ObjectiveThe EGF-EGFR signaling pathway plays a pivot role in regulating cell proliferation,survival,metastasis,and angiogenesis.The over-expression of EGFR on most human epithelial cancers makes it a well-characterized drug target for anti-tumor therapy.At present,two major categories of EGFR-targeted drugs,monoclonal antibodies and small molecular tyrosine kinase inhibitors,have been thoroughly investigated and approved in clinical use.However,drug resistance occurs in a significant proportion of patients due to various mechanisms,mainly including different mutations of the tyrosine kinase domain and compensatory activation of by-pass oncogenic pathways.One of those remarkable characteristics is that independent of ligand incentive,cancer cells can keep on proliferation,survival and anti-apoptosis.Therefore,to design novel EGFR-targeted drug is necessary and worth-while.Methods We originally constructed a recombinant protein,named as pLLO-hEGF,composed by functionally different two parts and expressed in a prokaryotic system.The N-terminal part contains three immunodominant epitopes from listeriolysin O (LLO),produced by Listeria monocytogenes(Lm),with an oligopeptide "GSGGSG" as the linker,while the C-terminal is the entire sequence of mature human EGF.MTT assay was used to analyze whether or not the fusion protein could potentially promote cancer cells to proliferate,and six human cancer cell lines of HCTll6,HT-29,A549,NCI-H157,MDA-MB-231 and SK-BR-3 were tested.Also,we observed the capacity of pLLO-hEGF binding to these cancer cells by immunofluorescent staining,and detected the immunogenicity to activate human PBMC by flow cytometry.Finally,the inhibitory effect to cancer cells was studied in vitro and vivo using human PBMC primed by pLLO-hEGF.Results The purified fusion protein could not accelerate cancer cells to proliferate and exhibited a great affinity to EGFR.Furthermore,pLLO-hEGF was able to promote amplification of CD3+ and CD4+T ceils in human PBMC in vitro,and the activated PMBC by pLLO-hEGF showed enhanced killing effects to EGFR-expressing cancer cells.Finally,the activatedlymphocytes could obviously suppress the growth of xenograft tumor of HCTll6,established in BALB/c-nude mice.Conclusion These findings suggest that pLLO-hEGF as a recombinant fusion protein,not only can efficiently bind to EGFR-expressing cancer cells,but also increase the immunogenicity of cancer cells by making them evident targets to immune cells.To design the recombinant molecule like pLLO-hEGF might represent an effective alternative to develop new EGFR-targeted drugs.