Abstract 12280: Bispecific Anti-GPIIb/IIIa Single Chain Antibodies for Stem Cell Homing
Stem cell therapy has been studied and applied in various clinical fields, including that of myocardial repair and atherosclerosis. However, the efficiency of regenerative cell delivery to areas of injury is low and represents one of the major, unresolved problems in stem cell therapy. A recombinant fusion construct (scFv_GPIIb/IIIa-Sca-1) was generated that contains two major components: A single-chain antibody (scFv) against the activated GPIIb/IIIa-receptor on platelets and a scFv against stem cell antigen-1 (Sca-1) on murine hematopoietic progenitor cells. Furthermore, Sca-1 was cloned and transfected into HEK293 cells thereby generating a stable cell line to be used as a tool for demonstrating specificity of stem cell targeting. Also a control fusion construct was created by subcloning a mutated, non-functional scFv, creating scFv_control-Sca−1. The bispecific scFv were produced in drosophila S2 cells and purified via immobilized metal ion affinity chromatography. Flow cytometry showed specific binding to activated platelets for scFv_GPIIb/IIIa-Sca-1 but not for scFv_control-Sca−1. In a static adhesion assay cell adhesion was increased 6-fold when compared to scFv_control-Sca-1 (figure 1). Furthermore, scFv_GPIIb/IIIa-Sca-1 promoted a 4.8-fold increase of cell adhesion under shear stress in a flow chamber model (figure 2). Intravital microscopy of mouse mesenteric arteries using GFP-labelled bone marrow-derived Sca-1+ stem cells and ferric chloride induced vascular injury demonstrated successful homing of stem cells in the presence of scFv_GPIIb/IIIa-Sca-1 but not scFv_control-Sca−1. In conclusion, beginning from the design of a recombinant protein, the production and purification, the functional in vitro evaluation and finally the in vivo demonstration of successful homing, the bispecific scFvGPIIb/IIIa-Sca-1 allows specific homing of Sca-1+ progenitor cells and promises to overcome a major hurdle in stem cell therapy.
- © 2010 by American Heart Association, Inc.