In Vitro Killing Of Chronic Myelogenous Leukemia Cells By T-lymphocyte Modified By Humanized Chimeric T Cell Receptors

Objectives: Chronic Myelogenous Leukemia (CML)is a clonalmalignant disease of hematopoietic stem cells. The commonly usedtreatments of CML include radiation therapy, chemotherapy, allogeneichematopoietic stem cell transplantation and BCR/ABL tyrosine kinasespecific inhibitors. Almost all of these therapies have certain limitations.This study is based on the development of scientific research aboutchimeric T cell receptors (chTCR) and immunological characteristics ofCML, utilizing humanized single chain variable fragment, hinge region ofCD8molecule, transmembrane and intracellular domain of human CD28molecule and intracellular signal transduction domain of human CD3ζchain to construct retrovirus, which can expressing humanized chimericTCR (hchTCR) molecules. In vitro killing effects of T lymphocytes fromhealthy donors, modified with the retrovirus, is then detected to verify theusage of this method to treat CML by adoptive immunotherapy.Methods: Leukemia cells from CML patients are used to immunizeBALB/c mice. Cell lines secreting monoclonal antibody specific for CMLleukemia cells are produced by hybridoma technology. CDR regions of the monoclonal antibody are confirmed by bioinfromation methods and linkedwith fragment regions from human antibody, which is highly homologouswith the murine monoclonal antibody, to construct humanized scFv.Retrovirus is constructed by fusing humanized scFv, hinge region of CD8molecule, transmembrane and intracellular domain of human CD28molecule and intracellular signal transduction domain of human CD3ζchain. T-lymphocytes from healthy donors are detected of the expressing ofhumanized chimeric TCR(hchTCR), after infected by retrovirus. Also, thecytotoxic effects, proliferation and cytokine secreting of T-lymphocytes,modified by humanized chimeric TCR and stimulated with CML leukemiacells are analyzed.Results: Cell line CMA1, which can constantly secreting monoclonalantibody specific for CML leukemia cells, was produced and the titre ofthis monoclonal antibody is more than1:1500. CDR regions of thismonoclonal antibody and humanized scFv sequence were obtained. Twocell lines PHA-hchCMLTCR and PHA-8283that can persistently produceretrovirus pBABEpuro-hchCMLTCR and pBABEpuro-8283are obtained.The cytotoxicity effects, cytokine secreting of IFN-γand proliferation ofT-lymphocytes from healthy donors, infected by pBABEpuro-hchCMLTCRand stimulated with CML leukemia cells, were confirmed by in vitroanalysis.Conclusions: T-lymphocytes from healthy donors, modified by humanized chimeric TCR can killing CML leukemia cells, secreting IFN-γand proliferation in vitro. This achievement indicates that this method canbe utilized to treat CML patients with adoptive immunotherapy.