Summary

The program Angiobodies 2.0 fits within the biomedical applications of biotechnology, specifically in the selection and engineering of recombinant antibodies (rAbs) with diagnostic and therapeutic potential against human diseases related to angiogenesis. The program Angiobodies 2.0 is a continuation of a previous program (Angiobodies; http://www.angiobodies.com/) funded under the 2006 call of Biosciences, Madrid. Compared to the first program, Angiobodies 2.0 focuses more on implementation of rAbs technologies developed in the context of the first program, such as trivalent form of rAbs (Trimerbodies), new bacterial display systems of rAbs in E. coli, lentiviral vectors for expression of rAbsa in the endothelium, or the systems of complete human IgG expression in cell culture in vitro. Angiobodies 2.0 starts with rAb candidates that have been selected during the previous program against molecular targets involved in angiogenesis (eg. Ephrin B2, p32/gC1qR, Calcineruina) and looks for their validation in vitro and in vivo, as well as their engineering to improve their potential in therapy and molecular diagnosis in vivo. Finally, Angiobodies 2.0 introduces a new goal to identify new "accessible" and "specific" molecular targets of pathological angiogenesis and inflammation, by selection of rAbs against pathological angiogenic vasculature using technologies, libraries of rAbs and in vivo animal models available within the program. Within this call for Biomedicine, Angiobodies program fits the priority area "therapeutic agents and carriers." Moreover, given the potential rABs as diagnostic tools in vivo, the project also complies with the priority area of "molecular diagnostics."

 

Strategic value and relevance of the scientific and technological research. Background.

Up to date monoclonal antibodies (MAbs), mainly from mice, have been an essential tool for research and diagnosis. However, the therapeutic and clinical value of MAbs have been restricted since they require “humanization” to diminish their immunogenicity.

rAbs have a higher biomedical potential since their technology make easier the generation of minimal fragments of human antibodies, and their genetic manipulation (affinity maturation and stability), production, conjugation and arming with effectors and toxins.

Further, rAbs can be reconstructed into complete Igs using appropriate vectors, and in novel multivalent and multi-specific fragments able to improve existing therapies and reagents for diagnosis and imaging.

Currently many rAbs are at various stages of clinical trial against inmunological, cardiovascular and tumor diseases . The technology of rAbs is also used to humanize and improve the affinity and specificity of new therapeutic MAbs.

 

Strategic Value

The strategic value of this project is enormous since the area of MAbs and rAbs has the greatest expansion and business growth in the biotecnological and pharmaceutical industry worldwide .

In 2004, the revenues from approved antibodies in clinic were 6 billion USD. Is estimated that rAbs will represent 30% of the whole biotech market in 2008. However this new market is unknown for Madrid-based companies mainly due to our low investment in R+D. Therefore, this project represent an opportunity for the birth in the Comunidad de Madrid of a network of research groups specialized in rAbs technologies for human therapy and diagnosis.

This network will nourish with state-of-the-art rAbs technologies the emerging biotech industry in Comunidad de Madrid, triggering the foundation and growth of biotech companies in our región and improving the transfer of research results to the clinic.

 

Recombinant antibodies, selection and biotechnological potential

Antibodies or immunoglobulins (Ig) play a central role in the immune system binding specifically to foreign molecules (antigens). The complex structure of natural Igs made unachievable their production in E. coli. This fact induced as an alternative the producción of small fragments that maintain intact their capacity to bind to the antigen. This was possible since antigen binding requires only the VH and VL domains from antibodies.

Several formats of rAbs have been developed. The single chain Fv (scFv) molecules are assembled by covalent attachment of the VH and VL domains by means of a flexible linker peptide. This configuration has a very small size(~30 kDa), allows the stoichiometric expression of both V domains, and maintains antigen binding and specificity. scFvs are the most frequent rAbs and had been obtained from mouse, rat, rabbit, and human Igs, having the latter the higher biotechnological potential for human therapy and diagnosis using imaging.

 

More recently even smaller functional rAbs have been developed (~15 kDa) made up of a single V domain V. The most common form of these single domain antibodies are obtained from a special kind of antibodies from camelids (camels, llamas, etc) lacking light chains, in such a way that an independent VH domain is responsible of binding to antigen. Camelbodies VHH (heavy chain only VH) have a very high similarity in sequence with human VH3 subfamily, allowing their use in human therapy and diagnosis. The advantages of VHH include a higher stability, solubility, and their tendency to recognize clefts and cavities in proteins, which makes them excellent inhibitors of enzymatic activities.

 

Libraries of scFv or VHH genes can be obtained from immunized animals with one or several antigens (immune libraries), or from non-immunized animals (naïve libraries). The standard technology for selection of rAbs with a given specificity is their display on the surface of filamentous bacteriophages (phage display). This technology is based on the physical association between a protein (phenotype) and its encoding gene (genotype) allowing the isolation of those rAbs in a library with a given antigen specificity through an immunopurification or “panning”. During panning the library of phages is incubated with the selected antigen and the bound phages are recovered and amplified in E. coli.

 

Thus a highly diverse population is transformed in a homogeneous population with a given binding specificity. Using phage display the isolation and development of new antibodies is faster and their ulterior manipulation is facilitated.

 

Angiogenesis-related pathologies

Angiogenesis is the process of formation of new blood vessels from pre-existing ones, and has a main role in different biological processes during embryonic development and postnatal life: reproduction, wound healing and inflammation. Although the molecular mechanisms responsible for the endothelial cell (EC) transition towards an angiogenic phenotype are not well known, the sequence of events leading to neo-vessel formation is well known . The aberrant growth of new blood vessels is common to a variety of pathological conditions: rheumatoid arthritis, diabetic retinopathy, tumor growth, and so on. Numerous experimental evidences suggest that progressive tumor growth depend on the constant recruitment of new blood vessels. Most tumors persist in situ in a quiescent state during long periods of time (months or years). The angiogenic switch affects the local balance between proand anti-angiogenic factors, and allows tumors to grow rapidly and to become clinically detectable. In the absence of supplementary vascularization tumor cells are subject to processes of necrosis and/or apoptosis, with the subsequent inhibition or limitation of tumor growth.

 

It has been also observed that the repeated administration of anti-angiogenic agents does not induce resistance, due to the fact that endothelial cells are genetically stable and their mutation rate is very low. On the contrary, resistance to chemotherapy protocols is a frequent problem due to the heterogeneity and genetic instability of neoplastic cells and their high rate of spontaneous mutations. Altogether, antiangiogenic therapies appear as a powerful alternative to conventional therapies that could substitute or complement current antitumoral therapies. Moreover, angiogenesis inhibitors could be applied to every type of solid tumor. Not surprisingly, in the last years great efforts have been devoted to the identification of agents able to inhibit this process. About 200 million patients could benefit of some type of antiangiogenic therapy; however, only 20 reagents of this kind are currently in clinical trials.