Exploring And Expanding Of The Covalent Binding Of PD-L1/PD-L2 With PD-1

BackgroundSo far,nearly 30%of targeted drugs on the market through covalent mechanisms play a role,but most are small molecules covalent drugs,the key in the development of small molecules covalent drugs is to a proper balance between the reactivity and selectivity,due to lack of selectivity,it often have off-target reactivity and high toxicity,which greatly limits its application value.Proteins have larger size and higher specificity than small molecules,but natural proteins lack the covalent binding mechanism with target proteins.The unnatural amino acid FSY with biological activity was inserted into the natural protein to form irreversible covalent binding with the target through SuFEx.In this paper,we prepared FSY modified pd-11(FSY),which is covalently cross-linked with pd-1 and has the potential to covalently activate the pd-1 immunosuppressive signaling pathway for the treatment of autoimmune diseases.In addition,FSY-modified pd-1(FSY)was combined with 4-1bbl,T7 peptide and cell-penetrating peptides to form fusion recombinant proteins,which can form covalent cross-linking with pd-11 and have the potential to play the role of fusion terminal proteins while blocking the pd-11/pd-1 signaling pathway,so as to enhance the anti-tumor effect of pd-1(FSY).Covalent protein drugs that are modified by unnatural amino acids have a promising prospect in the treatment of diseases.Objective1.FSY will be incorporated into PD-L1 and PD-L2,respectively,so that the resultant mutant proteins can covalently bind with PD-1 to develop potential treatment for autoimmune diseases.2.PD-1(FSY)mutants with covalent binding ability will be fused with other proteins or peptides,so as to not only exert the immunosuppressive function of PD-1(FSY),but also exploit the function of the fused protein/peptide,achieving effects of bifunctional protein drug with covalent binding ability,for enhance the efficacy of tumor therapy and expand its application scope.Methods1.Analyzed the crystal structure of the ligand-receptor complex and related literature to select the incorporation site.2.Literature review was conducted to select the possible proteins that could promote the blocking function of PD-1,and linkers were selected.3.PCR amplified the target sequence and ligated in vector PET26b.4.Expressed proteins in E.coli BL21(DE3)overnight and purified with Ni-NTA column;5.Renatured the purified proteins using serial dilatation in buffers containing 8 M,6 M,4 M,2 M,and 0 M urea for 4-6 h each and stirring at 4 ℃.Proteins were concentrated with ultrafiltration tube.6.Incubated PD-L1/PD-L2(FSY)protein and PD-1 WT protein in 50 mM Tris,200 mM NaCl,pH=8.0 buffer at 37℃ for 5 h to cross-link.7.Analyzed the protein samples with SDS-PAGE and Western Blot to detect the expression,purification and covalent cross-linking efficiency.Results1.SDS-PAGE and Western Blot results showed that among 20 different PD-L1(FSY)proteins,PD-L1(Y56FSY)formed covalent cross-linking with PD-1,and the cross-linking efficiency was 16.4%(0.16±0.02,P<0.0001)of that of the positive control.2.Among 5 different PD-L2(FSY)proteins,PD-L2(W110FSY)formed weak covalent cross-linking with PD-1,and the cross-linking efficiency was 7.2%(0.07±0.01,P<0.0001)of that of the positive control.3.The covalent cross-linking efficiency between PD-1(FSY)-(G4S)4G-4-lbbl fusion protein with PD-L1 was lower than that of the positive control,reaching 45.7%(0.46±0.04,P<0.01)of the latter,and the covalent cross-linking efficiencyPD-1(FSY)-(G4S)4G-T7 peptide reaching 49%(0.49±0.03,P<0.001)of the positive control.The two fusion proteins were unstable after refolding.In the fusion form of PD-1(FSY)-(G4S)2-membrane penetrating peptide,the covalent cross-linking efficiency of PD-1(FSY)-(G4S)2-p28 and P28-(G4S)2-PD-1(FSY)protein with PD-L1 protein was 97%(0.97±0.04,P>0.05)and 96.2%(0.96±0.02,0.05>P>0.01)of that of the positive control,and the protein stability and solubility were improved.Conclusion1.FSY incorporation into PD-L1 and PD-L2 had an impact on the expression level,purity and refolding of the protein,and no significant covalent cross-linking had been formed at all(FSY)sites,which were insufficient for subsequent testing.It is thus necessary to screen more sites to identify the appropriate FSY insertion site.2.Different fusion segments had different effects on the degree of covalent cross-linking of PD-1(FSY),the solubility of fusion protein,and the refolding of fusion protein.Steric hindrance effect could affect the covalent binding efficiency of FSY.The biological function of PD-1(FSY)-p28 fusion protein needs further test.