Generation and characterization of monospecific and bispecific hexavalent trimerbodies.

Here, we describe a new class of multivalent and multispecific antibody-based reagents for therapy. The molecules, termed "trimerbodies," use a modified version of the N-terminal trimerization region of human collagen XVIII noncollagenous 1 domain flanked by two flexible linkers as trimerizing scaffold. By fusing single-chain variable fragments (scFv) with the same or different specificity to both N- and C-terminus of the trimerizing scaffold domain, we produced monospecific or bispecific hexavalent molecules that were efficiently secreted as soluble proteins by transfected mammalian cells. A bispecific anti-laminin x anti-CD3 N-/C-trimerbody was found to be trimeric in solution, very efficient at recognizing purified plastic-immobilized laminin and CD3 expressed at the surface of T cells, and remarkably stable in human serum. The bispecificity was further demonstrated in T cell activation studies. In the presence of laminin-rich substrate, the bispecific anti-laminin x anti-CD3 N-/C-trimerbody stimulated a high percentage of human T cells to express surface activation markers. These results suggest that the trimerbody platform offers promising opportunities for the development of the next-generation therapeutic antibodies, i.e., multivalent and bispecific molecules with a format optimized for the desired pharmacokinetics and adapted to the pathological context.

The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype.

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, ß1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling.

Engineered human tumor xenografts with functional human vascular networks.

Animal models of human tumor angiogenesis would have multiple applications. However, they are extremely difficult to standardize. In this work, we tried to generate human tumor xenografts containing a human vascular bed in immunodeficient mice by subcutaneous co-implantation of matrigel-embedded human endothelial cells (EC), human mesenchymal stem cells (MSC) as a source of mural cells and HT1080 human fibrosarcoma cells. Unfortunately, in this context human EC were rapidly substituted by their murine counterparts, and by day 16 post-implantation human CD34 positive cells were hardly detectable in intratumoral vessels. In an attempt to inhibit host EC colonization of human xenografts and promote human EC grafting, we investigated the effect of radiation prior to implantation on the vascularization and growth of tumor xenografts. Nude mice underwent either localized (implantation area) or sublethal whole-body exposure to radiation. Localized radiation inhibited both human and murine neovascularization, and even the tumor growth rate was remarkably decreased when compared to control unirradiated mice and sublethally whole-body irradiated mice. Interestingly, numerous human vessels were detectable in sublethally irradiated mice at day 30, with murine EC only over passing human EC when spontaneous hematopoietic reconstitution has taken place. This observation strongly suggests the implication of bone marrow-derived murine endothelial precursors in tumor neovascularization. In summary, we have established a model of human tumor neovascularization that is amenable to both the study of molecular aspects in the angiogenic process and the evaluation of potential new antiangiogenic drugs.

Long-term in vivo imaging of human angiogenesis: critical role of bone marrow-derived mesenchymal stem cells for the generation of durable blood vessels.

Angiogenesis is a multistep process that encompasses complex molecular and cellular interactions that can not be recapitulated in vitro. Here, we demonstrate that vasculature generated from lentivirally transduced human primary endothelial cells expressing firefly luciferase and co-implanted with human bone marrow mesenchymal stem cells in immunodeficient mice can be assessed quantitatively by in vivo whole body bioluminescence imaging for more than 120 days. Luciferase activity correlated with the formation of a network of functional, mature blood vessels of human nature inside the implant that critically depend on the presence of mesenchymal stem cells. In summary, our study offers an unprecedented opportunity to perform long-term serial analysis of the molecular events involved in the angiogenic process and monitoring responses to anti-angiogenic agents.