人细胞融合肝癌疫苗的制备及临床前研究
摘要
原发性肝癌是我国常见、难治性恶性肿瘤之一，占我国恶性肿
瘤死亡率的第二位。手术切除仍为肝癌治疗的首选方法，但其疗效
仍欠满意，术后复发是影响其疗效的主要因素，研究和寻找抗肝癌
术后复发方法是近年来国内外学者共同关注的课题。本研究取手术
切除后的肝癌组织和自体脾脏，分离获得肝癌细胞和自体激活B细
胞。应用PEG融合技术，制备肝癌细胞与自体激活B细胞融合细胞，
经～（60）Co照射制成人细胞融合肝癌疫苗，应用流式细胞仪，检测三批
细胞融合率分别为66.84％，74.43％及76.55％。观察了肝癌细胞与
自身激活B细胞融合前后细胞表达gp75，MHC-I，MHC-Ⅱ及B7变化，
融合细胞不但表达肝癌细胞表面分子gp75，同时还高表达MHC-I，
MHC-Ⅱ及B7。对～（60）Co照射后融合细胞生长，致瘤性及细胞表型进
行观察。照射细胞失去增殖能力和裸鼠体内成瘤性，照射后3天内
细胞表型无明显变化，仍表达gp75，MHC-Ⅰ，MHC-Ⅱ及B7。人细
胞融合肝癌疫苗的无菌，热原质及内毒素试验结果均符合国家生物
制品制备和检定标准。人细胞融合肝癌疫苗中PEG、甘油及庆大霉
素残留量进行检测，结果分别为：PEG未检出，甘油残余量小于
0.02％，庆大霉素残余量小于1mg／L。样品中PEG、甘油及庆大霉素
均远低于其体内应用的有害剂量。上述试验结果提示该肿瘤疫苗的
有效性和安全性。
Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in Asian country. Its mortality takes the second place among malignant tumors in China. Although the surgical excision is considered the most effective therapy for early-stage HCC patients, only a very small proportion of patients with an operable primary lesion may transiently benefit from surgical treatment because of a high recurrence rate of cancer after operation. To study and search for an approach of anti-recurrence of RCC after operation is expected. In this study the purifed HCC cells and autologuous activated B cells from HCC tissue and autologous spleen were used. Hybrid cells were generated by fusion of human hepatocellular carcinoma cells (HCC) with autologous activated B cells of HCC patients and were irradiated by 60Co. Hybrid cells were determined by flow cytometry and the fused proportions were 66.84%,74.43% and 76.55%, respectively. The hybrid cells were able to express both gp75 molecules from human
HCC cells ,and MHC-I,MHC-II and B7 from B cells. 60Co-radiated fusion cells lose proliferative capacity ,but they have no apparent changes in immunogenicity-related phenotype MHC-I,MHC-II and B7 in subsequent 3 d. Asepsis, pyrogen
2
and endotoxin accord with the criterion of the national preparation and testing of bioproduction. Remnant PEG,glycerol and gentamicin were tested. The results were: PEG was not detected, remnant glycerol and gentamicin were less than 0.02% and 1mg/i, respectively. Remnant
PEG, glycerol and gentamicin in the sample are much less than the harmful dose in vivo. These results suggest the safty and efficiency of the hybrid cell vaccine.
瘤死亡率的第二位。手术切除仍为肝癌治疗的首选方法，但其疗效
仍欠满意，术后复发是影响其疗效的主要因素，研究和寻找抗肝癌
术后复发方法是近年来国内外学者共同关注的课题。本研究取手术
切除后的肝癌组织和自体脾脏，分离获得肝癌细胞和自体激活B细
胞。应用PEG融合技术，制备肝癌细胞与自体激活B细胞融合细胞，
经～（60）Co照射制成人细胞融合肝癌疫苗，应用流式细胞仪，检测三批
细胞融合率分别为66.84％，74.43％及76.55％。观察了肝癌细胞与
自身激活B细胞融合前后细胞表达gp75，MHC-I，MHC-Ⅱ及B7变化，
融合细胞不但表达肝癌细胞表面分子gp75，同时还高表达MHC-I，
MHC-Ⅱ及B7。对～（60）Co照射后融合细胞生长，致瘤性及细胞表型进
行观察。照射细胞失去增殖能力和裸鼠体内成瘤性，照射后3天内
细胞表型无明显变化，仍表达gp75，MHC-Ⅰ，MHC-Ⅱ及B7。人细
胞融合肝癌疫苗的无菌，热原质及内毒素试验结果均符合国家生物
制品制备和检定标准。人细胞融合肝癌疫苗中PEG、甘油及庆大霉
素残留量进行检测，结果分别为：PEG未检出，甘油残余量小于
0.02％，庆大霉素残余量小于1mg／L。样品中PEG、甘油及庆大霉素
均远低于其体内应用的有害剂量。上述试验结果提示该肿瘤疫苗的
有效性和安全性。
Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in Asian country. Its mortality takes the second place among malignant tumors in China. Although the surgical excision is considered the most effective therapy for early-stage HCC patients, only a very small proportion of patients with an operable primary lesion may transiently benefit from surgical treatment because of a high recurrence rate of cancer after operation. To study and search for an approach of anti-recurrence of RCC after operation is expected. In this study the purifed HCC cells and autologuous activated B cells from HCC tissue and autologous spleen were used. Hybrid cells were generated by fusion of human hepatocellular carcinoma cells (HCC) with autologous activated B cells of HCC patients and were irradiated by 60Co. Hybrid cells were determined by flow cytometry and the fused proportions were 66.84%,74.43% and 76.55%, respectively. The hybrid cells were able to express both gp75 molecules from human
HCC cells ,and MHC-I,MHC-II and B7 from B cells. 60Co-radiated fusion cells lose proliferative capacity ,but they have no apparent changes in immunogenicity-related phenotype MHC-I,MHC-II and B7 in subsequent 3 d. Asepsis, pyrogen
2
and endotoxin accord with the criterion of the national preparation and testing of bioproduction. Remnant PEG,glycerol and gentamicin were tested. The results were: PEG was not detected, remnant glycerol and gentamicin were less than 0.02% and 1mg/i, respectively. Remnant
PEG, glycerol and gentamicin in the sample are much less than the harmful dose in vivo. These results suggest the safty and efficiency of the hybrid cell vaccine.
引文
1． 汤钊猷.试论原发性肝癌复发转移的研究.中华实验外科杂志.1999;16(6) :485-6．
2． 李国强，孙跃明．肝癌切除后复发相关因素的研究进展．实用癌症杂志．2000;15(1) :105-7．
3． Guo Y, Wu M, Chen H et al.Effective tumor vaccine generated by fusion of hepatoma cells with activated B cells. Science, 1994; 263(5146) :518-20．
4． Cain JM, Howett MK. Preventing cervical cancer. Science. 2000;28 8(5472) : 1753-5．
5． Henry SH, Bosch FX, Troxell TC et al.Policy forum: public health. Reducing liver cancer-global control of aflatoxin.Science. 1999;286(5449) :2453-4．
6． Dunussi-Joannopoulos K, Krenger W, Weinstein HJ et al.CD8+ T cells activated during the course of murine acute myelogenous leukemia elicit therapeutic responses to late B7 vaccines after cytoreductive treatment.Blood. 1997;89(8) :2915-24．
7． Mosca PJ, Hobeika AC, Clay TM et al.Direct detection of cellular immune responses to cancer vaccines.Surgery. 2001;129(3) :248-54． .
8． You Z, Hester J, Rollins L et al.A retrogen strategy for presentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potent antitumor T helper and CTL responses.Cancer Res. 200 1;6 1(1) : 197-205．
9． Diehl L, den Boer AT, Schoenberger SP et al.CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.Nat Med. 1999;5(7) :774-9．
10． Ignatius R, Mahnke K, Rivera M et al.Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo.Blood. 2000;96(10) :3505-13．
11 . Reichardt VL, Okada CY, Liso A et al. Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma--a feasibility study.Blood. 1999;93(7) :2411-9．
12． Jager D, Jager E, Knuth A. Vaccination for malignant melanoma: recent developments.Oncology. 200 1 ;60( 1 ): 1-7．
13． de Gruijl TD, Curiel DT. Cancer vaccine strategies get bigger and better.Nat Med. 1999;5(10) :1124-5．
14． Pardoll DM. S. Cancer vaccines.Nat Med. 1998;4(5 Suppl):525-31．
15． Hislop AD, Good MF, Mateo L et al.Vaccine-induced cytotoxic T lymphocytes protect against retroviral challenge.Nat Med. 1998;4(10) : 1193-6．
16． Pardoll D. s Releasing the brakes on antitumor immune response.Science. 1996 22;271(5256) :1691．
17． Douin-Echinard V V, Bornes S, Rochaix P et al.The expression of CD70 and CD80 by gene-modified tumor cells induces an antitumor response depending on the MHC status.Cancer Gene Ther. 2000;7(12) : 1543-1556．
18． Torabi-Pour N, Nouri AM, Perrett D et al.Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line.Biomed Chromatogr. 2001;15(1) :18-24．
19． Todo T, Martuza RL, Dallman MJ et al. In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity.Cancer Res. 2001 ;61(1) : 153-61．
20． Dunussi-Joannopoulos K, Dranoff G, Weinstein HJ et al. Gene immunotherapy in murine acute myeloid leukemia: granulocyte-macrophage colony-stimulating factor tumor cell vaccines elicit more potent antitumor immunity compared with B7 family and other cytokine vaccines.Blood. 1998;91(1) :222-30．
21． Schendel DJ, Frankenberger B, Jantzer P et al.Expression of B7． 1 (CD80) in a renal cell carcinoma line allows expansion of tumor-associated cytotoxic T lymphocytes in the presence of an alloresponse.Gene Ther. 2000;7(23) :2007-14．
22． Machiels JP, Reilly RT, Emens LA et al.Cyclophosphamide, Doxorubicin, and Paclitaxel Enhance the Antitumor Immune Response of Granulocyte/Macrophage-Colony Stimulating Factor-secreting Whole-Cell Vaccines in HER-2/neu Tolerized Mice.Cancer Res. 2001;61(9) :3689-3697．
23． Borrello I, Sotomayor EM, Rattis FM et al.Sustaining the graft-versus-tumor effect through posttransplant immunization with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing tumor vaccines.Blood. 2000;95(10) :3011-9．
24． Waldmann TA, Dubois S, Tagaya Y.Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy.Immunity. 2001; 14(2) : 105-10．
25． Dunussi-Joannopoulos K, Runyon K, Erickson J et al.Vaccines with interleukin-12-transduced acute myeloid leukemia cells elicit very potent therapeutic and long-lasting protective immunity.Blood. 1999;94(12) :4263-73．
26． Schwarzenberger P, Huang W, Oliver P et al. Poly-L-lysine-based molecular conjugate vectors: a high efficiency gene transfer system for human progenitor and leukemia cells.Am J Med Sci. 2001;321(2) : 129-36．
27． Okada H, Attanucci J, Giezeman-Smits KM.Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.Cancer Res. 2001;61(6) :2625-31．
28． Todryk SM, Birchall LJ, Erlich R.Efficacy of cytokine gene transfection may differ for autologous and allogeneic tumour cell vaccines.Immunology. 2001; 102(2) : 190-8．
29． Guo YJ, Che XY, Shen F et al. Effective tumor vaccines generated by in vitro modification of tumor cells with cytokines and bispecific monoclonal antibodies.Nat Med. 1997;3(4) :451-5．
30． Chen Y, Emtage P, Zhu Q et al.Induction of ErbB-2/neu-specific protective and therapeutic antitumor immunity using genetically modified dendritic cells: enhanced efficacy by cotransduction of gene encoding IL-12． Gene Ther 2001 ;8(4) :316-23．
31． Hsu FJ, Benike C, Fagnoni F et al.Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.Nat Med. 1996;2(1) :52-8．
32． Lambert LA, Gibson GR, Maloney M et al. Intranodal immunization with tumor lysate-pulsed dendritic cells enhances protective antitumor immunity.Cancer Res. 2001;61(2) :641-6．
33． Kugler A, Stuhler G, Walden P et al.Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids.Nat Med. 2000;6(3) :332-6．
34． Novakovic S, Cegovnik U, Menart V.Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.Anticancer Res. 2001 ;21(1A):365-71．
35． Sotomayor EM, Borrello I, Tubb E et al. Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40． Nat Med. 1999;5(7) :780-7．
36． Herr W, Ranieri E, Olson W et al.Mature dendritic cells pulsed with freeze-thaw cell lysates define an effective in vitro vaccine designed to elicit EBV-specific CD4(+) and CD8(+) T lymphocyte responses.Blood. 2000;96(5) : 1857-64．
37． Williams N. s An immune boost to the war on cancer.Science. 1996 5;272(5258) :28-30．
38． Berd D. Autologous, hapten-modified vaccine as a treatment for human cancers.Vaccine. 2001; 19( 17-19) :2565-70．
39． Miconnet I, Coste I, Beermann F.Cancer vaccine design: a novel bacterial adjuvant for peptide-specific ctl induction.J Immunol. 2001;166(7) :4612-9．
40． Stubbs AC, Martin KS, Coeshott C.Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity.Nat Med. 2001;7(5) :625-9．
41． Levitsky HI. The best cytokine for the job.Nat Med. 1997;3(2) : 126．
42． Fujii S, Hamada H, Fujimoto K et al.Activated dendritic cells from bone marrow cells of mice receiving cytokine-expressing tumor cells are associated with the enhanced survival of mice bearing syngeneic tumors.Blood. 1999;93(12) :4328-35．
43． Decker T, Flohr T, Trautmann P et al.Role of accessory cells in cytokine production by T cells in chronic B-cell lymphocytic leukemia.Blood. 1995;86(3) : 1115-23．
44． Mayordomo JI, Zorina T, Storkus WJ et al. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.Nat Med. 1995;1(12) : 1297-302．
45． Pan ZK, Ikonomidis G, Lazenby A et al.A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours.Nat Med. 1995;1(5) :471-7．
46． Fong L, Brockstedt D, Benike C et al.Dendritic cells injected via different routes induce immunity in cancer patients.J Immunol. 2001;166(6) :4254-9．
47． Zirvogel L, Regnault A, Lozier A et al.Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.Nat Med. 1998;4(5) :594-600．
48． Nestle FO, Alijagic S, Gilliet M et al. Vaccination of melanoma patients with peptide-or tumor lysate-pulsed dendritic cells.Nat Med. 1998;4(3) :328-32．
49． Rosenberg SA, Yang JC, Schwartzentruber DJ.Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.Nat Med. 1998;4(3) :321-7．
50． Mendiratta SK, Thai G, Eslahi NK et al.Therapeutic tumor immunity induced by polyimmunization with melanoma antigens gp100 and TRP-2． Cancer Res. 2001;61(3) :859-63．
51． Timmerman JM, Levy R. Melanoma vaccines: prim and proper presentation.Nat Med. 1998;4(3) :269-70．
52． Huang TH, Wu PY, Lee CN et al.Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.Blood. 2000;96(12) :3663-70．
53． Huang TH, Wu PY, Lee CN et al. Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.Blood. 2000;96(12) :3663-70．
54． Bendandi M, Gocke CD, Kobrin CB et al.Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma.Nat Med. 1999;5(10) : 1171-7．
55． Lasek W, Mackiewicz A, Czajka A et al.Antitumor effects of the combination therapy with TNF-alpha gene-modified tumor cells and interleukin 12 in a melanoma model in mice.Cancer Gene Ther. 2000;7(12) :1581-1590．
56． Braly P. Preventing cervical cancer.Nat Med. 1996;2(7) :749-51．
57． Disis ML, Bernhard H, Shiota FM et al.Granulocyte-macrophage colony-stimulating factor: an effective adjuvant for protein and peptide-based vaccines.Blood. 1996;88(1) :202-10．
58． Seferian PG, Martinez ML. Immune stimulating activity of two new chitosan containing adjuvant formulations.Vaccine. 2000;19(6) :661-8．
59． Gao P, Uekusa Y, Nakajima C et al.Tumor vaccination that enhances antitumor T-cell responses does not inhibit the growth of established tumors even in combination with interleukin-12 treatment: the importance of inducing intratumoral T-cell migration.J Immunother. 2000;23(6) :643-53．
60． Vogel G. S.FDA weighs using tumor cell lines for vaccine development.Science. 1999 ;285(5435) : 1826-7．
61． Brossart P, Heinrich KS, Stuhler G et al.Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies.Blood. 1999;93( 12) :4309-17．
62． Freeman GJ,et al.Structure,expression and T cell costimuiating activity of the murine homologue of the human B lymphocyte activation antigen B7． J Exp Med 1991;174:625-631．
63． Freeman GJ,Freedman AS.Segil JM et aI.B7,a new member of the Ig superfamily with unique expression on activated and neoplastic B cells.J immunol 1989;143:2714-22．
64． Chen L,Ash S,Brad WA.et al.Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4． Cell 1992;7 1:1093-102．
65． Baskar S,Ostrand-Rosenberg S.Nabavi N et al.Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated MHC class II molecules. Proc Natl Acad Sci USA 1993; 90(12) :5687-90．
66． Marquardt H,et al.Purification and primary structure of a polypeptide with multiplication-stimulating activity from rat liver cell cultures.Homology with human insulin-like growth factor II. J Biol Chem 1981;256:6859-65．
67． Mueller DL,Jenkins MK and Schwartz RH.Clonal expansion versus functional clonal inactivation:A costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann Rev Immunol 1989;7:445-480．
68． Dolores J. Schendel and Bernd Gansbacher.Tumor-specific lysis of human renal cell carcinomas by rumor-infiltrating lymphocytesrmodulation of recognition through retroviral transuction of tumor cells with interleukin 2 complementary DNA and exogenous a-interferon treatment .Cancer Res 1993;53:4020-5．
69． Beniers AJ.et al.HLA-class I and class II expression on renal tumor xenografts and the relation to sensitivity for alpha-IFN,gamma-IFN and TNF. Int J Cancer 1991;48(5) :709-16．
70． Watanabe Y.Transfection of interferon gamma gene in animal tumors-a model for local cytokine production and tumor immunity. Semin Cancer Biol,1992;3(1) :43-6．
71． Weber JS,Rosenberg SA.Effects of murine tumor class I major histocompatibility complex expression on antitumor activity of tumor infiltrating lymphocytes.J Natl Cancer Inst 1990;82(9) :755-61．
72． Townsend SE and Allison JP. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science. 1 993;259:368-370．
73． 龚非力主编．基础免疫学．湖北：科学技术出版社，1998．
74． 彭朝晖，薛京伦，徐钤，郭亚军主编．基因治疗--基础与临床．北京：中国科学技术出版社，1994．
2． 李国强，孙跃明．肝癌切除后复发相关因素的研究进展．实用癌症杂志．2000;15(1) :105-7．
3． Guo Y, Wu M, Chen H et al.Effective tumor vaccine generated by fusion of hepatoma cells with activated B cells. Science, 1994; 263(5146) :518-20．
4． Cain JM, Howett MK. Preventing cervical cancer. Science. 2000;28 8(5472) : 1753-5．
5． Henry SH, Bosch FX, Troxell TC et al.Policy forum: public health. Reducing liver cancer-global control of aflatoxin.Science. 1999;286(5449) :2453-4．
6． Dunussi-Joannopoulos K, Krenger W, Weinstein HJ et al.CD8+ T cells activated during the course of murine acute myelogenous leukemia elicit therapeutic responses to late B7 vaccines after cytoreductive treatment.Blood. 1997;89(8) :2915-24．
7． Mosca PJ, Hobeika AC, Clay TM et al.Direct detection of cellular immune responses to cancer vaccines.Surgery. 2001;129(3) :248-54． .
8． You Z, Hester J, Rollins L et al.A retrogen strategy for presentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potent antitumor T helper and CTL responses.Cancer Res. 200 1;6 1(1) : 197-205．
9． Diehl L, den Boer AT, Schoenberger SP et al.CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.Nat Med. 1999;5(7) :774-9．
10． Ignatius R, Mahnke K, Rivera M et al.Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo.Blood. 2000;96(10) :3505-13．
11 . Reichardt VL, Okada CY, Liso A et al. Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma--a feasibility study.Blood. 1999;93(7) :2411-9．
12． Jager D, Jager E, Knuth A. Vaccination for malignant melanoma: recent developments.Oncology. 200 1 ;60( 1 ): 1-7．
13． de Gruijl TD, Curiel DT. Cancer vaccine strategies get bigger and better.Nat Med. 1999;5(10) :1124-5．
14． Pardoll DM. S. Cancer vaccines.Nat Med. 1998;4(5 Suppl):525-31．
15． Hislop AD, Good MF, Mateo L et al.Vaccine-induced cytotoxic T lymphocytes protect against retroviral challenge.Nat Med. 1998;4(10) : 1193-6．
16． Pardoll D. s Releasing the brakes on antitumor immune response.Science. 1996 22;271(5256) :1691．
17． Douin-Echinard V V, Bornes S, Rochaix P et al.The expression of CD70 and CD80 by gene-modified tumor cells induces an antitumor response depending on the MHC status.Cancer Gene Ther. 2000;7(12) : 1543-1556．
18． Torabi-Pour N, Nouri AM, Perrett D et al.Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line.Biomed Chromatogr. 2001;15(1) :18-24．
19． Todo T, Martuza RL, Dallman MJ et al. In situ expression of soluble B7-1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity.Cancer Res. 2001 ;61(1) : 153-61．
20． Dunussi-Joannopoulos K, Dranoff G, Weinstein HJ et al. Gene immunotherapy in murine acute myeloid leukemia: granulocyte-macrophage colony-stimulating factor tumor cell vaccines elicit more potent antitumor immunity compared with B7 family and other cytokine vaccines.Blood. 1998;91(1) :222-30．
21． Schendel DJ, Frankenberger B, Jantzer P et al.Expression of B7． 1 (CD80) in a renal cell carcinoma line allows expansion of tumor-associated cytotoxic T lymphocytes in the presence of an alloresponse.Gene Ther. 2000;7(23) :2007-14．
22． Machiels JP, Reilly RT, Emens LA et al.Cyclophosphamide, Doxorubicin, and Paclitaxel Enhance the Antitumor Immune Response of Granulocyte/Macrophage-Colony Stimulating Factor-secreting Whole-Cell Vaccines in HER-2/neu Tolerized Mice.Cancer Res. 2001;61(9) :3689-3697．
23． Borrello I, Sotomayor EM, Rattis FM et al.Sustaining the graft-versus-tumor effect through posttransplant immunization with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing tumor vaccines.Blood. 2000;95(10) :3011-9．
24． Waldmann TA, Dubois S, Tagaya Y.Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy.Immunity. 2001; 14(2) : 105-10．
25． Dunussi-Joannopoulos K, Runyon K, Erickson J et al.Vaccines with interleukin-12-transduced acute myeloid leukemia cells elicit very potent therapeutic and long-lasting protective immunity.Blood. 1999;94(12) :4263-73．
26． Schwarzenberger P, Huang W, Oliver P et al. Poly-L-lysine-based molecular conjugate vectors: a high efficiency gene transfer system for human progenitor and leukemia cells.Am J Med Sci. 2001;321(2) : 129-36．
27． Okada H, Attanucci J, Giezeman-Smits KM.Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.Cancer Res. 2001;61(6) :2625-31．
28． Todryk SM, Birchall LJ, Erlich R.Efficacy of cytokine gene transfection may differ for autologous and allogeneic tumour cell vaccines.Immunology. 2001; 102(2) : 190-8．
29． Guo YJ, Che XY, Shen F et al. Effective tumor vaccines generated by in vitro modification of tumor cells with cytokines and bispecific monoclonal antibodies.Nat Med. 1997;3(4) :451-5．
30． Chen Y, Emtage P, Zhu Q et al.Induction of ErbB-2/neu-specific protective and therapeutic antitumor immunity using genetically modified dendritic cells: enhanced efficacy by cotransduction of gene encoding IL-12． Gene Ther 2001 ;8(4) :316-23．
31． Hsu FJ, Benike C, Fagnoni F et al.Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.Nat Med. 1996;2(1) :52-8．
32． Lambert LA, Gibson GR, Maloney M et al. Intranodal immunization with tumor lysate-pulsed dendritic cells enhances protective antitumor immunity.Cancer Res. 2001;61(2) :641-6．
33． Kugler A, Stuhler G, Walden P et al.Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids.Nat Med. 2000;6(3) :332-6．
34． Novakovic S, Cegovnik U, Menart V.Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.Anticancer Res. 2001 ;21(1A):365-71．
35． Sotomayor EM, Borrello I, Tubb E et al. Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40． Nat Med. 1999;5(7) :780-7．
36． Herr W, Ranieri E, Olson W et al.Mature dendritic cells pulsed with freeze-thaw cell lysates define an effective in vitro vaccine designed to elicit EBV-specific CD4(+) and CD8(+) T lymphocyte responses.Blood. 2000;96(5) : 1857-64．
37． Williams N. s An immune boost to the war on cancer.Science. 1996 5;272(5258) :28-30．
38． Berd D. Autologous, hapten-modified vaccine as a treatment for human cancers.Vaccine. 2001; 19( 17-19) :2565-70．
39． Miconnet I, Coste I, Beermann F.Cancer vaccine design: a novel bacterial adjuvant for peptide-specific ctl induction.J Immunol. 2001;166(7) :4612-9．
40． Stubbs AC, Martin KS, Coeshott C.Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity.Nat Med. 2001;7(5) :625-9．
41． Levitsky HI. The best cytokine for the job.Nat Med. 1997;3(2) : 126．
42． Fujii S, Hamada H, Fujimoto K et al.Activated dendritic cells from bone marrow cells of mice receiving cytokine-expressing tumor cells are associated with the enhanced survival of mice bearing syngeneic tumors.Blood. 1999;93(12) :4328-35．
43． Decker T, Flohr T, Trautmann P et al.Role of accessory cells in cytokine production by T cells in chronic B-cell lymphocytic leukemia.Blood. 1995;86(3) : 1115-23．
44． Mayordomo JI, Zorina T, Storkus WJ et al. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.Nat Med. 1995;1(12) : 1297-302．
45． Pan ZK, Ikonomidis G, Lazenby A et al.A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours.Nat Med. 1995;1(5) :471-7．
46． Fong L, Brockstedt D, Benike C et al.Dendritic cells injected via different routes induce immunity in cancer patients.J Immunol. 2001;166(6) :4254-9．
47． Zirvogel L, Regnault A, Lozier A et al.Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.Nat Med. 1998;4(5) :594-600．
48． Nestle FO, Alijagic S, Gilliet M et al. Vaccination of melanoma patients with peptide-or tumor lysate-pulsed dendritic cells.Nat Med. 1998;4(3) :328-32．
49． Rosenberg SA, Yang JC, Schwartzentruber DJ.Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.Nat Med. 1998;4(3) :321-7．
50． Mendiratta SK, Thai G, Eslahi NK et al.Therapeutic tumor immunity induced by polyimmunization with melanoma antigens gp100 and TRP-2． Cancer Res. 2001;61(3) :859-63．
51． Timmerman JM, Levy R. Melanoma vaccines: prim and proper presentation.Nat Med. 1998;4(3) :269-70．
52． Huang TH, Wu PY, Lee CN et al.Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.Blood. 2000;96(12) :3663-70．
53． Huang TH, Wu PY, Lee CN et al. Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.Blood. 2000;96(12) :3663-70．
54． Bendandi M, Gocke CD, Kobrin CB et al.Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma.Nat Med. 1999;5(10) : 1171-7．
55． Lasek W, Mackiewicz A, Czajka A et al.Antitumor effects of the combination therapy with TNF-alpha gene-modified tumor cells and interleukin 12 in a melanoma model in mice.Cancer Gene Ther. 2000;7(12) :1581-1590．
56． Braly P. Preventing cervical cancer.Nat Med. 1996;2(7) :749-51．
57． Disis ML, Bernhard H, Shiota FM et al.Granulocyte-macrophage colony-stimulating factor: an effective adjuvant for protein and peptide-based vaccines.Blood. 1996;88(1) :202-10．
58． Seferian PG, Martinez ML. Immune stimulating activity of two new chitosan containing adjuvant formulations.Vaccine. 2000;19(6) :661-8．
59． Gao P, Uekusa Y, Nakajima C et al.Tumor vaccination that enhances antitumor T-cell responses does not inhibit the growth of established tumors even in combination with interleukin-12 treatment: the importance of inducing intratumoral T-cell migration.J Immunother. 2000;23(6) :643-53．
60． Vogel G. S.FDA weighs using tumor cell lines for vaccine development.Science. 1999 ;285(5435) : 1826-7．
61． Brossart P, Heinrich KS, Stuhler G et al.Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies.Blood. 1999;93( 12) :4309-17．
62． Freeman GJ,et al.Structure,expression and T cell costimuiating activity of the murine homologue of the human B lymphocyte activation antigen B7． J Exp Med 1991;174:625-631．
63． Freeman GJ,Freedman AS.Segil JM et aI.B7,a new member of the Ig superfamily with unique expression on activated and neoplastic B cells.J immunol 1989;143:2714-22．
64． Chen L,Ash S,Brad WA.et al.Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4． Cell 1992;7 1:1093-102．
65． Baskar S,Ostrand-Rosenberg S.Nabavi N et al.Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated MHC class II molecules. Proc Natl Acad Sci USA 1993; 90(12) :5687-90．
66． Marquardt H,et al.Purification and primary structure of a polypeptide with multiplication-stimulating activity from rat liver cell cultures.Homology with human insulin-like growth factor II. J Biol Chem 1981;256:6859-65．
67． Mueller DL,Jenkins MK and Schwartz RH.Clonal expansion versus functional clonal inactivation:A costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann Rev Immunol 1989;7:445-480．
68． Dolores J. Schendel and Bernd Gansbacher.Tumor-specific lysis of human renal cell carcinomas by rumor-infiltrating lymphocytesrmodulation of recognition through retroviral transuction of tumor cells with interleukin 2 complementary DNA and exogenous a-interferon treatment .Cancer Res 1993;53:4020-5．
69． Beniers AJ.et al.HLA-class I and class II expression on renal tumor xenografts and the relation to sensitivity for alpha-IFN,gamma-IFN and TNF. Int J Cancer 1991;48(5) :709-16．
70． Watanabe Y.Transfection of interferon gamma gene in animal tumors-a model for local cytokine production and tumor immunity. Semin Cancer Biol,1992;3(1) :43-6．
71． Weber JS,Rosenberg SA.Effects of murine tumor class I major histocompatibility complex expression on antitumor activity of tumor infiltrating lymphocytes.J Natl Cancer Inst 1990;82(9) :755-61．
72． Townsend SE and Allison JP. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science. 1 993;259:368-370．
73． 龚非力主编．基础免疫学．湖北：科学技术出版社，1998．
74． 彭朝晖，薛京伦，徐钤，郭亚军主编．基因治疗--基础与临床．北京：中国科学技术出版社，1994．