Preparation And Biological Function Of Anti-PD-1/IL-15/IL-15Rα Bifunctional Fusion Protein

Background and PurposeAdoptive cell therapy has become an important choice in the treatment of advanced malignant tumors.However,how to efficiently expand tumor antigen-specific T lymphocytes from tumor tissue or peripheral blood remains unclear.Recent studies have shown that the proportion and quantity of tumor antigen-specific T lymphocytes contained in CD8⁺PD-1⁺T cell subsets in tumor tissue or peripheral blood were much higher than those in CD8⁺PD-1^-T lymphocytes.However,PD-1⁺T cells are in an exhausted state and their proliferation ability is weak,and these cells are at the disadvantage of proliferation by conventional culture.Therefore,it is the key to optimize adoptive cell therapy to recover the function of CD8⁺PD-1⁺antigen-specific T cells in vitro and achieve massive expansion of this subset.The purpose of this study is to construct a fusion protein anti-PD-1/IL-15/IL-15Rαwith dual functions of anti-PD-1 antibody and IL-15/IL-15Rα[named PD-S15,a fusion protein containing the PD-1 specific single chain antibody（scFv）,IL-15 and the sushi domain of its receptor IL-15Rα],and then to investigate the capacity of PD-S15 fusion protein to targeted bind to PD-1 and expand NK/T cells in vitro,which lays a foundation for the selective expansion of CD8⁺PD-1⁺antigen-specific T lymphocytes from tumor tissues and even peripheral blood.Methods1.The human anti-PD-1（scFv）gene sequence and human IL-15/IL-15Rαfusion gene sequence was synthesized and cloned into the pUC57,respectively.The recombinant expression plasmid pUC57-PD-S15 was constructed by enzyme digestion and ligation of the two target genes.Then the recombinant plasmid was identified by enzyme digestion and Sequencing technology.2.The pUC57-PD-S15 plasmid was transfected into HEK293T cells by Lipofectamine method.The supernatant of cell culture medium was acquired.The expression of PD-S15 fusion protein in cell culture supernatants was detected by Western blot technology.Under the same conditions,the supernatant of cell culture medium transfected with empty vector pUC57 was used as control.3.Peripheral blood mononuclear cells were isolated from 3 tumor patients.Under the same conditions,PBMC from the same donor was incubated with pUC57/X-VIVO^TM15 medium and PD-S15/X-VIVO^TM15 medium,respectively.Then the capacity of PD-S15 fusion protein to bind to PD-1 molecule in vitro was detected by Flow cytometry.4.Peripheral blood mononuclear cells were isolated from 3 tumor patients,and labeled CFSE.The effects of PD-S15/X-VIVO^TM15 medium mixed at different proportion volume（1:19,1:9,1:4,1:1）on the proliferation of PBMC were detected by Microscopy and Flow cytometry,and pUC57/X-VIVO^TM15 medium mixed at 1:1volume was used as control.In addition,the proportion of CD3⁺CD8⁺,CD3⁺CD4⁺and CD3^-CD56⁺（NK）were detected by Flow cytometry in optimal hole of proliferation phenomenon on the zeroth and fifth day of culture,respectively.5.Tumor infiltrating lymphocyte（TIL）were isolated from three different patients.The effects of classical TIL culture method and PD-S15/X-VIVO^TM15medium mixed at 1:1 volume on the proliferation of TIL were detected by Microscopy and Cytometry.6.The above 2⁵ experiments were repeated independently for 3 times.SPSS21.0 statistical software was used for data analysis.The metrological data were expressed asx±s.The two groups were compared by paired t-test.P<0.05 was considered as statistically significant difference.Results1.The recombinant plasmid pUC57-PD-S15 was successfully constructed by identification of double enzyme digestion and Sequencing technology,and then successfully transfected into HEK293T cells.The relative molecular weight of the target protein was about 55 kD,which was in line with the expectation.The corresponding protein bands were not detected in the supernatant of cell culture medium transfected with empty vector pUC57.2.The results of Flow cytometry showed that all the PD-1 sites on the surface of PBMC were blocked by PD-S15/X-VIVO^TM15 medium.On the contrary,pUC57/X-VIVO^TM15 medium could not block the PD-1 sites on the PBMC surface.3.The results of Microscopy and Flow cytometry showed that PD-S15 fusion protein could stimulate PBMC activation and proliferation in vitro（P<0.05）,in a concentration-dependent manner（1:19 vs 1:9,P<0.05;1:9 vs 1:4,P<0.01;1:4 vs1:1,P>0.05）and the higher the concentration of PD-S15,the stronger the proliferation ability of PBMC.On the fifth day,immunophenotype analysis showed that the percentage of CD3⁺CD8⁺T cells was higher than that of pre-culture,and the difference was statistically significant（P<0.05）;and there was no statistical difference in the proportion of CD3^-CD56⁺（NK）cells comparing with that of pre-culture（P>0.05）;and there was no statistical difference in the proportion of CD3⁺CD4⁺T cells comparing with that of pre-culture（P>0.05）.4.The results of Microscopy and Cytometry showed that the efficiency of activation and amplification of T lymphocytes by PD-S15 culture method was better than that of classical TIL culture method in vitro（P<0.01）.ConclusionsIn this study,a novel bifunctional fusion protein PD-S15 is successfully prepared.It is proved that PD-S15 fusion protein can targeted bind to PD-1 and rapidly expand NK/T cells in vitro,which lays a foundation for the selective expansion of CD8⁺PD-1⁺antigen-specific T lymphocytes from tumor tissues and even peripheral blood.