| Background and Objective The cause of leukemia formation, growth, metastasis and relapse is that there is a group of leukemia stem cells (LSC) in vivo of patients. It is the same with the normal stem cells self-renewal and differentiation potential. It is only 0.25-1.0% in whole blood cells. Traditional treatment usually combined cytotoxic chemo- therapy with hematopoietic stem cell transplantation, but it will destroy normal cells while killing tumor cells. And it can only kill the most differentiated Cancer cells and hardly afect the tumor stem cells, cells. That is why most of the therapies do not achieve good results.Today, Cytokine fusion protein technology is a hot point in immunological research. The fusion of Cytokines and antibody fragment, can not only has antigen specific binding properties, also has multifunctional cytokine activity. Cytokines can be directed by antibodies to to the target, and then play a efficient biological function with reduceing systemic side effects.Immunotherapy based on peripheral blood lymphocytes (PBL) has become an important biological treatment of anti-tumor strategy. T cells in PBL directly kill tumor cells by exposure to the formation of T cell-tumor cell synapse-like structure. The complexity of the process of T cell recognizing tumor cells provides many opportunities for tumor cells to escape T cell recognition. CD3 are expressed on the T lymphocytes and NK cells.It can enrich cytolytic T lymphocytes.In this experiment, I have constructed the antiCD3/IL3 vector of which poducts are capable of redirecting cytotoxic T lymphocytes or NK cells towards leukemia stem cells cells that express tumour-associated antigens such as CD 123. we have also constructed antiCD3*/m2IL3,antiCD3/m2IL3,antiCD3*/⊿IL3,antiCD3/⊿IL3 to settle the problem of the loss of the C-terminal IL3 peptide.To improve fusion protein stability,I have introduced cysteine residues into the V domains of CD3 to promote the disulphide crosslinking of the dimer. Then expressed these vectors in E.coli 16C9, purified by 6×His-tag affinity chromatography and analyzed their biological activities in vitro.Methods Utilizing overlap PCR and PCR to construct the antiCD3/IL3 expressing vector pAYZCD3/IL3. Then introduced cysteine residues into the VL-100 and VH-44 domains of CD3 and built the expressing vector pAYZCD3*/IL3 of disulphide crosslinking of antiCD3/IL3 also with overlap PCR and PCR. We construct expressing vector pAYZantiCD3*/m2IL3, pAYZantiCD3/m2IL3, pAYZantiCD3*/⊿IL3, pAYZ antiCD3/⊿IL3 with the same method. Transformed these six vectors into E.coli 16C9 for expressing, respectively and purified the fusion proteins by 6 X His-tag affinity chromatography and confirmed the products by 12% SDS-PAGE and western-blot. Antigen binding activity of the fusion proteins to Jurkat cells which are CD3 positive or M07e cells which are CD 123 positive were analyzed by indirect immunological fluorescent assays or competitive immunological fluorescent assays with FACS. Fusion proteins were incubated at 37℃for various times in PBS containing 0.2%(w/v) human serum albumin (HSA) at a concentration of 20μg/mL. Comparing the remaining binding activity with Jurkat or M07e cells (1×106 cells) which were determined by flow cytometry.A non-radioactive cytotoxicity assay was performed to evaluate the efficacy of the fusion proteins in mediating T-cell cytotoxicity by fluorescent dyes Calcein-AM. Human peripheral blood lymphocytes (PBL) were isolated using Ficoll-Hypaque density-gradient centrifugation from heparinized blood of healthy volunteers and depleted of monocytes by adherence to plastic flasks. In vitro cytotoxity test was performed by using M07e and TF1 cells as targets and PBL as effectors firstly. The cytotoxity of effectors mediated by fusion proteins in different concentration and E/T ratio was compared to PBS, antiCD3/antiCD19. Additionally CD 123 negtive cells k562 was set up as unrelated target. Releasing of perforin and granzyme A of activated T cells were analyzed by qPCR.Result the DNA sequence of the expression vector containing antiCD3/IL3,antiCD3/m2IL3,antiCD3/⊿IL3 or disulphide stabilized antiCD3/IL3,antiCD3/m2IL3,antiCD3/⊿IL3 were correct. The yield of purified antiCD3/mIL3 was up to 1mg/L, the yield of purified antiCD3/⊿IL3 was up to 2mg/L. There were two bands of antiCD3/m2IL3,antiCD3/⊿IL3 at molecular weight 30kD and 27kD indicated by SDS-PAGE and one band at 30kD by western-blot because only the VH of the fusion proteins have the his6-tag.While there were several bands at 57kD,30 kD,27KD of antiCD3*/m2IL3,antiCD3*/⊿IL3 in the non-reducing SDS-PAGE and two in the reduing conditions which were located at 30kD and 27kD respectively. The fusion proteins can specifically bind to CD123+ M07e cells and CD3+ Jurkat cells while competitively inhibit binding of antiCD 123 and HIT3a to such cells. AntiCD3*/m2IL3,antiCD3*/⊿IL3 was much more stable than antiCD3/m2IL3,antiCD3/⊿IL3 evaluated by flow cytometry when incubatied in PBS containing 0.2%(w/v) HSA at 37℃for prolonged periods of time. The fusion proteins increased the cytotoxity of T cells in vitro apparently than the contrast groups (p<0.05). The release of perforin and Granzyme A of activated T cells were increased much more than the other groups (p<0.05).Conclusion Successfully constructed the expression vector of antiCD3/IL3 antiCD3/m2IL3,antiCD3/⊿IL3 and its disulphide stabilized configuration antiCD3*/ m2IL3,antiCD3*/⊿IL3, acquired the purified fusion proteins in high level yield with high specific affinity, Compared with the antiCD3/m2IL3,antiCD3/⊿IL3, the antiCD3*/m2IL3,antiCD3*/⊿IL3 obtained was more stable in human serum at 37℃, without loss of affinity or cytotoxicity activity in vitro. |
PDF Full Text Request