Antitumor Effect Of TCRγδ-Ig Fusion Protein Against Human Ovarian Carcinoma&Preparation And Characterization Of Functional Anti-hTCRγδAntibody And Single Chain Variable Fragment

Antibody (Ab)-based tumor-targeting immunotherapy has been rapidly developed during the last few decades. Trastuzumab and rituximab, two monoclonal antibodies used for the treatment of metastatic breast cancer and non-Hodgkin's lymphoma, respectively, have showed great clinical success. The identification of tumor-specific antigens is crucial for Ab-based tumor-targeting immunotherapy. However, only a limited number of tumor-specific antigens have been discovered so far, which greatly limits the use of Ab-based therapies in clinic.The ability of T cell receptors (TCRs) on tumor infiltrating lymphocytes (TILs) to specifically recognize tumor cells makes TCRs as ideal candidates for tumor-targeting applications. Several studies have attempted to develop novel TCRαβ-based antitumor strategies. The introduction of TCRy982into TCR-deficient JRT3-T3.5cells could enable the cells to possess the ability of tumor cell recognition and cytotoxicity, indicating the crucial role of TCRγ9δ2for y982T cells in fighting against tumors. The ability of specific recognition of tumor cells makes TCRγ9δ2available for tumor targeting therapies like Abs. Compared with TCRαβ, TCRγδ recognizes broader spectrum of tumor antigens, due to its human major histocompatibility complex (MHC)-independent manner. This may be a great advantage for the application of TCRγδ in tumor-targeting therapies.Our research focused on the study of application of TCRγδ in tumor-targeting therapies. We previously identified a complementarity determining region (CDR)3gene sequence, named OT3, in82chain from TILs in human ovarian epithelial carcinoma. TCRγ9δ2(OT3), containing the specific CDR3δ(OT3), was demonstrated to be a ovarian carcinoma specific TCR. A CDR38(OT3)-grafted Ab was constructed by grafting OT3 instead of the CDR3in the heavy chain of human IgG1. The CDR3δ(OT3)-grafted Ab showed specific binding activities to multiple tumor cell lines, but the affinities were low. To obtain an Ab-like TCRγδ-Ig fusion protein with high affinity, we constructed three TCRγδ-Ig fusion proteins by fusing the complete extracellular domains or V regions of TCRγ9δ2(OT3) to the constant domains of human IgG1. We analyzed the tumor-binding abilities of three TCRγδ-Ig fusion proteins and found TCRγ9δ2(OT3)-Fc possessing the best binding ability to tumor cell lines and tissues.In the first part of this study, we evaluated the antitumor effect of TCRy982(OT3)-Fc both in vitro and in vivo. The results showed that TCRγ9δ2(OT3)-Fc could bind to multiple tumor cell lines, including carcinoma of the ovary, cervix, lung, kidney and stomach. TCRγ9δ2(OT3)-Fc fusion protein mediated cytolysis by antibody-dependent cellular cytotoxicity (ADCC) in a dose dependent manner in vitro. TCRy982(OT3)-Fc fusion protein could also significantly inhibit tumor growth in vivo and provide a protection for the survival of animals in human ovarian cancer xenograft model. These data taken together demonstrate the feasibility of TCRγ9δ2(OT3)-Fc application for ovarian tumor-targeting therapy. TCRy982(OT3)-Fc fusion protein behaved as Abs but possessed the ability of TCRγδ to recognize antigens. Moreover, due to its broad spectrum of tumor recognition, TCRy982(OT3)-Fc has the potential to expand the range of tumors available for targeting therapies.In the second part of work, we mainly focused on the preparation and characterization of functional antibodies being able to amplify human γδT cells in vitro for adoptive immunotherapy. Based on their particular properties such as abundant interferon (IF)-γ secretion, potent cytotoxicity, and MHC-independent recognition of a broad spectrum of tumors, human γδT cells have been demonstrated as an attractive candidate for cancer immunotherapy. Unfortunately, γδT cells is a minor subset of T cells, constituting approximately1～0%of total T cells in human peripheral blood. Currently, there are mainly two methods to obtain y8T cells for adoptive cell therapy. One is to expand γδT cells by phosphoantigens such as bromohydrin pyrophosphate and1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate (HMBPP). Another is to expand y8T cells by immobilized anti-pan-TCRγδ Ab. Both methods require the participation of interleukin (IL)-2. In our laboratory, our research mainly focuses on Ab-expanded γδT cells in adoptive cellular therapy. Due to the human anti-mouse antibody (HAMA) response, the use of mouse-derived monoclonal antibody (mAb) in clinic treatments is limited. Therefore, in the second part of work, we proposed to develop a hybridoma cell line which could secrete a mAb against human TCRγδ. Thus, a humanized Ab and a single chain variable fragment (ScFv) were constructed and expressed. Biological function of the humanized Ab or ScFv was analyzed to detect whether they could substitute the mouse-derived Ab to expand human y8T cells.First, the BALB/c mice were immunized with TCRγ9δ2(OT3)-Fc. Hybridomas secreted the mAbs against human TCRγδ were selected by using traditional hybridoma methods. The Abs with the ability to expand y8T cells were identified.Subsequently, the genes of the antibody heavy (VH) and light chains (VL) were amplified by RT-PCR from the hybridoma cell line G5-4. Recombinant plasmids G5-4-pAc-K-CH3for the chimeric Ab and pET22b(+)-G5-4ScFv for ScFv were constructed. Then, the chimeric Abs were successfully expressed through baculovirus expression vector system in the sf9cells while the anti-hTCRγδ-ScFv (G5-4ScFv) was expressed in E.coli TransB (DE3).Finally, we focused on the biological function analysis of the G5-4ScFv. The results showed that G5-4ScFv could bind to γδ T cells and greatly inhibit the binding of a pan-TCRγδ antibody to y8T cells. The purity of y8T cells expanded by G5-4ScFv stimulation attained up to90%after culture for2weeks. The expanded γδT cells exhibited the abilities of IFN-y and TNF-a secretion and strong cytotoxicity against Daudi cells. Together, these results suggest that anti-hTCRγδ-ScFv with biological function has been successfully constructed and expressed in prokaryotic expression system, which provides the basis for further studying on its potential application in tumor immunotherapy.In summary, in the present study we have made several major discoveries as follows:1. We successfully constructed a TCRγ9δ2(OT3)-Fc fusion protein with both antigen-recognition properties of TCRγδ and effector functions like antibody.2. TCRy982(OT3)-Fc exhibited anti-tumor effects both in vitro and in vivo, indicating the feasibility of TCRy982(OT3)-Fc application for ovarian tumor targeting therapy.3. Due to its broad spectrum of tumor recognition, TCRγ9δ2(OT3)-Fc has the potential to expand the range of tumors available for targeting therapies.4. We have developed a hybridoma cell line, G5-4, which can secrete the mAb with the ability to expand human γδT cells.5. We constructed and expressed the chimeric Ab or ScFv of G5-4.6. G5-4ScFv possessed the properties to specifically bind to human TCRγδ and to efficiently expand γδT cells, indicating that it could substitute the mouse antibody to expand human γδT cells for adoptive immunotherapy.