Construction And Anit-tumor Effects Of The Recombinnat Antibody Targeting Lung Cancer

The carcinoembryonic antigen (CEA), known as an important clinical tumor marker, is a highly glycosylated oncofetal glycoprotein containing 50% carbohydrates with a molecular weight of about 180-200 kDa. CEA is a tumor-associated antigen uptrgulated in the majority of colon cancers, non-small cell lung cancers and half of all breast cancers. On the other hand, CEA is also present in some normal tissues. A lot of research demonstrated that, in normal tissues, CEA was mainly localized on the luminal surface of the single layer of columnar epithelial cells lining the upper parts of the crypts and was not directly in contact with blood flow or tissue fluid. While, in tumor tissues, CEA was localized on all sides of the cell membranes and was directly facing blood flow or tissue fluid. All these features made it an attractive target molecule for antibody-directed diagnosis, gene therapy and immunotherapy.Although monoclonal antibody was considered one of the preferred forms of cancer immunotherapy, many other methods have been proved more effective. Liu Y et al produced a dual functional protein that exhibited both tumor specific binding and killing activities by fusing the tumor-specific apoptosis-inducing molecule Apoptin to an anti-CEA single chain antibody. Alternatively, one study was to localize interleukin-2 (IL-2) to cancer cells by joining the IL-2 to the antibodies specific for tumor antigens. These studies indicated that antibody-directed targeting of the antitumoral molecule could alleviate the toxic side effects while enhancing the proteins deposition at tumor sites. Genetically engineered fusion proteins basing on tumor specific antibody have received more emphasis.The single-chain disulfide-stabilized Fv antibody (scdsFv), comprised of the variable heavy domain (VH) and the variable light domain (VL), is the smallest antibody fragment containing a complete antigen binding site. The engineered stabilizing disulfide bond in scdsFv can overcome the problem of aggregatjon at high concentrations of the basic single-chain Fv antibody (scFv). In comparison with the much larger IgG, scdsFv fragments have lower retention times in non-target tissues, a more rapid blood clearance and better tumor penetration in vivo. The use of labeled scdsFv that target the tumor-associated antigen has been described extensively.The selection of human anti-CEA single-chain antibody fragment (scFv) is a key step toward the development of new antitumoral agent designed for immunotherapy based on CEA. The engineered scFv of anti-CEA T84.66 antibody used for this research has been studied extensively since its discovery. Phase I clinical trials have demonstrated that the engineered scFv of anti-CEA T84.66 antibody was rapid tumor uptake, high tumor activity, fast blood clearance and suitable for clinical practice.Staphylococcal enterotoxin A (SEA), known as superantigens (SAgs) because of the ability to recognize Vβregion of T-lymphocyte receptors, is a one of the bacerial superantigen, involving in autoimmune and toxic shock disorders, that activates immune responses and induces production of various cytokines. Previous studies have indicated that SEA was the most potent stimulator of T cells and tumor necrosis factor (TNF). Furthermore, SEA stimulates T cells mainly in histocompatiblity complex classⅡrestricted manner and induces high levels of cytokines, such as TNF, IL-2 and interferon-y. These characteristic of SEA might be useful in therapy for regulating immune responses.In present study, we construted a chimeric protein, designated as SC-C/SEA that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A (SEA) to C-terminus of an anti-CEA single-chain disulfide-stabilized Fv antibody (scdsFv). The SC-C/SEA proteins were expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni2+ affinity chromatography column. SDS-PAGE and Western blotting revealed that the target protein was well-expressed. We demonstrated by immunofluorescence assays that SC-C/SEA could bind specifically to human lung carcinoma cells (A549), but almost not to human uterine cervix (HeLa). We also observed the anti-tumor activity of the recombinant proteins in vivo. The solid tumor model and the in situ model were established. The mean survival, the tumor growth, the suppression rate, the NK activity, the CTL activity and the cytokine levels of the animal models were evaluated. The results showed that the recombinant proteins inhibited the lung cell growth effectively. Although the injection of the recombinant adenoviruses did not lead to complete elimination of the tumors, effective inhibition was observed in the established solid tumor model and the in situ model. In summary, SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein was well expressed. Immunofluorescent and CTL assays indicated that SC-C/SEA could bind to CEA-positive cells specifically and induce CTL predominance. The SC-C/SEA protein produced in the work can be developed for potential use in CEA-targeted cancer immunotherapy.