高特异性HER2/neu手性多肽疫苗实验研究
|
摘要
细胞免疫是机体清除体内变异细胞的主要途径，其中细胞毒性T淋巴细胞(CTL)发挥关键作用。CTL的激活是通过识别抗原递呈细胞（APC）表面 MHCⅠ类分子递呈的抗原肽，被递呈的抗原一般为9～11个氨基酸的肽段。现已证实，合成肽不需要APC的加工、处理就能直接与APC表面的MHCⅠ类分子结合，与天然的内源性肽在激活免疫系统方面具有同等效力。肿瘤抗原肽的鉴定使直接应用明确的抗原肽作为肿瘤疫苗成为可能。但是合成肽疫苗分子较小，免疫原性较低，在体内半衰期短，容易被酶类降解,导致其诱导的特异性CTL存在时间短，效价低，不易达到使肿瘤消退所需的阈值。
为此本研究主要从以下两方面着手突破这种低应答状态：①在不影响合成多肽疫苗与MHC-Ⅰ类分子结合的前提下，在合成多肽中掺入D型氨基酸，使其局部的旋光性发生改变。合成的D型抗原肽对蛋白酶的降解具有抗性，其稳定性提高，体内的半衰期延长，免疫原性增强。②联合应用免疫佐剂GM-CSF，活化巨噬细胞和其它免疫反应细胞，以期获得较强的细胞免疫应答反应。
一．HER2/neu多肽疫苗序列的选择、设计与固相合成
1．多肽氨基酸序列的选择与设计：应用SYFPETTHI基序法预测系统进行
CTL表位初步预测，从抗原肽预测结果中选取两种区段P42-50 (HLYQGCQVV)、P782-790 ( VSRLLGICL)。因为MHC-Ⅰ类分子不同等位基因结合多肽的氨基酸残基变化主要局限在氨基末端，因此，在不改变多肽与MHC-Ⅰ类分子结合的前提下，改变多肽抗原的羧基端氨基酸，将P42-50的羧基端残基L-Leu替换为D-Leu , P782-790的羧基端残基L-Ser替换为D-Ser，合成了具有局部手性特征的多肽D-P42-50，D-P782-790，以期增强多肽在体内稳定性的同时提高其免疫原性。
2．多肽合成：采用Fmoc固相多肽合成策略，合成上述四种多肽，L-P42-50，，D-P42-50，L-P782-790和D-P782-790。
3．多肽的纯化及鉴定：利用中压反相液相层析对粗肽进行分析及纯化，L-P42-50、D-P42-50的纯度分别为99.2%及98.9% ，L-P782-790、D-P782-790的纯度分别为99.1%和99.0% 。利用激光解吸电离飞行时间质谱测定各色谱峰的分子量，经真空旋转蒸发及冻干获得纯品多肽。结果L-P42-50、D-P42-50总产率分别为63%及45%，，L-P782-790、D-P782-790总产率分别为58%及46%，。
二．合成多肽疫苗体内诱导CTL作用研究
小鼠乳腺癌模型的建立及多肽疫苗免疫 选用雌性BALB/c小鼠接种D2F2细胞,分别为对照组、GM-CSF组、L-P42-50、L-P42-50+ GM-CSF组、D-P42-50、D-P42-50+ GM-CSF组、L-P782-790、L-P782-790+ GM-CSF组、D-P782-790、D-P782-790+ GM-CSF组。
1．与对照组小鼠相比，L-P42-50+GM-CSF组、L-P782-790+GM-CSF组、D-P42-50+GM-CSF组和D-P782-790+GM-CSF组小鼠脾细胞重量增加差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），对淋巴细胞增殖的促进作用差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），分泌IFN-γ的T淋巴细胞数明显增多（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），对D2F2乳腺癌细胞的特异杀伤作用差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）。这四种多肽联合应用GM-CSF 都能促进小鼠T细胞增殖，促进T细胞分泌IFN-γ及激活CTL杀伤乳腺癌细胞，说明这两种预测的L型多肽在体内与MHC-Ⅰ类分子相结合产生复合物，与T细胞受体结合，激活CTL
细胞，对乳腺癌细胞产生杀伤作用。
2．D-P42-50+GM-CSF组和L-P42-50+GM-CSF组、D-P782-790+GM-CSF组L-P782-790+GM-CSF相比，小鼠脾细胞重量的增加差异显著（P﹤0.05, P﹤0.05），对淋巴细胞增殖的促进作用差异显著（P﹤0.05, P﹤0.05）,分泌γ-IFN的T淋巴细胞数增多（P﹤0.05, P﹤0.05）,诱导对D2F2乳腺癌细胞的特异杀伤作用也有显著差异（P﹤0.05, P﹤0.05）。D-P42-50和D-P782-790辅以GM-CSF可显著促进小鼠T细胞增殖，促进T细胞分泌IFN-γ及激活CTL杀伤乳腺癌细胞，说明这两种经改造的手性多肽与MHC-Ⅰ类分子结合的能力并没有发生改变，与L型多肽一样，在体内与MHC-Ⅰ类分子相结合产生复合物，与T细胞受体结合，激活CTL细胞，对乳腺癌细胞产生杀伤作用。D-P42-50和 D-P782-790多肽在局部上具有了手性特征，具有了不同于体内肿瘤细胞所表达的HER2/neu多肽的结构特征，在一定程度上增加了免疫原性，从而使其更能够有效地诱导CTL。本研究的淋巴细胞增殖实验和CTL杀伤活性测定结果也肯定了这一实验设计的可行性和有效性。
三．合成多肽疫苗体内抑瘤实验研究
BALB/c小鼠接种小鼠乳腺癌细胞D2F2后，进行多肽疫苗接种。接种肿瘤细胞42天后处死小鼠，取肿瘤组织进行光镜及电镜观察。
1．小鼠肿瘤组织体积和重量：与对照组小鼠相比，L-P42-50+GM-CSF组、L-P782-790+GM-CSF组、D-P42-50+GM-CSF组和D-P782-790+GM-CSF组的肿瘤重量和体积明显低于对照组，有显著性差异（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），而且D-P42-50+GM-CSF组和D-P782-790+GM-CSF组的肿瘤重量和体积与L-P42-50+GM-CSF组和L-P782-790+GM-CSF组相比，有显著差异（P﹤0.05, P﹤0.05）。NaCl组、GM-CSF 组、L-P42-50 组、D-P42-50组、L-P782-790组和 D-P782-790组小鼠与对照组相比无显?
Cell immunogy is the major to eliminate the tumor cells in the body,and cototo-
xic T cells(CTL) is the most immportant.CTL is activated by the compound of MHC-Ⅰmolecuar and antigen peptide which is presented by antigen presentation cells(APC).The length of the antigen peptide is most 9～11 amino acids.It is has been proved that synthesized peptide can combine with MHC-Ⅰmolecuar on the surface of APC directly without the processing of APC,have the same effect as the natual endogenous peptide.The identification of the tumor antigen peptide can apply the peptide as the tumor vaccine.But peptide molecular is small ,the immunogenicity of peptide is low,half-life time of peptide is short and the peptide is hydrolized by enzyme easily.All these can influence the existing time and valence of CTL.
Two methods were used to improve these situation : ①Change regional optical rotation by turn some L-peptide to D-peptide without altering the combination of peptide and MHC-Ⅰmolecuar. The synthesized D-peptide can be stable by resisting the hydrolization of the protein enzyme and increase the half-life time thus increade immunogenicity of peptide. ②Immunity ajuvant GM-CSF is used to activate the macrophage and other immunity cells and get the more powerful cell immunity.
1.The selection,designation and synthesis of the HER2/neu peptide:
(1) Design of the amino acid sequence of peptide: SYFPETTHI sequence
analysis system was used to predict MHC-Ⅰmolecular restricted CTL epi-position of HER2/neu protein ,and select two region P42-50 (HLYQGCQVV) and P782-790 ( VSRLLGICL).Because the change of combination region of the peptide with the different allel gene of MHC-Ⅰmolecular is locolized the N-terminal amino acid of the peptide, so we change the C-terminal amino acid without changing the combination of the peptide and the MHC-Ⅰmolecular.We substitute D-Leu for L-Leu for the P42-50, substitute D-Ser for L-Ser for P782-790,and get two chiral peptide vaccine D-P42-50,D-P782-790.
(2) Peptide synthesis: Peptides L-P42-50, D-P42-50,L-P782-790,D-P782-790 were synthesized with solid phase peptide synthesis method and Fmoc-strategy. Each amino acid was fully protected during the peptide synthesis procedure.
(3) The purification and identification of peptide:The peptide was purifed by middle pressure reverse liquid chromatography.And the purity of L-P42-50, D-P42-50 is 99.2%,98.9%, the purity of L-P782-790, D-P782-790 is 99.1%,99.0%. The fraction of interested was confirmed by measuring the molecular weight of each fraction with EMS. Then the crude peptide was purified on preparative RP-HPLC. The purified peptide was received through the collection of interested fraction and the disposal of vacuum rotation evaporation and ice freeze. The product of pure peptide L-P42-50, D-P42-50 is 63% and 45% ,the product of pure peptide L-P782-790,D-P782-790is 58% and 46% .
2. Study on induction of specific CTL in vivo by peptide vaccine immunization.
The construct of mouse breast tumor model and vaccinated with peptide vaccine:BALB/c mice were inoculated with mouse breast tumor cell line D2F2(which was transfected with full cDNA of human HER2/neu gene), then vaccinated with peptide vaccine and divided for ten group:Nacl group;GM-CSF group; L-P42-50 group;L-P42-50+ GM-CSF group;D-P42-50 group; D-P42-50+ GM-CSF group;L-P782-790
group; L-P782-790 + GM-CSF group; D-P782-790 group; D-P782-790 + GM-CSF group.
(1) Compared with the control group; L-P42-50+ GM-CSF group; D-P42-50+ GM-CSF group; L-P782-790+ GM-CSF group and D-P782-790 + GM-CSF group have significant difference on the increase of the weight of spleen（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）;promoting the proliferation of T lymphocytes（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）;the secreting γ-IFN of T cells（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）; and HER2/neu specific CTL（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）. The results indicated all these four peptides can form compound with MHC-Ⅰmolecular,bind to the T cell receptor,activated special CTL.
(2) Compared with L
为此本研究主要从以下两方面着手突破这种低应答状态：①在不影响合成多肽疫苗与MHC-Ⅰ类分子结合的前提下，在合成多肽中掺入D型氨基酸，使其局部的旋光性发生改变。合成的D型抗原肽对蛋白酶的降解具有抗性，其稳定性提高，体内的半衰期延长，免疫原性增强。②联合应用免疫佐剂GM-CSF，活化巨噬细胞和其它免疫反应细胞，以期获得较强的细胞免疫应答反应。
一．HER2/neu多肽疫苗序列的选择、设计与固相合成
1．多肽氨基酸序列的选择与设计：应用SYFPETTHI基序法预测系统进行
CTL表位初步预测，从抗原肽预测结果中选取两种区段P42-50 (HLYQGCQVV)、P782-790 ( VSRLLGICL)。因为MHC-Ⅰ类分子不同等位基因结合多肽的氨基酸残基变化主要局限在氨基末端，因此，在不改变多肽与MHC-Ⅰ类分子结合的前提下，改变多肽抗原的羧基端氨基酸，将P42-50的羧基端残基L-Leu替换为D-Leu , P782-790的羧基端残基L-Ser替换为D-Ser，合成了具有局部手性特征的多肽D-P42-50，D-P782-790，以期增强多肽在体内稳定性的同时提高其免疫原性。
2．多肽合成：采用Fmoc固相多肽合成策略，合成上述四种多肽，L-P42-50，，D-P42-50，L-P782-790和D-P782-790。
3．多肽的纯化及鉴定：利用中压反相液相层析对粗肽进行分析及纯化，L-P42-50、D-P42-50的纯度分别为99.2%及98.9% ，L-P782-790、D-P782-790的纯度分别为99.1%和99.0% 。利用激光解吸电离飞行时间质谱测定各色谱峰的分子量，经真空旋转蒸发及冻干获得纯品多肽。结果L-P42-50、D-P42-50总产率分别为63%及45%，，L-P782-790、D-P782-790总产率分别为58%及46%，。
二．合成多肽疫苗体内诱导CTL作用研究
小鼠乳腺癌模型的建立及多肽疫苗免疫 选用雌性BALB/c小鼠接种D2F2细胞,分别为对照组、GM-CSF组、L-P42-50、L-P42-50+ GM-CSF组、D-P42-50、D-P42-50+ GM-CSF组、L-P782-790、L-P782-790+ GM-CSF组、D-P782-790、D-P782-790+ GM-CSF组。
1．与对照组小鼠相比，L-P42-50+GM-CSF组、L-P782-790+GM-CSF组、D-P42-50+GM-CSF组和D-P782-790+GM-CSF组小鼠脾细胞重量增加差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），对淋巴细胞增殖的促进作用差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），分泌IFN-γ的T淋巴细胞数明显增多（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），对D2F2乳腺癌细胞的特异杀伤作用差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）。这四种多肽联合应用GM-CSF 都能促进小鼠T细胞增殖，促进T细胞分泌IFN-γ及激活CTL杀伤乳腺癌细胞，说明这两种预测的L型多肽在体内与MHC-Ⅰ类分子相结合产生复合物，与T细胞受体结合，激活CTL
细胞，对乳腺癌细胞产生杀伤作用。
2．D-P42-50+GM-CSF组和L-P42-50+GM-CSF组、D-P782-790+GM-CSF组L-P782-790+GM-CSF相比，小鼠脾细胞重量的增加差异显著（P﹤0.05, P﹤0.05），对淋巴细胞增殖的促进作用差异显著（P﹤0.05, P﹤0.05）,分泌γ-IFN的T淋巴细胞数增多（P﹤0.05, P﹤0.05）,诱导对D2F2乳腺癌细胞的特异杀伤作用也有显著差异（P﹤0.05, P﹤0.05）。D-P42-50和D-P782-790辅以GM-CSF可显著促进小鼠T细胞增殖，促进T细胞分泌IFN-γ及激活CTL杀伤乳腺癌细胞，说明这两种经改造的手性多肽与MHC-Ⅰ类分子结合的能力并没有发生改变，与L型多肽一样，在体内与MHC-Ⅰ类分子相结合产生复合物，与T细胞受体结合，激活CTL细胞，对乳腺癌细胞产生杀伤作用。D-P42-50和 D-P782-790多肽在局部上具有了手性特征，具有了不同于体内肿瘤细胞所表达的HER2/neu多肽的结构特征，在一定程度上增加了免疫原性，从而使其更能够有效地诱导CTL。本研究的淋巴细胞增殖实验和CTL杀伤活性测定结果也肯定了这一实验设计的可行性和有效性。
三．合成多肽疫苗体内抑瘤实验研究
BALB/c小鼠接种小鼠乳腺癌细胞D2F2后，进行多肽疫苗接种。接种肿瘤细胞42天后处死小鼠，取肿瘤组织进行光镜及电镜观察。
1．小鼠肿瘤组织体积和重量：与对照组小鼠相比，L-P42-50+GM-CSF组、L-P782-790+GM-CSF组、D-P42-50+GM-CSF组和D-P782-790+GM-CSF组的肿瘤重量和体积明显低于对照组，有显著性差异（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），而且D-P42-50+GM-CSF组和D-P782-790+GM-CSF组的肿瘤重量和体积与L-P42-50+GM-CSF组和L-P782-790+GM-CSF组相比，有显著差异（P﹤0.05, P﹤0.05）。NaCl组、GM-CSF 组、L-P42-50 组、D-P42-50组、L-P782-790组和 D-P782-790组小鼠与对照组相比无显?
Cell immunogy is the major to eliminate the tumor cells in the body,and cototo-
xic T cells(CTL) is the most immportant.CTL is activated by the compound of MHC-Ⅰmolecuar and antigen peptide which is presented by antigen presentation cells(APC).The length of the antigen peptide is most 9～11 amino acids.It is has been proved that synthesized peptide can combine with MHC-Ⅰmolecuar on the surface of APC directly without the processing of APC,have the same effect as the natual endogenous peptide.The identification of the tumor antigen peptide can apply the peptide as the tumor vaccine.But peptide molecular is small ,the immunogenicity of peptide is low,half-life time of peptide is short and the peptide is hydrolized by enzyme easily.All these can influence the existing time and valence of CTL.
Two methods were used to improve these situation : ①Change regional optical rotation by turn some L-peptide to D-peptide without altering the combination of peptide and MHC-Ⅰmolecuar. The synthesized D-peptide can be stable by resisting the hydrolization of the protein enzyme and increase the half-life time thus increade immunogenicity of peptide. ②Immunity ajuvant GM-CSF is used to activate the macrophage and other immunity cells and get the more powerful cell immunity.
1.The selection,designation and synthesis of the HER2/neu peptide:
(1) Design of the amino acid sequence of peptide: SYFPETTHI sequence
analysis system was used to predict MHC-Ⅰmolecular restricted CTL epi-position of HER2/neu protein ,and select two region P42-50 (HLYQGCQVV) and P782-790 ( VSRLLGICL).Because the change of combination region of the peptide with the different allel gene of MHC-Ⅰmolecular is locolized the N-terminal amino acid of the peptide, so we change the C-terminal amino acid without changing the combination of the peptide and the MHC-Ⅰmolecular.We substitute D-Leu for L-Leu for the P42-50, substitute D-Ser for L-Ser for P782-790,and get two chiral peptide vaccine D-P42-50,D-P782-790.
(2) Peptide synthesis: Peptides L-P42-50, D-P42-50,L-P782-790,D-P782-790 were synthesized with solid phase peptide synthesis method and Fmoc-strategy. Each amino acid was fully protected during the peptide synthesis procedure.
(3) The purification and identification of peptide:The peptide was purifed by middle pressure reverse liquid chromatography.And the purity of L-P42-50, D-P42-50 is 99.2%,98.9%, the purity of L-P782-790, D-P782-790 is 99.1%,99.0%. The fraction of interested was confirmed by measuring the molecular weight of each fraction with EMS. Then the crude peptide was purified on preparative RP-HPLC. The purified peptide was received through the collection of interested fraction and the disposal of vacuum rotation evaporation and ice freeze. The product of pure peptide L-P42-50, D-P42-50 is 63% and 45% ,the product of pure peptide L-P782-790,D-P782-790is 58% and 46% .
2. Study on induction of specific CTL in vivo by peptide vaccine immunization.
The construct of mouse breast tumor model and vaccinated with peptide vaccine:BALB/c mice were inoculated with mouse breast tumor cell line D2F2(which was transfected with full cDNA of human HER2/neu gene), then vaccinated with peptide vaccine and divided for ten group:Nacl group;GM-CSF group; L-P42-50 group;L-P42-50+ GM-CSF group;D-P42-50 group; D-P42-50+ GM-CSF group;L-P782-790
group; L-P782-790 + GM-CSF group; D-P782-790 group; D-P782-790 + GM-CSF group.
(1) Compared with the control group; L-P42-50+ GM-CSF group; D-P42-50+ GM-CSF group; L-P782-790+ GM-CSF group and D-P782-790 + GM-CSF group have significant difference on the increase of the weight of spleen（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）;promoting the proliferation of T lymphocytes（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）;the secreting γ-IFN of T cells（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）; and HER2/neu specific CTL（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）. The results indicated all these four peptides can form compound with MHC-Ⅰmolecular,bind to the T cell receptor,activated special CTL.
(2) Compared with L
引文
Boon T.Toward a genetic analysis of human tumor rejection antigens[J].Advances in Cancer Recearch,1992;58:177-210.
林雪颜，张玲.现代细胞与分子免疫学[M].第一版.北京：科学出版社,1999;373-379.
Anonymous.Interferon-alpha and surlval in metastatic renalcarcinoma: early results of a randomised controlled trial[J].Lancet,1999;353:14-17.
Belldegrum A,Plerce W,Kaboo N,et al. Interferon-alpha primed tumor- infiltrating lymphocytes combined with interleukin-2 and interferon- alpha as therapy for metastatic renal cell carcinoma[J]. J Urol,1993;150:1384-1390.
Oldham RK, Lewko WM,Good RW,Sharp E. Cancer biotherapy with Interferon interleukin-2 and tumor-derived cells(TDAC)[J]. Vivo,1994;8(5):653-663.
Hersh EM,Stopeck AT.Advances in the biological therapy and gene therapy of maligant disease [J]. Clin Cancer Res,1997;3:2623-2629.
Denton GW，Dorrant LG，Handcastle et al.Clinical outcome of colorectal cancer patients treated with human monoclonal antiidiotypic antibody[J]. Int J Cancer,1994;57(1):10-14 .
Sakakura C,Hagiwsia A,Tsujimoto H,et al.Inhibition of gastric cancer cell proliferation by antisense oligonuclootides targeting the messanger RNA encoding proliferating cell nuclear antigen[J]. Br Cancer ,1994;70:1060-1066.
Staveley O,Carrol K,Sotonmayor E,et,al.Induction of antigen- specific T cell anergy: An early event in the course of tumor progression[J]. Pro Natl Acad Sci USA,1998;95:1178-1183.
Vermorken JB,Clasessen AM,van Tinteren H,et al.Active specific immunotherapy for stageⅠand stageⅡ human colon cancer: a randomised trial[J]. Lancet, 1999;353:345-350.
Disis ML, Rinn K, Knutson KL, et al. Flt3 ligand as a vaccine adjuvant in association with Her-2/neu peptide–based vaccines in patient with HER-2/neu-overexpressing cancers [J]. Blood, 2002;99 (8): 2845- 2850.
Kato K, Cantwell MJ, Sharma S, et al. Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells [J]. J Clin Invest, 1998;5: 1133-1141.
Raychaudhuri S, Rock KL. Fully mobilizing host defense: building better
vaccine [J]. Nat Biotechnol, 1998;16: 1025-1029.
Reeves ME, Royal RE, Lam JS, et al. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene [J]. Cancer Res, 1996;56: 5672-5677.
Hsu FJ, Benike C, Fagnoni F, et al. Vaccination of patient with B-cell lymphoma using autologus antigen-pulsed dendritic cells [J]. Nat Med, 1996;2: 52-57.
Mediratta SK, Thai G, Eslahi NK, et al. Therapeution tumor immunity induce by polyimmunization with melanoma antigen gu100 and TRP-2 [J]. Cancer Res, 2001;61 (3): 859-863.
Chen BG, Shi Y, Smith JD, et al. The role of tumor necrosis factor–αin modulating the quantity of peripheral blood-derived, cytokine-derved human dendritic cells and its role in enhancing quality of dendritic cell function in presenting soluble antigens of CD+4T cell in vitro [J]. Blood, 1998;91: 4652-4661.
Paglia P, Chiodoni C, Rodolfo M, et al. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vitro [J]. J Exp Med, 1996;183: 317-322.
Strome SE, Voss S. Wilcox R, et al. strategies for antigen loading of dendritic cells to enhance the antitumor immune response [J]. Cancer Res, 2002;62 (6): 1884-1889.
Labeur M, Rosters B, Pers B, et al. Generation for tumor immunity by bone marrow –derived dendritic cells correlates with dendritic cell maturation stage [J].J Immunol,1999;162: 168-175.
Nikitina EY,Chada S,Muro-Cacho C,et al.An effective immunization and cancer treatment with activated dendritic cells transduced with full-length wild-type p53 [J].Gene,2002;9(5):345-352.
Specht JM, Wang G, Do MT, et al. Dencritic cells retrovirally transduced with a model antigen gene are therapeutically effective against establish pulmonary metastases [J]. J Exp Med, 1997;186: 1213-1221.
Song W, Kong HL, Garpenter H, et al. Dendritic cells genetically modified with a adenovirus vector encoding the DNA for a model antigen induce protective and therapeutic antitumor immunity [J]. Exp Med, 1997;186: 1247-1256.
Wang J, Saffold S, Cao XT, et al. Eliciting T cell immunity against prroly
immunogenic tumor by immunization with dendritic cell-tumor fusion vaccine [J]. J Immunol, 1998;161: 5561-5524.
Gong J,Chen D,Kashiwaka M,et al. Induction of antitumor activity by immunization with fusion of dendritic and carcinoma cells[J].1997;3:558-562.
Ayyoub M, Mazarguil H, Monsarrat B, et al. A structure-based approach to designing non-natural peptides that can activate antimelanoma cytotoxic T cells [J]. J Biol Chem, 1999;274: 10227-10231.
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 [J].Cancer Research,2001;61:197-202.
Tan M C, Mommaas A M, Drijfhout J W, et al. Mannose receptor mediated uptake of antigens strongly enhanced HLA class restricted antigen presentation by cultured dendritic cells[J]. Eur J Immunol. 1997;23:3217-3222.
Binder RJ, Blachere NE, Srivastava PK et,al.Heat shock protein-chaperoned pepides but not free peptide introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules [J]. J Biol Chem, 2001;276(20): 17163-17171.
Moroiy，Dessel R et al. Induction of cellular immunity by immunization with novel hybrid peptides complexeed to heat shock protein 70 [J]. proc Natl Scad Sci USA, 2000;97(7): 3485-3489.
王洪琦. 热休克蛋白在肿瘤应用基础领域中的研究进展 [J].中华肿瘤杂志，2001;23（2）：93-95.
Park JH, Kim CJ, Lee JH, Shin SH, et al. Effiective immunotherapy of cancer by DNA vaccine [J]. Mol Cells, 1999;9 (4): 384-388.
De Giovanni C, Nanni P, Forni Get al. The prospects for cancer gene therapy [J]. Int J Immunopharmacol, 2002;22 (12): 1025-1030.
Wolkers MC, Toebes M, Okabe M, et al. Optimizing the efficacy of pepitope- directed DNA vaccination [J]. Cancer Gene Ther, 2002;9 (4): 330-337.
Kontani K, Taguchi O, Ozaki Y, et al. Novel vaccination protocol consisting of injecting MUCI DNA and nonprimed dendritic cells at the same region greatly enhanced MUCI-specific antitumor immunith in a murine model [J]. Cancer Gene Ther, 2002;9 (4): 330-337.
Guo C, Ding J, Yu Z, et al. Development of oral DNA vaccine based on MG
(7)-Ag mimotope of gastric cancer [J]. Zhonghua Zhong Liu Za Zhi, 2002;24(2): 110-113.
Bhattacharya C, M Nath, Baral R, et al. Counterpoint. Cancer vaccines: Single–epitope anti-idiotype vaccine versus multiple- epitope antigen vaccine [J]. Cancer Immunother, 2000;49: 133-141.
Tripathi PK, Qin H, Bhattacharya-Chatterjee M, et al. Construction and characterization of a chimeric fusion protein consisting of an anti-idiotype antibody mimicking a breast cancer-associated antigen and the cytokine GM-CSF [J]. Hybridoma, 1999;18 (2): 193-202.
刘宪玲. 抗独特型抗体疫苗在肿瘤治疗中的应用进展 [J]. 国外医学肿瘤分册，1997;27（1）;3-8.
Bhattacharya C, M Nath, Baral R, et al. Cancer vaccines:Single–epitope anti- idiotype vaccine versus multiple- epitope antigen vaccine [J]. Cancer Immunother, 2000;49: 133-141
Hung MC,Lau YK.Basic science of HER2/neu:a review[J].Semin Oncol,1999;26(4 suppl 12): 51-59
Shih C,Padhy LC,Murray M,et al.Transforming genes of carcimomas and neuroblastomas introduced into mouse fiboblasts[J].Nature,1981;290:261-264
Coussens L,Teresa LY, Liao YC,et al.Tyrosine kinase receptor with extensive homolgy to EGF receptor shares chromosomal location with neu oncogene[J]. Science, 1985;30(4730):1132-1139
Wang S,Saboorian MH ,Frenkel E,et al.Laboratory assessment of the status of HER2/neu protein and oncogene in breast cancer specimens : comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays[J]. J Clin Pathol,2000;53(5):374-381
Samata A,Levea C M ,Dougall WC ,et al .Ligand and p185/neu density govern receptor interactions and tyrosine kinase activation[J].Proc Natl Acad Sci USA,1994;91(5):1711-1715
Hynes NE,Stern DF.The biology of p185/neu /HER2 and its role in cancer [J].Biochim Biophys Acta,1994,198(1):165-184
Christianson TA,Doherty JK, Lin YJ , et al . NH2-terminal truncated HER2/ neu protein:relationship with shedding of the extracellular domain and with prognostic factors in breast cancer[J].Cancer Res,1999;58 (22):5123-5129
HoangM P,SahinA A,Ordonez NG et al.HER2-neu gene amplification
compared with HER-2/neu protein overexpression and interobserver in invasive breast carcinoma.Am J Clin Pathol[J],2000;113(6):852-856.
Pawlowski V,Revillion F,Hornez L,et al.A real time one-step reverse transcriptase polymerse chain reaction method to quantify c-erbB2 expression in human breast cancer[J].Cancer Detect Prev,2000;24(3)∶212-216
Samata A, Levea CM, Dougall WC, et al. Ligand and p185/neu density govern receptor interactions and tyrosine kinase activation[J].Proc Natl Acad Sci USA , 1994; 91 (5):1711-1715
Weiner DB,Liu J,Cohen JA,et al.A point mutation in the neu oncogene mimics ligand induction of receptor aggregation[J].Nature,1989;339: 230-231
Porta DG,Beerli RR,Daly JM ,et al.ErbB-2,the preferred heterodimerization partner of all ErbB receptors,is a mediator of lateral signaling[J]. J EMBO, 1997;16(7):1647-1655
Klapper LN,Waterman H,Sela M,et al.Tumor-inhibitory antibodies to HER2/ ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2[J]. Cancer Res,2000;60(13):3384-3388
.Levkowit G,Waterman Ettenberg SA,et al.Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1[J].Mol Cell,1999;4(6):1029-1040.
Wang S,Saboorian MH,Frenkel E,et al.Laboratory assessment of the status of HER2/neu protein and oncogene in breast cancer specimens: comparised immunohistochemistry assay with fluorescence in situ hybridisation assays[J].J Clin Pathol, 2000;53（5）: 374-378
Ozer E,Sis B, Ozen E,et al. BRCAA 1,c-erbB-2, and H-ras gene expression in young women with breast cancer. An immunohistochemical Study[J]. App l Immunoh istochem Molecul Morphol, 2000; 8(1)∶12-17
Pawlow skiV ,Revillion F,Hornez L,et al. A real-time one-step reverset ranscrip tase-polymerse chain reaction method to quantify c-erbB2 expression in human breast cancer[J]. Cancer Detect Prev, 2000; 24(3)∶212-217
Levkowitz G, Waterman H , Ettenberg SA , et al . Ubiquitin ligase activity and tyrosine phosphorylation underlie suppres2 sion of growth factor signaling by c2Cbl/ Sli21[J ] . Mol Cell , 1999 ; 4(6) :1029 - 1040
Kiyokawa N,Karunagaran D,Lee EK,et al.Involvement of cdc2-mediated phosphorylation in the cell cycle-dependent regulation of p185 neu[J].
Oncogene, 1997;15:2633-2641.
Kiyokawa,Lee EK,Karunagaran D,et al.Mitosis-specific negative regulation of epidermal growth factor receptor,triggered by a decrease in ligand binding and dimerization,can be overcomed by overexpression of receptor[J].J Biol Chem,1997;272: 18656-18665
Lane HA , Beuvink I , Motoyama AB , et al . ErbB potentiates breast tumor proliferation through modulation of p27Kip12Cdk2 complex formation : receptor overexpression does not determine growth dependency[J].Mol Cell Biol, 2000 ;20(9) :3210 - 3223
Pegram MD,Baly D,Wirth C,et al.Antibody dependent cell-mediated cytotoxicity in breast cancer patients in phase Ⅲ clinical trials of a humanized anti HER-antibody [J] .Proc Am Assoc Cancer Res , 1997;38: 602-607
Bookman MA,Darcy KM,Clarke2Pearson D ,et al .Evalu2 ation of monoclonal humanized anti2HER2 antibody , trastuzumab , in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2 : a phase Ⅱtrial of the Gynecologic Oncology Group [J ] . J Clin Oncol , 2003 ; 21 (2) :283 - 290
Burstein HJ , Harris LN , Gelman R , et al . Preoperative therapy with trastuzumab and paclitaxel followed by sequen2 tial adjuvant doxorubicin/ cyclophosphamide for HER2 over2 expressing stage Ⅱor Ⅲ breast cancer : a pilot study[J ] . J Clin Oncol , 2003; 21 (1) :46 - 53.
Fleming GF , Meropol NJ , Rosner GL , et al . A phase Ⅰtrial of escalating doses of trastuzumab combined with daily subcutaneous interleukin 2 : report of cancer and leukemia group B 9661[J ] . Clin Cancer Res,2002;8 (12) : 3718 –3727.
Fry DW, Bridges AJ , Denny WA , et al . Specific , irre2 versible inactivation of the epidermal growth factor receptor and erbB22 , by a new class of tyrosine kinase inhibitor[J ] [J]. Proc Natl Acad Sci USA, 1998;95 ( 20):12022 -12027.
Zhang ZG, Cai HB,Bai L , et al. A comparison between the yield of the attachment of Boc-AA-O- Cs+ to Merrifield resin and of Fmoc-AA-OH to Wang resin[J]. Chinese J Org Chem,2000;20(3):419-423.
黄惟德，陈长庆主编.多肽合成﹙M﹚.第一版.北京：科学出版,1985，144-149
Swinnen D, Hilvert D. Facile, Fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters[J]. Org Lett, 2000;2(16):2439-2444.
Nakahara Y,Ando S, Ho Y, et al. New allylester linker and solid-phase block synthesis of the serglycin core region[J]. Bio sci Bio technol Biochem, 2001;65(6):1358-1364.
Wade WS, Yang F, Sowin TJ. Application of base cleavable safety catch linkers to solid phase library production[J]. J Comb Chem,2000;2(3):266-272.
张志刚，何素婷，何明华等. DCM和DMF对DCC-HOBt系统催化酯化及酰化反应的影响[J].生物化学杂志， 1997;13(2): 177-180.
GA. Pietersz, V. Apostolopoulos, IFC Mckenzie. Generation of cellular immune responses to antigenic tumor peptides[J]. Cell Mol Life Sci, 2000; 57: 290-310.
张若蘅，徐筱杰，唐有祺. 应用反相高效液相色谱法分析纯化化学合成肽. 色谱[J], 1996;14(3): 182-184.
陈执中.基质辅助激光解吸离子化—飞行时间质谱法在生化和基因工程药物分析研究中的应用进展[J]. 药物生物技术，2000; 7(2): 122-125.
沈关心,周汝麟主编 现代免疫学实验技术(第一版)[M].武汉:湖北科学技术出版社.1998.P18-20.
Eggers M, Boes Fabian ,Ruppert T, et al. The cleavage preference of the proteasome governs the yield of antigenic peptides[J]. J.Exp. Med, 1995;182:1865-1870.
Disis ML,Grabstein KH.Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine[J].Clin Cancer Res,1999;5(6):1289-1297.
Geysen HM,Macfarian R,Rodda SJ,et al.A period delineation of a peptide which mimics a discontinuous antigenic determinant[J].Molecular Imunology,1986;23(7)709-715.
Bjorkman P.J,and P.Parham.Structure,function and diversity of class I major histocompatibility complex molecules[J].Ann Rev Bioche,1990;(59)253-288.
Ayyoub M, Mazarguil H, Monsarrat B. et al. A Struture Based approach to designing nonnatural peptides that can activate ant-melanoma cytotoxic T cells[J]. J.Biol Chem, 1999;74(15):10227-10234.
Reynolds SR, Oratz R.,Shapiro RL,.et al. Stimulation of CD8+ T cell responses to MAGE-3and Melana/MART-1 by immunization to a polyvalent melanoma vaccine[J]. Int J. Cancer,1997;72(6):972-976.
Parkhurst MR, Salgaller ML, Southwood S, et al. Improved induction of
melanoma reactive CTL with peptides from the melanoma antigen gp100 modified at HLA.a.0201 binding residues[J]. J. Jmmunol, 1996;157(6):2539 –2548.
Porta D G, Beerli R R , Daly J M , et al . ErbB22 , the preferred heterodimeri- zation partner of all ErbB receptors , is a mediator of lateral signaling[J]. J EMBO, 1997 ; 16 (7) : 1647-1655.
Madden,D.The three dimension structures of peptide-MHCⅠcomlex[J].Annual Review of Immunology,1995;13:587-622.
Sela, M., Zisman, E. Different roles of D-amino acids in immune phenomena. Faseb journal : the official publication of the federation of american societies for experiment [J].Biology; 1997;11(6):449-456 .
Afonso LC. The adjuvant effect of interleukin-12 in a vaccine against leishm[J]. Science,1994;263(5144):235-237.
Nencioni L, Villa L, Tagliabue A, et al. In-vivo immunostimulating activity of the 163-171 peptide of human IL-1-beta[J]. J. Immunology, 1987;139(3): 800 –804.
Duits AJ., Van Puijenbroek, Andre, Vermeulen, Hans,et al. Immunoadjuvant activity of a liposomal IL-6 formulation[J]. Vaccine , 1993;11(7):777-781..
Tarr PE. Granulocyte macrophage colony stimulating factor and the immune system[J]. Med Oncol, 1996;13: 133-138.
Dranoff G, Jaffee E, Lazenby A, et al. Vaccination with irradiated tumor cell engineered to secrete murine GM-CSF stimulates potent, specific and long lasting antitumor immunity[J]. Proc Natl Acad Sci USA,1993;90:3539-3543.
单梅梅.细胞毒T细胞在病毒性肝炎发病机理中作用的若干研究进展[J]. 国外医学:流行病学传染病学分册,1998;25(1):12-17.
Altenschmidt U, Klundt E, Groner B et al.Adoptive transfer of in vitro-targeted, activated T lymphocytes results in total tumor regression[J].J Immunol, 1997; 159(11):5509-5515.
柳忠辉,吕昌龙.免疫学常用实验技术 [M].北京：科学出版社，2002:109-113.
Knutson KL, Schiffman K, Disis ML,et al. Immunization with a HER-2/neu helper peptide vaccine generates HER-2/neu CD8 T-cell immunity in cancer patients[J]. J.Clin.Invest,2001;107:477-484.
沈关心, 周汝麟.现代免疫学实验技术[M].湖北：湖北科学技术出版社,1998:333-336.
Ruff MR, Farrar WL, Pert CB. Interferon-γ and granulocyte-macrophage colony- stimulating factor inhibit growth and induce antigens characteristic of myeloid differentiation in small-cell lung cell lines[J]. Proc Natl Acad Sci USA, 1986;83:6613-6613.
Sensi M, Farina C,Maccalli C, et al. Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine[J]. J Clin Invest,1997;99:710-717.
Berd D,Maguire HC,Schuchter LM,et al .Autologous hapten-modified melanoma vaccine as postsurgical adjuvant treatment after resection of nodal metastases[J]. J Clin Oncol,1997;15:2359-2370.
Lindaner M,Stanislawski T, Haubler A, et al. The molecular basis of cancer immunotherapy by cytotoxic T lymphocytes[J] . J. Mol. Med,1998;76:32-37.
Jager E, Ringho?er M, Dienes HP,et al., Granulocyte-macrophage-colony stimulating factor enhances immune responses to melanoma associated peptides in vivo[J]. Int J Cancer,1996;67:54-62.
Soo Kie, Kim SK，Ragupathi G, Cappello S，et al. Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates[J]. Vaccine,2001;19:530-537.
Kawakami Y, Eliyahu S, Jenning C,et al. Recongnition of multiple epitopes in the human melanoma antigen gp100 by tumor infiltrating T lymphocytes associated with in vivo tumor regression[J]. J.Immunol.,1995;154(8):3961 –3968.
Chang EY, Chen CH, Ji H, et al. Antigen specific cancer immunotherapy using a GM-CSF secreting allogenic tumor cell based vaccine[J]. Int.J.Cancer, 2000;86(5):725-730.
常乃柏,孙芾,顾惜春. 粒-巨噬细胞集落刺激因子对外周血T细胞和NK细胞的作用[J]. 中华血液学杂志. 1999;20(12):35-37.
Kapoor Detal. Granulocyte-macrophage colony-stimulating factor enhances the efficacy of hepatitis B virus vaccine in previously unvaccinated haemodialysis patients[J]. J. Viral Hepat,1999;6(5):405 –409.
Sub KW. Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine[J].J. Surg. Oncol, 1999;72(4):218-224.
108． Farrar,M.A.,Schreiber R.D.The molecular cell biology of interferon-γ and its receptor.Ann Rev Immuno,1993;(11):571-611.
林雪颜，张玲.现代细胞与分子免疫学[M].第一版.北京：科学出版社,1999;373-379.
Anonymous.Interferon-alpha and surlval in metastatic renalcarcinoma: early results of a randomised controlled trial[J].Lancet,1999;353:14-17.
Belldegrum A,Plerce W,Kaboo N,et al. Interferon-alpha primed tumor- infiltrating lymphocytes combined with interleukin-2 and interferon- alpha as therapy for metastatic renal cell carcinoma[J]. J Urol,1993;150:1384-1390.
Oldham RK, Lewko WM,Good RW,Sharp E. Cancer biotherapy with Interferon interleukin-2 and tumor-derived cells(TDAC)[J]. Vivo,1994;8(5):653-663.
Hersh EM,Stopeck AT.Advances in the biological therapy and gene therapy of maligant disease [J]. Clin Cancer Res,1997;3:2623-2629.
Denton GW，Dorrant LG，Handcastle et al.Clinical outcome of colorectal cancer patients treated with human monoclonal antiidiotypic antibody[J]. Int J Cancer,1994;57(1):10-14 .
Sakakura C,Hagiwsia A,Tsujimoto H,et al.Inhibition of gastric cancer cell proliferation by antisense oligonuclootides targeting the messanger RNA encoding proliferating cell nuclear antigen[J]. Br Cancer ,1994;70:1060-1066.
Staveley O,Carrol K,Sotonmayor E,et,al.Induction of antigen- specific T cell anergy: An early event in the course of tumor progression[J]. Pro Natl Acad Sci USA,1998;95:1178-1183.
Vermorken JB,Clasessen AM,van Tinteren H,et al.Active specific immunotherapy for stageⅠand stageⅡ human colon cancer: a randomised trial[J]. Lancet, 1999;353:345-350.
Disis ML, Rinn K, Knutson KL, et al. Flt3 ligand as a vaccine adjuvant in association with Her-2/neu peptide–based vaccines in patient with HER-2/neu-overexpressing cancers [J]. Blood, 2002;99 (8): 2845- 2850.
Kato K, Cantwell MJ, Sharma S, et al. Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells [J]. J Clin Invest, 1998;5: 1133-1141.
Raychaudhuri S, Rock KL. Fully mobilizing host defense: building better
vaccine [J]. Nat Biotechnol, 1998;16: 1025-1029.
Reeves ME, Royal RE, Lam JS, et al. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene [J]. Cancer Res, 1996;56: 5672-5677.
Hsu FJ, Benike C, Fagnoni F, et al. Vaccination of patient with B-cell lymphoma using autologus antigen-pulsed dendritic cells [J]. Nat Med, 1996;2: 52-57.
Mediratta SK, Thai G, Eslahi NK, et al. Therapeution tumor immunity induce by polyimmunization with melanoma antigen gu100 and TRP-2 [J]. Cancer Res, 2001;61 (3): 859-863.
Chen BG, Shi Y, Smith JD, et al. The role of tumor necrosis factor–αin modulating the quantity of peripheral blood-derived, cytokine-derved human dendritic cells and its role in enhancing quality of dendritic cell function in presenting soluble antigens of CD+4T cell in vitro [J]. Blood, 1998;91: 4652-4661.
Paglia P, Chiodoni C, Rodolfo M, et al. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vitro [J]. J Exp Med, 1996;183: 317-322.
Strome SE, Voss S. Wilcox R, et al. strategies for antigen loading of dendritic cells to enhance the antitumor immune response [J]. Cancer Res, 2002;62 (6): 1884-1889.
Labeur M, Rosters B, Pers B, et al. Generation for tumor immunity by bone marrow –derived dendritic cells correlates with dendritic cell maturation stage [J].J Immunol,1999;162: 168-175.
Nikitina EY,Chada S,Muro-Cacho C,et al.An effective immunization and cancer treatment with activated dendritic cells transduced with full-length wild-type p53 [J].Gene,2002;9(5):345-352.
Specht JM, Wang G, Do MT, et al. Dencritic cells retrovirally transduced with a model antigen gene are therapeutically effective against establish pulmonary metastases [J]. J Exp Med, 1997;186: 1213-1221.
Song W, Kong HL, Garpenter H, et al. Dendritic cells genetically modified with a adenovirus vector encoding the DNA for a model antigen induce protective and therapeutic antitumor immunity [J]. Exp Med, 1997;186: 1247-1256.
Wang J, Saffold S, Cao XT, et al. Eliciting T cell immunity against prroly
immunogenic tumor by immunization with dendritic cell-tumor fusion vaccine [J]. J Immunol, 1998;161: 5561-5524.
Gong J,Chen D,Kashiwaka M,et al. Induction of antitumor activity by immunization with fusion of dendritic and carcinoma cells[J].1997;3:558-562.
Ayyoub M, Mazarguil H, Monsarrat B, et al. A structure-based approach to designing non-natural peptides that can activate antimelanoma cytotoxic T cells [J]. J Biol Chem, 1999;274: 10227-10231.
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 [J].Cancer Research,2001;61:197-202.
Tan M C, Mommaas A M, Drijfhout J W, et al. Mannose receptor mediated uptake of antigens strongly enhanced HLA class restricted antigen presentation by cultured dendritic cells[J]. Eur J Immunol. 1997;23:3217-3222.
Binder RJ, Blachere NE, Srivastava PK et,al.Heat shock protein-chaperoned pepides but not free peptide introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules [J]. J Biol Chem, 2001;276(20): 17163-17171.
Moroiy，Dessel R et al. Induction of cellular immunity by immunization with novel hybrid peptides complexeed to heat shock protein 70 [J]. proc Natl Scad Sci USA, 2000;97(7): 3485-3489.
王洪琦. 热休克蛋白在肿瘤应用基础领域中的研究进展 [J].中华肿瘤杂志，2001;23（2）：93-95.
Park JH, Kim CJ, Lee JH, Shin SH, et al. Effiective immunotherapy of cancer by DNA vaccine [J]. Mol Cells, 1999;9 (4): 384-388.
De Giovanni C, Nanni P, Forni Get al. The prospects for cancer gene therapy [J]. Int J Immunopharmacol, 2002;22 (12): 1025-1030.
Wolkers MC, Toebes M, Okabe M, et al. Optimizing the efficacy of pepitope- directed DNA vaccination [J]. Cancer Gene Ther, 2002;9 (4): 330-337.
Kontani K, Taguchi O, Ozaki Y, et al. Novel vaccination protocol consisting of injecting MUCI DNA and nonprimed dendritic cells at the same region greatly enhanced MUCI-specific antitumor immunith in a murine model [J]. Cancer Gene Ther, 2002;9 (4): 330-337.
Guo C, Ding J, Yu Z, et al. Development of oral DNA vaccine based on MG
(7)-Ag mimotope of gastric cancer [J]. Zhonghua Zhong Liu Za Zhi, 2002;24(2): 110-113.
Bhattacharya C, M Nath, Baral R, et al. Counterpoint. Cancer vaccines: Single–epitope anti-idiotype vaccine versus multiple- epitope antigen vaccine [J]. Cancer Immunother, 2000;49: 133-141.
Tripathi PK, Qin H, Bhattacharya-Chatterjee M, et al. Construction and characterization of a chimeric fusion protein consisting of an anti-idiotype antibody mimicking a breast cancer-associated antigen and the cytokine GM-CSF [J]. Hybridoma, 1999;18 (2): 193-202.
刘宪玲. 抗独特型抗体疫苗在肿瘤治疗中的应用进展 [J]. 国外医学肿瘤分册，1997;27（1）;3-8.
Bhattacharya C, M Nath, Baral R, et al. Cancer vaccines:Single–epitope anti- idiotype vaccine versus multiple- epitope antigen vaccine [J]. Cancer Immunother, 2000;49: 133-141
Hung MC,Lau YK.Basic science of HER2/neu:a review[J].Semin Oncol,1999;26(4 suppl 12): 51-59
Shih C,Padhy LC,Murray M,et al.Transforming genes of carcimomas and neuroblastomas introduced into mouse fiboblasts[J].Nature,1981;290:261-264
Coussens L,Teresa LY, Liao YC,et al.Tyrosine kinase receptor with extensive homolgy to EGF receptor shares chromosomal location with neu oncogene[J]. Science, 1985;30(4730):1132-1139
Wang S,Saboorian MH ,Frenkel E,et al.Laboratory assessment of the status of HER2/neu protein and oncogene in breast cancer specimens : comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays[J]. J Clin Pathol,2000;53(5):374-381
Samata A,Levea C M ,Dougall WC ,et al .Ligand and p185/neu density govern receptor interactions and tyrosine kinase activation[J].Proc Natl Acad Sci USA,1994;91(5):1711-1715
Hynes NE,Stern DF.The biology of p185/neu /HER2 and its role in cancer [J].Biochim Biophys Acta,1994,198(1):165-184
Christianson TA,Doherty JK, Lin YJ , et al . NH2-terminal truncated HER2/ neu protein:relationship with shedding of the extracellular domain and with prognostic factors in breast cancer[J].Cancer Res,1999;58 (22):5123-5129
HoangM P,SahinA A,Ordonez NG et al.HER2-neu gene amplification
compared with HER-2/neu protein overexpression and interobserver in invasive breast carcinoma.Am J Clin Pathol[J],2000;113(6):852-856.
Pawlowski V,Revillion F,Hornez L,et al.A real time one-step reverse transcriptase polymerse chain reaction method to quantify c-erbB2 expression in human breast cancer[J].Cancer Detect Prev,2000;24(3)∶212-216
Samata A, Levea CM, Dougall WC, et al. Ligand and p185/neu density govern receptor interactions and tyrosine kinase activation[J].Proc Natl Acad Sci USA , 1994; 91 (5):1711-1715
Weiner DB,Liu J,Cohen JA,et al.A point mutation in the neu oncogene mimics ligand induction of receptor aggregation[J].Nature,1989;339: 230-231
Porta DG,Beerli RR,Daly JM ,et al.ErbB-2,the preferred heterodimerization partner of all ErbB receptors,is a mediator of lateral signaling[J]. J EMBO, 1997;16(7):1647-1655
Klapper LN,Waterman H,Sela M,et al.Tumor-inhibitory antibodies to HER2/ ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2[J]. Cancer Res,2000;60(13):3384-3388
.Levkowit G,Waterman Ettenberg SA,et al.Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1[J].Mol Cell,1999;4(6):1029-1040.
Wang S,Saboorian MH,Frenkel E,et al.Laboratory assessment of the status of HER2/neu protein and oncogene in breast cancer specimens: comparised immunohistochemistry assay with fluorescence in situ hybridisation assays[J].J Clin Pathol, 2000;53（5）: 374-378
Ozer E,Sis B, Ozen E,et al. BRCAA 1,c-erbB-2, and H-ras gene expression in young women with breast cancer. An immunohistochemical Study[J]. App l Immunoh istochem Molecul Morphol, 2000; 8(1)∶12-17
Pawlow skiV ,Revillion F,Hornez L,et al. A real-time one-step reverset ranscrip tase-polymerse chain reaction method to quantify c-erbB2 expression in human breast cancer[J]. Cancer Detect Prev, 2000; 24(3)∶212-217
Levkowitz G, Waterman H , Ettenberg SA , et al . Ubiquitin ligase activity and tyrosine phosphorylation underlie suppres2 sion of growth factor signaling by c2Cbl/ Sli21[J ] . Mol Cell , 1999 ; 4(6) :1029 - 1040
Kiyokawa N,Karunagaran D,Lee EK,et al.Involvement of cdc2-mediated phosphorylation in the cell cycle-dependent regulation of p185 neu[J].
Oncogene, 1997;15:2633-2641.
Kiyokawa,Lee EK,Karunagaran D,et al.Mitosis-specific negative regulation of epidermal growth factor receptor,triggered by a decrease in ligand binding and dimerization,can be overcomed by overexpression of receptor[J].J Biol Chem,1997;272: 18656-18665
Lane HA , Beuvink I , Motoyama AB , et al . ErbB potentiates breast tumor proliferation through modulation of p27Kip12Cdk2 complex formation : receptor overexpression does not determine growth dependency[J].Mol Cell Biol, 2000 ;20(9) :3210 - 3223
Pegram MD,Baly D,Wirth C,et al.Antibody dependent cell-mediated cytotoxicity in breast cancer patients in phase Ⅲ clinical trials of a humanized anti HER-antibody [J] .Proc Am Assoc Cancer Res , 1997;38: 602-607
Bookman MA,Darcy KM,Clarke2Pearson D ,et al .Evalu2 ation of monoclonal humanized anti2HER2 antibody , trastuzumab , in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2 : a phase Ⅱtrial of the Gynecologic Oncology Group [J ] . J Clin Oncol , 2003 ; 21 (2) :283 - 290
Burstein HJ , Harris LN , Gelman R , et al . Preoperative therapy with trastuzumab and paclitaxel followed by sequen2 tial adjuvant doxorubicin/ cyclophosphamide for HER2 over2 expressing stage Ⅱor Ⅲ breast cancer : a pilot study[J ] . J Clin Oncol , 2003; 21 (1) :46 - 53.
Fleming GF , Meropol NJ , Rosner GL , et al . A phase Ⅰtrial of escalating doses of trastuzumab combined with daily subcutaneous interleukin 2 : report of cancer and leukemia group B 9661[J ] . Clin Cancer Res,2002;8 (12) : 3718 –3727.
Fry DW, Bridges AJ , Denny WA , et al . Specific , irre2 versible inactivation of the epidermal growth factor receptor and erbB22 , by a new class of tyrosine kinase inhibitor[J ] [J]. Proc Natl Acad Sci USA, 1998;95 ( 20):12022 -12027.
Zhang ZG, Cai HB,Bai L , et al. A comparison between the yield of the attachment of Boc-AA-O- Cs+ to Merrifield resin and of Fmoc-AA-OH to Wang resin[J]. Chinese J Org Chem,2000;20(3):419-423.
黄惟德，陈长庆主编.多肽合成﹙M﹚.第一版.北京：科学出版,1985，144-149
Swinnen D, Hilvert D. Facile, Fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters[J]. Org Lett, 2000;2(16):2439-2444.
Nakahara Y,Ando S, Ho Y, et al. New allylester linker and solid-phase block synthesis of the serglycin core region[J]. Bio sci Bio technol Biochem, 2001;65(6):1358-1364.
Wade WS, Yang F, Sowin TJ. Application of base cleavable safety catch linkers to solid phase library production[J]. J Comb Chem,2000;2(3):266-272.
张志刚，何素婷，何明华等. DCM和DMF对DCC-HOBt系统催化酯化及酰化反应的影响[J].生物化学杂志， 1997;13(2): 177-180.
GA. Pietersz, V. Apostolopoulos, IFC Mckenzie. Generation of cellular immune responses to antigenic tumor peptides[J]. Cell Mol Life Sci, 2000; 57: 290-310.
张若蘅，徐筱杰，唐有祺. 应用反相高效液相色谱法分析纯化化学合成肽. 色谱[J], 1996;14(3): 182-184.
陈执中.基质辅助激光解吸离子化—飞行时间质谱法在生化和基因工程药物分析研究中的应用进展[J]. 药物生物技术，2000; 7(2): 122-125.
沈关心,周汝麟主编 现代免疫学实验技术(第一版)[M].武汉:湖北科学技术出版社.1998.P18-20.
Eggers M, Boes Fabian ,Ruppert T, et al. The cleavage preference of the proteasome governs the yield of antigenic peptides[J]. J.Exp. Med, 1995;182:1865-1870.
Disis ML,Grabstein KH.Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine[J].Clin Cancer Res,1999;5(6):1289-1297.
Geysen HM,Macfarian R,Rodda SJ,et al.A period delineation of a peptide which mimics a discontinuous antigenic determinant[J].Molecular Imunology,1986;23(7)709-715.
Bjorkman P.J,and P.Parham.Structure,function and diversity of class I major histocompatibility complex molecules[J].Ann Rev Bioche,1990;(59)253-288.
Ayyoub M, Mazarguil H, Monsarrat B. et al. A Struture Based approach to designing nonnatural peptides that can activate ant-melanoma cytotoxic T cells[J]. J.Biol Chem, 1999;74(15):10227-10234.
Reynolds SR, Oratz R.,Shapiro RL,.et al. Stimulation of CD8+ T cell responses to MAGE-3and Melana/MART-1 by immunization to a polyvalent melanoma vaccine[J]. Int J. Cancer,1997;72(6):972-976.
Parkhurst MR, Salgaller ML, Southwood S, et al. Improved induction of
melanoma reactive CTL with peptides from the melanoma antigen gp100 modified at HLA.a.0201 binding residues[J]. J. Jmmunol, 1996;157(6):2539 –2548.
Porta D G, Beerli R R , Daly J M , et al . ErbB22 , the preferred heterodimeri- zation partner of all ErbB receptors , is a mediator of lateral signaling[J]. J EMBO, 1997 ; 16 (7) : 1647-1655.
Madden,D.The three dimension structures of peptide-MHCⅠcomlex[J].Annual Review of Immunology,1995;13:587-622.
Sela, M., Zisman, E. Different roles of D-amino acids in immune phenomena. Faseb journal : the official publication of the federation of american societies for experiment [J].Biology; 1997;11(6):449-456 .
Afonso LC. The adjuvant effect of interleukin-12 in a vaccine against leishm[J]. Science,1994;263(5144):235-237.
Nencioni L, Villa L, Tagliabue A, et al. In-vivo immunostimulating activity of the 163-171 peptide of human IL-1-beta[J]. J. Immunology, 1987;139(3): 800 –804.
Duits AJ., Van Puijenbroek, Andre, Vermeulen, Hans,et al. Immunoadjuvant activity of a liposomal IL-6 formulation[J]. Vaccine , 1993;11(7):777-781..
Tarr PE. Granulocyte macrophage colony stimulating factor and the immune system[J]. Med Oncol, 1996;13: 133-138.
Dranoff G, Jaffee E, Lazenby A, et al. Vaccination with irradiated tumor cell engineered to secrete murine GM-CSF stimulates potent, specific and long lasting antitumor immunity[J]. Proc Natl Acad Sci USA,1993;90:3539-3543.
单梅梅.细胞毒T细胞在病毒性肝炎发病机理中作用的若干研究进展[J]. 国外医学:流行病学传染病学分册,1998;25(1):12-17.
Altenschmidt U, Klundt E, Groner B et al.Adoptive transfer of in vitro-targeted, activated T lymphocytes results in total tumor regression[J].J Immunol, 1997; 159(11):5509-5515.
柳忠辉,吕昌龙.免疫学常用实验技术 [M].北京：科学出版社，2002:109-113.
Knutson KL, Schiffman K, Disis ML,et al. Immunization with a HER-2/neu helper peptide vaccine generates HER-2/neu CD8 T-cell immunity in cancer patients[J]. J.Clin.Invest,2001;107:477-484.
沈关心, 周汝麟.现代免疫学实验技术[M].湖北：湖北科学技术出版社,1998:333-336.
Ruff MR, Farrar WL, Pert CB. Interferon-γ and granulocyte-macrophage colony- stimulating factor inhibit growth and induce antigens characteristic of myeloid differentiation in small-cell lung cell lines[J]. Proc Natl Acad Sci USA, 1986;83:6613-6613.
Sensi M, Farina C,Maccalli C, et al. Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine[J]. J Clin Invest,1997;99:710-717.
Berd D,Maguire HC,Schuchter LM,et al .Autologous hapten-modified melanoma vaccine as postsurgical adjuvant treatment after resection of nodal metastases[J]. J Clin Oncol,1997;15:2359-2370.
Lindaner M,Stanislawski T, Haubler A, et al. The molecular basis of cancer immunotherapy by cytotoxic T lymphocytes[J] . J. Mol. Med,1998;76:32-37.
Jager E, Ringho?er M, Dienes HP,et al., Granulocyte-macrophage-colony stimulating factor enhances immune responses to melanoma associated peptides in vivo[J]. Int J Cancer,1996;67:54-62.
Soo Kie, Kim SK，Ragupathi G, Cappello S，et al. Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates[J]. Vaccine,2001;19:530-537.
Kawakami Y, Eliyahu S, Jenning C,et al. Recongnition of multiple epitopes in the human melanoma antigen gp100 by tumor infiltrating T lymphocytes associated with in vivo tumor regression[J]. J.Immunol.,1995;154(8):3961 –3968.
Chang EY, Chen CH, Ji H, et al. Antigen specific cancer immunotherapy using a GM-CSF secreting allogenic tumor cell based vaccine[J]. Int.J.Cancer, 2000;86(5):725-730.
常乃柏,孙芾,顾惜春. 粒-巨噬细胞集落刺激因子对外周血T细胞和NK细胞的作用[J]. 中华血液学杂志. 1999;20(12):35-37.
Kapoor Detal. Granulocyte-macrophage colony-stimulating factor enhances the efficacy of hepatitis B virus vaccine in previously unvaccinated haemodialysis patients[J]. J. Viral Hepat,1999;6(5):405 –409.
Sub KW. Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine[J].J. Surg. Oncol, 1999;72(4):218-224.
108． Farrar,M.A.,Schreiber R.D.The molecular cell biology of interferon-γ and its receptor.Ann Rev Immuno,1993;(11):571-611.