Design And Optimization Of High Affinity Anatgonists Against PD-1 Receptor

The Fc fusion protein is a novel structural protein,which is fused by Fc fragment of human antibody as a scaffold and exhibits the structural domains of biologically active polypeptides or functional proteins through conjugated peptides.Its structure,function,expression system,purification process and pharmacological research are all similar to those of antibody drugs,therefore they are considered as broad-based antibody drugs.At present,11 Fc fusion protein drugs have been approved by the FDA,e.g.Etanercept and Abecept,etc.The native PD-1/PD-L1 has an intermolecular low affinity of about 10^-66 M.Wild-type PD-1 or PD-L1as an antagonist molecule cannot effectively compete to reverse the PD-1/PD-L1 inhibition signals in the tumor microenvironment.Similar to the antibody drugs,PD-1 or PD-L1 mutants can effectively block PD-1/PD-L1 signals if they are artificially modified with higher affinity(e.g.up to 10^-9-10^-1010 M).The engineered high-affinity receptor or ligand possesses overlapped epitope with that of the natural PD-1/PD-L1,so its blocking effect might be superior to that of antibodies theoretically.In this study,based on the structure of the wild-type PD-1 molecule,we used the computer-aided molecular analysis and design methods to simulate and dock the PD-1/PD-L1 structure,following the principle of keeping the epitope in PD-L1 recognized by PD-1.At the same time,the key pharmaceutical characters?e.g.affinity,stability,etc.?were considered to obtain the virtual library of PD-1 mutants.With the help of mammalian cell display technology,the mutanted PD-1 genes in the library were displayed on the mammalian cell surface.Then the high-throughput flow cytometry method was used to screen and enrich positive clones to improve the maturity and stability of PD-1 molecule in vitro.Furthermore,in vivo and in vitro models were established to evaluate the biological activity of the candidates.The details of the research results are as follows:1.Design and Construction of PD-1 High-affinity Mutant Library?1?Based on the crystal structure of PD-1 and PD-L1,the spatial structure of PD-1/PD-L1 interaction complex was reasonably constructed by computer-aided molecular simulation and docking technology.The key amino acids in PD-1/PD-L1 complex were theoretically analyzed by computer graphics distance geometry and interaction modes?intermolecular hydrogen bonding,Van der Waals interaction,etc.?,according to which five mutants,M1-M5,were designed using the alanine replacement,in which M3 is a control mutant?negative and invalid mutation?.Experiments indicated that M1,M2,M4,and M5 were indeed the key epitopes of PD-1 recognized by PD-L1,confirming the rationality of the theoretical model of PD-1/PD-L1 structure.Mutations were then designed by considering the mode of amino acid action and the physicochemical properties of amino acids,and the virtual library including all mutations was obtained.?2?According to the library design,specific primers were synthesized,and the DNA sequences were subcloned to the membrane display vector?PFRT-TM?and then transferred to the cells to construct the library.We selected 100 clones randomly for sequencing using the multiple sequence alignment analysis,from which 71 correct clones were analyzed to have the mutation sites in agreement with the theoretical design.The frequency of amino acids appeared uniformly.At the same time,71 sequences were available in diversity and didn't contain repetitive sequences,indicating that the effective ratio of the library is⁷1%,which meets the experimental requirement.?3?The plasmids of the library were stably transfected into CHO-Flp-In cells?site-directed integration?,and the stable expression cells of exogenous proteins were screened out by adding hygromycin B.2.Screening of High-affinity PD-1 Mutants and Preliminary Identification of CandidatesThe positive cells with high affinity were enriched by cell sorting.During three rounds of screening,the concentration of PD-L1-biotin was reduced from 10?g/mL,5?g/mL to 2?g/mL in order to obtain the best clones.The genome of the final cells was extracted,and the target genes were amplified by PCR using specific primers.Then genes were subcloned into the existing vector PFRT-KIgG1/Fc,and the positive clones were sequenced after ligation and transformation steps to know the candidate sequences.The candidate sequences were then compared with the maternal sequence,and 9 representative sequences were preliminarily selected by cluster analysis.The extracellular segment target proteins was prepared in CHO-S expression system,and purified to obtain the PD-1 mutants.Finally,463 was chosen for further research by binding ELISA,competitive ELISA,and affinity assay,which had an affinity increase of about 1000-fold than the Wild-type PD-1.3.The Bioactivity Evaluation of High Affinity PD-1 Mutant 463According to ELISA results,463 could specifically recognize PD-1 ligand PD-L1/PD-L2.Compared with wild-type PD-1,the binding activity of 463 to PD-L1/PD-L2 was significantly increased.Competitive ELISA results showed that 463 could effectively compete to block the binding of PD-1 to PD-L1,while wild-type PD-1 could not.It concluded that the blocking activity of 463 was about 668-fold higher than that of the parent protein.Experiments of in vitro PBMC activation experiments showed that 463 could reverse the inhibition of T cell activation.CD3 and CD28 stimulated T cell activation,and IFN-?expression was upregulated.However,T cell activation was inhibited by PD-1/PD-L1 pathway.At the same time,IFN-?secretion decreased.The candidate 463 and the positive antibody drug Opdivo could block the inhibitory PD-1/PD-L1 signaling pathway at the concentrations above 2.5?g/mL and re-activate the T cells to secrete IFN--?in a dose-dependent manner.In exograft transgenic mice model,the satisfactory inhibition ratios of tumor growth were 72%in the10mg/kg of 463 group,43%in the 2mg/kg of 463 group,and 77%in the 10mg/kg of Atezolizumab?positive antibody?group,compared with 14%in the 10mg/kg of wild-type PD-1 group.The high-dose of463 had a similar anti-tumor effect to the antibody drug Atezolizumab,which was much better than the wild-type PD-1 protein.In conclusion,the candidate PD-1 mutant 463 had good biological activity both in vivo and in vitro,indicating certain values and potential for further exploitation and application.