Generation of single-chain bispecific green fluorescent protein fusion antibodies for imaging of antibody-induced T cell synapses

• Published in: Analytical biochemistry 2012-04, Vol.423 (2), p.261-268
• Main Authors: Stamova, Slava, Feldmann, Anja, Cartellieri, Marc, Arndt, Claudia, Koristka, Stefanie, Apel, Falko, Wehner, Rebekka, Schmitz, Marc, Bornhäuser, Martin, von Bonin, Malte, Ehninger, Gerhard, Bartsch, Holger, Bachmann, Michael
• Format: Article
• Language: English
• Subjects: Antibodies, Bispecific - genetics; Antibodies, Bispecific - immunology; Antibodies, Bispecific - metabolism; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - metabolism; Antigens, CD - immunology; Antigens, Differentiation, Myelomonocytic - immunology; Bispecific antibodies; CD3 Complex - immunology; CD33; Cell Line, Tumor; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Immunological Synapses - metabolism; Immunological Synapses - pathology; La antigen; Microscopy, Confocal; Prostate-Specific Antigen - immunology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Retargeting; Sialic Acid Binding Ig-like Lectin 3; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; Tumor immunology
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2011.12.042

There is growing interest in the development of novel single-chain bispecific antibodies for retargeting of immune effector T cells to tumor cells. Until today, functional fusion constructs consisting of a single-chain bispecific antibody and a fluorescent protein were not reported. Such molecules could be useful for an in vivo visualization of this retargeting process. Recently, we established two novel single-chain bispecific antibodies. One is capable of retargeting T cells to CD33, and the other is capable of retargeting T cells to the prostate stem cell antigen (PSCA). CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). The PSCA is a potential target on prostate cancer cells. Flanking the reading frame encoding the green fluorescent protein (GFP) with a recently described novel helical linker element allowed us to establish novel single-chain bispecific fusion antibodies. These fluorescent fusion antibodies were useful to efficiently retarget T cells to the respective tumor cells and visualize the formation of immune synapses between effector and target cells.