HER2靶向的嵌合性T细胞受体基因在淋巴造血细胞中的表达及其抗肿瘤效应的研究
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摘要
目的和意义:
HER2/neu 癌基因在许多恶性肿瘤细胞表面均有高表达,其中包括乳腺癌、
卵巢癌、直肠癌、胰腺癌及前列腺癌等。已有众多的研究结果证实由 HER2/neu
癌基因表达的 p185HER2 是免疫治疗乳腺癌等恶性肿瘤的极佳靶位点。在新确
诊的乳腺癌中约有 20-40%患者有 HER2/neu 基因的扩增或过表达。回顾性分析
表明此类患者对联合化疗的反应差、复发率高、总的长期生存率低。体外研究
及动物体内实验结果证实 p185HER2 在乳腺癌的发病及临床进程中发挥重要作
用。p185HER2 特异性单克隆抗体(mAb)对表达 p185HER2 的肿瘤细胞系在体外
生长有明显的抑制作用。目前 p185HER2 靶向的人源化的单克隆抗体 Herceptin
已成功应用于临床并在 HER2 阳性的乳腺癌患者取得了良好的疗效。
嵌合性 T 细胞受体(chTCR)是一种应用基因工程技术重组的免疫受体,它包
括细胞膜外的抗原识别区和细胞膜内的信号传导区两部分。目前最为常用的膜
外的抗原识别区为单链免疫球蛋白可变区片段(scFv),即由多肽将 mAb 的重链
可变区与轻链可变区相连接而成。scFv 与 mAb 的可变区对相应抗原具有相似的
特异性和亲合性。激活细胞的信号传导肽则分别选用 T 细胞受体(TCR)中的
ζ 链以及与其有类似作用的 FcεR1γ 链,由此重组而成的嵌合性受体 scFv-ζ/γ
chTCR 可象 mAb 一样特异性地与相应抗原结合,然后经由信号传导肽激活相应
的效应细胞。与天然的 TCR 相比,chTCR 的最大优点是与抗原结合时可不受
MHC 的限制。将 chTCR 导入 T 细胞或杂交瘤细胞系中,则所转染的细胞系即
具有相应的抗原特异性,并可让该细胞系在受到相应抗原刺激时释放细胞因子、
表现出细胞杀伤活性。
4
第二军医大学 博士学位论文 中文摘要
本研究期望:1)应用 CD3/ CD28 单抗激活淋巴细胞、CH296 包被和离心
转染等,建立一种能将 p185HER2 特异的 chTCR 基因通过逆转录病毒(RV)载
体高效转染淋巴细胞的方法。2)研究转染 chTCR 的淋巴细胞能否特异性杀伤
表达 p185HER2 的肿瘤细胞。3)比较 ζ 和 γ 信号传导肽在同一 chTCR 结构中
发挥信号传导的作用。4)应用逆转录病毒载体将 chTCR 转染小鼠造血干细胞,
用转基因造血干细胞移植的受体小鼠在造血重建后,其淋巴细胞是否对相应的
肿瘤细胞具有排斥作用。
方法和结果:
将 p185HER2 特异性的 mAb(N29)的 scFv 基因与 ζ 和 γ 信号传导肽基因
分别构建入逆转录病毒载体的骨架 pRET6 得到逆转录病毒载体 pRET6 N29γ和
pRET6 N29ζ。乒乓转染法将载体转入包装细胞系 PG13、GP+E86 成为稳定、高
效的产病毒细胞系,病毒滴度约为 1×106CFU/ml。健康人志愿者静脉血淋巴细
胞及小鼠脾脏淋巴细胞经 Ficoll 分离后过尼龙柱去除单核细胞,纯化的淋巴细
胞经包被在培养板上的抗 CD3、抗 CD28 单抗活化并加入 IL-2 培养后用于基因
转染实验。分别将活化的淋巴细胞和病毒上清加入 CH296 包被的培养板,离心
转染。转染效率为 50%-80%,转染后的淋巴细胞经流式细胞分析 CD8+ 、CD4+
的 T-细胞分别占 80%和 20%。转基因的淋巴细胞分别与高表达 p185HER2 的肿
瘤细胞系 SK-OV-3 及不表达 p185HER2 的 MCF7 共培养,检测培养上清中细胞
因子 GM-CSF、TNF-α、IFN-γ的浓度,结果表明转染 chTCR 的淋巴细胞与高
表达 p185HER2 的 SK-OV-3 共培养组三种细胞因子浓度显著高于对照组。转染
载体 pRET6 N29γ的淋巴细胞在细胞因子释放方面明显优于转染 pRET6 N29ζ
者。
将 HER2/neu 基因构建入逆转录病毒载体的骨架 pRET6 得到逆转录病毒载
体 pRET6HER2 ,将载体转入包装细胞系 GP+E86 成为稳定、高效的产病毒细
胞系,收集病毒上清分别转染小鼠肿瘤细胞系 MCA-205 和 MT-901,转基因细
胞系皮下及静脉注射后得到表达人p185HER2的小鼠皮下肿瘤和肺转移瘤模型。
含 pRET6 N29γ和 pRET6 N29ζ病毒上清分别转染小鼠骨髓造血细胞后移植
给经ΤΒΙ 9.0 Gy 预处理的同系小鼠,移植后 6 个月时分别经皮下或静脉接种转
5
第二军医大学 博士学位论文 中文摘要
染 HER2 的同系小鼠肿瘤细胞系 MCA-205/ HER2 和 MT-901/ HER2。结果,小
鼠皮下肿瘤 MT-901/ HER2 生长明显较对照组缓慢;在 MCA-205/ HER2 肺转移
瘤模型中,肺内肿瘤结节数显著较对照组少。
结论:
1.本研究应用重组 DNA 技术构建的逆转录病毒载体 pRET6 N29γ和 pRET6
N29ζ,将逆转录病毒载体 pRET6 N29γ和 pRET6 N29ζ转染经抗 CD3、抗 CD28
单抗活化的人或小鼠淋巴细胞后,转基因的淋巴细胞可特异性地识别相应的肿
瘤抗原 p185HER2,并通过释放 Th1 类细胞因子 GM-CSF、TNF-α、IFN-γ及
CTL 对表达 p185HER2 的肿瘤细胞产生明显的杀伤作用。
2.我们通过不断的改良,应用相应的抗 CD3、抗 CD28 单抗激活人或小鼠
淋巴细胞,使淋巴细胞处于最佳的活化状态,随后采用 RetroNectin (CH296)
包被的培养板并结合离心转染法,使人淋巴细胞的基因转染效率稳定保持在
50%至 60%;小鼠淋巴细胞的基因转染效率稳定在 80%以上。高效的基因?
The HER2/neu oncogene is overexpressed in a variety of human malignant
disorders, including breast cancer, ovarian cancer, colon cancer, pancreatic cancer,
and non-small cell lung cancer. Several lines of evidence suggest that p185HER2 is
an excellent target for immune based therapy of breast cancer. HER2/neu is
amplified or overexpressed in approximately 20-40% of newly diagnosed human
breast cancers. Overexpression of HER2/neu is a predictive marker of therapeutic
response, identifying patients at increased risk for early disease relapse and reduced
overall survival. Studies in cultured cell lines and animal models suggest a direct
role for HER2 in the pathogenesis and aggressive clinical behavior of these tumors.
Antibodies specific for p185HER2 inhibit the growth of cell lines and tumors
expressing elevated levels of the receptor. Clinical trials of anti-p185HER2 mAb
(Herceptin), administered to women with metastatic breast cancer, have documented
clinical responses with minimal toxicity.
Chimeric T-cell receptors (chTCR) are recombinant immune receptors
composed of two domains - an extracellular antigen binding domain and an
intracellular signaling domain. The most commonly employed antigen targeting
domain is a single chain Fv (scFv), whereby the VL and VH domains of a monoclonal
antibody (mAb) are tethered in a single polypeptide by a flexible linker. scFv
regions exhibit similar specificities and affinities compared to normal antibody
variable regions. Cellular activation is achieved by coupling the antigen binding
domain to an intracellular signaling chain. The ζ-chain of the T-cell receptor and
the FcεR1γ-chain are closely related and are capable of activating T-cells and/or
myeloid cells when incorporated in chimeric receptor constructs. Thus, in a single
molecule, the scFv-ζ/γ chTCR combines the exquisite antigen specificity of a mAb
and signal transduction elements necessary to activate effector cell function. The
antigenic target is an intact cell surface protein defined by a mAb. A significant
potential advantage of this design is the lack of MHC restriction and/or need for
antigen processing.
Such chTCR molecules have been successfully developed to target a variety of
tumor antigens, including the Mov18-defined ovarian tumor antigen, TAG-72, CEA,
and p185HER2. Introduction of the scFv-ζ/γ construct into T cell lines or
hybridomas has resulted in a functioning chTCR with antigen specificity, as defined
by cytokine release and/or cytotoxicity following stimulation of the transduced
effector cells with the appropriate antigen-bearing target cell, and antigen specific
regression of tumor in animal models of T-cell adoptive immunotherapy.
Objective:
1. To demonstrate if the anti-CD3/anti-CD28 activated human or murine
T-cells could be efficiently transduced with a p185HER2 specific chTCR constructs.
2. To investigate if retroviral gene transfer of a chTCR could efficiently
redirects primary human T-cell responses to p185HER2 positive tumor cells.
3. To compare two different signal transduction chains in our construct side by
side and find out which chain is more efficient regarding cytokine release and
cytotoxicity.
4. To define the antigen specific anti-tumor effects carried by the chTCR gene
modified hematopoietic stem cells in vivo in a mouse model.
Methods and results:
We have developed and tested chimeric T-cell receptors (TCR) specific for
p185HER2, the product of the HER2/neu gene. Retroviral vectors expressing the
N29γ or N29ζ receptors were constructed in pRET6, a bicistronic MPSV-based
backbone developed in our laboratory. Amphotropic viral producer cells were
established in the GALV-based PG13, GP+E86 packaging cell line. Ficoll purified
human peripheral blood lymphocytes (PBL) and murine spleen lymphocytes were
virally transduced using an optimized protocol incorporating activation with
immobilized anti-CD3/anti-CD28 monoclonal antibodies, followed by viral infection
in the presenc
HER2/neu 癌基因在许多恶性肿瘤细胞表面均有高表达,其中包括乳腺癌、
卵巢癌、直肠癌、胰腺癌及前列腺癌等。已有众多的研究结果证实由 HER2/neu
癌基因表达的 p185HER2 是免疫治疗乳腺癌等恶性肿瘤的极佳靶位点。在新确
诊的乳腺癌中约有 20-40%患者有 HER2/neu 基因的扩增或过表达。回顾性分析
表明此类患者对联合化疗的反应差、复发率高、总的长期生存率低。体外研究
及动物体内实验结果证实 p185HER2 在乳腺癌的发病及临床进程中发挥重要作
用。p185HER2 特异性单克隆抗体(mAb)对表达 p185HER2 的肿瘤细胞系在体外
生长有明显的抑制作用。目前 p185HER2 靶向的人源化的单克隆抗体 Herceptin
已成功应用于临床并在 HER2 阳性的乳腺癌患者取得了良好的疗效。
嵌合性 T 细胞受体(chTCR)是一种应用基因工程技术重组的免疫受体,它包
括细胞膜外的抗原识别区和细胞膜内的信号传导区两部分。目前最为常用的膜
外的抗原识别区为单链免疫球蛋白可变区片段(scFv),即由多肽将 mAb 的重链
可变区与轻链可变区相连接而成。scFv 与 mAb 的可变区对相应抗原具有相似的
特异性和亲合性。激活细胞的信号传导肽则分别选用 T 细胞受体(TCR)中的
ζ 链以及与其有类似作用的 FcεR1γ 链,由此重组而成的嵌合性受体 scFv-ζ/γ
chTCR 可象 mAb 一样特异性地与相应抗原结合,然后经由信号传导肽激活相应
的效应细胞。与天然的 TCR 相比,chTCR 的最大优点是与抗原结合时可不受
MHC 的限制。将 chTCR 导入 T 细胞或杂交瘤细胞系中,则所转染的细胞系即
具有相应的抗原特异性,并可让该细胞系在受到相应抗原刺激时释放细胞因子、
表现出细胞杀伤活性。
4
第二军医大学 博士学位论文 中文摘要
本研究期望:1)应用 CD3/ CD28 单抗激活淋巴细胞、CH296 包被和离心
转染等,建立一种能将 p185HER2 特异的 chTCR 基因通过逆转录病毒(RV)载
体高效转染淋巴细胞的方法。2)研究转染 chTCR 的淋巴细胞能否特异性杀伤
表达 p185HER2 的肿瘤细胞。3)比较 ζ 和 γ 信号传导肽在同一 chTCR 结构中
发挥信号传导的作用。4)应用逆转录病毒载体将 chTCR 转染小鼠造血干细胞,
用转基因造血干细胞移植的受体小鼠在造血重建后,其淋巴细胞是否对相应的
肿瘤细胞具有排斥作用。
方法和结果:
将 p185HER2 特异性的 mAb(N29)的 scFv 基因与 ζ 和 γ 信号传导肽基因
分别构建入逆转录病毒载体的骨架 pRET6 得到逆转录病毒载体 pRET6 N29γ和
pRET6 N29ζ。乒乓转染法将载体转入包装细胞系 PG13、GP+E86 成为稳定、高
效的产病毒细胞系,病毒滴度约为 1×106CFU/ml。健康人志愿者静脉血淋巴细
胞及小鼠脾脏淋巴细胞经 Ficoll 分离后过尼龙柱去除单核细胞,纯化的淋巴细
胞经包被在培养板上的抗 CD3、抗 CD28 单抗活化并加入 IL-2 培养后用于基因
转染实验。分别将活化的淋巴细胞和病毒上清加入 CH296 包被的培养板,离心
转染。转染效率为 50%-80%,转染后的淋巴细胞经流式细胞分析 CD8+ 、CD4+
的 T-细胞分别占 80%和 20%。转基因的淋巴细胞分别与高表达 p185HER2 的肿
瘤细胞系 SK-OV-3 及不表达 p185HER2 的 MCF7 共培养,检测培养上清中细胞
因子 GM-CSF、TNF-α、IFN-γ的浓度,结果表明转染 chTCR 的淋巴细胞与高
表达 p185HER2 的 SK-OV-3 共培养组三种细胞因子浓度显著高于对照组。转染
载体 pRET6 N29γ的淋巴细胞在细胞因子释放方面明显优于转染 pRET6 N29ζ
者。
将 HER2/neu 基因构建入逆转录病毒载体的骨架 pRET6 得到逆转录病毒载
体 pRET6HER2 ,将载体转入包装细胞系 GP+E86 成为稳定、高效的产病毒细
胞系,收集病毒上清分别转染小鼠肿瘤细胞系 MCA-205 和 MT-901,转基因细
胞系皮下及静脉注射后得到表达人p185HER2的小鼠皮下肿瘤和肺转移瘤模型。
含 pRET6 N29γ和 pRET6 N29ζ病毒上清分别转染小鼠骨髓造血细胞后移植
给经ΤΒΙ 9.0 Gy 预处理的同系小鼠,移植后 6 个月时分别经皮下或静脉接种转
5
第二军医大学 博士学位论文 中文摘要
染 HER2 的同系小鼠肿瘤细胞系 MCA-205/ HER2 和 MT-901/ HER2。结果,小
鼠皮下肿瘤 MT-901/ HER2 生长明显较对照组缓慢;在 MCA-205/ HER2 肺转移
瘤模型中,肺内肿瘤结节数显著较对照组少。
结论:
1.本研究应用重组 DNA 技术构建的逆转录病毒载体 pRET6 N29γ和 pRET6
N29ζ,将逆转录病毒载体 pRET6 N29γ和 pRET6 N29ζ转染经抗 CD3、抗 CD28
单抗活化的人或小鼠淋巴细胞后,转基因的淋巴细胞可特异性地识别相应的肿
瘤抗原 p185HER2,并通过释放 Th1 类细胞因子 GM-CSF、TNF-α、IFN-γ及
CTL 对表达 p185HER2 的肿瘤细胞产生明显的杀伤作用。
2.我们通过不断的改良,应用相应的抗 CD3、抗 CD28 单抗激活人或小鼠
淋巴细胞,使淋巴细胞处于最佳的活化状态,随后采用 RetroNectin (CH296)
包被的培养板并结合离心转染法,使人淋巴细胞的基因转染效率稳定保持在
50%至 60%;小鼠淋巴细胞的基因转染效率稳定在 80%以上。高效的基因?
The HER2/neu oncogene is overexpressed in a variety of human malignant
disorders, including breast cancer, ovarian cancer, colon cancer, pancreatic cancer,
and non-small cell lung cancer. Several lines of evidence suggest that p185HER2 is
an excellent target for immune based therapy of breast cancer. HER2/neu is
amplified or overexpressed in approximately 20-40% of newly diagnosed human
breast cancers. Overexpression of HER2/neu is a predictive marker of therapeutic
response, identifying patients at increased risk for early disease relapse and reduced
overall survival. Studies in cultured cell lines and animal models suggest a direct
role for HER2 in the pathogenesis and aggressive clinical behavior of these tumors.
Antibodies specific for p185HER2 inhibit the growth of cell lines and tumors
expressing elevated levels of the receptor. Clinical trials of anti-p185HER2 mAb
(Herceptin), administered to women with metastatic breast cancer, have documented
clinical responses with minimal toxicity.
Chimeric T-cell receptors (chTCR) are recombinant immune receptors
composed of two domains - an extracellular antigen binding domain and an
intracellular signaling domain. The most commonly employed antigen targeting
domain is a single chain Fv (scFv), whereby the VL and VH domains of a monoclonal
antibody (mAb) are tethered in a single polypeptide by a flexible linker. scFv
regions exhibit similar specificities and affinities compared to normal antibody
variable regions. Cellular activation is achieved by coupling the antigen binding
domain to an intracellular signaling chain. The ζ-chain of the T-cell receptor and
the FcεR1γ-chain are closely related and are capable of activating T-cells and/or
myeloid cells when incorporated in chimeric receptor constructs. Thus, in a single
molecule, the scFv-ζ/γ chTCR combines the exquisite antigen specificity of a mAb
and signal transduction elements necessary to activate effector cell function. The
antigenic target is an intact cell surface protein defined by a mAb. A significant
potential advantage of this design is the lack of MHC restriction and/or need for
antigen processing.
Such chTCR molecules have been successfully developed to target a variety of
tumor antigens, including the Mov18-defined ovarian tumor antigen, TAG-72, CEA,
and p185HER2. Introduction of the scFv-ζ/γ construct into T cell lines or
hybridomas has resulted in a functioning chTCR with antigen specificity, as defined
by cytokine release and/or cytotoxicity following stimulation of the transduced
effector cells with the appropriate antigen-bearing target cell, and antigen specific
regression of tumor in animal models of T-cell adoptive immunotherapy.
Objective:
1. To demonstrate if the anti-CD3/anti-CD28 activated human or murine
T-cells could be efficiently transduced with a p185HER2 specific chTCR constructs.
2. To investigate if retroviral gene transfer of a chTCR could efficiently
redirects primary human T-cell responses to p185HER2 positive tumor cells.
3. To compare two different signal transduction chains in our construct side by
side and find out which chain is more efficient regarding cytokine release and
cytotoxicity.
4. To define the antigen specific anti-tumor effects carried by the chTCR gene
modified hematopoietic stem cells in vivo in a mouse model.
Methods and results:
We have developed and tested chimeric T-cell receptors (TCR) specific for
p185HER2, the product of the HER2/neu gene. Retroviral vectors expressing the
N29γ or N29ζ receptors were constructed in pRET6, a bicistronic MPSV-based
backbone developed in our laboratory. Amphotropic viral producer cells were
established in the GALV-based PG13, GP+E86 packaging cell line. Ficoll purified
human peripheral blood lymphocytes (PBL) and murine spleen lymphocytes were
virally transduced using an optimized protocol incorporating activation with
immobilized anti-CD3/anti-CD28 monoclonal antibodies, followed by viral infection
in the presenc
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26
第二军医大学 博士学位论文 第一部分
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10. Stoolman LM.Adhesion molecules controlling lymphocyte migration. Cell,1989;56:1989
11. Nakagomi H, Petersson M, Magnusson I, et al. Decreased expression of the
signal-transducing zeta chains in tumor-infiltrating T-cells and NK cells of patients with
colorectal carcinoma. Cancer Res, 1993;53:5610-2
12. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin/T cell receptor chimeric
molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA,
1989;86:10024
13. Gross G, Gorochov G, Waks T, et al. Generation of effector T cells expressing chimeric T
cell receptors with antibody type specificity. Transplant Proc, 1989;21:127
14. Bird RE, Hardman KD, Jacobson JW, et al. Single chain antigen binding proteins. Science,
1988;242:423
15. Hwu P, Shafer GE, Treisman J, et al. Lysis of ovarian cancer cells by human lymphocytes
redirected with a chimeric gene composed of an antibody variable region and the Fc
receptorγ chain. J Exp Med, 1993;178:361
16. Riviere I, Brose K, and Mulligan RC. Effects of retroviral vector design on expression of
human adenosine deaminase in murine bone marrow transplant recipients engrafted with
genetically modified cells. Proc Natl Acad Sci USA, 1995;92:6733-7
17. Challita P, Skelton D, El-Khoueiry A, et al. Multiple modifications in cis elements of the
long terminal repeat of retroviral vectors lead to increased expression and decreased DNA
methylation in embryonic carcinoma cells. J Virology, 1995;69:748-55
18. Robbins PB, Yu XJ, Skelton DM, et al. Increased probability of expression from modified
retroviral vectors in embryonal stem cells and embryonal carcinoma cells. J Virology,
1997;71:9466-74
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第二军医大学 博士学位论文 第一部分
19. Robbins PB, Skelton DC, Yu XJ, et al. Consistent, persistent expression from modified
retroviral vectors in murine hematopoietic stem cells. Proc Natl Acad Sci USA,
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transcribed in embryonal carcinoma stem cells have gained a functional Sp1 binding site. J
Virology,1991;65:1803-11
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leukemia virus long terminal repeat in embryonal stem cells. J virology, 1991;65:4961-98
22. Adam MA, Ramesh N, Miller AD, et al. Internal initiation of translation in retroviral vectors
carrying picomavirus 5’nontranslated regions. J Virol, 1991;65:4985
23. Morgan RA, Couture L, Elroy-stein O, et al. Retroviral vectors containing putative internal
ribosome entry sites: Development of a polycistronic gene transfer system and applications
to human gene therapy. Nucleic Acids Res. 1992;20:1293
24. Sugimoto Y, Aksentijevich I, Gottesman MM, et al. Efficient expression of drug-selectable
genes in retroviral vectors under control of an internal ribosomal entry site. Biotechnology.
1994;12:694
25. Levenson VV, Transue EDE, and Roninson IB. Internal ribosomal entry site-containing
retroviral vectors with green fluorescent protein and drug resistance markers. Human Gene
Ther,1998;9:1233
26. Riddell SR, Elliott M, Lewinsohn DA, et al. T-cell mediated rejection of gene-modified
HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nature Med, 1996;2:216
27. Xu L, Yee JK, Wolff JA, et al. Factors affecting long-term stability of Moloney murine
leukemia virus-based vectors. Virology, 1989;171:331
28. von Melcher H, and Houseman D. The expression of neomycin phosphotransferase in
human promyelocytic leukemia cells (HL-60) delays their differentiation. Oncogene,
1988;2:137
29. Onodera M, Yachie A, Nelson DM, et al. A simple and reliable method for screening
retroviral producer clones without selectable markers. Human Gene Ther, 1997;8:1189
30. Bowles NE, Eisensmith RC, Mohuiddin R, et al. A simple and efficient method for the
concentration and purification of recombinant retrovirus for increased hepatocyte
transduction in vitro. Hum Gene Ther, 1996; 7:1735
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Practice of Oncology. DeVita VTJ, Hellman S, Rosenberg SA, editors. JB Lippincott Co.,
Philadelphia. 1993; P293
8. Rosenberg SA. Cell transfer therapy: Clinical applications. In Biologic Therapy of Cancer.
DeVita VTJ, Hellman S, Rosenberg SA, editors. JB Lippincott Co., Philadelphia. 1994;
P487
9. Riethmuller G. Monoclonal antibodies in cancer therapy. Curr Opin Immunol, 1993;5:732
10. Stoolman LM.Adhesion molecules controlling lymphocyte migration. Cell,1989;56:1989
11. Nakagomi H, Petersson M, Magnusson I, et al. Decreased expression of the
signal-transducing zeta chains in tumor-infiltrating T-cells and NK cells of patients with
colorectal carcinoma. Cancer Res, 1993;53:5610-2
12. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin/T cell receptor chimeric
molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci USA,
1989;86:10024
13. Gross G, Gorochov G, Waks T, et al. Generation of effector T cells expressing chimeric T
cell receptors with antibody type specificity. Transplant Proc, 1989;21:127
14. Bird RE, Hardman KD, Jacobson JW, et al. Single chain antigen binding proteins. Science,
1988;242:423
15. Hwu P, Shafer GE, Treisman J, et al. Lysis of ovarian cancer cells by human lymphocytes
redirected with a chimeric gene composed of an antibody variable region and the Fc
receptorγ chain. J Exp Med, 1993;178:361
16. Riviere I, Brose K, and Mulligan RC. Effects of retroviral vector design on expression of
human adenosine deaminase in murine bone marrow transplant recipients engrafted with
genetically modified cells. Proc Natl Acad Sci USA, 1995;92:6733-7
17. Challita P, Skelton D, El-Khoueiry A, et al. Multiple modifications in cis elements of the
long terminal repeat of retroviral vectors lead to increased expression and decreased DNA
methylation in embryonic carcinoma cells. J Virology, 1995;69:748-55
18. Robbins PB, Yu XJ, Skelton DM, et al. Increased probability of expression from modified
retroviral vectors in embryonal stem cells and embryonal carcinoma cells. J Virology,
1997;71:9466-74
27
第二军医大学 博士学位论文 第一部分
19. Robbins PB, Skelton DC, Yu XJ, et al. Consistent, persistent expression from modified
retroviral vectors in murine hematopoietic stem cells. Proc Natl Acad Sci USA,
1998;95:10182-7
20. Prince VE, and Rigby PJ. Derivatives of moloney murine sarcoma virus capable of being
transcribed in embryonal carcinoma stem cells have gained a functional Sp1 binding site. J
Virology,1991;65:1803-11
21. Grez M, Zornig M, Nowock J, et al. A single point mutation activates the moloney murine
leukemia virus long terminal repeat in embryonal stem cells. J virology, 1991;65:4961-98
22. Adam MA, Ramesh N, Miller AD, et al. Internal initiation of translation in retroviral vectors
carrying picomavirus 5’nontranslated regions. J Virol, 1991;65:4985
23. Morgan RA, Couture L, Elroy-stein O, et al. Retroviral vectors containing putative internal
ribosome entry sites: Development of a polycistronic gene transfer system and applications
to human gene therapy. Nucleic Acids Res. 1992;20:1293
24. Sugimoto Y, Aksentijevich I, Gottesman MM, et al. Efficient expression of drug-selectable
genes in retroviral vectors under control of an internal ribosomal entry site. Biotechnology.
1994;12:694
25. Levenson VV, Transue EDE, and Roninson IB. Internal ribosomal entry site-containing
retroviral vectors with green fluorescent protein and drug resistance markers. Human Gene
Ther,1998;9:1233
26. Riddell SR, Elliott M, Lewinsohn DA, et al. T-cell mediated rejection of gene-modified
HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nature Med, 1996;2:216
27. Xu L, Yee JK, Wolff JA, et al. Factors affecting long-term stability of Moloney murine
leukemia virus-based vectors. Virology, 1989;171:331
28. von Melcher H, and Houseman D. The expression of neomycin phosphotransferase in
human promyelocytic leukemia cells (HL-60) delays their differentiation. Oncogene,
1988;2:137
29. Onodera M, Yachie A, Nelson DM, et al. A simple and reliable method for screening
retroviral producer clones without selectable markers. Human Gene Ther, 1997;8:1189
30. Bowles NE, Eisensmith RC, Mohuiddin R, et al. A simple and efficient method for the
concentration and purification of recombinant retrovirus for increased hepatocyte
transduction in vitro. Hum Gene Ther, 1996; 7:1735
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