LC-ESI-MS/MS on a purified recombinant protein sample to confirm its amino acid sequence and a certain cleavage site

The study entails the design, production and in vitro testing of a novel recombinant anti-B7-H3 affibody coupled with a small cytotoxic protein, with considerable potential in Acute Myeloid Leukemia (AML) immunotherapy. The protein construct was expressed in E. coli. After affinity purification and validation of its sequence through LC-MS/MS, the obtained protein’s efficacy was tested on AML representative B7-H3-positive THP-1 cells and B7-H3-negative RAJI cells. The IC50 of the cytotoxic protein was determined through MTS assay and its apoptotic and necrotic activities on the two cell lines were evaluated through flow cytometry and Western blot. Taken together, the results show that the cytotoxic anti-B7-H3 affibody we produced possesses highly necrotic and apoptotic targeted effects that are specific to only B7-H3-expressing THP-1 cells and not the RAJI control line. Moreover, these observed effects are significantly more potent than the effects of the cytotoxin alone, as they are described in previous studies. In conclusion, we designed, produced and evaluated a novel anti-B7-H3 cytotoxic affibody that from these preliminary in vitro data, shows high potential for translation to the immunotherapy of AML.