Inhibition of CTLA-۴ receptor on the surface of T cells using a recombinant protein for cancer immunotherapy

Immune checkpoints are molecules with the inhibitory role to prevent an immune response to destroy healthy cells in the body. In cancer immunotherapy with blocking these inhibitory receptors, the immune system is activated and as a result the cancer cells are killed .One of the inhibitory receptors in the surface of T cells that is the target of many cancer immunotherapy methods is CTLA-۴. So far, many monoclonal antibodies have been produced to block CTLA-۴; by inhibiting this receptor, the T cell remains active. Due to the costly production of antibodies and the side effects reported in their usage, in this study we tried to design and produce a recombinant protein with high affinity for CTLA-۴ by using the natural ligands of this receptor.CD۸۰ is a transmembrane protein in the surface of B cells and monocytes and binds naturally to CTLA-۴. In this study an extracellular domain of this protein was extracted from PDB data bank and amino acids ۲۹, ۳۱ and ۳۳ were mutated by using R software. After calculating the binding energy of each mutant with foldX software, the best variant was selected and its gene was cloned in pET۲۲b vector. Finally, corresponding protein was expressed in E. coli. The results showed that the variants with point mutations R۲۹Y, Y۳۱R and Q۳۳K and the binding energy of -۲۱.۴۳ kcal/mol is the best protein for binding to CTLA-۴. Also, recombinant protein produced in E. coli was purified and subsequently its secondary structure was determined by CD spectroscopy. The results showed that produced protein has a secondary structure with acceptable similarity to native CD۸۰; so, it can be considered as a drug candidate for CTLA-۴ inhibition and cancer immunotherapy.

کلیدواژه ها:

CD۸۰ ، checkpoint inhibitors ، E. coli