Engineering and Characterization of Recombinant Chimeric Dimeric and Trimeric Proteins of Murine GITRL Fused to the Fc Region of Human Immunoglobulin

Abstract

Background & Aim: Tumor necrosis factor (TNF) superfamily (TNFSF) comprises different ligands and receptors with pivotal roles in cell proliferation, differentiation, survival, and apoptosis. TNF Ligands are produced as functional trimeric-transmembrane and soluble proteins, with the exception of the dimeric murine glucocorticoid-induced TNF receptor ligand (mGITRL). GITR-GITRL interaction contributes to controlling the immune response and holds the potential for being a promising anti-cancer and autoimmune disease immunotherapy. Production of dimeric and trimeric recombinant mGITRL is a key step towards preclinical investigations of the possible therapeutic characteristics of GITRL. Aim: Production, characterization and evaluation of dimeric versus trimeric recombinant GITRL proteins, based on binding to GITR and T cell activation. Methods: Two chimeric recombinant proteins were designed and produced in the Chinese Hamster Ovary (CHO) cell line. The dimer protein encompassed the ectodomain of mGITRL fused to the fragment crystallizable region (Fc region) of human immun oglobulin hIgG1, whereas trimeric ligand was produced via co-expression of [hFc.mGITRL] and [mGITRL.HA], and selection of high co-expressing positive clones. Ligands binding to mGITR expressed in human Jurkat cells was assessed using flow cytometry and co-immunoprecipitation assays, and functionality was evaluated by means of T cell proliferation. Results: A dimeric (hFc. mGITRL)2 and trimeric (hFc. mGITR)2:mGITRL.HA proteins were produced, and highly expressing clones were selected and expanded. Both proteins were capable of binding to mGITR and activating naïve T lymphocytes CD4+CD25- to proliferate, with superiority of the trimer. Conclusions: Our findings prove comparable functionality of murine dimeric and trimeric ligands, and establish for the dimeric Fc fusion GITRL production and purification for in vivo testing to investigate the potential of GITRL as tumor immunotherapy.