基于FIt3L基因和Anti-CTLA4抗体的抗肿瘤免疫联合治疗的实验研究
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摘要
T细胞介导的抗肿瘤细胞免疫在机体排斥肿瘤方面起着关键作用，是抗肿瘤
免疫治疗最重要的靶点。
细胞毒性T淋巴细胞相关抗原4（cytotoxic T lymphocyte associated antigen 4，
CTLA-4），是共刺激分子B7的受体，主要表达于激活的T细胞表面，与B7结
合后能抑制小鼠和人的T细胞的激活，在T细胞活化中起负调节作用。用抗
CTLA4抗体阻断CTLA-4与B7的结合，在体内外均能促进T细胞的增殖和活化
能力，从而增强T细胞的免疫反应，并能够产生良好的抗肿瘤效果。
另一个重要的提升T细胞抗肿瘤免疫反应的策略是以分泌细胞因子的肿瘤细
胞作为肿瘤疫苗，通过对T细胞和APC产生旁分泌效应，达到抗肿瘤的目的。
FL（fms-like tyrosine kinase receptor-3 ligand，fms样酪氨酸激酶受体3配体，Flt3
Ligand，FL）是一种新近发现的细胞因子。在体内外均能够诱导树突状细胞（DC）
和许多造血早期干细胞的增殖和活化，是目前发现的最为重要的DC生长因子之
一。而DC是体内一种重要的专职抗原提呈细胞（APC），在肿瘤抗原激发特异
性免疫的过程中具有重要作用，并且荷瘤宿主DC功能一般都低下。FL可以通
过促进体内抗肿瘤免疫效应细胞（DC、NK细胞、CTL等）的增殖、分化和成
熟，产生显著的抗肿瘤效应。
然而，对一些低免疫原性、高恶性的肿瘤，单一的免疫治疗策略并不能达到
理想的抗肿瘤效果。而且，目前关于anti-CTLA4抗体的抗肿瘤机制还未彻底明
了，存在着一些不同的认识。为此，我们研究了anti-CTLA4抗体与FL瘤苗和
B7瘤苗间可能存在的协同效应，进一步探讨了anti-CTLA4抗体的抗肿瘤作用机
制，以期为肝癌及其他低免疫原性肿瘤的临床免疫和基因治疗提供新的思路和实
验依据。
我们构建了含有一个FL基因和一个GFP基因真核表达质粒pGFP-FL，并
建立稳定转染有该质粒的小鼠肝癌细胞系Hepa1-6（H2），检测到了FL和GFP
基因在其中的高效表达，以之作为能够分泌FL细胞因子的瘤苗；利用商品化的
能够分泌抗小鼠CTLA4抗体的杂交瘤，制备、纯化得到了anti-muCTLA4抗体，
用于后续的动物实验；同时还在本所以往的工作基础上，用经典的单克隆抗体制
备方法，获得了一株可以抗人CTLA4的单克隆抗体杂交瘤7H9B5A8，为进一步
发展人源化抗人CTLA4单抗，用于临床抗肿瘤免疫治疗的研究工作提供了便利。
在对荷瘤Hepa1-6的C57BL/J小鼠的免疫治疗中，我们发现，单用
anti-muCTLA4抗体、FL瘤苗、B7瘤苗或野生型Hepa1-6瘤苗均不能完全排斥
Hepa1-6肿瘤；anti-muCTLA4抗体与FL瘤苗则可以使肿瘤完全消退；而联合应
用anti-muCTLA4抗体与野生型Hepa1-6瘤苗，或B7瘤苗与FL瘤苗，却未见有
协同效应。值得注意的是，在我们的实验中发现anti-muCTLA4抗体与B7瘤苗
联合治疗小鼠肿瘤具有很好的协同效应，这与1998年Hurwitz等在类似的研究
中所得出的结论不同，即他们认为anti-muCTLA4抗体与B7瘤苗间没有协同抗
小鼠肿瘤作用。另外，经不同方法治疗有效的小鼠均能够抵抗在肿瘤消退后30
天时，10倍于初次荷瘤剂量的野生型Hepa1-6肿瘤细胞的攻击。
综合国内外同类研究和我们的实验结果，我们认为，anti-CTLA4抗体在抗
肿瘤免疫中是通过多机制同时作用的。既可以通过阻断宿主体内APC上的B7
分子和肿瘤特异性T细胞表面的CTLA4分子的结合，增强宿主本身APC和T
细胞间的共刺激作用来达到抗肿瘤效应的；又可以通过促进未发生免疫耐受的肿
中立摘要
瘤特异性T细胞亚群的增殖与活化，放大T细胞与APC间的有效作用，来提升
机体的抗肿瘤兔疫效应。anh（TLA抗体与FL瘤苗，或7i（TLA抗体与B7
瘤苗联合治疗，都是一种较好的抗肿瘤兔疫联合治疗策略。这些结果也提示，最
有效的肿瘤疫苗协同治疗策略应该是那些既能够增强宿主的APC功能，同时又
能够阻断T细胞活化过程中的抑制信号，并能够刺激宿主体内尚未发生免疫耐受
的T细胞的增殖的方法。
另外，考虑到将来可能的肿瘤易感人群的预防接种问题，我们还对发展口服
型真核表达核酸疫苗用于肿瘤预防进行了初步的实验研究。
我们设计以GFP作为TAA，利用pEGFP－NI（GFP”，FL”）和pGFP－FL（GFP”，
FL勺两种真核表达质粒为核酸疫苗，采用减毒伤寒沙门氏菌SL7207作疫苗的
口服型载体，对小鼠进行口服兔疫，观察口服疫苗对GFP＋的HI肿瘤细胞
（Heyal6／pEGFP－NI）的预防作用。这样的研究目前还为发现有相同报道。
我们发现，野生型的SL7207和SL7207／pEGFP－NI均不能诱发小鼠的保护性
免疫，而SL7207／pGFP－FL则可以在部分小鼠（2／10）中诱发保护性免疫，使小
鼠免生肿瘤，并能抵抗在初次荷瘤攻击后40天，10倍于首次剂量的HI肿瘤细
胞的再攻击。这两只小鼠的脾淋巴细胞，体外对HI肿瘤细胞的刺激有明显的增
殖效应，而且流式细胞术检测到了其中分别有 17．4％和 16．29％的 GFP＋细胞的存
在。同时在体外实验中也发现，我们构建的两种口服型核酸疫苗?
T cell mediated immunity plays a key role in antitumor responses, and it is also
the major target of antitumor immunotherapy.
CTLA-4, a second T cell receptor for B7, which is expressed on the surface of
activated T cells, has been shown to play an inhibitory role in regulation of T cell
responses by preventing full T cell activation or terminating T cell responses. Also, it
has been shown that antibody-mediated CTLA-4 blockade can augment host
antitumor responses by prolonging T cell activity and/or facilitating antigen-specific T
cell costimulatory activation.
Another approach taken to raise antitumor immune responses has been to use
cytokine-expressing tumor cells as vaccines, which in turn have paracrine effects on T
cells or APCs. F1t3 Ligand(FL), a newly found cytokine, which is one of the most
important Dendritic Cells (DCs) growth factors, can expand and activate DCs and
some hemaprogenitors in vitro and in vivo. While DCs, the most important APCs, are
instrumental for the activation of tumor antigen-specific immune response. Recently,
it has been reported that FL can promote antitumor activity through inducing a
profound expansion of antitumor effect cells (DCs, NKs, CTLs).
However, it has been shown that these treatment regimens alone cannot induce
complete tumor regression in wealdy immunogenic tumors. And the exact
mechanism(s) involved in the antitumor responses elicited by CTLA-4 blockade
remains to be elucidated, there also exists different opinions about it. In the present
study, we test the hypothesis that FL- and B7-transduced tumor vaccine therapy,
combining with CTLA-4 blockade, should enhance antitumor responses in the
treatment of poorly immunogenic tumor. We argue the exact mechanism(s) involved
in the antitumor responses elicited by anti-CTLA-4; try to open up a novel avenue for
the treatment of weakly immunogenic tumors.
We constructed a plasmid pGFP-FL, containing eukaryotic expressing GFP and
FL gene, and established a Hepal-6 murine hepatoma cell line (H2) harboring the
plasmid, which can express both GFP and FL. The H2 cells were further used as
FL-expressing tumor vaccines. We also prepared purified anti-muCTLA4 antibodies
using a commercial hybridoma cell line, which can secrete anti-muCTLA4.
Simultaneously, based on our former works, we produced an anti-huCTLA4-secreting
7H9B5A8 hybridoma cell line using the classical protocols for producing monoclonal
antibodies. This cell line may be beneficial to the future researches about humanizing
murine anti-huCTLA4 for clinical antitumor immunotherapy.
In the treatment of Hepal-6 bearing C57BL/J mice, we found that, FL- and
B7-expressing tumor vaccine, wide type Hepal-6 vaccine, or anti-muCTLA-4 alone
all showed inefficient effect against Hepal-6 tumors. And Hepal-6 tumors can be
fully rejected using both anti-CTLA4 and FL-expressing tumor vaccines, while no
synergizing effects were found between anti-CTLA4 and Hepal-6 vaccine, or FL- and
B7-expressing tumor vaccines. Notably, we also found that anti-CTLA4 can well
synergize with B7-expressing tumor vaccines. This result is different with that of
Hurwitz et at obtained from a similar research in 1998, in which they found that
anti-CTLA4 can not synergize with B7-expressing tumor vaccines. And, all mice that
displayed complete tumor rejection in different treatment groups were immune to
rechallenge of 10-fold Hepal-6 cells when take 30 days post tumor regression.
Taken together the results of others and our relevant research, we propose that,
anti-CTLA4 can simultaneously e
免疫治疗最重要的靶点。
细胞毒性T淋巴细胞相关抗原4（cytotoxic T lymphocyte associated antigen 4，
CTLA-4），是共刺激分子B7的受体，主要表达于激活的T细胞表面，与B7结
合后能抑制小鼠和人的T细胞的激活，在T细胞活化中起负调节作用。用抗
CTLA4抗体阻断CTLA-4与B7的结合，在体内外均能促进T细胞的增殖和活化
能力，从而增强T细胞的免疫反应，并能够产生良好的抗肿瘤效果。
另一个重要的提升T细胞抗肿瘤免疫反应的策略是以分泌细胞因子的肿瘤细
胞作为肿瘤疫苗，通过对T细胞和APC产生旁分泌效应，达到抗肿瘤的目的。
FL（fms-like tyrosine kinase receptor-3 ligand，fms样酪氨酸激酶受体3配体，Flt3
Ligand，FL）是一种新近发现的细胞因子。在体内外均能够诱导树突状细胞（DC）
和许多造血早期干细胞的增殖和活化，是目前发现的最为重要的DC生长因子之
一。而DC是体内一种重要的专职抗原提呈细胞（APC），在肿瘤抗原激发特异
性免疫的过程中具有重要作用，并且荷瘤宿主DC功能一般都低下。FL可以通
过促进体内抗肿瘤免疫效应细胞（DC、NK细胞、CTL等）的增殖、分化和成
熟，产生显著的抗肿瘤效应。
然而，对一些低免疫原性、高恶性的肿瘤，单一的免疫治疗策略并不能达到
理想的抗肿瘤效果。而且，目前关于anti-CTLA4抗体的抗肿瘤机制还未彻底明
了，存在着一些不同的认识。为此，我们研究了anti-CTLA4抗体与FL瘤苗和
B7瘤苗间可能存在的协同效应，进一步探讨了anti-CTLA4抗体的抗肿瘤作用机
制，以期为肝癌及其他低免疫原性肿瘤的临床免疫和基因治疗提供新的思路和实
验依据。
我们构建了含有一个FL基因和一个GFP基因真核表达质粒pGFP-FL，并
建立稳定转染有该质粒的小鼠肝癌细胞系Hepa1-6（H2），检测到了FL和GFP
基因在其中的高效表达，以之作为能够分泌FL细胞因子的瘤苗；利用商品化的
能够分泌抗小鼠CTLA4抗体的杂交瘤，制备、纯化得到了anti-muCTLA4抗体，
用于后续的动物实验；同时还在本所以往的工作基础上，用经典的单克隆抗体制
备方法，获得了一株可以抗人CTLA4的单克隆抗体杂交瘤7H9B5A8，为进一步
发展人源化抗人CTLA4单抗，用于临床抗肿瘤免疫治疗的研究工作提供了便利。
在对荷瘤Hepa1-6的C57BL/J小鼠的免疫治疗中，我们发现，单用
anti-muCTLA4抗体、FL瘤苗、B7瘤苗或野生型Hepa1-6瘤苗均不能完全排斥
Hepa1-6肿瘤；anti-muCTLA4抗体与FL瘤苗则可以使肿瘤完全消退；而联合应
用anti-muCTLA4抗体与野生型Hepa1-6瘤苗，或B7瘤苗与FL瘤苗，却未见有
协同效应。值得注意的是，在我们的实验中发现anti-muCTLA4抗体与B7瘤苗
联合治疗小鼠肿瘤具有很好的协同效应，这与1998年Hurwitz等在类似的研究
中所得出的结论不同，即他们认为anti-muCTLA4抗体与B7瘤苗间没有协同抗
小鼠肿瘤作用。另外，经不同方法治疗有效的小鼠均能够抵抗在肿瘤消退后30
天时，10倍于初次荷瘤剂量的野生型Hepa1-6肿瘤细胞的攻击。
综合国内外同类研究和我们的实验结果，我们认为，anti-CTLA4抗体在抗
肿瘤免疫中是通过多机制同时作用的。既可以通过阻断宿主体内APC上的B7
分子和肿瘤特异性T细胞表面的CTLA4分子的结合，增强宿主本身APC和T
细胞间的共刺激作用来达到抗肿瘤效应的；又可以通过促进未发生免疫耐受的肿
中立摘要
瘤特异性T细胞亚群的增殖与活化，放大T细胞与APC间的有效作用，来提升
机体的抗肿瘤兔疫效应。anh（TLA抗体与FL瘤苗，或7i（TLA抗体与B7
瘤苗联合治疗，都是一种较好的抗肿瘤兔疫联合治疗策略。这些结果也提示，最
有效的肿瘤疫苗协同治疗策略应该是那些既能够增强宿主的APC功能，同时又
能够阻断T细胞活化过程中的抑制信号，并能够刺激宿主体内尚未发生免疫耐受
的T细胞的增殖的方法。
另外，考虑到将来可能的肿瘤易感人群的预防接种问题，我们还对发展口服
型真核表达核酸疫苗用于肿瘤预防进行了初步的实验研究。
我们设计以GFP作为TAA，利用pEGFP－NI（GFP”，FL”）和pGFP－FL（GFP”，
FL勺两种真核表达质粒为核酸疫苗，采用减毒伤寒沙门氏菌SL7207作疫苗的
口服型载体，对小鼠进行口服兔疫，观察口服疫苗对GFP＋的HI肿瘤细胞
（Heyal6／pEGFP－NI）的预防作用。这样的研究目前还为发现有相同报道。
我们发现，野生型的SL7207和SL7207／pEGFP－NI均不能诱发小鼠的保护性
免疫，而SL7207／pGFP－FL则可以在部分小鼠（2／10）中诱发保护性免疫，使小
鼠免生肿瘤，并能抵抗在初次荷瘤攻击后40天，10倍于首次剂量的HI肿瘤细
胞的再攻击。这两只小鼠的脾淋巴细胞，体外对HI肿瘤细胞的刺激有明显的增
殖效应，而且流式细胞术检测到了其中分别有 17．4％和 16．29％的 GFP＋细胞的存
在。同时在体外实验中也发现，我们构建的两种口服型核酸疫苗?
T cell mediated immunity plays a key role in antitumor responses, and it is also
the major target of antitumor immunotherapy.
CTLA-4, a second T cell receptor for B7, which is expressed on the surface of
activated T cells, has been shown to play an inhibitory role in regulation of T cell
responses by preventing full T cell activation or terminating T cell responses. Also, it
has been shown that antibody-mediated CTLA-4 blockade can augment host
antitumor responses by prolonging T cell activity and/or facilitating antigen-specific T
cell costimulatory activation.
Another approach taken to raise antitumor immune responses has been to use
cytokine-expressing tumor cells as vaccines, which in turn have paracrine effects on T
cells or APCs. F1t3 Ligand(FL), a newly found cytokine, which is one of the most
important Dendritic Cells (DCs) growth factors, can expand and activate DCs and
some hemaprogenitors in vitro and in vivo. While DCs, the most important APCs, are
instrumental for the activation of tumor antigen-specific immune response. Recently,
it has been reported that FL can promote antitumor activity through inducing a
profound expansion of antitumor effect cells (DCs, NKs, CTLs).
However, it has been shown that these treatment regimens alone cannot induce
complete tumor regression in wealdy immunogenic tumors. And the exact
mechanism(s) involved in the antitumor responses elicited by CTLA-4 blockade
remains to be elucidated, there also exists different opinions about it. In the present
study, we test the hypothesis that FL- and B7-transduced tumor vaccine therapy,
combining with CTLA-4 blockade, should enhance antitumor responses in the
treatment of poorly immunogenic tumor. We argue the exact mechanism(s) involved
in the antitumor responses elicited by anti-CTLA-4; try to open up a novel avenue for
the treatment of weakly immunogenic tumors.
We constructed a plasmid pGFP-FL, containing eukaryotic expressing GFP and
FL gene, and established a Hepal-6 murine hepatoma cell line (H2) harboring the
plasmid, which can express both GFP and FL. The H2 cells were further used as
FL-expressing tumor vaccines. We also prepared purified anti-muCTLA4 antibodies
using a commercial hybridoma cell line, which can secrete anti-muCTLA4.
Simultaneously, based on our former works, we produced an anti-huCTLA4-secreting
7H9B5A8 hybridoma cell line using the classical protocols for producing monoclonal
antibodies. This cell line may be beneficial to the future researches about humanizing
murine anti-huCTLA4 for clinical antitumor immunotherapy.
In the treatment of Hepal-6 bearing C57BL/J mice, we found that, FL- and
B7-expressing tumor vaccine, wide type Hepal-6 vaccine, or anti-muCTLA-4 alone
all showed inefficient effect against Hepal-6 tumors. And Hepal-6 tumors can be
fully rejected using both anti-CTLA4 and FL-expressing tumor vaccines, while no
synergizing effects were found between anti-CTLA4 and Hepal-6 vaccine, or FL- and
B7-expressing tumor vaccines. Notably, we also found that anti-CTLA4 can well
synergize with B7-expressing tumor vaccines. This result is different with that of
Hurwitz et at obtained from a similar research in 1998, in which they found that
anti-CTLA4 can not synergize with B7-expressing tumor vaccines. And, all mice that
displayed complete tumor rejection in different treatment groups were immune to
rechallenge of 10-fold Hepal-6 cells when take 30 days post tumor regression.
Taken together the results of others and our relevant research, we propose that,
anti-CTLA4 can simultaneously e
引文
1 Lynch DH, Andreasen A, Maraskovsky E，et al．Flt3 ligand induces tumor regression and antitumor immune responses in vivo．Nat Med 1997；3(6) ：625-31
2 Braun SE，Chen K, Blazar BR, et al．FL antitumoractivity in a murine breast cancer model：a comparison with GM-CSF and a potential mechanism of action．Hum Gene Ther 1999；10(13) ： 2141-51
3 Heim R, Cubitt AB，Tsien RY．Improved green fluorescence．Nature 1995；373：663．
4 J．萨姆布鲁克， E．F．弗里奇，T．曼尼阿蒂斯 著，金冬雁，黎孟枫 译。《分子克隆实验 指向》，科学出版社，1992年第二版。
5 鄂征 主编，《组织培养技术》， 人民卫生出版社，1993年第2版
6 Shih PJ, Evansta K，Schifferli K，et al． High efficiency transfection with minimal optimization using the lipofectamin plus ~TM reagent．Focus (GIBCO)1997；19(3) ：52-56．
2 Braun SE，Chen K, Blazar BR, et al．FL antitumoractivity in a murine breast cancer model：a comparison with GM-CSF and a potential mechanism of action．Hum Gene Ther 1999；10(13) ： 2141-51
3 Heim R, Cubitt AB，Tsien RY．Improved green fluorescence．Nature 1995；373：663．
4 J．萨姆布鲁克， E．F．弗里奇，T．曼尼阿蒂斯 著，金冬雁，黎孟枫 译。《分子克隆实验 指向》，科学出版社，1992年第二版。
5 鄂征 主编，《组织培养技术》， 人民卫生出版社，1993年第2版
6 Shih PJ, Evansta K，Schifferli K，et al． High efficiency transfection with minimal optimization using the lipofectamin plus ~TM reagent．Focus (GIBCO)1997；19(3) ：52-56．