Toward the epitope mapping of a single chain antibody fragment against the CD133 receptor


Puig Freixa, Martí


Glioblastoma multiforme (GBM) is the most malignant primary brain tumour and is currently incurable. One of the main obstacles for the treatment of GBM is the presence of cancer stem cells (CSCs), which have chemoresistance and the capacity to induce tumour recurrence. Therapies that aim to eliminate CSCs face many challenges, such as the need to cross the BBB and to target these cells selectively. The main problem in targeting therapies to CSC is that receptors overexpressed in these cells are also present in healthy stem cells. Aiming to meet this challenge, we are working toward the generation of a conditionally-activated antibody binding CD133, the most well studied extracellular marker on CSCs.
Protease-sensitive masked antibodies are one of the most selective approaches to render antibodies activatable since proteases are overexpressed in the tumour microenvironment compared to healthy tissues. One of the most commonly used strategy in protease-sensitive antibodies relies on the use of an N-terminal extension involving a protease-labile sequence followed by an epitope or epitope-mimetic peptide that will block the antibody binding site.
In this thesis we aim to identify the epitope of an antibody derivative against CD133 to enable the generation of a conditionally-activated scFv targeting CD133. This work is structured in two parts: First, the production of CD133 extracellular domains and second, the epitope mapping of the selected antibody derivative was pursued. In the first part, for the production of the ECDs constructs, cloning, transformation and expression were performed, as well as the characterization of antibody binding via western blot. In the second part, a protocol for antigen tryptic digestion was developed and, finally, epitope extraction and excision mapping approaches were applied to identify the epitope.



Oller-Salvia, Benjamí
Lecina Veciana, Martí


IQS SE - Undergraduate Program in Biotechnology