Antitumor Activity Of A Novel DC-targeting Bifunctiona Fusion Protein

Dendritic cells are professional antigen presenting cells with potent immune stimulating functions and are effective in activating host immune response through initiation of naive T cell activation.Dendritic cells play an important role in induction of host anti-tumor specific immunity,which has been intensively investigated due to its potential clinical applications as an immune stimulator for stimulation of host antitumor immune response in cancer hosts.It has also been demonstrated that DC vaccines generated by different antigen loading approaches can stimulate antigen specific T cells with induction of antitumor immunity,which can recognize and kill tumor cells. However,the broad applications of these approaches are limited due to time consuming and complexity.It is an urgent need to explore simple and more effective approach to generate DC vaccines for treatment of cancers.Previous work indicated that fusion of tumor antigen with a fragment of antibody was able to target DCs in vivo for effectively delivering antigen into DCs through antibody mediated internalization and presentation of antigens.The in vivo modified DCs effectively presented targeted antigen on the cell surface and,however,were unable to stimulate antigen specific immunity when administrated into animals.More importantly,the DCs generated by this approach induced antigen specific tolerance in mice.The mechanism by which the antigen specific tolerances could be induced was considered as the targeted DCs might be immature DCs(iDCs) but not mature DCs (mDCs),which induced the deletion of activated T cells or induction of anergy.On the basis of previous works,the current study has developed a novel DC targeted fusion vaccine so called FL-Fc-αCD11c-TRP2_180-188.FL is an effective growth factor which stimulates proliferation of most bone marrow cells,especially for maturation of DCs and releasing the mDC cells with strong antigen presentation function and NK cells with killing activity into blood circulation from peripheral immune organs.It will be a great idea for development of effective fusion vaccines by fusion of FL as immune stimulator with anti-CD11c antibody(αCD11c) and TRP2_180-188 as a targeting and antigen resource.CD11c is a cell surface antigen specifically expressed on DCs and can be targeted by the antibody portion of this fusion protein.Insertion of Fc fragment of immunoglubin between extracellular domain of FL andαCD11c could prolonge the half life of the fusion proteins and maintain flexibility of the fusion proteins,which facilitated the bindings of both FL and CD11c antibody with the antigens on the cellular membrane.This study will focus on the investigation of the mechanisms by which the fusion vaccines induce antitumor immunity and optimize administration routes which are more effective in stimulating host immune responses.Cloning of genes encoding fusion protein and construction of expression vector:The V_H and V_L of Hamster anti-mouse CD11c were cloned from hybridoma cell line (N418) by 5' RACE and overlap PCR technology.TRP2_180-188 was genetically fused in frame to 3' termius of single chain antibody by(Gly₄Ser)₃ The previously constructed FL-Fc fusion gene was fused to 5' end ofαCD11c-TRP2_180-188 via a(Gly₄Ser)₃ linker by overlap PCR.The fusion gene of FL-Fc-αCD11c-TRP2_180-188 with the size of 2190bp was inserted into pcDNA3.1(+)vector.The expression vector was characterized by digestion with restrictive enzymes and stored for further use.The expression and purification of fusion protein and biological assay:CHO cells were transfected with FL-Fc-αCD11c-TRP2_180-188/pcDNA3.1(+)vector using lipofectamine 2000 reagent.The expressed protein could be detected within 72 hours after transfection.The cell clones with higher expression levels were selected by the Dot-Blot assay and expended in serum-free medium.The FL-Fc-αCD11c-TRP2_180-188 fusion protein was purified by protein A affinity column from the culture supernatant. SDS-PAGE and Western Blot showed a single band with a molecular mass of 80KDa. Mouse dendritic cells were generated from bone marrow u and 90%of the obtained cells were CD11c positive.The resulting fusion protein could was demonstrated to be able to specifically bind to DCs indicated by the flow cytometery assay. Induction of antitumor immunity by the fusion protein:C57BL/6 mice were immunized by the fusion protein and lymph nodes were taken 8 h after immunization.The monocytes were obtained from the lymph nodes and analyzed by flow cytometery with antibodies specifically for CD11c,FL and CD86 antigen.The results showed that the number of CD11c⁺ FL⁺,CD11c⁺ CD86⁺ cells significantly increased in FL-Fc-αCD11c-TRP2_180-188 treated animals.The lymphocytes from the fusion protein treated animals had a proliferated response to DCs loaded with TRP2_180-188 antigen and could secrete substantial amounts of IFNγand TNFα.The data demonstrates that FL-Fc-αCD11c-TRP2_180-188 can specifically target dendritic cells when administrated into animals and elicit a Th1 mediated T cell response,which are associated with induction of host antitumor immunity.Prevention and treatment of melanoma in mouse model system:B16/F1 is a mouse melanoma cell line which express TRP2 tumor antigen on the cell surface.1×10⁴ B16/F1 cells were inoculated subcutaneously into C57BL/6 mice.The animals were divided into three groups one week after tumor cell injection and treated subcutaneously with 100μg FL-Fc-αCD11c-TRP2_180-188,mixture ofαCD11c-TRP2_180-188 and FL-Fc and PBS individually.Total two injections were given weekly and tumor growth and size were monitored and recorded for fifty days.The results showed that the immunization with FL-Fc-αCD11c-TRP2_180-188 was effective in inhibiting tumor growth. The T cells obtained from the animals treated with fusion protein,but not from the control groups had a specifically killing activity to B16/F1 melanoma cell line.In summary,we have successfully developed a DC targeted fusion protein, FL-Fc-αCD11c-TRP2_180-188,which consists of three functional portions,FL for immuno-stimulation,CD11c for DC targeting and TRP2 for tumor antigen.The generated fusion protein can specifically target DCs and delivery tumor antigen into targeted DCs. Then DCs will present the tumor antigen on the cell surface.More importantly,the fusion protein can function as an immuno-stimulator to facilitate DCs maturation and releasing from bone marrow to peripheral blood,which greatly enhance host immune responses to TRP2 antigen.Immunization with the fusion proteins can elicit host antitumor immunity which is mainly mediated by Th1 T cells.The antitumor immunity elicited by immunization with the fusion proteins is both preventive and curative.This novel approach may provide a novel strategy for cancer immunotherapy.