CPP及ER滞留信号在肿瘤治疗性肽疫苗中的修饰研究
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摘要
伴随人类肿瘤抗原的不断揭示，治疗性肿瘤疫苗成为肿瘤生物治疗研究的热点课题之一。以往研究表明：细胞毒性T淋巴细胞(Cytotoxic T Lymphocytes，CTL)介导的特异性细胞免疫在机体控制肿瘤中起重要作用。因此，有效激发特异性CTL应答是治疗性肿瘤疫苗研究的重要目标。CTL通过其T细胞受体(T cell receptor，TCR)特异性识别抗原提呈细胞(antigen-presenting cell，APC)(如肿瘤细胞)表面的主要组织相容性复合体(Major Histocompatibility Complex，MHC)-Ⅰ类分子-肽复合物，然后自身得以活化，并最终通过多种机制杀死靶细胞。这些与MHC-Ⅰ类分子结合的长度为8～10个氨基酸的短肽被定义为CTL表位(epitope)。目前众多的肿瘤抗原CTL表位相继被鉴定，使得基于CTL表位的肽疫苗成为治疗性肿瘤疫苗研制的一个重要方向。由于肽疫苗以天然氨基酸为原料，可通过全自动化学合成手段大规模生产，纯化和质控相当方便，而且对于机体相对安全，因此受到人们的广泛重视并呈现出极大的发展空间。但由于存在免疫原性弱、半衰期短及不易被APC摄取等缺陷，肽疫苗很难在体内诱导出有效的CTL反应，故其临床应用受到很大限制。为弥补这些不足，人们尝试了多种策略对肽疫苗进行改造和修饰，如对与MHC-Ⅰ类分子结合的锚点氨基酸进行置换、在多肽N、C末端添加糖脂类基团或进行甲基化“加帽”修饰等，但在诱导特异性CTL效应方面仍没有太大改善。
研究表明，大多数与MHC-Ⅰ类分子结合的短肽来源于胞浆内合成的内源性蛋白；而外源性抗原如可溶性蛋白或多肽被APC以胞饮方式内吞后，绝大多数进入MHC-Ⅱ类抗原呈递途径以激活CD4~+辅助性T细胞(T helper cell，Th)，仅有少量抗原可被MHC-Ⅰ类分子呈递，即“交叉呈递(cross—presentation)”。因此内源性蛋白抗原激发抗原特异性CTL应答的能力远远强于外源性抗原。
第三军医大学博士学位论文
对于肤疫苗而言，如何将外源性抗原肤有效投入APC内MHC一I类抗原呈递途径
是激发特异性CTL应答的关键。一方面讲，被MHC一工类分子提呈的抗原肤大多数来
源于胞浆中合成的蛋白，所以理论上，若促进外源性抗原肤进入APC胞浆，则可望相
应增强抗原肤被MHC一I类分子呈递的效率。近年来，在细胞生物、基因治疗及药物体
内转运等研究领域中出现了细胞膜通透性肚(eell一Penetrating pePtides，ePP)的研究
热点。CPP属于小分子肤类，长度为几个到几十个氨基酸不等。研究证实:CPP可有
效携带蛋白、多肤、DNA甚至颗粒性物质等，通过受体依赖或非依赖方式，穿过大多
数活细胞的质膜进入胞浆，并在一定浓度范围内对细胞无任何毒副作用。目前己有多
种蛋白来源的cPPs得到证实，这些蛋白包括果蝇的antennapedia蛋白、单纯疙疹病毒
的vP22蛋白及I型人类免疫缺陷病毒(Hlv一1)的Tat蛋白。Tat蛋白是Hry一1的转
录活化因子，长度为86~roZ个氨基酸，该蛋白可自由穿过活细胞质膜，并且整个蛋
白或片段(第37一72位氨基酸)可定位于胞浆及胞核。研究表明，Tat蛋白的碱性区
(第49一58位氨基酸)对其实现细胞内在化起关键作用，利用来源于该蛋白碱性区的
肤片段可将其它蛋白有效导入活细胞内。迄今为止，研究报道有数种长短不一的源于
Tat的短肤显示出较强的穿膜功能，其中最短的片段是Ta如_57。我们设想，若将Ta晒_57
与抗原肤连接后，可望借助Ta伽一5:的穿膜活性，将抗原肤直接带入细胞浆，最终使其
有效进入APC内MHC一I类抗原呈递途径。
另一方面，从进化选择角度来讲，内质网(endoplasmic retieulum，ER)是MHc-
I类分子提呈抗原肤的最佳场所。因为ER腔内不仅存在着大量新生成的可接纳抗原
肤的MHC一I类分子，以及促进抗原肤与MHC一I类分子结合的分子伴侣如免疫球蛋
白重链结合蛋白(immunoglobulin heavy ehain一binding Protein，Bip)、pZ一微球蛋白
(beta一2一mieroglobulin，p Zm)、钙连接蛋白(ea一nexin)、钙网蛋白(ealretieulin)及
taPasin等，而且还具有丰富的蛋白酶，可对抗原肤前体分子进行加工修剪。所以促进
外源性抗原肚靶向转运至ER腔内，可望增强抗原肤被MHC一I类分子提呈的效率。
研究表明，抗原加工相关转运体(transporter assoeiated with antigen proeessing，T^P)
是在抗原肤前体分子从胞浆到ER的转运过程中起关键作用的分子伴侣，但在TAP缺
陷的细胞系中发现某些抗原如一些膜蛋白、病毒结构蛋白等仍然可被MHC一I类分子
第三军医大学博士学位论文
有效提呈，说明还存在一些TAP非依赖的
的ER转位信号肤介导了此尸nAP非依赖性
ER转运机制。实验证实，位于蛋白N末端
MHC一I类抗原提呈。研究发现，
原N末端与ER转位信号连接后，可使该抗原直接跨过ER膜进入ER腔内
将目的抗
，从而被
ER腔内新生成的MHC一I类分子有效呈递。这种ER转位信号发挥其转位功能的前提
是它所引导的蛋白抗原必须定位于胞浆，所以N末端ER转位信号仅应用于DNA重
组疫苗的研制，而在肤疫苗研制中的应用受到限制。
另一种ER靶向性转运信号是位于蛋白C末端、以赖氨酸一天冬氨酸一谷氨酸一亮氨
酸(Lys一AsP一Glu一Leu，KDEL)为代表的ER滞留信号。ER定居蛋白的C末端都具有
KDEL或类似
The antigen specific CD8+ cytotoxic T lymphocytes (CTLs) are thought to play a key role in tumor immunotherapy. The stimulation of specific CTL therefore represents one major goal in the design of vaccines for tumor immunotherapy. CTLs recognize short peptides of 8~10 amino acids (derived from tumor-associated antigen (TAA), known as CTL epitopes) in the context of Major Histocompatibility Complex (MHC) class I molecules on the surface of antigen-presenting cells (APCs) such as malignant cells. As the result of the specific interaction between the T cell receptor (TCR) and MHC class I /peptide complexes, CTLs are able to kill target cells expressing TAAs. In consequence, manufacturing these TAAs derived-epitopes by conventional organic peptide synthesis methods is relatively simple, expedient, and cost-effective way to produce safe, therapeutic tumor vaccines. Identification of TAAs and CTL epitopes provides more opportunities for the development of synthetic peptide vaccines. Unfortunately, peptide vaccines
are always unsuccessful at stimulating CTL response, either because of their inherent lack of immunogenicity, or because of their rapid biodegradation, or because of poor uptaken by APCs.
Generally, the majority of MHC class I-associated peptides are derived from the cytosolic degradation of endogenous protein. Exogenous proteins, however, enter the MHC class II-presentation pathway through endocytosis and are degraded in lysosomes, thus the resulting peptides generally do not contribute to MHC class I-associated presentation. A few of exogenous soluble antigens can find their way to associate with MHC class I
molecules (i.e., "cross-presentation"), but this is not an effective way to activate CTL. This basic dichotomy explains why antigen-specific CD8+ CTLs are generated most effectively against intracellular antigens or when immunogens are delivered into the cytosol, e.g., using viral vectors, and why immunization with soluble proteins or peptides rarely induces CTL.
In the case of peptide-based vaccines, the way of delivering exogenous peptides into the MHC class I presentation pathway is critical to elicite CTL responses. On the one hand, an appropriate delivery system may represent a method to transport exogenous antigenic peptides into the cytosol of APCs. Over the past few years, there has been an increased interest in using cell-penetrating peptides (CPP) to transport exogenous molecules into living cells. Several CPPs have been identified from proteins, including antennapedia protein of Drosophila, VP22 proteins of herpes simplex virus and Tat protein of HIV-1. Among them, the short carriers derived from Tat protein have been well studied for the transduction of biologically active proteins into cells both in vitro and in vivo. The region of Tat necessary for translocation has been identified as a short basic sequence corresponding to residues 49~57 (RKKRRQRRR). We hypothesized that exogenous synthetic peptides fused with HIV-Tat49-57 sequence could efficiently cross the cell membrane and be transported rapidly from the extracellular milieu into the cytosol of most cells, and then directly enter into classic MHC class I presentation pathway.
On the other hand, the association of antigenic peptides with MHC class I molecules in the ER is, in some respects, a curious evolutionary choice, because nascent peptide-receptive MHC class I molecules and molecules chaperones such as immunoglobulin heavy chain-binding protein (BiP), calnexin, calreticulin and Tapasin, which are beneficial for peptide assembling with MHC molecules, are present in the ER at very high concentration. Thus an appropriate antigen delivery system may also represent a method to increase the number of defined epitopes in the ER lumen. It's well known that an important step in MHC class I presentation pathway is the translocation of processed
proteins from the cytosol across the endoplasmic reticulum (ER) membrane mediated by transporter associated with antigen processing (TAP). Functional TAP is required for the optimal assembly of
研究表明，大多数与MHC-Ⅰ类分子结合的短肽来源于胞浆内合成的内源性蛋白；而外源性抗原如可溶性蛋白或多肽被APC以胞饮方式内吞后，绝大多数进入MHC-Ⅱ类抗原呈递途径以激活CD4~+辅助性T细胞(T helper cell，Th)，仅有少量抗原可被MHC-Ⅰ类分子呈递，即“交叉呈递(cross—presentation)”。因此内源性蛋白抗原激发抗原特异性CTL应答的能力远远强于外源性抗原。
第三军医大学博士学位论文
对于肤疫苗而言，如何将外源性抗原肤有效投入APC内MHC一I类抗原呈递途径
是激发特异性CTL应答的关键。一方面讲，被MHC一工类分子提呈的抗原肤大多数来
源于胞浆中合成的蛋白，所以理论上，若促进外源性抗原肤进入APC胞浆，则可望相
应增强抗原肤被MHC一I类分子呈递的效率。近年来，在细胞生物、基因治疗及药物体
内转运等研究领域中出现了细胞膜通透性肚(eell一Penetrating pePtides，ePP)的研究
热点。CPP属于小分子肤类，长度为几个到几十个氨基酸不等。研究证实:CPP可有
效携带蛋白、多肤、DNA甚至颗粒性物质等，通过受体依赖或非依赖方式，穿过大多
数活细胞的质膜进入胞浆，并在一定浓度范围内对细胞无任何毒副作用。目前己有多
种蛋白来源的cPPs得到证实，这些蛋白包括果蝇的antennapedia蛋白、单纯疙疹病毒
的vP22蛋白及I型人类免疫缺陷病毒(Hlv一1)的Tat蛋白。Tat蛋白是Hry一1的转
录活化因子，长度为86~roZ个氨基酸，该蛋白可自由穿过活细胞质膜，并且整个蛋
白或片段(第37一72位氨基酸)可定位于胞浆及胞核。研究表明，Tat蛋白的碱性区
(第49一58位氨基酸)对其实现细胞内在化起关键作用，利用来源于该蛋白碱性区的
肤片段可将其它蛋白有效导入活细胞内。迄今为止，研究报道有数种长短不一的源于
Tat的短肤显示出较强的穿膜功能，其中最短的片段是Ta如_57。我们设想，若将Ta晒_57
与抗原肤连接后，可望借助Ta伽一5:的穿膜活性，将抗原肤直接带入细胞浆，最终使其
有效进入APC内MHC一I类抗原呈递途径。
另一方面，从进化选择角度来讲，内质网(endoplasmic retieulum，ER)是MHc-
I类分子提呈抗原肤的最佳场所。因为ER腔内不仅存在着大量新生成的可接纳抗原
肤的MHC一I类分子，以及促进抗原肤与MHC一I类分子结合的分子伴侣如免疫球蛋
白重链结合蛋白(immunoglobulin heavy ehain一binding Protein，Bip)、pZ一微球蛋白
(beta一2一mieroglobulin，p Zm)、钙连接蛋白(ea一nexin)、钙网蛋白(ealretieulin)及
taPasin等，而且还具有丰富的蛋白酶，可对抗原肤前体分子进行加工修剪。所以促进
外源性抗原肚靶向转运至ER腔内，可望增强抗原肤被MHC一I类分子提呈的效率。
研究表明，抗原加工相关转运体(transporter assoeiated with antigen proeessing，T^P)
是在抗原肤前体分子从胞浆到ER的转运过程中起关键作用的分子伴侣，但在TAP缺
陷的细胞系中发现某些抗原如一些膜蛋白、病毒结构蛋白等仍然可被MHC一I类分子
第三军医大学博士学位论文
有效提呈，说明还存在一些TAP非依赖的
的ER转位信号肤介导了此尸nAP非依赖性
ER转运机制。实验证实，位于蛋白N末端
MHC一I类抗原提呈。研究发现，
原N末端与ER转位信号连接后，可使该抗原直接跨过ER膜进入ER腔内
将目的抗
，从而被
ER腔内新生成的MHC一I类分子有效呈递。这种ER转位信号发挥其转位功能的前提
是它所引导的蛋白抗原必须定位于胞浆，所以N末端ER转位信号仅应用于DNA重
组疫苗的研制，而在肤疫苗研制中的应用受到限制。
另一种ER靶向性转运信号是位于蛋白C末端、以赖氨酸一天冬氨酸一谷氨酸一亮氨
酸(Lys一AsP一Glu一Leu，KDEL)为代表的ER滞留信号。ER定居蛋白的C末端都具有
KDEL或类似
The antigen specific CD8+ cytotoxic T lymphocytes (CTLs) are thought to play a key role in tumor immunotherapy. The stimulation of specific CTL therefore represents one major goal in the design of vaccines for tumor immunotherapy. CTLs recognize short peptides of 8~10 amino acids (derived from tumor-associated antigen (TAA), known as CTL epitopes) in the context of Major Histocompatibility Complex (MHC) class I molecules on the surface of antigen-presenting cells (APCs) such as malignant cells. As the result of the specific interaction between the T cell receptor (TCR) and MHC class I /peptide complexes, CTLs are able to kill target cells expressing TAAs. In consequence, manufacturing these TAAs derived-epitopes by conventional organic peptide synthesis methods is relatively simple, expedient, and cost-effective way to produce safe, therapeutic tumor vaccines. Identification of TAAs and CTL epitopes provides more opportunities for the development of synthetic peptide vaccines. Unfortunately, peptide vaccines
are always unsuccessful at stimulating CTL response, either because of their inherent lack of immunogenicity, or because of their rapid biodegradation, or because of poor uptaken by APCs.
Generally, the majority of MHC class I-associated peptides are derived from the cytosolic degradation of endogenous protein. Exogenous proteins, however, enter the MHC class II-presentation pathway through endocytosis and are degraded in lysosomes, thus the resulting peptides generally do not contribute to MHC class I-associated presentation. A few of exogenous soluble antigens can find their way to associate with MHC class I
molecules (i.e., "cross-presentation"), but this is not an effective way to activate CTL. This basic dichotomy explains why antigen-specific CD8+ CTLs are generated most effectively against intracellular antigens or when immunogens are delivered into the cytosol, e.g., using viral vectors, and why immunization with soluble proteins or peptides rarely induces CTL.
In the case of peptide-based vaccines, the way of delivering exogenous peptides into the MHC class I presentation pathway is critical to elicite CTL responses. On the one hand, an appropriate delivery system may represent a method to transport exogenous antigenic peptides into the cytosol of APCs. Over the past few years, there has been an increased interest in using cell-penetrating peptides (CPP) to transport exogenous molecules into living cells. Several CPPs have been identified from proteins, including antennapedia protein of Drosophila, VP22 proteins of herpes simplex virus and Tat protein of HIV-1. Among them, the short carriers derived from Tat protein have been well studied for the transduction of biologically active proteins into cells both in vitro and in vivo. The region of Tat necessary for translocation has been identified as a short basic sequence corresponding to residues 49~57 (RKKRRQRRR). We hypothesized that exogenous synthetic peptides fused with HIV-Tat49-57 sequence could efficiently cross the cell membrane and be transported rapidly from the extracellular milieu into the cytosol of most cells, and then directly enter into classic MHC class I presentation pathway.
On the other hand, the association of antigenic peptides with MHC class I molecules in the ER is, in some respects, a curious evolutionary choice, because nascent peptide-receptive MHC class I molecules and molecules chaperones such as immunoglobulin heavy chain-binding protein (BiP), calnexin, calreticulin and Tapasin, which are beneficial for peptide assembling with MHC molecules, are present in the ER at very high concentration. Thus an appropriate antigen delivery system may also represent a method to increase the number of defined epitopes in the ER lumen. It's well known that an important step in MHC class I presentation pathway is the translocation of processed
proteins from the cytosol across the endoplasmic reticulum (ER) membrane mediated by transporter associated with antigen processing (TAP). Functional TAP is required for the optimal assembly of
引文
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28. Yang B, Hahn YS, Hahn CS, Braciale TJ. The requirement for proteasome activity class Ⅰ major histocompatibility complex antigen presentation is indicated by the length of preprocessed antigen [J]. J Exp Med. 1996, 183(4): 1545-1552.
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30. Lu J, Wettstein PJ, Higashimoto Y, Appella E, Celis E. TAP-independent presentation of CTL epitopes by Trojan antigens [J]. J Immunol. 2001, 166(12): 7063-7071.
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32. Bullock TN, Colella TA, Engelhard VH. The density of peptides displayed by dendritic cells affects immune responses to human tyrosinase and gp100 in HLA-A2 transgenic mice [J]. J Immunol. 2000, 164(5): 2354-2361.
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35. Parkhurst MR, Salgaller ML, Southwood S, Robbins PF, Sette A, Rosenberg SA, Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues [J]. J Immunol. 1996, 157(6): 2539-2548.
36. Uger RA, Chart SM, Barber BH. Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes [J]. J Immunol. 1999, 162(10): 6024-6028.
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40. Uger RA, Chan SM, Barber BH. Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes [J]. J Immunol. 1999, 162(10): 6024-6028.
41. Diedrich G, Bangia N, Pan M, Cresswell P. A role for calnexin in the assembly of the MHC class I loading complex in the endoplasmic reticulum [J]. J Immunol, 2001, 166(3): 1703-1709.
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28. Yang B, Hahn YS, Hahn CS, Braciale TJ. The requirement for proteasome activity class Ⅰ major histocompatibility complex antigen presentation is indicated by the length of preprocessed antigen [J]. J Exp Med. 1996, 183(4): 1545-1552.
29. Mo XY, Cascio P, Lemerise K, Goldberg AL, Rock K. Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides [J]. J Immunol. 1999, 163(11): 5851-5859.
30. Lu J, Wettstein PJ, Higashimoto Y, Appella E, Celis E. TAP-independent presentation of CTL epitopes by Trojan antigens [J]. J Immunol. 2001, 166(12): 7063-7071.
31. Serwold T, Gaw S, Shastri N. ER aminopeptidases generate a unique pool of peptides for MHC class Ⅰ molecules [J]. Nat Immunol. 2001, 2(7): 644-651.
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34. Hahn YS, Hahn CS, Braciale TJ. Endogenous presentation of a nascent antigenic epitope to CD8~+ CTL is more efficient than exogenous presentation [J]. Immunol Cell Biol. 1996, 74(5): 394-400.
35. Parkhurst MR, Salgaller ML, Southwood S, Robbins PF, Sette A, Rosenberg SA, Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues [J]. J Immunol. 1996, 157(6): 2539-2548.
36. Uger RA, Chart SM, Barber BH. Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes [J]. J Immunol. 1999, 162(10): 6024-6028.
37. Townsend A, Ohlen C, Bastin J, Ljunggren HG, Foster L, Karre K. Association of class I major histocompatibility heavy and light chains induced by viral peptides [J]. Nature. 1989, 340(6233): 443-448.
38. Levitt JM, Howell DD, Rodgers JR, Rich RR. Exogenous peptides enter the endoplasmic reticulum of TAP-deficient cells and induce the maaturation of nascentMHC class I molecules [J]. Eur J Immunol. 2001, 31 (4): 1181-1190.
39. Binder R J, Blachere NE, Srivastava PK. Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules [J]. J Biol Chem. 2001, 276(20): 17163-17171.
40. Uger RA, Chan SM, Barber BH. Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes [J]. J Immunol. 1999, 162(10): 6024-6028.
41. Diedrich G, Bangia N, Pan M, Cresswell P. A role for calnexin in the assembly of the MHC class I loading complex in the endoplasmic reticulum [J]. J Immunol, 2001, 166(3): 1703-1709.