Screening And Evaluation Of Human-derived Protein Scaffold As A Novel Type Of PD-L1 Inhibitor

In recent years,cancer immunotherapy has gradually become a very important strategy of cancer treatment.Many novel drugs and methods were constantly being developed,these drugs and techniques achieved good therapeutic effects in clinical trials.Although monoclonal antibodies were widely used in clinical trials,their shortcomings had always restricted their development and application,and even plagued the entire biopharmaceutical field,for example,immunogenicity,complex production processes,and high costs greatly limit the application of antibodies.The shortcomings of antibodies have prompted people to turn their attention to small-molecule compounds.However,small-molecule compounds are not derived from humans,problems such as immunogenicity will inevitably arise,and they are also limited by factors such as molecular weight.The existing drug types are very single,and there is a lack of new drug types and sources in the field of cancer immunotherapy even extending to cancer targeted therapy.This paper proposes a new type of therapeutic protein drug for research and development,using the human-derived protein scaffold as a new type of therapeutic drug,which can fundamentally solve the problem of drug immunogenicity and minimize adverse reactions and side effects during clinical use.In this paper,firstly,a computer program was used to grab the qualified human proteins in the PDB and established a human protein scaffolds library.The PD-L1 in the classic signaling pathway PD-1/PD-L1 in cancer immunotherapy was selected as the target.The feasibility and development potential of human-derived protein scaffolds as a novel type of therapeutic drug were explored by the screening and biological activity evaluation.Based on the above research background,this paper used the molecular docking program Patch Dock to screen for PD-L1 inhibitors with research and development potential in the constructed human protein scaffolds library and set the screening conditions,such as scoring,PD-L1 binding conformation,original cell location,original function,molecular weight,and finally two human protein scaffolds were selected as examples for biological activity evaluation.The selected protein scaffolds are both derived from the nucleus,and when combined with PD-L1 as a therapeutic drug,it will not cause the body's inherent function disorder,and at the same time will not produce immunogenicity.The protein scaffolds were constructed on the E.coli expression vectors,and tags for expression and purification were added to become recombinant protein scaffolds,which are labeled as rhsRuvBL2 and rU1 snRNPA,respectively.After E.coli expression and nickel ion affinity chromatography,the protein scaffolds rhsRuvBL2 and rU1 snRNPA were successfully expressed and purified,and identified by SDS-PAGE and WB,the quantified protein scaffolds rhsRuvBL2 and rU1 snRNPA were used for biological activity evaluation to evaluate whether they are truly capable of becoming PD-L1 inhibitors.Firstly,whether the protein scaffolds rhsRuvBL2 and rU1 snRNPA could inhibit the interaction of PD-1/PD-L1 was explored.A platform based on SERS is established in this paper for detecting protein interactions.The results showed that rhsRuvBL2 and rU1 snRNPA can reduce the intensity of SERS characteristic peaks and could inhibit the interaction of PD-1/PD-L1.To further proved that rhsRuvBL2 and rU1 snRNPA inhibit the interaction of PD-1/PD-L1 by binding to PD-L1,this paper chose the ELISA for detection.The results showed that rhsRuvBL2 and rU1 snRNPA can bind to PD-L1 with EC50 of 135.3n M and 55.17 n M,respectively.At the same time,rhsRuvBL2 and rU1 snRNPA can compete with PD-1 to bind PD-L1,with IC50 of66.29 n M and 18.06 n M,respectively.To evaluate whether rhsRuvBL2 and rU1 snRNPA could reactivate T cells by pre-activated CD4+ T cells and PD-L1 expressing cells in co-culture experiments,the results showed that the level of the cytokines IFN-? and TNF-? secreted by CD4+ T cells in the group added rhsRuvBL2 or rU1 snRNPA was significantly increased,and positively correlated with the concentration,indicating that rhsRuvBL2 and rU1 snRNPA can reactivate T cell.The results of in vitro anti-tumor experiments proved that rhsRuvBL2 and rU1 snRNPA could kill human melanoma A375 cells and human breast cancer MDA-MB-231 cells relying on T cells.The results conducted in human immune system-melanoma-immune-deficient mice showed that rhsRuvBL2 and rU1 snRNPA could resist melanoma in vivo and will not cause damage to major organs.The above results can prove that rhsRuvBL2 and rU1 snRNPA have biological activity and can be used for further research and development.In addition,this paper also used site-directed mutagenesis to design three variants of rhsRuvBL2 or rU1 snRNPA to further explore the mechanism of the protein scaffolds.The results showed that rhsRuvBL2 and rU1 snRNPA were indeed dependent on the ability to compete with PD-1 to bind to PD-L1.The variant A32 D of rU1 snRNPA may be candidate protein scaffolds worthy of further study.In summary,this paper used PD-1/PD-L1 as a research model to use the human protein scaffold as a novel type of PD-L1 inhibitor and achieved ideal biological activity was achieved.Not only the selected protein scaffolds rhsRuvBL2 and rU1 snRNPA can be used for subsequent research and development,but more importantly,it proved the feasibility of human-derived protein scaffolds as therapeutic protein drugs,providing novel types of drug screening and development for the cancer treatment field.