P067 Binding of infliximab (IFX) to human serum exosomes.
B. Hernández-Breijo1, M. Chaparro*2, A. Paradela3, I.D. Román1, D. Cano-Martínez1, P. Sanmartín-Salinas1, J.P. Gisbert2, L. Guijarro1
1Universidad de Alcalá and CIBERehd, Systems Biology, Alcalá de Henares, Spain, 2Hospital Universitario de La Princesa, IIS-IP and CIBERehd, Gastroenterology Unit, Madrid, Spain, 3Centro Nacional de Biotecnología, Proteomic service, Madrid, Spain
The loss of response to infliximab has been associated with the presence of antidrug antibodies (ATI). However, other protein complexes in blood could bind this drug blocking its effect. Several studies have suggested that the exosomes (30-100 nm) present in plasma could bind biological drugs decreasing their bioavailability.
Aims: To analyze the molecules that bind IFX. To assess the interaction of IFX with exoxomes. To know the composition of exoxomes by proteomic methods
Human sera from healthy volunteers (n=3) were analized. The samples were incubated with IFX-alexa 488 and the mixture was injected onto SE-HPLC column. Exosomes were isolated from human serum by Size Exclusion-HPLC using a Yarra3000 column from Phenomenex, USA. The void volume from the chromatographic profile was submitted to ultracentrifugation (160,000 g at 4ºC for 90 min). The pellet was analysed by western blot and using a Nano LC ESI-MSMS shotgun proteomics approach. In the last method was used an Eksigent 1D- nano HPLC coupled via a nanospray source to a 5600TripleTOF QTOF mass spectrometer (ABSciex, Framinghan, MA, USA).
We observed IFX-alexa 488 bound to protein complexes that eluted in the void volume, indicating a diameter (greater than 30 nm) consistent with the size of exosomes. After ultracentrifugation of this last fraction, the pellet was characterized by western blot against CD63 (a classical marker of exosomes). Another part of the sample was used to identify by proteomic Methods, other molecules that co-eluted with IFX-alexa 488. Interestingly, we observed new proteins in the exosomes, as polymeric immunoglobulin receptor (PIGR), fibulin-1, utrophin, complement factor H, dermcidin, immunoglobulin J chain, among others. As predictable, we also identified proteins previously described in exosomes such as keratins, Ig alpha-2 chain C region, Ig gamma-4 chain C region, or thrombospondin-1. At present, we have immunoprecipitated the protein complexes from exosomes using IFX aiming to define news targets of this therapeutic antibody.
Our study demonstrated that IFX was able to bind to human serum exosomes, which were characterized by immunological and proteomic Methods. That should be taken into account when measuring antibodies to IFX as they could be wrongly considered ATI. The study of the structure and the functions of these exosomes could be important to evaluate the bioavailability and efficacy of IFX in patients with inflammatory bowel disease