Cloning and recombinant expression of the Immunoglobulin-like variable domains of humans PD-1 and PD-L1 immunoreceptors

Abstract

In cancer immunotherapy, the use of monoclonal antibodies (MAbs) that block the interaction of PD-1/PD-L1 immunoreceptors improves patient treatment. For MAb production, the availability of the protein antigen is a critical aspect. In this scenario, biomedical biotechnology allows the production of almost inexhaustible quantities of recombinant versions of these proteins. In this context, our objective was to clone and express the immunoglobulin-like variable domains (IgV) of PD-1 and PD-L1, because the interaction between them occurs among their IgV extracellular domains, specific primers were designed to clone them. Once cloned, they were individually ligated to pcDNA6 and introduced into Escherichia coli, and positive transformants were defined by PCR and sequencing. With the respective transformants, pilot expressions induced by 1 mM IPTG were performed, and the optimal time of expression was defined by electrophoresis in 15 % polyacrylamide gels. The cloning results were products of 375 bp for PD-1 and 357 bp for PD-L1, whose sequences showed 99% identity for PD-1 (GenBank OM363223) and 100% for PD-L1 (GenBank OM363224). Both proteins showed an optimal expression time of 4 h post-induction and ~14 kDa consistent with estimates. In conclusion, the cloning and recombinant expression of the IgV extracellular domains of PD-1 and PD-L1 was achieved, which will allow the production of a MAb or other immunotherapeutic tool against cancer, something that has not yet been achieved in Mexico.