Pro-domain-dependent folding and co-receptor-mediated targeting to optimize an antagonistic TGF-β monomer for gene-based delivery

Abstract Transforming growth factor-beta (TGF-β), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-β inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-β (mmTGF-β), which potently and specifically inhibits TGF-β activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-β was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-β pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-β. Furthermore, we show that fusion of mmTGF-β to a CD44 binding domain enhances the inhibitory potential of mmTGF-β on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-β for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.