AntibodyCytokine Fusion Proteins with Members of the TNF-Family

The generation of antibody fusion proteins for target-directed delivery of cytokines constitutes a promising immunotherapeutic approach. The fusion of recombinant antibodies to members of the TNF-family that differ clearly in their soluble and membrane-bound forms for their receptor activating capacities is therefore of special interest because binding of the antibody moiety implies the presentation of the cytokine in a membrane-associated form, affecting directly the cytokine activity. Here we describe the generation and characterization of two of these antibody–cytokine fusion proteins composed of a tumor specific scFv and the extracellular domain of CD95L (scFv-CD95L) or CD137L (scFv-CD137L), respectively. Protocols are indicated for cloning, expression in mammalian cells, purification, and functional characterization in terms of costimulatory and cytotoxicity properties.