title: Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications.
abstract: Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells.
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain.
authors: Marta
authors: M
authors: Compte
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain.
authors: Ana
authors: A
authors: Alvarez-Cienfuegos
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain.
authors: Natalia
authors: N
authors: Nuñez-Prado
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain.
authors: Noelia
authors: N
authors: Sainz-Pastor
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain.
authors: Ana
authors: A
authors: Blanco-Toribio
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain.
authors: Nuria
authors: N
authors: Pescador
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain.
authors: Laura
authors: L
authors: Sanz
authors: Molecular Immunology Unit; Hospital Universitario Puerta de Hierro; Madrid, Spain ; BCE Protein Engineering; Department of Engineering; Aarhus University; Aarhus, Denmark.
authors: Luis
authors: L
authors: Alvarez-Vallina
keywords: bispecific antibody; diabody; gene therapy; in vivo secretion; tandem-scFv