Synthesis,Construction And Characteriazation Of ATRA-resistant APL Cell Line Targeting Peptides And Peptibodies

Acute promyelocytic leukemia is an M3 subtype of acute myeloid leukemia.All-trans retinoic acid(ATRA)in combination with arsenic trioxide have become a paradigm of targeted therapy for acute promyelocytic leukemia(APL).Although the targeted therapy can result in both the complete remission rate of 90%and the cure rate of nearly 80%,some patients cannot be cured because of ATRA resistance.Thus,ATRA resistance has become one key issue to be solved in APL diagnosis and treatment process.Peptides targeting multidrug-resistant tumor cells have the potential to guide chemotherapeutic agents into the tumor tissues,which not only improve the accumulation of drugs in targeting sites,but also reduce systemic toxicity.But,to date,no peptide ligands with affinity for the multidrug resistance(MDR)APL cells have been developed.Phage display is a powerful technique for the selection and identification of peptide ligands specifically directed against live whole cells.At present,a variety of tumor-targeting polypeptides have been obtained through this technology,and used in the diagnosis and treatment of cancer.Fc-based fusion protein(peptibody)is composed of an immunoglobin Fc domain that is directly linked to a polypeptide.Peptibody retains the binding specificity of the polypeptide to the target molecule.The presence of the Fc domain enables it to interact with Fc-receptors(FcRs)on immune cells,then enhances antibody-dependent cell-mediated cytotoxicity(ADCC)or opsonization.At the same time,it increases plasma half-life of peptibody,and easier to purify.All the characteristics promote its application in oncological therapies.In the present study,the acute promyelocytic leukemia cell line HL-60 was screened in vitro by concentration increasing intermittent induction method,and the corresponding drug resistant cell line was named as HL-60R.HL-60 cells were used as the negative sieve cells and HL-60R cells were screened by whole cell ablation.Four phage clones specifically binding to HL-60R cells were obtained and named as No.26,No.27,No.5R5 and No.5R13.Their exogenous dodecapeptide sequences were as following:pepNo.27(SPTSSFQVGTFA),pepNo.26(TLTTHGRLFESN),pepNo.5R5(WAITKPAPAAHP)and pepNo.5R13(TSTTFKSHLDHH).In addition,the binding specificity of phage-peptide and drug-resistant cell lines was preliminarily verified.Based on this,the corresponding fluorescent labeling polypeptide was further synthesized according to the peptide sequence shown by phage-peptide cloning,and the specificity of cell binding was confirmed.The main test results are as follows:1.According to phage-peptide cloning No.26,No.27,No.5R5,No.5R13 exogenous insert sequence,commissioned by the company to synthesize the corresponding fluorescent labeled polypeptide.To make the synthetic peptides coherent with the corresponding phage polypeptides,we added the GGGSK sequence at the carboxy terminus of the synthetic polypeptide to mimic the free ends of the phage PIII protein and conjugated fluorescein 5-TAMRA to lysine,the corresponding fluorescent labeling test peptide and control peptide(GGGAGGGAGGGAK)were synthesized.Flow cytometry and cellular immunofluorescence showed that the four test peptides were specifically bound to HL-60R cells and did not bind to other tumor cells and human embryonic kidney cells.The results of the competitive inhibition test showed that the test peptide of uncoupled fluorescein could compete effectively with the corresponding fluorescently labeled polypeptide,further confirming the binding specificity of pepNo.27,pepNo.26,pepNo.5R5,pepNo.5R13.The results of internalization showed that the fluorescent peptide pepNo.27 could be internalized into the cytoplasm under the condition of prolonging the incubation time.PepNo.5R13 and normal human peripheral blood nucleated cells do not combine,suggesting that it has clinical application potential.2.According to pepNo.27,pepNo.26 sequence,designed and synthesized at both ends with EcoRI and NcoI cleavage site of the target fragment,and EcoRI and NcoI double digested pFUSE-hIgG2-Fc2 plasmid linked to transformed into DH5a,picking positive colony to extract the plasmid sequencing.The recombinant plasmid pFUSE-hIgG2-Fc2-26 and pFUSE-hIgG2-Fc2-27 were successfully constructed.The recombinant plasmid was transfected into 293T cells for eukaryotic expression.The supernatant was collected and the Peptibody-26,Peptibody-27,successfully expressed.3.Bioinformatics prediction pepNo.27,pepNo.26,pepNo.5R5,pepNo.5R13 four peptide physical and chemical properties and three-dimensional structure.Literatures were searched for leukemia drug resistance-related molecules,and their subcellular localization was identified by the GeneCards database to determine the presence of candidate molecules in the cell membrane.PepSite2 software was used to predict the docking of peptides with them.Based on the docking P value,the candidate target protein NCAM and pepNo.5R5 were docked(P<0.05).The potential for binding of the candidate target protein P-gp to the four targeting peptides was pepNo.26(P =0.04336),pepNo.27(P = 0.04421),pepNo.5R5(P = 0.03271),pepNo.5R13(P =0.02861).The binding possibilities of the candidate target protein Bcl-2 and pepNo.26,pepNo.5R5 and pepNo.5R13 were pepNo.26(P = 0.02514),pepNo.5R5(P =0.008697)and pepNo.5R13(P = 0.03774).In conclusion,the phage clones obtained from the previous screening showed the exogenous polypeptide sequences,and four fluorescent groups labeled with 5-TAMRA were synthesized.The binding specificity of the four polypeptides and the acute promyelocytic leukemia drug resistant cell line HL-60R was further confirmed by experiments such as immunofluorescence binding assay,competitive inhibition assay,flow cytometry and other experiments.At the same time,we also successfully constructed pepNo.26,pepNo.27 corresponding peptides and expressed them in eukaryotic cells.Bioinformatics predicts the possibility of binding between candidate target proteins and four targeting peptides that NCAM is paired with pepNo.5R5(P<0.05).The candidate target protein P-gp binds to the four targeting peptides.The candidate target protein Bcl-2 binds to pepNo.26,pepNo.5R5 and pepNo.5R13.These results will help to provide a new detection and targeted therapy for ATRA drug-resistant APL,to explore the ATRA-resistant mechanism of APL cells,and to lay an important experimental basis on the development of new ATRA drug resistance reversal agents.