J Pharm Pharmacogn Res 2(Suppl. 1): S3, 2014
Special supplement with the abstract book of LATINFARMA 2013
CO 004: In Silico DESIGN AND FUNCTIONAL CHARACTERIZATION OF A TGFβ1 MUTEIN, ANTAGONISTIC OF TGFβ SIGNALING, WITH ANTITUMOR AND IMMUNOMODULATORS PROPERTIES
Corría Osorio J, Pérez Rodríguez S, Carmenate Portilla T, León Monzón K.System Biology Department. Center of Molecular Immunology, Havana, Cuba. Email: firstname.lastname@example.org
Introduction: Transforming growth factor β (TGFβ) is pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. There are three isoforms: TGFβ1, TGFβ2, and TGFβ3. They interact on the cell surface with the TβRII receptor, TβRIII receptor and several of the type I receptors that have been described (ALK5, ALK1, ALK2, ALK3); ALK5 is the clasic type I receptor of these ligands. It has been proposed that the ligands bind first to TβRIII and then this receptor transfers them to TβRII receptor. TGFβ1 and TGFβ3 can bind to TβRII receptor with high affinity (5-30 pM) whereas TGFβ2 has 1000 fold less affinity. ALK5 is recruited and activated in a highly cooperative manner by the TβRII/TGFβ complex, forming the heterotrimeric signaling complex. The ALK5 signaling plays a pivotal role in regulating the pathogenesis of a wide variety of disorders including cancer. Blockade of TGFβ signaling has been reported to be efficacious in the suppression of tumor growth and metastasis in animal models and clinical trials. In the present work we described for first time, a TGFβ1 mutein that behave as an antagonistic of the TGFβ signaling and has antitumor and immunemodulators effects in vitro.
Material and methods: This mutein was designed using several bioinformatics tool such as 3DCoffee and clustalX for the evolutionary analysis, the web server Protein Interaction Calculator was used to determine principals interactions in the interface, and Rosetta to determine hot spot residues and destabilizing mutations This mutein was fused to a human IgG1 Fc region with a set of mutations in the the Cγ2 domain that abrogates the binding with Fc gamma receptors except the neonatal. The fusion to Fc region was useful for expressing, for first time, the TGFβ domain without its latency associated propeptide (LAP) that is very important because the mutein can be obtained in its active form to interact with TβRII receptor. TGFβ1 mutein.Fc was obtained using a lentiviral transduction protocol in CHO-K1 cells. Evaluation of the antagonistic effects was done in IL-2– dependent CTLL-2 cell proliferation assay, TGFβ1-induced Treg differentiations assays and in vitro tumor cell migration assay.
Results: TGFβ1Mut_Fc had less biological activity than recombinant TGFβ1 and blocked TGFβ1-stimulated inhibition of IL-2–dependent CTLL-2 cell proliferation. Also, TGFβ1Mut_Fc inhibited the generation of foxp3 positive CD4+ T cells induced by wtTGFβ1. Finally, TGFβ1Mut_Fc inhibited tumor cell migration in vitro.
Conclusions: The TGFβ1 mutein.Fc behaves as an antagonistic of TGFβ signaling and that suggest that the destabilizing mutations predicted by using bioinformatics tool affect interaction between TGFβ1 and ALK5. These data provide a foundation to support using TGFβ1 mutein_Fc as a therapeutic agent for TGFβ-dependent cancer with metastatic and immuneregulatory capacity.