• antibody
• cancer
• mass-spectrometry
• proteomics
• targeted therapy

The experimental activities have been performed in the Philochem laboratories.
Philochem is a fully owned daughter company of Philogen and represents the Discovery Unit of the Philogen group.
In this thesis project we set up a mass spectrometry-based (MS) method that allows us to quantify the abundance of antibody-based therapeutics in tumor and healthy tissues after administration in a mouse model. These in vivo biodistribution experiments are necessary to evaluate the selective accumulation of our molecules of interest at the tumor site. This targeted tumor delivery should prevent their off-target effects and related toxicity. In the company, these experiments are currently performed using radiolabelled surrogates, however this procedure does require a stable labelled antibody and the expensive use of facilities that allow to handle mice injected with radioactive compounds.
The aim of the thesis was to achieve MS-based quantification results as accurate as radioactivity-based ones. This non-radioactive analytical method will enable company’s scientists to screen for different antibody-based products without, or with a reduced use of radioactivity. As starting model for our analysis, we have chosen full immunoglobulins G (IgGs) and IgG fragments such as the tandem diabody format.
The goal of our experimental workflow was to extract proteins from tissue samples using mild lysis conditions to avoid conformational changes in the analyte (our IgG or diabody of interest). Later, an enrichment step was performed using Protein A (Staphylococcus aureus). Finally, samples were digested with trypsin and resulting peptides analysed by mass spectrometry. To compensate variability introduced by the sample preparation, we decided to add an internal standard (IS). As IS we decided to use a different biotherapeutic in the same format and with low sequence homology to our target protein. In specific, we used a monoclonal antibody carrying a kappa light chain as IS for an analyte with lambda light chain and vice versa.
To optimize our methodology, we firstly performed experiments in which we “spikedin” different amounts of our antibodies in “blank” tissues to assess if the signal detected by the MS was proportional to the amount of protein injected. The results of these experiments showed good linearity between injected analytes and measured signals for all antibody-formats that were tested. For the tandem diabody format, the MS signal detected was lower when compared to the IgGs signal. This was probably due to the fact that the affinity of the tandem diabody for protein A is lower than the affinity of an IgG. For this reason, we also started investigating alternative enrichment protocols.
Then, encouraged by these positive results we moved to in-vivo experiments. To validate our MS measurements and to benchmark our results with radioactivity-based quantifications, we choose well characterized antibody (L19) of our company for which radioactive data were available. The antibody biodistributions were measured at two different time point (24h and 48h after IgG administration) and three mice per each time point were analysed. The biodistribution data that we obtain showed a selective targeting at the tumor site of the antibody. These results were further validated by comparing our MS biodistribution data with radioactive data.