A Study About The Responses Of T -Cells Against Hematologic Malignancies Mediated By TfR-BiTE

ObjectiveTransferrin Receptor?TfR,CD71?is a protein involved in the uptake of transferrin-bound iron into cells by endocytosis and is of great significance for cell proliferation.The expression of TfR on cells is very low under normal circumstances.While iron metabolism of tumor cells has changed,so the TfR expression level is significantly up-regulate on tumor cells.In this study,a bispecific antibody against TfR was designed and purified,and its efficacy in killing tumor cells was preliminarily verified through cell lines and clinical sample experiments in vitro to lay a foundation for TfR as a tumor treatment target.Methods1.The expression vector Single-TfR-CD3-His was constructed and transfected into DG44 cells.Cell line with high expression was selected and cultured in large scale.The target antibody was purified by Ni²⁺affinity chromatography and identified.2.In vitro,cell lines flow assay is used to detect the killing effect of T cells mediated by TfR-BiTE on blood tumor cells from different cell sources.3.Clinical samples of leukemia patients were collected and the expression of TfR level of tumor cells was detected.The killing effect of T cells mediated by TfR-BiTE on primary leukemia cells was detected by flow cytometry.Results1.A total of 0.8ml of TfR-BiTE was purified with a concentration of2.17mg/ml and a molecular weight of about 55kDa.2.TfR was highly expressed in all blood tumors,and TfR-BiTE can mediate T cells to kill all kinds of blood tumor cell lines:the maximum killing rate for AML cell line KG-1a reached about 40%,and the maximum killing rate for lymphocyte-derived tumor cell lines?MOLT-4 and U266?exceeded80%.Changing effector cells into activated T cells can achieve obvious killing effect under the condition of low efficiency target ratio and low antibody concentration.3.Individual differences in the expression of TfR of tumor cells in clinical leukemia patients were significant,with the positive rate ranging from 12.1%to 81.7%.T cells activated by TfR-BiTE were not effective in killing AML patients,but achieved a killing rate of 80%for B-ALL.ConclusionsIn vitro cell line and clinical sample experiments confirmed that the TfR-BiTE produced in our laboratory can activate T cells and play a killing effect on blood tumors.If we can solve the problems of antibody's production,purity,affinity and so on,TfR-BiTE has the potential to be used in clinical immunotherapy of tumors.