Study On The Design And Antitumor Activity Of PD-1/PD-L1 Peptide Inhibitors

In the past ten years,cancer immunotherapy has made great progress,especially the blocking of immune checkpoints is more prominent,and the inhibition of PD-1/PD-L1 is more concerned.And the PD-1/PD-L1 antibody has developed rapidly.The FDAapproved PD-1 antibody and PD-L1 antibody are also prominent in the treatment of melanoma,urothelial carcinoma and glioblastoma.These antibody drugs have high affinity and specificity.However,the drawbacks of antibody drugs are not negligible,including high cost,immune-related toxicity,pharmacokinetics and insufficient tumor penetration.which hinder the wider clinical application of antibody drugs.Therefor,the search for effective small molecule inhibitors has become a hot spot in current research.Small molecule inhibitors can make up for the deficiency of antibody drugs.Small molecule inhibitors can be divided into peptide and non-peptide inhibitors,among which peptide small molecule inhibition has better biocompatibility,so the main purpose of this paper is to design a polypeptide of less than 20 amino acids and study its blocking effect and anti-tumor activity.We found that the binding interface of PD-1 / PD-L1 complex is a hairpin structure,and a 14 amino acid short peptide(p1)was identified at its core.We performed a series of transformations on the sequence,including a Stable hairpin structure(p2-p4),cyclic peptide(p5),weakening the hairpin structure(p6-p8)and introducing a non-natural amino acid(p9-p12).A total of 12 peptide inhibitors were designed.The peptide was synthesized by Fmoc solid phase peptide synthesis and detected by HPLC and MALDITOFMS-MS;PD-1 and PD-L1 were used as the stationary phase,and the polypeptide inhibitor(p1-p12)was used as the mobile phase.The binding constant of the peptide to PD-1/PD-L1 was detected by the biomolecular interaction system(BLI);the toxicity of the peptide inhibitor(p1-p12)to T cells and HCT-116 cells was detected by MTT assay;A simple immunosuppressive model was established by co-incubation of T cells with HCT-116 cells,followed by the addition of a peptide inhibitor(p1-p12),collection of cell culture media,and detection of changes in IL-2 secretion by T cells by ELISA to assess its activity in re-activating immune cells.By detecting the KD value,it was found that the affinity of p1 and PD-1 was comparable to that of its natural ligand,and the binding constant of p3 to PD-1 was 1.8 ?M stronger than that of PD-L1,and the binding constant with PD-L1 was 2.06,which is close to the binding constant of PD-1 and PD-L1;the sequence obtained by engineering p3(p6-p8)has a binding constant of 5 or even 70 times greater than that of PD-1 and PD-L1;then we introduced unnatural amino acids(p9-p12)and found that their affinity constants with PD-1 and PD-L1 are slightly higher than p3,but the method of introducing unnatural amino acid may contribute to the study of future stability;The MTT assay showed that the peptide p1-p12 was not toxic to both cells when administered to 2 ?M.Most peptide inhibitors showed weak toxicity when administered to 4 ?M or 8 ?M,so we chose 2 ?M for the next concentration of the drug concentration.In the T cell reactivation experiment,we found that except for p5 and p10,other peptides can achieve the effect of re-activating T cells,with an average increase of about 30%,and the highest level reached 48%.From the above data,the following conclusions were obtained.We designed a short peptide with good affinity to PD-1/PD-L1,and these short peptides have certain activities to reactivate T cells;We can also initially conclude that the core hairpin structure of the peptide designed for PD-1/PD-L1 may be a key factor affecting the stability of its binding.