Preparation Of The Immunotoxin T-CUS And Detection Of Its Anti-breast Cancer Activity In Vitro

Objective Immunotoxin(IMT) which is known as the biological missile refers to the immune complex of monoclonal antibody and Cytotoxin(CTX),that can be directed against the target cells. The immunotoxin, conjugate of Trastuzumab and Cucurmosin, was prepared by this study, aiming to make full advantage of the former’s high targeting, the latter’s high activity and eliminate many defects such as the former’s low efficacy and the latter’s high side effects. The experiment was designed to detect the binding activity of the immunotoxin on targeted cells and killing activity in vitro, for seeking a new treatment method of HER-2-positive breast cancer.Methods 1.The preparation of immunotoxin: The two immunotoxins T-CUS-PDP and T-CUS-2-IT were successively prepared by the two crosslinking agent SPDP and 2-IT. 2.Isolation and Purification of immunotoxin: The purifed immunotoxin was got by affinity chromatography and cation exchange column using Ni Sepharose 6 Fast Flow and Sp- Sepharose Fast Flow respectively,following the identification by SDS-PAGE and quantification by BAC protein assay. 3.Detection the binding activity: ELISA assay was used to detect the binding activity of Trastuzumab and the above immunotoxins on these cells.4.SRB assay was used to detect the cell-killing effects of CUS,CUS-PDP and CUS-2-IT on BT-474 and HCC1937 in vitro.5.SRB assay was used to detect the cell-killing effect of T and T-PDP on BT-474 and HCC1937 in vitro when exposed to high and low concentration groups.6. SRB assay was used to compare the cell-killing effect of CUS,T,T-CUS-PDP,T-CUS-2-IT and CUS cooperated with T on BT-474 in vitro.Results 1.The immunotoxins was constructed by the crosslinking agents SPDP,2-IT,then purified successively using Ni Sepharose 6 Fast Flow and Sp-Sepharose Fast Flow,the result of SDS-PAGE showed that the electrophoretic band of immunotoxins was appeared,and the immunotoxins was constructed successfully. 2.The binding activity of the two prepared immunotoxins with BT-474 had no significant change in comparison with Trastuzumab. 3. The IC50 of CUS on BT-474 and HCC1937 rose from 0.325μg/ml to 1.736μg/ml and 0.298μg/ml to 0.904μg/ml respectively after crosslinking with SPDP, with a loss of activity about 80% and70% respectively. Nevertheless after crosslinking with 2-IT, the IC50 of CUS on BT-474 and HCC1937 rose from 0.325μg/ml to 0.365μg/ml and 0.298μg/ml to 0.316 μg/ml respectively, both with a retention of activity about 90%. 4.The high and low concentration groups of Trastuzumab after been crossed linked with SPDP kept most of the activity on BT-474. The inhibition ratio of T and T-PDP on BT-474 were above 80% and showed no significant difference.Nevertheless, the IC50 of T and T-PDP on BT-474 were 0.124μg/ml and 0.143μg/ml respectively,showing that T-PDP kept most of the activity of the antibody. In addition,T and T-PDP had no significant inhibition effect on HCC1937 and couldn’t reach IC50,neither in high concentration groups nor in low groups. 5. The IC50 of immunotoxins T-CUS-PDP and T-CUS-2-IT on BT-474 were 0.011μg/ml and 0.008μg/ml respectively;the results of the combination of CUS and T showed that the effect of immunotoxin on BT-474 wasn’t simply an addition of effect of Trastuzumab and CUS used alone, and the killing effect of CUS on BT-474 could be greatly improved when coupled with Trastuzumab.Conclusion 1.The immunotoxin constructed by T and CUS could be successfully achieved with SPDP and 2-IT as crosslinking agents. 2.The purification of immunotoxin was hard and the new ways of purification should be explored in the future research.3.The immune activity of two immunotoxins detected by ELISA assay showed no significant difference compared with that before crosslinking and kept the previous activity. 4.The activity of CUS cross-linked with SPDP decreased significantly compared that with 2-IT, while the activity of immunotoxin prepared by T and CUS with SPDP and 2-IT as couplants on BT-474 was enhanced than respectively using CUS or T alone.This study provides the new reference for seeking a new treatment method of HER-2-positive breast cancer.