The Experimental Study On The Treatment Of Colorectal Cancer Combined α-gal Epitopes With Immunocytes

Background:The carbohydrate structure, a-gal epitope, is abundantly produced in non-primate. It interacts specifically with anti-Gal antibody. The synthesis of the a-gal epitope is catalyzed by enzyme α1,3GT encoded by GGTA1 gene. The evolutionary event resulted in the absence of the a-gal epitope in human. But human can produce the anti-Gal antibody. The expression difference of a-gal/anti-Gal in species has opened the way of treatment for cancers. Studies have confirmed the anti-tumor effect in animal models such as melanoma and pancreatic cancer. This is also a good new strategy for immunotherapy of colorectal cancer. However, the results of a-gal glycolipids used in clinical trials are likely to vary from one patient to the other. The treatment of a-gal glycolipids depends on the potency of the immune function in the individual patient, but the DC and T cell in cancer patients are not enough to stimulate the effective anti-tumor immune response. In recent years, the adoptive cellular immunotherapy has made considerable progress, it can be combined with the a-gal treatment to enhance the anti-tumor effect. On one hand, a-gal epitope can be used to enhance the immunogenicity of tumor cells and induce stronger DC-CTL vaccine to reconstruct the immune system of patients. On the other hand, in situ conversion of tumor lesions into autologous vaccine by expressing a-gal epitope though "virus-gene" therapy could be targeted by DC-CTL vaccine. The method may enhance anti-tumor response in multiple steps of recognition, presentation and effect.Objectives:To enhance the immunogenicity of tumor by inducing the tumor cell or tissue to stably express a-gal epitope and enhance the effect of adoptive cellular immunotherapy by developing new specific DC-CTL vaccine. To explore the feasibility and therapeutic strategy of combination therapy of a-gal and newtype DC-CTL in the treatment of colorectal cancer and to lay a foundation for further research and clinical application.Methods:1) Lentiviral vector carrying GGTA1 gene (Lenti-GGTA1) and control lentivirus (Lenti-control) being constructed through plasmids recombination and viral packaging were used to respectively infect human colorectal cancer cell SW620 and human gastric cancer cell BGC823 in order to establish stable cell lines expressing a-gal epitope. Western Blot identified the gene expression. Fluorescence microscopy observed the expression of a-gal epitope. Flow cytometry detected the positive expression rate.2) Two AAV vectors were constructed, including AAV-GGTA1 and AAV-control. A nude mouse model of subcutaneous xenotransplanted tumor was established. The ability to induce the expression of a-gal epitope in autologous tumor was observed by multi-point intratumoral injection with AAV-GGTA1, AAV-control and PBS. Western Blot was used to verify the expression of fusion protein. Immunofluorescent staining with frozen sections was used to observe the expression of α-gal epitope in tumor tissue.3) DC and CTL were isolated and cultured from healthy volunteer’s PBMCs. The DC were pulsed with different total tumor cell lysate antigens according to normal tumor cell (SW620-normal), control group infected by AAV-control (SW620-control) and a-gal experimental group (SW620-a-gal) and then stimulated CTL cells. The cell phenotype was tested by flow cytometry. The secretion of IL-12 and IL-10 by DC as well as TNF-a and IL-4 by CTL were detected by ELISA. The in vitro cytotoxicity of different CTLs was determined by LDH release assay.Results:1) After endonuclease, PCR and Western Blot identification, the Lenti-GGTA1 and Lenti-control were constructed successfully and achieved ideal titers. The stable cell lines SW620-a-gal and BGC823-a-gal were constructed successfully by infecting SW620 at 200 of MOI and BGC823 at 40 of MOI. The a-gal epitopes were widely expressed on tumor cell membrane and the positive expression rate was more than 97%.2) The AAV-GGTA1 and AAV-control were constructed successfully after endonuclease and identification of PCR and sequencing. The titers met the requirement of experiment. Nude mouse model of subcutaneous xenotransplanted tumor was established successfully and the tumor initiation rate was 100%. The fusion protein was expressed in AAV-GGTA1 group. The expression of a-gal in tumor tissues of AAV-GGTA1 group was observed by immunofluorescent staining.3) a-gal modified antigen pulsed DC had not only a significant higher ratio of CD80+, HLA-DR+and secretion of IL-12 (P<0.05), but also a significant lower secretion of IL-10 (P<0.05) in comparison with control group and normal group. CTL being stimulated by a-gal modified antigen pulsed DC had a significant higher ratio of CD8+T cells, NKT cells and the secretion level of TNF-a as well as a lower ratio of Tregs (P<0.05). The killing ability of CTL stimulated by a-gal-modified antigen pulsed DC was enhanced.Conclusions:1) In vitro experiment, the lentivirus can induce human tumor cell to stably express the a-gal epitopes and improve the immunogenicity of tumor cell.2) In vivo experiment, the AAV can be used to induce the human CRC tissue widely express the a-gal epitopes and improve the immunogenicity of tumor tissue.3) DC vaccine can be induced by a-gal modified tumor cell antigen. The method can not only effectively activate the second signal system and increase the DC activation level and the secretion of IL-12, but also decrease the secretion of IL-10. Stronger CTL can be stimulated by such DC vaccine. The ratio of CD8 positive T cells, NKT cells and the secretion of TNF-a were increased and the ratio of Treg cells was reduced.