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, three molecules [Fibroblast growth factor receptor 3 (FGFR3), ephrin B2 and gastrin] 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).

 

Our main target, FGFR3, belongs to the tyrosine-kinase receptor family, which plays a role in the translocation of signals for many cellular functionalities incluing embryo development, wound healing, hematopoiesis and angiogenesis. Deregulation of FGF signalling 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 1. Internalization of immunotoxin into RT112 visualized by confocal microscopy.

 

Also, the group is interested in the development of recombinant antibodies as delivery vehicles for agents such as radionuclides to target tissues, for radioimmunoimaging and radioimmunotherapy. To this end, antibodies must be engineered to select formats with optimized in vivo kinetics properties. Intact mAbs possess a long circulating half-lives resulting in a high background activity for extended periods of time, on the contrary, small fragments show high clearance rates despite of having higher tumor penetration and better tissue distribution. So, small to intermediate mAb fragments such as minibodies or small immunoproteins (scFvs fused to single constant domains of IgGs) should be the best candidates for imaging tumors. Our group is involved in the construction of optimized antibody fragments coupled to positron emitters for positron emission tomography (PET).

 

Figure 2. Schematic showing domain composition of engineered fragments

 

For gastrin project, different collections of gastrin-neutralizing antibodies (from human and murine libraries) have been prepared with the aim of identifying the sequences of the complementary determining regions (CDR) responsible for the binding to the gastrin. Once those sequences have been identified, the final goal is to synthesise binding bodies that mimic the CDRs for therapy of pancreatic cancer.