PB0078 - Study of new single domain of antibodies against Factor IX as diagnostic agents

Background: Single domain of antibodies (sdAb) or nanobodies are the smallest biological entities able to target specifically with a strong affinity their antigen. SdAb have several advantages; small size, robust behavior, easy production and engineering make them a very attractive tools for the diagnosis.
In hemostasis, some sdAb have been already characterized, mainly for treatment and research. But there are few nanobodies characterized for the diagnostic purposes, only against VWF, FVIIa and FXIIa and Fibrinogen.

Aims: Our objective is to develop new nanobodies against FIX(a) as new hemostasis tools. We characterized them on their specificity, affinity and potential impact on coagulation.

Methods: After production of 8 nanobodies in bacteria and purification on cobalt column, different ELISA were performed against FIX and FIXa and other antigens to ensure the specificity. The affinity for FIX and FIXa was measured on bio-layer interferometry, Octet® system. The potential FIXa inhibition activity by sdAb has been tested by chromogenic assay and Nanobodies' effect on coagulation has been evaluated using the aPTT (activated partial thromboplastin time) test with different reagents.

Results: Most of the nanobodies exhibit a great specificity against Factor IX and IXa, with various responses to the antigens. The affinity of nanobodies obtained from the octet system is really different between the sdAb ranging from 3nM to 350nM depending to the clone. None of the sdAb seems to have an impact in activity assay of FIXa but the F02 clone increases significantly the coagulation time independently of aPTT reagent.
Moreover, the F02 is the sdAb the most interesting for the diagnostic agent which present the best specificity and affinity.

Conclusion(s): We have obtained nanobodies against FIX and FIXa very specific better than antibodies and recognizing many species from FIX. The F02 clone is the best sdAb with the better affinity and it is very interesting for diagnostic agent