Abstract
Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.
Highlights
The coming of monoclonal antibodies into therapy has led to a real revolution
Based on previous reports showing immunoglobulin expression on exosomes purified from either cell line supernatant or body fluids[32,38,40,41], we preliminary evaluated the presence of exosomes in the supernatant of the OKT3 hybridoma cell line (IgG2a positive), or P3X63 (IgG2a negative) or human primary macrophages through nanoparticle tracking analysis (NTA)[49,50], confirming the presence of extracellular vesicles (EVs) with size within the typical range of nanovesicles/exosomes (Supplementary Figure S2)
To provide more specific information on EVs released by OKT3 hybridoma cell line, we characterised our samples for the expression of typical exosomal markers, (i.e. CD9, CD81 and Tsg101), using two different techniques: nanoscale flow cytometry for surface markers CD9 and CD81 and western blot analysis for the expression of cytosolic protein Tsg[101]
Summary
The coming of monoclonal antibodies into therapy has led to a real revolution Because of their high specificity and affinity for the target molecules, monoclonal antibodies have attracted the interest of the pharmaceutical industry, representing the fastestgrowing branch of therapeutic proteins and biotechnological research[1]. Many steps forward have been made so far, starting from the first in vitro production of monoclonal antibodies in mice[2], to the subsequent development of chimeric anti-human and humanised antibodies to improve half-life and reduce adverse effects[3,4]. Subsequent technological advances have led to the production of single chain fragment variable (scFv) antibodies[10], more advantageous than monoclonal antibodies in tumour therapy due to their small size, high affinity for the specific target, faster penetration of tissues and faster clearance[11,12]. Studies about the immunogenicity of mAbs have led to the development of in silico and in vitro instruments able to predict the functioning of the antibodies generated before their use in the clinic[13,14,15,16,17]
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