The novel multispecies Fc-specific Pseudomonas exotoxin A fusion protein α-Fc-ETA′ enables screening of antibodies for immunotoxin development

• A novel multispecies Fc-specific Pseudomonas exotoxin A fusion protein was generated.
• α-Fc-ETA′ enables screening of mouse and human antibodies for immunotoxin design.
• Promising antibody candidates can be identified early in drug development.

Immunoconjugates that deliver cytotoxic payloads to cancer cells represent a promising class of therapeutic agents which are intensively investigated in various clinical applications. Prerequisites for the generation of effective immunoconjugates are antibodies which efficiently deliver the respective cytotoxic payload. To facilitate the selection of human or mouse antibodies that display favorable characteristics as immunotoxins, we developed a novel Pseudomonas exotoxin A (ETA)-based screening protein. The α-Fc-ETA′ consists of a multispecies-specific Fc-binding domain antibody genetically fused to a truncated ETA version (ETA′). α-Fc-ETA′ non-covalently bound to human and mouse antibodies but did not form immune complexes with bovine immunoglobulins. In combination with antibodies harboring human or mouse Fc domains α-Fc-ETA′ inhibited proliferation of antigen-expressing tumor cells. The cytotoxic effects were strictly antibody dependent and were observed with low α-Fc-ETA′ concentrations. Mouse antibodies directed against CD7 and CD317/HM1.24 that previously had been used for the generation of functional recombinant immunotoxins, also showed activity in combination with α-Fc-ETA′ by inhibiting growth of antigen-positive myeloma and leukemia cell lines. In contrast, α-kappa-ETA′, a similarly designed human kappa light chain-specific fusion protein, was only specifically active in combination with antibodies containing a human kappa light chain. Thus, the novel α-Fc-ETA′ fusion protein is broadly applicable in screening antibodies and Fc-containing antibody derivatives from different species to select for candidates with favorable characteristics for immunotoxin development.