Antibody–Cytokine 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.
KeywordsFusion Protein Cell Culture Supernatant Eukaryotic Expression Vector Crystal Violet Solution Flag Peptide
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