1-antitrypsin (A1A) and clusterin. Therefore, our goal is to obtain specific nanobodies (NanoAbs) against putative AD biomarkers (Aβ 1-42 peptide, particularly oligomeric forms, A2M and A1AT in this preliminary phase), as new tools for research and diagnosis of AD. For this purpose, VHH immune libraries were constructed from the blood of two llamas (Lama glama) immunized with each antigen. The "display" of VHH on the surface of phages enables the selection of specific NanoAbs against each Ag. The selected NanoAbs are expressed as soluble proteins in bacteria and then purified for characterization of their reactivity against putative biomarkers. For Aβ 1-42 peptide, a single change variable fragment (scFv) NUsc1-Ab specific for oligomeric forms-will also be used. The obtained nanoAbs and this scFv, would constitute a panel of recombinant Abs to perform determinations in plasma and CSF of patients (from FLENI) in the near future, with the objective of developing an immunoassay for early diagnosis of AD.The superior biological properties exhibited by nanoAbs make them a great tool for investigation and diagnosis; hence, nanoAbs against relevant biomarkers in AD could be successfully used in an ELISA-type diagnostic immunoassay.