Our main area of interest is to provide new ways of producing human antibodies in vitro by using phage display technology. Over the last few years, our group has worked in several projects focused on developing tumour-specific recombinant antibody single-chain fragments (scFvs) for a variety of therapeutic and diagnostic applications. Initially, two molecules [Fibroblast growth factor receptor 3 (FGFR3) and ephrin B2] were selected as therapeutic targets in diverse tumors and several specific panels of scFv fragment were generated by using different phage display libraries, Tomlinson I+J (MRC) and Mehta libraries (Dana Farber Cancer Institute).

 

Figure 1. Diagram showing domain composition of engineered fragments

 

FGFR3 belongs to the tyrosine-kinase receptor family and plays a crucial role in the translocation of signals for many cellular functions including embryo development, wound healing, hematopoiesis and angiogenesis. Deregulation of FGF signaling leads to different syndromes related to the bone development, multiple myeloma and cervix, hepato- and bladder carcinomas. For FGFR3 project and their applications in bladder cancer therapy, we have obtained a collection of 6 different scFvs specific for FGFR3. After an extensive characterization of the antibodies, two of them showed a promising therapeutic potential since both were able to block cellular proliferation of FGFR3-overexpressing bladder carcinoma cells (RT112) in a dose- and ligand-dependent manner. In order to potentiate this inhibitory effect we decided to follow a new approach based on the conjugation of effector molecules as toxins, in collaboration with Dr. M. Rosenblum (MD Anderson Cancer Center, Houston). To this end, we have conjugated these antibodies to the plant toxin r-gelonin. The resulting immunotoxins have shown an enhanced inhibitory activity mediated by apoptosis in vitro and in vivo in a xenograft mouse model suggesting a therapeutic application.

 

Figure 2. Internalization of immunotoxin into RT112 visualized by confocal microscopy.

 

Also, the group is interested in other targets involved in angiogenesis such as ephrinB2. The ephrins and their receptors (Eph) have recently emerged as attractive therapeutic targets, since they are involved in critical pathways for the development and maturation of the blood and lymphatic vascular systems. In fact, we have demonstrated that the antibody-based blocking of ephrinB2 may represent an effective strategy for the development of antiangiogenic and antitumoural therapies. Two highly-specific human scFvs against ephrin-B2 were generated by a phage display approach and characterized in depth. These specific antibodies were able to suppress endothelial cell migration and tube formation in in vitro assays. In addition, systemic treatment of mice xenografted with pancreatic, lung or colon carcinoma cells resulted in a significant inhibition of tumor growths, accompanied by a drastic reduction in the number of blood and lymphatic vessels. In light of these results, those ephrinB2-specific scFvs may be considered as promising candidates to develop improved antiangiogenic therapies in cancer.

 

Figure 3. Inhibition of tumor growth in mice xenografted with colon carcinoma cells and treated with ephrinB2-specific scFvs