The Expression Of Bispecific Immunological Directing Agent CAtin And Its Anti-tumor Effects In Vivo And In Vitro

The Expression of Bispecific Immunological Directing Agent CAtin and its Anti-tumor Effects in Vivo and in VitroWe designed and synthesized a compatible scdsFv gene sequence to allow the production of the recombinant protein for the targeting therapy to CEA-expressing tumor cells. In the resulting molecule, the heavy and light chains of Fv fragment were connected to each other by both an interdomain disulfide bond and a 15-amino acid linker. The Fv gene sequences used in the construct were derived from an anti-CEA scFv assembled from T84.66 scFvs in VL to VH orientation. With the purpose of disulfide stabilization of the scFv, the disulfide bond-stabilized antibody fragment, dsFv, was generated by mutations of the amino acid Arg (106) Cys of the VL and Gln (43) Cys of the VH domain. To generate the single-chain disulfide-stabilized Fv antibody, the VL and VH fragments were genetically connected by the sequences coding for a (GGGGS)3 flexible polypeptide linker. To improve the specificity and efficacy of the antitumor response, the Apoptin gene sequence was amplified from the plasmid pVAX1-Apoptin by PCR and fused to C-terminus of the scdsFv via another 15-amino acid linker. Final DNA sequencing of the construct confirmed the sequences published for T84.66 scFvs and also for Apoptin gene (data not shown). pET28a(+) vector was used to construct the effective expression system for the recombinant protein CAtin. By the His-tag fused with C-terminus of the CAtin (VL-linker-VH-Apoptin) gene fragment, the expressed protein could be detected by HRP labeled anti-His tag monoclonal antibody in Western blotting, Immunofluorescence or Flow cytometric analysis.The expression plasmid pCAtin was transformed into the E. coli strain BL21 and selected on LB plates containing ampicillin. The transformants were then conformed by digestion using restriction enzymes (Nco I and Not I) and transformed into the E. coli BL21. The production of the CAtin was performed as described above. SDS-PAGE analysis of the samples indicated that the recombinant protein was successfully expressed. The band of approximately 43 kDa was detected, which corresponds to the full-length fusion protein of CAtin. The recombinant protein was found only in the inclusion body fraction. CAtin was then efficiently purified by immobilized metal ion-affinity chromatography (Ni2+-chelating) column and refolded by a step-down urea concentration strategy.To evaluate the CEA-positive tumor targeting property of CAtin, human colon carcinoma cell LoVo was analyzed by immunofluorescence and human adenocarcinoma of the uterine cervix Hela was used as negative control. LoVo and Hela cells were plated on coverslides the day before assay and incubated with or without CAtin for 2 h at 37℃. The slides were then incubated with mouse anti-His tag monoclonal antibody and FITC-labeled goat anti-mouse IgG respectively. The LoVo cells incubated with CAtin exhibited strong membrane staining. In contrast, no staining was visualized in the Hela cells incubated with CAtin as well as the LoVo and Hela cells incubated without CAtin under the same conditions. The comparison of the image of LoVo and Hela cells incubated with or without CAtin demonstrated the high uptake onto CEA-positive cells and CEA-specificity of CAtin.The CEA-positive cells targeting effect of CAtin expressed by the E. coli was evaluated using human CEA-positive cell line LoVo and CEA-negative control Hela cells as described above. LoVo and Hela cells were incubated with or without 30μg CAtin purified and refolded for 2 h followed by FITC-labeled anti-His antibody, respectively.35.59% of the LoVo cells cocultured with CAtin and FITC-labeled anti-His antibody exhibited positive staining, with regard to which, only 7.23% of Hela cells treated with the same conditions displayed positive staining.The cell transduction and anti-tumor effects of CAtin in LoVo cells were identified by MTT. The results showed that, except for CAtin, other treatments (CEA-EGFP and CEA-scdsFv) did not kill LoVo cells in varying degrees. In the certain treatment doses, the anti-tumor effects of CAtin were heightened with the extending of treatment time, and resulted in some time-effect relationship. And the experiments also identified that the suppression rates of CAtin were concerned with treatment doses. By using AO/EB assay, DAPI assay, Annexin V assay and Caspases activity assay, we analyzed the mechanism of the anti-tumor effects mediated by CAtin. The results showed that the recombinant protein CAtin can induce apoptosis of LoVo cells.We also constrcted the BALB/c nude mice model bearing LoVo by transplanting LoVo cells into the right hind limb of the mice. And then, the in vivo anti-tumor effects of CAtin were observed through the mice model. The results showed that CAtin treatment group demonstrates varying degree anti-tumor effects. The suppression rates of CAtin group were 24.85%, which significant higher than the other groups (Saline, CEA-scdsFv and CEA-EGFP). We, then, observed the mean survival of the mice model. The results showed that the mean survival rates of CAtin treatment group was 66.7%, and much higher that Saline, CEA-scdsFv and CEA-EGFP treatment groups.In summary, the recombinant protein CAtin in this study, which has both CEA specific recognition and apoptosis induction penetration abilities, can suppress LoVo cells and inhibit solid tumor in animal model. The study provides a novel method for the cancer bio-therapy.