表达嵌合T细胞受体的T淋巴细胞介导的肿瘤过继性免疫治疗作用机理研究
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摘要
有效的抗肿瘤免疫需要机体的免疫系统能够识别肿瘤抗原,并最终利用免疫效应机制清除肿瘤细胞。在人们寻找肿瘤抗原的几十年中,发现大多数的肿瘤抗原是自身抗原,它们异位表达或过表达于肿瘤细胞,能诱导免疫系统产生忽视、克隆清除、和免疫耐受等来阻碍机体的抗肿瘤免疫反应。另外,肿瘤细胞还能通过降低肿瘤抗原和MHC分子的表达、通过分泌抑制性的细胞因子等机制逃脱机体的免疫监视。既使肿瘤抗原能够被识别,并诱导出肿瘤特异性的CTL,也不一定完全清除肿瘤细胞,因为CTL必须有足够的数量迁移到肿瘤部位来发挥细胞毒作用,并能克服肿瘤细胞逃脱免疫监视的种种机制。过继性免疫治疗是通过给荷瘤机体输注抗肿瘤免疫效应细胞,如致敏或激活的淋巴细胞的方法来杀伤肿瘤。这些方法曾成功地应用于黑色素瘤和肾细胞癌的治疗,然而对于大多数实体肿瘤如卵巢癌、结肠癌、乳腺癌来说并未取得满意的治疗效果,主要因为分离和培养足够数量的肿瘤特异性的淋巴细胞非常困难。另外,实体肿瘤的屏障作用和注入体内肿瘤特异性抗体的快速清除,使得体液免疫在肿瘤免疫治疗中的作用受到很大的限制。为此,有研究者将抗体对肿瘤抗原的高度亲和性和T淋巴细胞的杀伤作用相结合,利用基因工程技术,构建嵌合型的人工T细胞受体(CARs),它包括识别肿瘤相关抗原的单链抗体、共刺激分子和T细胞的活化CD3ζ链,通过逆转录病毒载体,高效转染T淋巴细胞。使得T细胞能以MHC非限制性的方式识别并杀伤肿瘤细胞。越来越多的证据表明,CD4~+T细胞在肿瘤免疫治疗中发挥重要的作用,而肿瘤特异性的CD4~+T细胞的获得非常困难。由于嵌合型的T细胞受体不仅能转染CD8~+T细胞,还能转染CD4~+T细胞,且两者均能被相应的抗原分子激活,因此在抗肿瘤免疫中发挥重要的作用。据Hiroshi报道,用CARs修饰的Thl细胞与Tcl细胞相比,能分泌更多的IL-2和IFN-γ等细胞因子,将Thl与Tcl联合应用,在BALB/cRAG-2~(-/-)小鼠体内能起到协同治疗肿瘤的目的。
erbB2癌基因是表皮生长因子受体家族成员,是具有酪氨酸激酶活性的,分子量为185kDa的跨膜糖蛋白。Her2/neu普遍表达于上皮性肿瘤,过表达于约30%的卵巢癌和乳腺癌,并且这种过表达往往预示着肿瘤的高恶性度和险恶的预后。由于在乳腺癌和卵巢癌患者体内能检测到抗erbB2特异性的抗体和T淋巴细胞,因
Effective tumor immunity requires immune recognition of tumor antigens and the tumor eradication by immune effector cells. Over the past decades, immunologists and oncologists have been searching for tumor-specific antigens. However, most antigens that have been identified so far are self-antigens, many of which are either exotically expressed or over-expressed than in normal tissues. Tumor cells have the abilities to escape from the immune response through mechanisms such as anergy, apoptosis and suppression. Although tumor antigens can be recognized, they are not sufficient to enable tumor eradication until the primed cells expand to sufficient numbers, migrate to tumor sites, mature into effector cells and overcome the tumor's abilities of escaping from the immune surveillance. The infusion of tumor-specific T cells into the tumor-bearing host referred to as adoptive immunotherapy , such as lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes (TIL) can be highly effective in the destruction of tumor burdens in some patients with melanoma and renal carcinoma. However, this therapy has been unsuccessful in the treatment of more prevalent cancers such as ovarian, breast cancers and colon carcinoma. Three problems have commonly precluded more universally effective adoptive immunotherapy including ①difficulties in isolating and culturing tumor specific T cells from all tumor types; ② poor tumor localization of ex vivo-modified lymphocytes upon adoptive transfer; and ③ IL-2 toxicity. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effectors of the immune system. Attempts to improve the efficacy of adoptive immunotherapy have led to the development of novel strategies that combine advantages of T cell-based and antibody-based immunotherapy by gene engineering technology to construct the chimeric artificial T cell receptor which comprises antibody fragments specific for tumor-associated antigens and a cellular activation motif. The artificial chimeric antigen receptors (CARs) will be transduced into T cells by retroviral transduction system and the antigen recognition is therefore
not restricted to major histocompatibility complex. This strategy will be of great value for adoptive tumor immunotherapy. On the other hand, increasing evidence from animal studies and clinical trials indicated that CD4+ T cell plays a central role in orchestrating host immune responses against cancer. Adoptive transfer using tumor-specific Thl and Tel cells is a promising therapeutic strategy for tumor immunotherapy. However, its application has been hampered because of difficulties in generating tumor-specific Thl cells from tumor patients. To overcome this problem, we transfected CD4+ and CD8+ T cells with CARs to generate both helper and CTL responses in this projects.ErbB2 or Her-2/neu, a member of the epidermal growth factor receptor family with tyrosine kinase activity, encodes an 185kDa transmembrane protein receptor. HER-2/neu is ubiquitously expressed in many epithelial tumors and known to be over-expressed in approximately 30% of all ovarian and breast cancers, 35-45% of all pancreatic carcinomas and up to 90% of colorectal carcinomas and this overexpression was shown to correlate with aggressiveness of malignancy and poor prognoses. Because erbB2-specific antibody and T cells are detected in breast and ovarian cancer patients, erbB2 is recognized as a target of immunotherapy.In part I , we first constructed a retroviral vector which expressed the chimeric scFv-anti-erbB2-CD28- ?> receptor by gene engineering technology. For detecting purpose, we cloned a c-myc tag epitope (EIKLISEEDL) at the C terminus of VL. Anti-erbB2 scFv was obtained from VH and VL by overlapping PCR, CD28 molecule and CD3 ￡ cytoplasmic domain were obtained by PCR, respectively. An appropriate spacer of outer cytoplasmic CD28 was inserted between scFv and membrane to keep its right folding, and the intracellular domain of CD3 ￡ chain and CD28 domain as the activation motif and costimulation motif, respectively, could redirect the specificity of a transfected mouse T lymphocytes towards erbB2-expression tumor cells. The chimeric antigen receptors(CARs) were composed of scFv-anti-erbB2> CD28 molecule and cytoplasmic regions of ￡ chain. A three-plasmid expression system was used to generate MLV-derived retroviral vector particles by transient transfection. Viral
supernatant was harvested at 48h after transfection, passed through a 0.45 v m membrane, and refrigerated. Concentrated viral stocks were prepared by centrifugation of viral supernatant in an SA-300 rotor at 50000g, 4°C, for 1.5h. Virus was resuspended in an appropriate volume of 1640 and stored at -80°C. Viral titers were estimated by transduction of NIH3T3 cells.In part II, we engineered the chimeric antigen receptor on mouse T lymphocytes and conducted experiments in vitro. Firstly, the CD4+ and CD8+ T cells were sorted by MACS separators and the purity was up to 90%. After stimulated by ConA or antibodies, the cells were infected by condensed virus. The transduction efficiency of retroviral vectors containing scFv-CD28- 4 or a mock gene into CD4+ and CD8+ T cells were determined by measuring the percentage of anti-c-myc-FITC cells using flow cytometry, ranged from 35% to 45%, and CD4+ and CD8+ T cells subsets were transduced at similar efficiencies. 1 > We detected the antigen-specific binding ability of gene modified T cells. We stained T-CARs and T-mock cells with Her2/Ig, respectively. T-CARs conjugated with Her2/Ig detected by flow cytometry. 2> We evaluated the ability of transduced CD4+ and CD8+ T cells to mediate specific target cell lysis in a non-radioactivation cytotoxicity assay. T cells expressing scFv-anti-erbB2 receptor were able to lyse the erbB2+ tumor cells D2F2/E2 and SK-BR-3, but not erbB2" MCF-7 and D2F2/E2-mask cell lines. Mock-transduced T cells did not lyse D2F2/E2, SK-BR-3 > D2F2/D2-mask or MCF-7 cells. CD8+T-CARs cells exhibited a stronger cytotoxicity compared with CD4+ T-CARs cells, indicating the cytotoxicity was antigen-dependent and CARs induced. 3 > We also demonstrated the ability of gene-modified T cells to expand in culture after specific ligation with irradiated erbB2+ tumor target for 72 hours. Compared with antibody-mediated stimulation of endogenous CD3 and CD28 receptors expressed on the same T cells, the gene-modified T cells have an equivalent proliferation ability. 4, The capacity of CD4+and CD8+ T-CARs cells to produce cytokines in response to erbB2-expressing tumor cells were examined by measuring cytokine levels in the culture supernatant. CD4+ and CD8+ T-CARs cells produced high levels of IFN- y and GM-CSF when they were cocultured with erbB+ D2F2/E2 and SK-BR-3 target cells. T cells transduced with mock virus
secret only background level of cytokines when incubated with D2F2/E2 or MCF-7 cells. In addition, These CD4+ or CD8+T-CARs cells did not produce IL-4, IL-5 or IL-10 in response to stimulation by erbB+ tumor cells(data not shown), indicating that CD4+ and CD8+ T-CARs were successfully induced from nonspecifically activated T cells in Th 1-polariaing conditions. CD4+and CD8+T-CARs cells did not produce IFN-Y and GM-CSF in response to erbB2' MCF-7 and D2F2/E2-mask cells, indicating that its production was induced in an antigen-specific manner.In part III, the antitumor efficacy of gene-modified T cells was evaluated against the D2F2/E2 tumor cell line following adoptive transfer. In the first model, 1X105 D2F2/E2 tumor cells were injected subcutaneously into 3 groups of BALB/c nude mice(n=10). Enriched CD3+ T cells from BALB/c mice(transduced with the scFv-CD28- C receptor or mock transduced T cells ) were injected intravenously into tumor-inoculated mice at 6 hours(day 0, 5X106) and 24 hours(day 1, 5X106). Only tumor cells were injected and non-T cells were transferred as control. In the second model, 1X105 D2F2/E2 tumor cells were injected subcutaneously into 3 groups of BALB/c mice(n=10). The treatment regimen was as the same as in the first model. After adoptive transfer of T cells, tumor growth in mice was measured daily by Caliper Square along the perpendicular axes of the tumor. The data were recorded as mean volume+ SEM. Tumor growth in mice was observed daily for at least 60 days. After the initial tumor challenge, tumor free mice were rechallenged with 1 X 105 D2F2/E2 tumor cells in the opposite flank. Naive mice were also injected with 1 X 105 D2F2/E2 cells to verify tumor growth. In BALB/c nude mice, although CD3+ T-CARs cells strongly inhibited tumor growth in vivo, only 2 out of 10 mice completely eradicated the tumor. In BALB/c syngeneic mice, 7 out of 10 completely rejected the tumors, and then we rechallenged them with 1 X 105 D2F2/E2 in the opposite flank of the tumor free mice. Any tumors were not palpated for 4 weeks observation in rechallenged BALB/c mice and two BALB/c nude mice all grew tumors. Spleen T cells were harvested from tumor free mice and control mice; cytotoxicity capacity of T cells was determined in non-radioactivation cytotoxicity assay. T cells coming from tumor-free mice were able to lyse not only D2F2/E2 cells but also D2F2/E2-mask cells, although the lysis function
to D2F2/E2 was stronger than to D2F2/E2-mask cells. We also evaluated IFN- Y and GM-CSF secretion when T cells cocultured with D2F2/E2, D2F2/E2-mask cells in 12 well plates for 20 hours. Supernatants were harvested and IFN- y and GM-CSF secretion by transduced T cells were dertermined by ELISA. T cell coming from tumor-free mice were able to produce GM-CSF and IFN- Y when cocultured with D2F2/E2^ D2F2/E2-mask cells. While T cells coming from control mice were unable to produce any cytokines. Histologically, tumors treated by T-CARs showed massive destruction accompanied by a large degree of lymphocytic infiltration in BALB/c mice. Immunohistochemistry assays showed that massive gene modified T cells detected by anti-c-myc-biotin staining in tumor site, lymph nodes, spleen and thymus. Tumors treated by T-mock cells were conserved with no evidence of lymphocytes infiltration and tissue destruction.To investigate whether these CARs were involved in immune synapse when T cells were stimulated by corresponding antigens. In part IV, we studied the colocalization of CARs with lipid rafts using a raft marker fluorescein isothiocyanate (FITC)-labled cholera toxin (CTx) B subunit, which binds GM1 glycosphingolipid, and anti-c-myc-CY3 binds to CARs. In unstimulated T-CARs cells, the surface distribution of FITC and CY3 appeared homogeneous. When these T cells were cultured with precoated Her2/Ig protein, within 30 min FITC and CY3 redistributed to form a dense cap. However, T-mock cells had not any changes whether stimulated or unstimulated by antigens. So we think maybe the scFv-CD28- C chimera synergistic the signaling activities of endogeneous TCR and CD28 signaling pathways. Further exploration will be made about the signal transduction by the CARs in the future.In this experiment, the artificial TCR was transduced into primary mouse T lymphocytes by retroviral transduction system and antigen recognition is therefore not restricted to MHC. CD4+ T cells in this process played a very important value. The CD8+ T-CARs cells transfused into mice could directly kill the target cells, while the CD4+ T-CARs have many anti-tumor effects mainly induced by cytokines, such as IFN-Y and GM-CSF with inflammatory and antitumor potentials. In the last but not the least, the mechanism of the function played by CARs was determined by confocol
erbB2癌基因是表皮生长因子受体家族成员,是具有酪氨酸激酶活性的,分子量为185kDa的跨膜糖蛋白。Her2/neu普遍表达于上皮性肿瘤,过表达于约30%的卵巢癌和乳腺癌,并且这种过表达往往预示着肿瘤的高恶性度和险恶的预后。由于在乳腺癌和卵巢癌患者体内能检测到抗erbB2特异性的抗体和T淋巴细胞,因
Effective tumor immunity requires immune recognition of tumor antigens and the tumor eradication by immune effector cells. Over the past decades, immunologists and oncologists have been searching for tumor-specific antigens. However, most antigens that have been identified so far are self-antigens, many of which are either exotically expressed or over-expressed than in normal tissues. Tumor cells have the abilities to escape from the immune response through mechanisms such as anergy, apoptosis and suppression. Although tumor antigens can be recognized, they are not sufficient to enable tumor eradication until the primed cells expand to sufficient numbers, migrate to tumor sites, mature into effector cells and overcome the tumor's abilities of escaping from the immune surveillance. The infusion of tumor-specific T cells into the tumor-bearing host referred to as adoptive immunotherapy , such as lymphokine-activated killer (LAK) cells and tumor-infiltrating lymphocytes (TIL) can be highly effective in the destruction of tumor burdens in some patients with melanoma and renal carcinoma. However, this therapy has been unsuccessful in the treatment of more prevalent cancers such as ovarian, breast cancers and colon carcinoma. Three problems have commonly precluded more universally effective adoptive immunotherapy including ①difficulties in isolating and culturing tumor specific T cells from all tumor types; ② poor tumor localization of ex vivo-modified lymphocytes upon adoptive transfer; and ③ IL-2 toxicity. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effectors of the immune system. Attempts to improve the efficacy of adoptive immunotherapy have led to the development of novel strategies that combine advantages of T cell-based and antibody-based immunotherapy by gene engineering technology to construct the chimeric artificial T cell receptor which comprises antibody fragments specific for tumor-associated antigens and a cellular activation motif. The artificial chimeric antigen receptors (CARs) will be transduced into T cells by retroviral transduction system and the antigen recognition is therefore
not restricted to major histocompatibility complex. This strategy will be of great value for adoptive tumor immunotherapy. On the other hand, increasing evidence from animal studies and clinical trials indicated that CD4+ T cell plays a central role in orchestrating host immune responses against cancer. Adoptive transfer using tumor-specific Thl and Tel cells is a promising therapeutic strategy for tumor immunotherapy. However, its application has been hampered because of difficulties in generating tumor-specific Thl cells from tumor patients. To overcome this problem, we transfected CD4+ and CD8+ T cells with CARs to generate both helper and CTL responses in this projects.ErbB2 or Her-2/neu, a member of the epidermal growth factor receptor family with tyrosine kinase activity, encodes an 185kDa transmembrane protein receptor. HER-2/neu is ubiquitously expressed in many epithelial tumors and known to be over-expressed in approximately 30% of all ovarian and breast cancers, 35-45% of all pancreatic carcinomas and up to 90% of colorectal carcinomas and this overexpression was shown to correlate with aggressiveness of malignancy and poor prognoses. Because erbB2-specific antibody and T cells are detected in breast and ovarian cancer patients, erbB2 is recognized as a target of immunotherapy.In part I , we first constructed a retroviral vector which expressed the chimeric scFv-anti-erbB2-CD28- ?> receptor by gene engineering technology. For detecting purpose, we cloned a c-myc tag epitope (EIKLISEEDL) at the C terminus of VL. Anti-erbB2 scFv was obtained from VH and VL by overlapping PCR, CD28 molecule and CD3 ￡ cytoplasmic domain were obtained by PCR, respectively. An appropriate spacer of outer cytoplasmic CD28 was inserted between scFv and membrane to keep its right folding, and the intracellular domain of CD3 ￡ chain and CD28 domain as the activation motif and costimulation motif, respectively, could redirect the specificity of a transfected mouse T lymphocytes towards erbB2-expression tumor cells. The chimeric antigen receptors(CARs) were composed of scFv-anti-erbB2> CD28 molecule and cytoplasmic regions of ￡ chain. A three-plasmid expression system was used to generate MLV-derived retroviral vector particles by transient transfection. Viral
supernatant was harvested at 48h after transfection, passed through a 0.45 v m membrane, and refrigerated. Concentrated viral stocks were prepared by centrifugation of viral supernatant in an SA-300 rotor at 50000g, 4°C, for 1.5h. Virus was resuspended in an appropriate volume of 1640 and stored at -80°C. Viral titers were estimated by transduction of NIH3T3 cells.In part II, we engineered the chimeric antigen receptor on mouse T lymphocytes and conducted experiments in vitro. Firstly, the CD4+ and CD8+ T cells were sorted by MACS separators and the purity was up to 90%. After stimulated by ConA or antibodies, the cells were infected by condensed virus. The transduction efficiency of retroviral vectors containing scFv-CD28- 4 or a mock gene into CD4+ and CD8+ T cells were determined by measuring the percentage of anti-c-myc-FITC cells using flow cytometry, ranged from 35% to 45%, and CD4+ and CD8+ T cells subsets were transduced at similar efficiencies. 1 > We detected the antigen-specific binding ability of gene modified T cells. We stained T-CARs and T-mock cells with Her2/Ig, respectively. T-CARs conjugated with Her2/Ig detected by flow cytometry. 2> We evaluated the ability of transduced CD4+ and CD8+ T cells to mediate specific target cell lysis in a non-radioactivation cytotoxicity assay. T cells expressing scFv-anti-erbB2 receptor were able to lyse the erbB2+ tumor cells D2F2/E2 and SK-BR-3, but not erbB2" MCF-7 and D2F2/E2-mask cell lines. Mock-transduced T cells did not lyse D2F2/E2, SK-BR-3 > D2F2/D2-mask or MCF-7 cells. CD8+T-CARs cells exhibited a stronger cytotoxicity compared with CD4+ T-CARs cells, indicating the cytotoxicity was antigen-dependent and CARs induced. 3 > We also demonstrated the ability of gene-modified T cells to expand in culture after specific ligation with irradiated erbB2+ tumor target for 72 hours. Compared with antibody-mediated stimulation of endogenous CD3 and CD28 receptors expressed on the same T cells, the gene-modified T cells have an equivalent proliferation ability. 4, The capacity of CD4+and CD8+ T-CARs cells to produce cytokines in response to erbB2-expressing tumor cells were examined by measuring cytokine levels in the culture supernatant. CD4+ and CD8+ T-CARs cells produced high levels of IFN- y and GM-CSF when they were cocultured with erbB+ D2F2/E2 and SK-BR-3 target cells. T cells transduced with mock virus
secret only background level of cytokines when incubated with D2F2/E2 or MCF-7 cells. In addition, These CD4+ or CD8+T-CARs cells did not produce IL-4, IL-5 or IL-10 in response to stimulation by erbB+ tumor cells(data not shown), indicating that CD4+ and CD8+ T-CARs were successfully induced from nonspecifically activated T cells in Th 1-polariaing conditions. CD4+and CD8+T-CARs cells did not produce IFN-Y and GM-CSF in response to erbB2' MCF-7 and D2F2/E2-mask cells, indicating that its production was induced in an antigen-specific manner.In part III, the antitumor efficacy of gene-modified T cells was evaluated against the D2F2/E2 tumor cell line following adoptive transfer. In the first model, 1X105 D2F2/E2 tumor cells were injected subcutaneously into 3 groups of BALB/c nude mice(n=10). Enriched CD3+ T cells from BALB/c mice(transduced with the scFv-CD28- C receptor or mock transduced T cells ) were injected intravenously into tumor-inoculated mice at 6 hours(day 0, 5X106) and 24 hours(day 1, 5X106). Only tumor cells were injected and non-T cells were transferred as control. In the second model, 1X105 D2F2/E2 tumor cells were injected subcutaneously into 3 groups of BALB/c mice(n=10). The treatment regimen was as the same as in the first model. After adoptive transfer of T cells, tumor growth in mice was measured daily by Caliper Square along the perpendicular axes of the tumor. The data were recorded as mean volume+ SEM. Tumor growth in mice was observed daily for at least 60 days. After the initial tumor challenge, tumor free mice were rechallenged with 1 X 105 D2F2/E2 tumor cells in the opposite flank. Naive mice were also injected with 1 X 105 D2F2/E2 cells to verify tumor growth. In BALB/c nude mice, although CD3+ T-CARs cells strongly inhibited tumor growth in vivo, only 2 out of 10 mice completely eradicated the tumor. In BALB/c syngeneic mice, 7 out of 10 completely rejected the tumors, and then we rechallenged them with 1 X 105 D2F2/E2 in the opposite flank of the tumor free mice. Any tumors were not palpated for 4 weeks observation in rechallenged BALB/c mice and two BALB/c nude mice all grew tumors. Spleen T cells were harvested from tumor free mice and control mice; cytotoxicity capacity of T cells was determined in non-radioactivation cytotoxicity assay. T cells coming from tumor-free mice were able to lyse not only D2F2/E2 cells but also D2F2/E2-mask cells, although the lysis function
to D2F2/E2 was stronger than to D2F2/E2-mask cells. We also evaluated IFN- Y and GM-CSF secretion when T cells cocultured with D2F2/E2, D2F2/E2-mask cells in 12 well plates for 20 hours. Supernatants were harvested and IFN- y and GM-CSF secretion by transduced T cells were dertermined by ELISA. T cell coming from tumor-free mice were able to produce GM-CSF and IFN- Y when cocultured with D2F2/E2^ D2F2/E2-mask cells. While T cells coming from control mice were unable to produce any cytokines. Histologically, tumors treated by T-CARs showed massive destruction accompanied by a large degree of lymphocytic infiltration in BALB/c mice. Immunohistochemistry assays showed that massive gene modified T cells detected by anti-c-myc-biotin staining in tumor site, lymph nodes, spleen and thymus. Tumors treated by T-mock cells were conserved with no evidence of lymphocytes infiltration and tissue destruction.To investigate whether these CARs were involved in immune synapse when T cells were stimulated by corresponding antigens. In part IV, we studied the colocalization of CARs with lipid rafts using a raft marker fluorescein isothiocyanate (FITC)-labled cholera toxin (CTx) B subunit, which binds GM1 glycosphingolipid, and anti-c-myc-CY3 binds to CARs. In unstimulated T-CARs cells, the surface distribution of FITC and CY3 appeared homogeneous. When these T cells were cultured with precoated Her2/Ig protein, within 30 min FITC and CY3 redistributed to form a dense cap. However, T-mock cells had not any changes whether stimulated or unstimulated by antigens. So we think maybe the scFv-CD28- C chimera synergistic the signaling activities of endogeneous TCR and CD28 signaling pathways. Further exploration will be made about the signal transduction by the CARs in the future.In this experiment, the artificial TCR was transduced into primary mouse T lymphocytes by retroviral transduction system and antigen recognition is therefore not restricted to MHC. CD4+ T cells in this process played a very important value. The CD8+ T-CARs cells transfused into mice could directly kill the target cells, while the CD4+ T-CARs have many anti-tumor effects mainly induced by cytokines, such as IFN-Y and GM-CSF with inflammatory and antitumor potentials. In the last but not the least, the mechanism of the function played by CARs was determined by confocol
引文
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21、 Disis, M.L., E.Calenoff,G.McLaughlin, and A.E.Murphy. et al. Existent T-cell and antibody immunity to Her-2/neu protein in patients with breast cancer. Cancer Res. 1994;54:16.
22、 Peoples,G.E.,P.S.Goedegebuure,and T.J.Eberlein. et al. Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same Her2/neu-derived peptide. Proc. Natl. Acad. Sci. USA 1995;92:432.
23、 Fisk, B.,B.Anderson, and C.GIoannides. Identification of naturally processed human ovarian peptides recognized by tumor-associated CD8+ cytotoxicity T lymphocytes. Cancer res.l997;57:87.
24、 Pages JC, Bru T. Tool box for retrovectorologists. J Gene med 2004;6:S67-S82.
25、 Cone RD, Mulligan RC. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host tange. Proc Natl Acad Sci USA.1984;81:6349-6353.
26、 Cavazzana-Calvo M et al. Gene therapy of human severe combined with nonmyeloablative conditioning. Science.2002;288:669-672.
27、 Kay MA, Glorioso JC and Naldini L et al. Viral vectors for gene therapy: the art
of turning infectious agents into vehicles of therapeutics. Nat. med. 2001;7:33-40.
28、 Lois C, Refae Y, Qin XF, et al. Retrovirus as tools to study the immune system. Curr opin Immunol. 2001; 13:496-504.
29、 Naldini L, Blomer U, Gallay P. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vectors. Science. 1996;272:263-267.
30、 Pringle CR. Virus taxonomy-1999. The universal system of virus taxonomy, updated to include the new proposals ratified by the International Committee on Taxonomy of Viruses. Arch Virol 1999; 144:421-429.
31、 De Felipe P, Izquierdo M. Retroviral vectors for gene therapy. Hum. Gene Ther. 2000;11:1921-1931.
32、 Burns JC, Friedmann T, Driever W. et al.Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmamalian cells. Proc. Natl. Acad. Sci. USA 1993;90:8033-8037.
33、 Lavillette D, Russell SJ, Cosset FL. Retargeting gene delivery using surface-engineered retroviral vector particles. Curr Opin Biotechnol. 2001 Oct; 12(5):461-6. review.
34、 Brocker T, Karjalainen K. Adoptive tumor immunity mediated by lymphocytes bearing modified antigen-specific receptors. Adv Immunol. 1998;68:257.
35、 Eshhar Z. tumor-specific T-bodies: towards clinical applications. Cancer Immunol Immunother.l997;45:131.
36、 Hombach, A., A.Wieczarkowiecz, T. Marquardt,C. Heuser,L. Usai,C. Pohl,B- Seliger, and H.Abken. Tumor-specific T cell activation by recombinant immunoreceptors: CD3 (?) signaling and Cd28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 (?) signaling receptor molecule. J.Immunol.2001; 167:6123
37、 Maher, J.,Brentjens R J and Sadelain M. Human T-lymphocytes cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor. Nature Biotechnol.2002;20:70-75.
38、 Eshhar. Z,Waks,T. Gross. G. et al. Specific activation and targeting of cytotoxic lymphocytes through chimetic single chains consisting of antibody-binding domains and a gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc. Natl Acad Sci USA 1993:90:720-724.
39、 Altenschmidt, U.,Klundt,E.,Groner B. Adoptive transfer of in vivo-targeted,activated T lymphocytes results in total tumor regression. J.Immunol. 1997; 159:5509-5515.
40、 Darcy,P.K. et al. Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineering CTL. J. Immunol.2000; 164:3705-3712.
41、 Haynes, N.M. et al. Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors. Blood 2002; 1000:3155-3163.
42、 Mcguinness.r.p. et al. Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor.Hum Gene Ther.l999;10:165-173.
43、 Hwu,P. et al. In vivo anti-tumor activity of T cells redirected with chimeric antibody/T-cell receptor genes. Cancer res. 1995; 55:3369-3373.
44、 Brentjens R.,Latouche,J.B.,Sadelain, M. et al. In vivo anti-tumor activity of genetically modified T cells is dependent on the method of ex vivo T cell expansion. Blood. 2002; 100:577a.
45、 Hombach A,, Wieczarkowiecz A, Marquardt T, et al. Tumor-specific T cell activation by recombinant immunoreceptors: CD3 (?) signaling and CD28 stimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD33 signaling receptor molecule. J immunol. 2001;167:6123.
46、 Geiger TL, Leitenberg D, Flavell RA. The TCR zeta-chain immunoreceptor tyrosine-based activation motifs are sufficient for the activation and differentiation of primary T lymphocytes. J Immunol. 1999; 162:5931-5939.
47、 Cohen, PA.et al. CD4+ T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection.Crit.Rev.Immunol. 2000; 20:17-56.
48、 Pardall, D.M. and Topalian, SL. The role of CD4+ Tcell responses in antitumor immunity. Curr. Opin. Immunol. 1998; 10:588-594.
49、 Mumberg, D. et al. CD4+ T cells eliminates MHC class II-negative cancer cells in vivo by indirect effects of IFN- Y .Proc. Natl.Acad.Sci. USA 1999; 96:8633-8638.
50、 Qin Z, Blankenstein T. CD4+ T cells mediated tumor rejection involved inhibition of angiogenesis that is dependent on IFN- y receptor expression by nonhematopoietic cells. Imunity. 2000; 12:677-686.
51、 Hiroshi G, Takemasa T, Yoshinori S, et al. Generation and targeting of human tumor-specific Tcl and Thl cells transduced with a lentivirals containing a chimeric immunoglobulin T-cell receptor. Cancer research. 2004;64:1490-1495.
52、 Pawelec C, Zeuthen J, Kiessling R. Escape from host antitumor immunity. Crit Rev Onbog.
1997;8:111.
53、 Shinkai Y, Ma A, Cheng HL, et al. CD3 epsilon and CD3 zeta cytoplasmic domains can independently generate signals for T cell development and function. Immunity. 1995;2:401.
54、 Davis MM, Boniface JJ, Reich Z et al. Ligand recognition by alpha beta T cell receptors. Annu Rev Immynol. 1998; 16:523.
55、 Boon, T. et al. Tumor antigens recognized by T lymphocytes. Annu. Rev. Immunol. 1994; 12:337-365.
56、 Rosenberg, S.A. A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity. 1999; 10:281-187.
57、 Wang, R-F. and Rosenberg, S.A. Human tumor antigens for cancer vaccine development. Immunol. Rev. 1999; 170:85-100.
58、 Rosenberg, S.A. et al. Immunologic and therapeutic evaluation of a synthetic tumor associated peptide vaccine for the treatment of patients with metastatic melanoma. Nat. med. 1998; 4:321-327.
59、 Nestle, F.O. et al. Vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells. Nat. Med. 1998; 4:328-332.
60、 Marchand, M. et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigen peptide encoded by gene MAGE-3 and presented by HLA-Al. Int. J. Cancer. 1999; 80:219-230.
61、 Toes, R.E. et al. CD4+T cells and their role in antitumor immune responses. J. Exp. Med. 1999; 189:753-756.
62、 Cnhen, RA. et al. CD4+T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection. Crit. Rev. Immunol.2000; 20:17-56.
63、 Roberts MR,Qin L,Zhang D, et al. Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors. Blood 1994,84:2878-2889.
64、 Sherrington, C.S.(1897) The central nervous system. In A Text-Book of Physiology(Vol. 3,7th edn)(Foster, M., ed.), Macmillan
65、 Norcross, M.A. (1984) A synapse basis for T-lymphocytes activation. Ann. .Immunol. (Paris) 135D,113-134
66、 Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell, 1994, 76:241-251.
67、 Kupher. A. and Singer. S.J. The specific interaction of helper T cells and antigen-presenting B
2、Mule JJ, Shu S, Schwartz SL et al. Successful adoptive immunotherapy of established metastases with LAK and recombinant interleukin 2. Science., 1984,225:1489-89.
3、Rosenberg SA, Spiess PJ, Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science., 1986,233:1318-1321.
4、Rosengerg SA. Use of tumor-infiltrating lymphocytes and IL-2 in the immunotherapy of patients with metastatic melanoma. N. Engl. J. Med., 1988; 319:1676.
5、Rosenberg SA. The immunotherapy and gene therapy of cancer. J. Clin. Oncol.,1992;10:180.
6、Kuwana Y, Asakura y, Utsonmiya N. et al. Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions. Biochem Biophys Res Common 1989,149:960.
7、Brocker T, Karjalainen K. Adoptive tumor immunity mediated by lymphocytes bearing modified antigen-specific receptors. Adv Immunol 1998,68:257.
8、Eshhar Z. tumor-specific T-bodies: towards clinical applications. Cancer Immunol Immunother 1997,45:131.
9、Irving BV, Weiss A. The cytoplasmic domain of the T cell receptor ζ chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 1991,64:891.
10、Letourneur F, Klausner RD. T cell and basophile activation through the cytoplasmic tail of T-cell receptor ζ family proteins.Proc Natl Acad Sci USA 1991,88:8905.
11、Romec C, Seed B. Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 1991,64:1037.
12、John maher, Renier J. Brentijens and Michel Sadelain. et al. Human T-lymphocytes cytotoxicity and proliferation directed by a single chimeric TCR ζ/CD28 receptor. Nature biotechnology. 2002; 20:70-75.
13、Finney H M, Lawson A D and Bebbington C R. et al. Chimeric receptors providing both primary and costimulatory signaling in T cell from a single gene product. J Immunol. 1998; 161:2791-2797.
14、Haynes, NM. Trapani, JA. and Darcy PK. et al. Rejection of synngeneic colon carcinoma by CTLs expressing single-chain antibody receptor codelivering CD28 costimulation. 2002; 169:5780-5786.
15、 Ben-EFRAIM S: Cancer immunotherapy: hopes and pitfalls. Anti-cancer Res 16: 3235-3240, 1996.
16、 DUDLEY ME: Cell transfer therapy: basic principles and preclinical studies. In: Principles and Practice of the Biologic Therapy of Cancer. SA ROSENBERG, Lippincott Williams and Wilkins, Philadelphia, 2000, pp305-321.
17、 Hung MC. Lau YK. Basic science of HER2/neu: a review. Semin oncol. 1999, 26:51-59.
18、 Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, Press MR Studies of the HER2/neu proto-oncogene in human breast and ovarian cancer. Science(Washington DC). 1989;244:707-710.
19、 Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Voelter J, Pegram M, Baselga J, Norton L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpress HER2. N. Engl J Med. 1990;344:783.
20、 Diss, M.L.,S.M.PuPa and M.A.Cheever. et al. High-titer Her-2/neu protein-specific antibody can be detected in patients with early-stage breast cancer. J. Clin. Oncol. 1997;15:3363.
21、 Disis, M.L., E.Calenoff,G.McLaughlin, and A.E.Murphy. et al. Existent T-cell and antibody immunity to Her-2/neu protein in patients with breast cancer. Cancer Res. 1994;54:16.
22、 Peoples,G.E.,P.S.Goedegebuure,and T.J.Eberlein. et al. Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same Her2/neu-derived peptide. Proc. Natl. Acad. Sci. USA 1995;92:432.
23、 Fisk, B.,B.Anderson, and C.GIoannides. Identification of naturally processed human ovarian peptides recognized by tumor-associated CD8+ cytotoxicity T lymphocytes. Cancer res.l997;57:87.
24、 Pages JC, Bru T. Tool box for retrovectorologists. J Gene med 2004;6:S67-S82.
25、 Cone RD, Mulligan RC. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host tange. Proc Natl Acad Sci USA.1984;81:6349-6353.
26、 Cavazzana-Calvo M et al. Gene therapy of human severe combined with nonmyeloablative conditioning. Science.2002;288:669-672.
27、 Kay MA, Glorioso JC and Naldini L et al. Viral vectors for gene therapy: the art
of turning infectious agents into vehicles of therapeutics. Nat. med. 2001;7:33-40.
28、 Lois C, Refae Y, Qin XF, et al. Retrovirus as tools to study the immune system. Curr opin Immunol. 2001; 13:496-504.
29、 Naldini L, Blomer U, Gallay P. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vectors. Science. 1996;272:263-267.
30、 Pringle CR. Virus taxonomy-1999. The universal system of virus taxonomy, updated to include the new proposals ratified by the International Committee on Taxonomy of Viruses. Arch Virol 1999; 144:421-429.
31、 De Felipe P, Izquierdo M. Retroviral vectors for gene therapy. Hum. Gene Ther. 2000;11:1921-1931.
32、 Burns JC, Friedmann T, Driever W. et al.Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmamalian cells. Proc. Natl. Acad. Sci. USA 1993;90:8033-8037.
33、 Lavillette D, Russell SJ, Cosset FL. Retargeting gene delivery using surface-engineered retroviral vector particles. Curr Opin Biotechnol. 2001 Oct; 12(5):461-6. review.
34、 Brocker T, Karjalainen K. Adoptive tumor immunity mediated by lymphocytes bearing modified antigen-specific receptors. Adv Immunol. 1998;68:257.
35、 Eshhar Z. tumor-specific T-bodies: towards clinical applications. Cancer Immunol Immunother.l997;45:131.
36、 Hombach, A., A.Wieczarkowiecz, T. Marquardt,C. Heuser,L. Usai,C. Pohl,B- Seliger, and H.Abken. Tumor-specific T cell activation by recombinant immunoreceptors: CD3 (?) signaling and Cd28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 (?) signaling receptor molecule. J.Immunol.2001; 167:6123
37、 Maher, J.,Brentjens R J and Sadelain M. Human T-lymphocytes cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor. Nature Biotechnol.2002;20:70-75.
38、 Eshhar. Z,Waks,T. Gross. G. et al. Specific activation and targeting of cytotoxic lymphocytes through chimetic single chains consisting of antibody-binding domains and a gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc. Natl Acad Sci USA 1993:90:720-724.
39、 Altenschmidt, U.,Klundt,E.,Groner B. Adoptive transfer of in vivo-targeted,activated T lymphocytes results in total tumor regression. J.Immunol. 1997; 159:5509-5515.
40、 Darcy,P.K. et al. Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineering CTL. J. Immunol.2000; 164:3705-3712.
41、 Haynes, N.M. et al. Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors. Blood 2002; 1000:3155-3163.
42、 Mcguinness.r.p. et al. Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor.Hum Gene Ther.l999;10:165-173.
43、 Hwu,P. et al. In vivo anti-tumor activity of T cells redirected with chimeric antibody/T-cell receptor genes. Cancer res. 1995; 55:3369-3373.
44、 Brentjens R.,Latouche,J.B.,Sadelain, M. et al. In vivo anti-tumor activity of genetically modified T cells is dependent on the method of ex vivo T cell expansion. Blood. 2002; 100:577a.
45、 Hombach A,, Wieczarkowiecz A, Marquardt T, et al. Tumor-specific T cell activation by recombinant immunoreceptors: CD3 (?) signaling and CD28 stimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD33 signaling receptor molecule. J immunol. 2001;167:6123.
46、 Geiger TL, Leitenberg D, Flavell RA. The TCR zeta-chain immunoreceptor tyrosine-based activation motifs are sufficient for the activation and differentiation of primary T lymphocytes. J Immunol. 1999; 162:5931-5939.
47、 Cohen, PA.et al. CD4+ T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection.Crit.Rev.Immunol. 2000; 20:17-56.
48、 Pardall, D.M. and Topalian, SL. The role of CD4+ Tcell responses in antitumor immunity. Curr. Opin. Immunol. 1998; 10:588-594.
49、 Mumberg, D. et al. CD4+ T cells eliminates MHC class II-negative cancer cells in vivo by indirect effects of IFN- Y .Proc. Natl.Acad.Sci. USA 1999; 96:8633-8638.
50、 Qin Z, Blankenstein T. CD4+ T cells mediated tumor rejection involved inhibition of angiogenesis that is dependent on IFN- y receptor expression by nonhematopoietic cells. Imunity. 2000; 12:677-686.
51、 Hiroshi G, Takemasa T, Yoshinori S, et al. Generation and targeting of human tumor-specific Tcl and Thl cells transduced with a lentivirals containing a chimeric immunoglobulin T-cell receptor. Cancer research. 2004;64:1490-1495.
52、 Pawelec C, Zeuthen J, Kiessling R. Escape from host antitumor immunity. Crit Rev Onbog.
1997;8:111.
53、 Shinkai Y, Ma A, Cheng HL, et al. CD3 epsilon and CD3 zeta cytoplasmic domains can independently generate signals for T cell development and function. Immunity. 1995;2:401.
54、 Davis MM, Boniface JJ, Reich Z et al. Ligand recognition by alpha beta T cell receptors. Annu Rev Immynol. 1998; 16:523.
55、 Boon, T. et al. Tumor antigens recognized by T lymphocytes. Annu. Rev. Immunol. 1994; 12:337-365.
56、 Rosenberg, S.A. A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity. 1999; 10:281-187.
57、 Wang, R-F. and Rosenberg, S.A. Human tumor antigens for cancer vaccine development. Immunol. Rev. 1999; 170:85-100.
58、 Rosenberg, S.A. et al. Immunologic and therapeutic evaluation of a synthetic tumor associated peptide vaccine for the treatment of patients with metastatic melanoma. Nat. med. 1998; 4:321-327.
59、 Nestle, F.O. et al. Vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells. Nat. Med. 1998; 4:328-332.
60、 Marchand, M. et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigen peptide encoded by gene MAGE-3 and presented by HLA-Al. Int. J. Cancer. 1999; 80:219-230.
61、 Toes, R.E. et al. CD4+T cells and their role in antitumor immune responses. J. Exp. Med. 1999; 189:753-756.
62、 Cnhen, RA. et al. CD4+T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection. Crit. Rev. Immunol.2000; 20:17-56.
63、 Roberts MR,Qin L,Zhang D, et al. Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors. Blood 1994,84:2878-2889.
64、 Sherrington, C.S.(1897) The central nervous system. In A Text-Book of Physiology(Vol. 3,7th edn)(Foster, M., ed.), Macmillan
65、 Norcross, M.A. (1984) A synapse basis for T-lymphocytes activation. Ann. .Immunol. (Paris) 135D,113-134
66、 Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell, 1994, 76:241-251.
67、 Kupher. A. and Singer. S.J. The specific interaction of helper T cells and antigen-presenting B