Small Molecular Peptide-ScFv αvβ3 Conjugates Specifically Inhibit Lung Cancer Cell Growth In Vitro And In Vivo

Background:Integrin αvβ3(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 αvβ3)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 αvβ3 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 αvβ3 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 α3β1 aberrantly expressed on the surface of lung cancer cells to construct cdGIGPQc-ScFv αvβ3 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 αvβ3We 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 αvβ3 ScFv was artificially synthesized,recombinant plasmid pET-28a-ScFv αvβ3 was transferred into E.coli BL21 and induced to express.Purified ScFv αvβ3 was connected with peptide cdGIGPQc and cNAQAEQc respectively through SMCC.2.Cell-binding assay of cdGIGPQc-scFv αvβ3Immunofluorescence technique was used to explore whether cdGIGPQc-scFvαvβ3 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 αvβ3 and cNAQAEQc-ScFv αvβ3.3.The αvβ3-binding property and anti-tumor growth effects of cdGIGPQc-ScFvαvβ3 in vitroThe binding abilities of ScFv αvβ3 and cdGIGPQc-ScFv αvβ3 to human integrin αvβ3 were checked by Western Blot assay.The cytotoxicity of cdGIGPQc-ScFv αvβ3 in vitro was evaluated via CCK-8 assays and compared with cNAQAEQc-ScFv αvβ3.4.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 αvβ3,cNAQAEQc-ScFv αvβ3 and ScFvαvβ3 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 αvβ3Specific 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 αvβ3 and cNAQAEQc-ScFv αvβ3 shared a similar molecule weight slightly larger than ScFvαvβ3.2、Binding ability of cdGIGPQc-ScFv to lung cancer cells in vitroThe fluorescent images demonstrated that FITC-cdGIGPQc-ScFv αvβ3 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 αvβ3 exhibited a similar binding rate close to 100%as FITC-cdGIGPQc,while FITC-cNAQAEQc-ScFv αvβ3 holding a much lower positive rate(about 50%)as well as fluorescence intensity.3、ITG αvβ3-binding activity of ScFv αvβ3 and cdGIGPQc-ScFv αvβ3 in vitroWestern Blot assay revealed that both ScFv αvβ3 and cdGIGPQc-ScFv αvβ3 were able to recognize integrin αvβ3 as primary antibodies through antigen-antibody reaction,resulting in identical protein bands with the mAb of αvβ3.4、Inhibition of cancer cell proliferation by cdGIGPQc-ScFv αvβ3 in vitroThe results of CCK-8 assays showed that cdGIGPQc-ScFv αvβ3 had a lower average IC50 value and inhibited tumor cell proliferation more dramatically under indicated concentrations,while the semblable dose-dependent curves of ScFv αvβ3 and cNAQAEQc-ScFv αvβ3 suggested a similar inhibitory effect between them two.When the concentration increased to 5 μM,cdGIGPQc-ScFv αvβ3 was able to kill almost all the NSCLC cells after 24 h of treatment,while ScFv αvβ3 and cNAQAEQc-ScFv αvβ3 presented much lower inhibitive rates around 40%.Apparently,cdGIGPQc-ScFv αvβ3 could efficiently inhibit proliferation of A549 and L78 cells at micromolar concentrations.5、Inhibition of tumor growth by cdGIGPQc-ScFv αvβ3 in vivoMice with xenograft were followed for 3 weeks after treatment.ScFv αvβ3 and cNAQAEQc-ScFv αvβ3 showed a similar anti-tumor growth activity,while cdGIGPQc-ScFv αvβ3 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 αvβ3 group compared with any other groups.And the simultaneously decreased trend of CD31 and Ki67 demonstrated that cdGIGPQc-ScFv αvβ3 can decrease the tumor microvessel density and suppress cell proliferation more effectively,thus exerted a superior antitumor effect to ScFv αvβ3 and cNAQAEQc-ScFv αvβ3.6、Security of cdGIGPQc-ScFv αvβ3There was no obvious change in mice weight or general activity when treated with cdGIGPQc-ScFv αvβ3.In addition,no obvious influence on major organs was found after H&E staining.Conclusions:1.cdGIGPQc-ScFv αvβ3 conjugates have been successfully constructed.2.cdGIGPQc-ScFv αvβ3 conjugates retained the specific lung cancer cell-binding ability of cdGIGPQc.3.cdGIGPQc-ScFv αvβ3 conjugates were able to inhibit the growth of lung cancer both in vitro and in vivo4.cdGIGPQc-ScFv αvβ3 retarded tumor growth more vigorously through inhibiting angiogenesis and tumor growth.5.The little influence of cdGIGPQc-ScFv αvβ3 on normal vital organs implied its low toxicity and good security in vivo.