Bifunctional Antibody Fragment-Based Fusion Proteins for the Targeted Elimination of Pathogenic T-Ce

Pathogenic effector T cells are key contributors to autoimmune diseases such as systemic lupus erythematosus (SLE). General inhibition of T cells using, e.g., methotrexate, prednisolone, or TNF blockers, has prominent therapeutic effects frequently at the cost of severe long-term side effects and toxicity. Therefore, targeted strategies that can selectively inhibit or eliminate pathogenic T cells are sought after as a new approach to safely block perpetual inflammatory T-cell responses and inhibit concomitant progressive tissue destruction. Of particular interest in this respect is the use of the so-called single-chain fragments of variable region (scFv) antibody fragments for the targeted reactivation of Fas-dependent activation-induced cell death (AICD). Recently, we demonstrated that a recombinant fusion protein comprising a T-cell-targeted anti-CD7 scFv antibody fragment genetically fused to soluble FasL (sFasL) can eliminate synovial fluid T cells in the absence of activity toward resting peripheral blood cells. Here, we describe a detailed protocol for construction and preclinical evaluation of such scFv:FasL fusion proteins that may be used to selectively eliminate pathogenic immune cells.