Design,Expression,functional Verification And Molecular Dynamics Analysis Of Cell-penetrating Peptide Conjugated Antibody

Breast cancer(BC)is the most common type of cancer among women and is the second leading cause of cancer related deaths.Based on both molecular and histological evidences,BC could be categorized into three groups;BC expressing hormone receptor(estrogen receptor(ER+)or progesterone receptor(PR+)),BC expressing human epidermal receptor 2(HER2+)and triple-negative breast cancer(TNBC)(ER-,PR-,HER2-).Approximately 25% of breast cancers are Her2-positive.Her2-positive breast cancer has been attracting attention because of its high invasiveness and poor prognosis.Conventional tumor treatment methods mainly include surgery,chemotherapy,and radiotherapy.Surgical resection and chemotherapy usually cause greater damage.Traditional chemotherapeutic drugs used in cancer treatment are usually some cytotoxic molecules,like platinum drugs,alkylating agents and paclitaxel,which have systemic toxicity due to lack of targeting.Trastuzumab,a humanized monoclonal antibody,targeting Her2 approved by FDA in 1998.With the application of trastuzumab,the prognosis of patients with early Her2-positive breast cancer has been significantly improved.It is currently the first-line targeted drug for the treatment of Her2-positive breast cancer.The effective rate of trastuzumab for Her2-positive advanced breast cancer is only 20%-30%,and the maximum effective rate of combined chemotherapy is only 60%.Due to a series of adverse reactions such as drug resistance and cardiotoxicity,at least 70% of Her2 positive breast cancer patients have disease progression.How to improve the internalization ability of targeted drugs has become the direction of developing new Her2 drugs.Cell penetrating peptides(CPPs)are a class of short peptides which can penetrate biofilms and deliver various foreign substances into cells.This efficient transport system is unaffected by cell types and has low cytotoxicity.CPPs could be categorized based on the physicochemical properties into three classes: Cationic,amphipathic and hydrophobic.The use of CPPs to increase the delivery of antibody drugs in vivo is the preferred solution to resolve antibody drug resistance and increase the effectiveness of antibody drugs.In recent years,single chain antibody(sc Fv)is widely used in the field of anti-intracellular antigens owing to it's small molecular,strong ability of penetration and short halflives in blood.It has been reported that CPPs are used for in vivo delivery antibody and clearance of intracellular targets.BP16 is a cationic CPP selected from a library of cecropin-melittin hybrids.S413 is a chimeric peptide,including a 13 amino acid penetrate sequence derived from the dermaseptin S4,and a nuclear localization sequence derived from SV40-T antigen,capable of penetrating into intact cells Accumulates in the nucleus.MAP is a secondary amphiphilic CPP,which includes a hydrophilic part and a hydrophobic part.In this paper,cell penetrating peptides BP16,MAP and S413 were coupled to the trastuzumab sc Fv screened by our laboratory to obtain the recombinant protein CPP-sc Fv.The results showed that conjugated CPP can increase the transmembrane effect of sc Fv and increase the apoptosis of Her2-positive breast cancer cells.Through the study on the design,expression,functional verification and molecular dynamics analysis of the recombinant protein CPP-sc Fv.MAP(Amphiphilic CPP)interacts strongly with phospholipid membranes,easily forms a more stable structure,and coupled with sc Fv,which effectively improves the anti-tumor ability.The specific research results are as follows:1.Constructed recombinant protein BP16-sc Fv,S413-sc Fv,MAP-sc Fv expression vectors by overlap PCR and achieve expression in E.coli BL21(DE3)strains.The purity of the recombinant protein obtained by nickel affinity chromatography is above 90%,which meets the requirements of subsequent biological experiments.2.The interaction of recombinant proteins sc Fv,BP16-sc Fv,S413-sc Fv,and MAP-sc Fv with Her2 antigen was detected by MST.The results showed that the presence of CPP did not affect the affinity between sc Fv and Her2 Antigen.The endocytosis of the recombinant protein was detected,and obvious endocytosis of the recombinant protein was observed in Her2-positive breast cancer cells.indicating that sc Fv and CPP-sc Fv all bind to cells through Her2 specific binding.Compared with sc Fv,CPP-sc Fv has more obvious endocytosis effect,and the most significant one is MAP-sc Fv,is three times of sc Fv.Then we tested the cytotoxicity of the recombinant protein.The results show that compared with sc Fv alone,sc Fv coupled with CPP can significantly reduce its IC50 value,have greater cytotoxicity,and have a stronger ability to induce apoptosis,and the effect of MAP-sc Fv is more obvious,its IC50 value was reduced by 7.7 times.3.Molecular dynamics simulation results show that in the 2000 ns simulation,with the increase of simulation time,MAP is gradually inserted into the membrane,and eventually submerged in the double-layer membrane,while S413 and BP16 are only adsorbed on the membrane surface,the insertion depth is far less than MAP.After the three CPPs interact with the phospholipid membrane,the phospholipid membrane has a tendency to expand laterally and compress vertically.Among them,the trend of MAP is the most significant,with the strongest effect on phospholipid membrane,followed by S413,BP16 is relatively poor.4.By calculating the total potential energy of the model constructed by homology modeling,the results show that MAP-sc Fv has the lowest total potential energy,MAP conjugated sc Fv is more stable.In summary,the CPP-sc Fv prepared in this paper has good anti-tumor effect in vitro,and the amphiphilic transmembrane peptide represented by MAP will lay a theoretical foundation for the development of new Her2-positive breast cancer targeted drugs.