Formulation development of a recombinant VAR2-antiCD3 protein for cancer immunotherapy

Abstract: The use of immunotherapy for treating cancer has received a lot of attention in the recent years. The therapy is based on activating or deactivating the patient’s own immune system to help fight the disease. One way of doing this, is using bispecific molecules that can target both cancer cells and T-cells simultaneously. In 2003, a researcher found that the surface protein VAR2, expressed on malaria infected erythrocytes, could also specifically bind cancer cells. An idea came to mind to combine this protein with a scFv of the antibody aCD3, for immunotherapy targeting cancer. This rVAR2-aCD3 fusion protein was the start of the company VAR2 Pharmaceuticals. Today, the product has been developed and the company is hoping to start clinical trials in the near future. The aim of this master thesis was to select the best candidate for a frozen-liquid rVAR2-aCD3 buffer formulation to be used in clinical phase I studies. The method included 10 analytical techniques measuring different properties correlated to stability. SE-HPLC and SDS-PAGE were used to examine aggregation, degradation and contamination. Size distribution was studied using DLS and SEC-MALS. Furthermore, SEC-MALS and MS was used to determine the molar mass. Protein concentration was measured in NanoDrop and unfolding correlated to temperature was examined in NanoDSF. ELISA and FACS were used to investigate binding. Lastly, a fractionation of monomer and dimer was performed using NGC. At the start of the study, the stability effect on the protein of six different buffers with pH ranging from 3-8, stored in -80°C versus 4°C respectively, were examined. A final formulation was systematically down selected through three consecutive sub studies, investigating pH dependence, tonicity and physical stress. In the initial study it was found that low pH (3.3-4.5) seemed to destabilize the protein. Using NanoDSF, it was also found that the unfolding process related to increased temperature seemed to be pH dependent. Buffers with a pH <7 tended to unfold in two steps whereas buffers with a pH >7 unfolded in one single step. From the tonicity study, it was found that both sucrose and NaCl could work as tonicity providers to the fusion protein. However, the combination of histidine buffer and sucrose seemed to have negative effects on the desalting process. Also, it was seen that the samples stored in -80°C were more stable than the samples stored in 4°C. The formulation with PBS, tris-HCl with sucrose and histidine buffer with NaCl showed to be stable all through the 5 weeks of which the tonicity sub-study was performed. In the final stress study, it was found that the protein was stable towards freezing and thawing up to 6 times. Furthermore, storage at room temperature and rotation caused aggregation and degradation which was found in SDS-PAGE and SE-HPLC measurements. The histidine buffer with NaCl seemed to be most stress resistant with the least aggregation and degradation products forming, keeping the highest monomer content over time and showing the highest binding towards the desired ligand decorin. Hence, histidine buffer with 150mM NaCl was chosen as the best candidate for a frozen-liquid rVAR2-aCD3 buffer formulation to be used in clinical phase I studies.