Small Molecular Peptide-ScFv ?v?3 Conjugates Specifically Inhibit Lung Cancer Cell Growth In Vitro And In Vivo

Background:Integrin ?v?3(ITG)is highly expressed in various cancers and promotes tumor growth[1],thus is considered a major target for anti-angiogensis therapy[2-5].The single chain fragment variable of which(ScFv ?v?3)has been reported to inhibit tumor growth both in vitro and in vivo.Unlike full length mAbs,ScFvs are recombinant antibody fragments just composed of connected variable light(VL)and variable heavy(VH)while retaining both the original antigen-binding capacity and biological function[7].They have lower production cost,better tuMor microcirculation penetration and less immunogenicity due to the lack of Fc portion[8,9].Here,we conjugated cdGIGPQc which can exclusively bind to NSCLC cells according to our previous study synthesized by SPPS with ScFv ?v?3 expressed in E.coli BL21(DE3)to develop a novel lung cancer specific targeted drug.cdGIGPQc was supposed to enhance the binding specificity of ScFv ?v?3 to lung cancers and the recombinant protein was expected to characterized by more specific tumor targeting and faster tumor penetration.Objects:This paper is based on the previously found peptide cdGIGPQc which can bind specifically to integrin ?3?1 aberrantly expressed on the surface of lung cancer cells to construct cdGIGPQc-ScFv ?v?3 and finish the preclinical research.To provide a new target drug and efficient technique for lung cancer treatment.Materials and methods:1.Construction of cdGIGPQc-ScFv ?v?3We synthesized small molecular peptide cdGIGPQc(cd)and non-related peptide cNAQAEQc(cN)by solid-phase peptide synthesis(SPPS).After the DNA sequence of anti-human integrin ?v?3 ScFv was artificially synthesized,recombinant plasmid pET-28a-ScFv ?v?3 was transferred into E.coli BL21 and induced to express.Purified ScFv ?v?3 was connected with peptide cdGIGPQc and cNAQAEQc respectively through SMCC.2.Cell-binding assay of cdGIGPQc-scFv ?v?3Immunofluorescence technique was used to explore whether cdGIGPQc-scFv?v?3 was able to bind lung cancer cells as well as cdGIGPQc.And flow cytometry was next used to quantified the different binding affinity against lung cancer cells between cdGIGPQc-ScFv ?v?3 and cNAQAEQc-ScFv ?v?3.3.The ?v?3-binding property and anti-tumor growth effects of cdGIGPQc-ScFv?v?3 in vitroThe binding abilities of ScFv ?v?3 and cdGIGPQc-ScFv ?v?3 to human integrin ?v?3 were checked by Western Blot assay.The cytotoxicity of cdGIGPQc-ScFv ?v?3 in vitro was evaluated via CCK-8 assays and compared with cNAQAEQc-ScFv ?v?3.4.Therapeutic efficacy of cdGIGPQc-ScFv in vivoNude-mice xenograft models of A549 and L78 cells were established to analyze the therapeutic effects of cdGIGPQc-ScFv ?v?3,cNAQAEQc-ScFv ?v?3 and ScFv?v?3 on non small cell lung cancer.And immunohistochemistry analysis of harvested tumors was performed to verify the functional mechanism.5.Toxicity and security analysis of cdGIGPQc-ScFv in vivoThe general condition of mice under treatment was evaluated and compared with that before treatment.The potential effect or toxity on vital organs,including heart,lung,liver,kidney and spleen,was analyzed with hematoxylin-eosin staining(H&E staining).Results:1?Construction of cdGIGPQc-ScFv ?v?3Specific peptide cdGIGPQc and nonspecific peptide cNAQAEQc were synthesized by SPPS before identified and purified by ESI-MS and HPLC.The purity of cdGIGPQc and cNAQAEQc were 96.45%and 95.08%,respectively.Purified ScFv avP3 protein migrated as a single band of 26.5 KDa when demonstrated by SDS-PAGE and Coomassie Blue staining.Recombinant cdGIGPQc-ScFv ?v?3 and cNAQAEQc-ScFv ?v?3 shared a similar molecule weight slightly larger than ScFv?v?3.2?Binding ability of cdGIGPQc-ScFv to lung cancer cells in vitroThe fluorescent images demonstrated that FITC-cdGIGPQc-ScFv ?v?3 was able to bind A549 and L78 lung cancer cells like the positive control FITC-cdGIGPQc,with an apparently higher affinity than FITC-cNAQAEQc-ScFv avP3.Consistently in the flow cytometry assays,FITC-cdGIGPQc-ScFv ?v?3 exhibited a similar binding rate close to 100%as FITC-cdGIGPQc,while FITC-cNAQAEQc-ScFv ?v?3 holding a much lower positive rate(about 50%)as well as fluorescence intensity.3?ITG ?v?3-binding activity of ScFv ?v?3 and cdGIGPQc-ScFv ?v?3 in vitroWestern Blot assay revealed that both ScFv ?v?3 and cdGIGPQc-ScFv ?v?3 were able to recognize integrin ?v?3 as primary antibodies through antigen-antibody reaction,resulting in identical protein bands with the mAb of ?v?3.4?Inhibition of cancer cell proliferation by cdGIGPQc-ScFv ?v?3 in vitroThe results of CCK-8 assays showed that cdGIGPQc-ScFv ?v?3 had a lower average IC50 value and inhibited tumor cell proliferation more dramatically under indicated concentrations,while the semblable dose-dependent curves of ScFv ?v?3 and cNAQAEQc-ScFv ?v?3 suggested a similar inhibitory effect between them two.When the concentration increased to 5 ?M,cdGIGPQc-ScFv ?v?3 was able to kill almost all the NSCLC cells after 24 h of treatment,while ScFv ?v?3 and cNAQAEQc-ScFv ?v?3 presented much lower inhibitive rates around 40%.Apparently,cdGIGPQc-ScFv ?v?3 could efficiently inhibit proliferation of A549 and L78 cells at micromolar concentrations.5?Inhibition of tumor growth by cdGIGPQc-ScFv ?v?3 in vivoMice with xenograft were followed for 3 weeks after treatment.ScFv ?v?3 and cNAQAEQc-ScFv ?v?3 showed a similar anti-tumor growth activity,while cdGIGPQc-ScFv ?v?3 retarded tumor growth more significantly,considering both the smaller volume and lower weight of tumor(p<0.05).For immunohistochemistry,the IOD/Area values of CD31 and Ki67 were significantly lower in the cdGIGPQc-ScFv ?v?3 group compared with any other groups.And the simultaneously decreased trend of CD31 and Ki67 demonstrated that cdGIGPQc-ScFv ?v?3 can decrease the tumor microvessel density and suppress cell proliferation more effectively,thus exerted a superior antitumor effect to ScFv ?v?3 and cNAQAEQc-ScFv ?v?3.6?Security of cdGIGPQc-ScFv ?v?3There was no obvious change in mice weight or general activity when treated with cdGIGPQc-ScFv ?v?3.In addition,no obvious influence on major organs was found after H&E staining.Conclusions:1.cdGIGPQc-ScFv ?v?3 conjugates have been successfully constructed.2.cdGIGPQc-ScFv ?v?3 conjugates retained the specific lung cancer cell-binding ability of cdGIGPQc.3.cdGIGPQc-ScFv ?v?3 conjugates were able to inhibit the growth of lung cancer both in vitro and in vivo4.cdGIGPQc-ScFv ?v?3 retarded tumor growth more vigorously through inhibiting angiogenesis and tumor growth.5.The little influence of cdGIGPQc-ScFv ?v?3 on normal vital organs implied its low toxicity and good security in vivo.