增殖病毒对肿瘤的治疗作用

目的：探讨ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫诱导的特异性细胞免疫应答及其在体内的抗瘤效应。方法：克隆并构建人ＨＥＲ２／ｎｅｕ原癌基因及其胞外区片段表达载体,分别转染ＳＰ２／０细胞以制备杀伤靶细胞。将质粒ＤＮＡ免疫小鼠,观察其诱导的细胞免疫应答,同时分析免疫动物体内抗瘤效应。结果：体外获得稳定表达ＨＥＲ２／ｎｅｕ基因的ＳＰ２／０靶细胞。目的基因ＤＮＡ免疫后,免疫鼠脾细胞在体外可检测到特异性杀伤作用,体内肿瘤细胞接种后可发现肿瘤结节出现时间延迟,肿瘤生长缓慢。结论： ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫可诱导出特异细胞免疫应答,并具有一定抗瘤效应。     

分次小剂量放射免疫治疗预防和控制荷瘤小鼠肺转移的实验研究 *

目的：探讨ＨＥＲ２／ｎｅｎ的癌基因胞外区片段ＤＮＡ免疫诱导的特异性细胞免疫应答及其在体内的抗瘤效应。方法：克隆并构建人ＨＥＲ２／ｎｅｎ原癌基因及其胞外区片表达载体,分别转染ＳＰ２／０细胞以制备样伤靶细胞。将质粒ＤＮＡ免疫小鼠,观察其诱导的细胞免疫应答,同时分析免疫动物体内抗瘤效应。结果：体外获得稳定表达ＨＥＲ２／ｎｅｎ基因的ＳＰ２／０靶细胞。目的基因ＤＮＡ免疫后,免疫鼠脾细胞在体外可检测到特异性杀     

黑色素瘤B16细胞热休克蛋白—抗原肽复合物的体内外抑？…

目的：黑色素瘤Ｂ１６细胞胞浆ＨＳＰ－抗原肽复合物（ＨＡＣ）的制备、免疫原性的诱导及其抑瘤作用的研究。方法：采用Ｔｒｉｓ－ＨＣＩ提取和Ｓｅｐｈａｃｒｙｌ　Ｓ－２００凝胶过滤制备Ｂ１６细胞ＨＡＣ,通过Ｃ５７ＢＬ／６Ｊ小鼠体内诱导特异性ＣＴＬ,再经体内、体外实验检测其抑癌作用。结果：凝胶过滤获得的含６０～９７ｋＤ蛋白的４１,４７和５３管的ＨＡＣ可以降低肿瘤发生率、延长肿瘤出现时间及降低小鼠死亡率。结论：Ｂ１６细胞胞浆中的６０～９７ｋＤ的ＨＡＣ具有免疫原性及抑瘤作用,为制备肿瘤疫苗提供了实验依据。     

小鼠骨髓瘤杂交细胞在动物体内的抗肿瘤保护作用

将小鼠ＳＰ２／０骨髓瘤细胞与脂多糖（ＬＰＳ）活化的同系动物来源的Ｂ淋巴细胞融合，获得１Ｆ１１和２Ａ６杂交细胞。采用荷瘤小鼠模型，观察了１Ｆ１１、２Ａ６杂交细胞的免疫治疗对小鼠存活率、小鼠脾ＣＴＬ细胞体外杀瘤活性的影响。结果显示，腹腔荷骨髓瘤小鼠经ＳＰ２／０杂交细胞治疗后第３周时存活率均为５０．０％，对照组小鼠全部死亡；１Ｆ１１、２Ａ６治疗组分别有３０．０％、２０．０％小鼠长期存活；杂交细胞治疗组小鼠脾ＣＴＬ细胞杀伤ＳＰ２／０细胞活性显著高于对照组（Ｐ＜０．０５）。结果表明，１Ｆ１１和２Ａ６杂交细胞具有一定的体内抗肿瘤治疗作用，提示肿瘤细胞与活化Ｂ淋巴细胞的杂交细胞可作为一种肿瘤疫苗用于肿瘤的防治研究     

重组人白介素2-卡介苗对荷瘤小鼠腹腔巨噬细胞体外杀伤活性的影响 *

目的：研究ＨＥＲ２／ｎｅｕ原癌基因胞外区片段（ＥＣＤ）ＤＮＡ直接肌肉注射在小鼠体内表达,体液免疫状况,利用小鼠流产模型观察其体内效应。方法：克隆并构建了人及大鼠ＨＥＲ２／ｎｅｕ原癌基因胞外区片段真核表达载体ｐＣＤＮＡ３－Ｘ,采用肌肉直接注射的方法免疫小鼠,分析目的基因在小鼠体内的表达及其诱导的兔疫应答。结果：在质粒ＤＮＡ直接注射部位可检测到目的基因的表达,并诱导了针对ＨＥＲ２／ｎｅｕ的抗体反应;同时诱发小鼠流产,使免疫鼠产仔数减少,可观察到流产结节形成。结论：通过基因免疫,可诱导针对ＨＥＲ２／ｎｅｕ原癌基因产物的有效免疫应答。     

小鼠B16黑色素瘤细胞HACs的提纯及其抑瘤作用和机理的探讨

目的：从小鼠Ｂ１６黑色素瘤细胞胞浆中提纯热休克蛋白抗原肽复合物（ｈｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ－ａｎｔｉｇｅｎ ｐｅｐｔｉｄｅ ｃｏｍｐｌｅｓｅｓ．ＨＡＣＣｓ）,并观察其抑瘤作用及探索其机制。方法：ＣＮＢｒ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析法纯纯Ｂ１６ ＨＡＣｓ,应用体内免疫法检测ＨＡＣ的抑瘤作用,结晶紫染色法检测γ－ＩＦＮ分泌活性,ＣｏｎＡ诱导的淋巴母细胞增殖法检测ＩＬ－２分泌活性,＾３Ｈ－     

HSV-tk/GCV协同放射治疗黑色素瘤的实验研究 *

目的：了解ＨＳＶ－ｔｋ／ＧＣＶ系统协同放射治疗对小鼠黑色素瘤的联合杀伤作用。方法：通过逆转录病毒介导,将潮霉素磷酸转移酶和ＨＳＶ－ｔｋ的融合基因（ｈｙｔｋ）导入黑色素瘤细胞中并获得表达,通过体外和Ｃ５７ＢＬ／６小鼠动物实验,检测 ＧＣＶ对转基因细胞的体内外杀伤作用及联合应用放射治疗对黑色素瘤的治疗作用。结果：ＰＣＲ、ＲＮＡ Ｄｏｔ ｂｌｏｔｔｉｎｇ、免疫组化检测证实ＨｙＴＫ在转基因细胞的表达,体内外实验示ＧＣＶ对Ｂ１６／ＨｙＴＫ细胞有明显的杀伤作用,而对野生型Ｂ１６细胞无明显杀伤作用（Ｐ＜０．０５）;联合应用低剂量ＧＣＶ可使转入ｈｙｔｋ基因的黑色素瘤细胞对放疗的敏感性明显增高（ＳＥＲ＝１．６２）,体内实验也证实协同疗法可延缓荷瘤小鼠的肿瘤生长。结论：ＨＳＶ－ｔｋ／ＧＣＶ系统协同放射治疗有望成为黑色素瘤基因治疗的一种有效方法。     

AK细胞及环磷酰胺对肝癌的实验研究

为研究１种新的过继免疫化疗治疗肝癌的方法，采用肝癌细胞株Ｈ２２接种于近交系Ｂａｌｂ／ｃ小鼠皮下，制成肿瘤模型。全胜、肿瘤活化的杀伤细胞（ＡＫ细胞）及环磷酰胺（Ｃｙ）进行治疗，检测小鼠脾淋巴细胞ＮＫ、ＬＡＫ及ＣＴＬ活笥，用流式细胞仪检测Ｌ３Ｔ４ｙｔ－２亚群含量结果表明在过继免疫化疗组小鼠ＬＡＫ及ＣＴＬ活性明显高于其它治疗组（Ｐ〈０．０１），生存期也明显高于其它各治疗组（Ｐ〈０．０１）。该研究过继免疫     

人鼠T－B细胞杂交瘤瘤苗对小鼠骨髓瘤的抗瘤效应

Ｐ３是用人Ｔ细胞与小鼠Ｓｐ２／０骨髓瘤细胞融合而成的人鼠Ｔ－Ｂ细胞杂交瘤，其表面具有人Ｔ细胞Ｅ玫瑰花受体标志，我们将Ｐ３作为肿瘤疫苗进行了初步的抗肿瘤实验研究，结果表明：Ｐ３细胞在ＢＡＬＢ／ｃ小鼠体内失去了成瘤性。Ｐ３肿瘤疫苗有较好的治疗肿瘤的作用，实验组瘤重７４７±１８２ｍｇ，显著轻于对照组瘤重２７２５±９５２ｍｇ，Ｐ＜０．０００１。在肿瘤预防实验中，Ｐ３瘤苗存活期为：３１．６±６．８天，显著长于对照组的２４．０±２．１天，Ｐ＜０．０００１。用ＢＣＧ和ＩＬ－２可强化Ｐ３瘤苗的抗肿瘤效应，表现为Ｐ３＋ＢＣＧ＋ＩＬ－２组小鼠存活期显著延长，并有２／８的小鼠存活期大于６０天，且无瘤生长。     

抗人肝癌细胞膜单克隆抗体HCMP—1的制备及其生物学特性的初步鉴定

用超选择免疫法，诱导ＢＡＬＢ／ｃ小鼠对正常人肝细胞抗原产生选择性低兔疫反应后，二步腹腔注射人肝癌细胞悬液，２－４周后用人肝癌细胞作强化免疫动物１次，取出脾细胞与ＳＰ２／０骨髓瘤细胞用ＰＥＧ法融合制备杂交瘤。采用细胞抗原ＥＬＩＳＡ法，同时测定培养上清对肝癌细胞及正常肝细胞的免疫反应，筛出一株能稳定分泌抗人肝癌细胞膜抗原的单克隆抗体的杂交瘤细胞株，ＨＣＭＰ－１ＭＡｂ是一种ＩｇＧ２亚型的单抗，它与ＨＢｓ     

HER2/neu胞外区片段基因免疫诱导特异细胞免疫及其抗瘤效应研究 *

目的：了解鱼精蛋白应用于血管内皮生长因子受体介导的靶向性非病毒载体的可行性。方法：ＣＶ１,ＣＶ２靶向性非病毒载体来比较多聚赖氨酸与鱼精蛋白对靶向性基因转移复合体携带ＤＮＡ的能力及体外基因转移效率的影响。结果：在Ａ３７５细胞中,鱼业 白与多聚赖氨酸参与形成的复合基因导入率都为５０％左右。在ＡＢＡＥ细胞中,鱼精蛋白参与形成的复合体基因导入率只有２０％左右,而多聚赖酸可达７０％左右。鱼精蛋白参与形成的复     

Her2／ECD －sf162／TM嵌合 VLPs 的制备及抗肿瘤免疫效果研究

目的：成功制备 Her2胞外段基因与猴免疫缺陷病毒（simian immunodeficiency virus,SIV）包膜蛋白sf162跨膜区基因融合的 Her2／ECD －sf162／TM病毒样颗粒（VLPs）,并在小鼠体内进行初步的抗肿瘤免疫效果研究。方法：利用前期构建好的 Her2／neu 与 SIV －gag 嵌合的表达载体 Her2／ECD －sf162／TM,制备 Her2／neu 与 SIV －gag 嵌合型 VLPs 疫苗,并用该疫苗免疫小鼠。结果：VLPs 可成功激发小鼠体内免疫应答反应,产生血清抗 VLPs 的抗体;VLP 免疫后接种 Her2／neu ＋小鼠乳腺癌细胞 EMT6,结果显示该疫苗可有效抑制肿瘤生长;同时,VLP 对荷瘤鼠治疗结果也显示,该疫苗可在一定程度上抑制肿瘤生长。结论：VLPs 疫苗具有良好的免疫原性,且免疫后对肿瘤攻击具有保护作用。     

抗双链DNA自身抗体的抗肿瘤作用

目的　体内外探讨抗双链DNA(anti dsDNA)自身抗体对肿瘤生长的影响。方法　用灭活的肿瘤细胞免疫BALB/c小鼠 ,于初次免疫后第 8周 ,分别用SP 2 / 0和Wehi1 6 4肿瘤细胞攻击。将肿瘤细胞与免疫血清先行孵育后再接种正常小鼠 ,分别观察肿瘤的生长情况。结果　体内研究结果显示 ,经肿瘤细胞免疫诱导产生的anti dsDNA自身抗体具有抑制肿瘤生长的作用。体外研究结果显示 ,anti dsDNA自身抗体对肿瘤细胞的生长有抑制作用 ,其机制可能与诱导肿瘤细胞凋亡有关 ,且其诱导SP 2 / 0和Wehi 1 6 4肿瘤细胞发生凋亡的能力与亲和力有显著相关性 (r=0 .990 ,P <0 .0 1和r =0 .90 1 ,P <0 .0 5 )。结论　Anti dsDNA自身抗体具有抗肿瘤作用。     

Defining promiscuous MHC class II helper T-cell epitopes for the HER2/neu tumor antigen

It is accepted that both helper and CTLs play a critical role in immune antitumor responses. Thus, the design of effective immune-based therapies for cancer relies in the identification of relevant tumor-associated antigens (TAAs) capable of eliciting strong helper and cytotoxic T-cell responses against tumor cells. The product of the HER2/neu oncogene is considered as a prototype TAA, because it is found overexpressed in a large variety of malignancies, whereas normal cells only produce low levels of this product. Several cytotoxic T-cell epitopes for HER2/neu have been identified that enable the design of peptide-based therapeutic vaccines for tumors expressing this TAA. Nevertheless, it is expected that inclusion of peptide epitopes capable of eliciting HER2/neu-specific T helper responses into these vaccines may enhance their effectiveness in the clinic. We describe here a strategy to identify helper T-cell epitopes for HER2/neu that focuses on peptides predicted to bind to numerous histocompatibility alleles (promiscuous epitopes), which would encourage their use in therapeutic vaccines for the general cancer patient population. Following this approach, we successfully identified several peptides that elicited T helper (CD4+) proliferative responses to peptides derived from HER2/neu. Most of the T-cell responses appeared to reflect a low affinity for antigen, which could be the result of immune tolerance because HER2/neu is expressed in low levels in normal cells and possibly including lymphocytes and monocytes. Interestingly, one of these peptides, HER2(883), was recognized by T cells in the context of either HLA-DR1, HLA-DR4, HLA-DR52, and HLA-DR53, indicating a high degree of histocompatibility promiscuity. Furthermore, T cells that reacted with peptide HER2(883) could also recognize antigen-presenting cells that process HER2/neu recombinant protein. These results may be relevant for the design of more effective therapeutic vaccines for tumors expressing the HER2/neu oncogene product.     

P5 HER2/neu-derived peptide conjugated to liposomes containing MPL adjuvant as an effective prophylactic vaccine formulation for breast cancer

Vaccines containing synthetic peptides derived from tumor-associated antigens (TAA) can elicit potent cytotoxic T lymphocyte (CTL) response if they are formulated in an optimal vaccine delivery system. The aim of this study was to develop a simple and effective lipid-based vaccine delivery system using P5 HER2/neu-derived peptide conjugated to Maleimide-PEG2000-DSPE. The conjugated lipid was then incorporated into liposomes composed of DMPC:DMPG:Chol:DOPE containing Monophosphoryl lipid A (MPL) (Lip-DOPE-P5-MPL). Different liposome formulations were prepared and characterized for their physicochemical properties. To evaluate anti-tumoral efficacy, BALB/c mice were immunized subcutaneously 3 times in two-week intervals and the generated immune response was studied. The results demonstrated that Lip-DOPE-P5-MPL induced a significantly higher IFN-γ production by CD8+ T cells intracellularly which represents higher CTL response in comparison with other control formulations. CTL response induced by this formulation caused the lowest tumor size and the longest survival time in a mice model of TUBO tumor. The encouraging results achieved by Lip-DOPE-P5-MPL formulation could make it a promising candidate in developing effective vaccines against Her2 positive breast cancers.     

异位hCGβ基因免疫诱导的特异性抗肿瘤免疫应答

目的　观察异位hCGβ基因免疫诱导的肿瘤特异性免疫应答及其抗肿瘤作用 ,为肿瘤的免疫生物治疗寻求新途径。方法　构建含hCGβ编码基因的质粒TR4 2 1 hCGβ ,对BALB/c小鼠实施基因免疫 ,并以空质粒为对照。采用ELISA法和3 H TdR掺入法分别检测免疫小鼠血清中特异性抗hCGβ IgG抗体及其对肿瘤细胞体外生长的抑制作用 ;特异抗原体外刺激免疫小鼠脾细胞后 ,用3 H TdR掺入法和3 H TdR释放法分别检测其特异性增殖活性和细胞毒活性 ;皮下接种SP2 /0 hCGβ细胞攻击免疫小鼠 ,以成瘤率及实体瘤重量评估体内抗瘤效果。结果　全部TR4 2 1 hCGβ质粒免疫小鼠均产生高水平的抗hCGβ IgG抗体 ,该抗体可抑制肿瘤细胞的体外生长 ,与对照血清相比 ,差异有显著差性 (P <0 .0 5 ) ;hCGβ蛋白、灭活SP2 /0 hCGβ细胞以及两者混合均能刺激TR4 2 1 hCGβ质粒免疫小鼠脾细胞的体外增殖 (SI值分别为 1.5 3、1.81和 2 .0 5 ) ,与空质粒免疫小鼠相比 ,差异有显著性 (P <0 .0 1) ;特异抗原刺激的TR4 2 1 hCGβ质粒免疫小鼠脾细胞对SP2 /0 hCGβ细胞的细胞毒活性明显高于SP2 /0细胞(P <0 .0 1) ;空质粒免疫小鼠接种SP2 /0 hCGβ细胞后成瘤率为 10 0 % ,瘤重达 3.37g ,而TR4 2 1 hCGβ质粒免疫小鼠的成瘤率为 16 .6 6 % ,瘤重为     

A HER2/NEU-derived peptide, a K(d)-restricted murine tumor rejection antigen, induces HER2-specific HLA-A2402-restricted CD8(+) cytotoxic T lymphocytes

We have identified an H-2K(d)-binding peptide, HER2p780 (PYVSRLLGI), derived from murine HER2/neu (HER2), that can induce HER2-specific murine cytotoxic T lymphocytes (CTL). Weekly vaccination of BALB/c mice by syngeneic dendritic cells pulsed with HER2p780 peptide, entirely common to murine and human HER2, suppressed growth of pretransplanted HER2-expressing syngeneic tumors. A HER2-expressing human cancer cell line SKOV3 transfected with murine H-2K(d) cDNA could also be lysed by HER2p780-specific murine CTLs, indicating that human HER2-expressing cancer cells can process and present the cognate peptide in the context of H-2K(d). Since H-2K(d) and HLA-A2402 molecules have similar anchor motifs, the possibility of inducing HER2-specific CTL activity with HER2p780 in HLA-A2402 individuals was examined. CD8(+) CTL clones specific for HER2-expressing cancer cell lines were established from peripheral blood lymphocytes with HLA-A2402 by repeatedly sensitizing with peptide-pulsed autologous dendritic cells as well as peripheral blood mononuclear cells. Detailed analysis of their specificity revealed that the cytotoxicity of CTL clones is specific for the cognate peptide with HLA-A2402 restriction. The results suggest that HER2p780 is a unique peptide that may function as a tumor rejection antigen peptide in HLA-A2402 individuals, as it was directly proven here to function in a murine tumor system.     

T cells sensitized with breast tumor progenitor cell vaccine have therapeutic activity against spontaneous HER2/neu tumors

Cancer progenitor cells are critical for tumor initiation and recurrence so they are an important therapeutic target. We tested whether T cells could recognize tumor antigens expressed by breast cancer progenitor cells and acquire therapeutic activity against established metastases or delay onset of spontaneous tumors. Breast tumors were derived from HER2/neu transgenic mice and propagated in vitro under conditions that selected progenitor cells which were then used as an irradiated whole cell vaccine. A minor subset of recently sensitized T cells was isolated from vaccine-draining lymph nodes then activated in vitro to achieve numerical expansion. We show that the tumor progenitor cell vaccines reversed tolerance to a known HER2/neu epitope, otherwise inhibited by Treg cells. Additional shared tumor antigens were recognized because a Neuneg subclone also induced a Th1 type immune response against breast tumors. Adoptive transfer of in vitro activated lymph node T cells-mediated regression of established metastases from multiple independently derived breast tumor lines. Moreover, adoptive transfer of effector T cells into Neu-tolerant mice, months before the onset of spontaneous tumors, significantly postponed tumor development. Interestingly, T-cell-mediated lysis of metastases stimulated an IgG response to HER2/neu as well as other shared antigens. In summary, tumor progenitor cells contain shared antigens which can lead to a cross-protective T-cell response. Moreover, antigens acquired during immune-mediated tumor destruction are presented in a manner conducive to reversal of tolerance and Ig class switching. These complementary effector mechanisms might augment therapy by eliminating refractory breast cancer stem cells.     

Dendritic cells pulsed with HER-2/neu-derived peptides can induce specific T-cell responses in patients with gastric cancer.

PURPOSE: We have previously reported (K. Kono et al., Int. J. Cancer, 78: 202-208, 1998) that HER-2/neu-derived peptides are naturally processed as tumor-associated antigens recognized by tumor-specific, human leukocyte antigen (HLA)-A2-restricted CTLs in gastric cancer. In the present study, we described a Phase-1 vaccination trial in gastric cancer patients using dendritic cells (DCs) pulsed with the immunodominant HER-2/neu(p369) peptides. EXPERIMENTAL DESIGN: Nine enrolled patients, who had HER-2/neu-overexpressing tumors and who were HLA-A2 positive, received four vaccinations by DCs pulsed with HER-2(p369) peptide at 2-week intervals intradermally. RESULTS: There were no serious adverse effects noted in the immunized patients. Peripheral blood mononuclear cells, preimmunization and after the fourth immunization, were cultured with autologous, HER-2(p369)-pulsed antigen-presenting cells for 12 days. Thereafter, peptide specificity was evaluated by IFN-gamma secretion assay from cultured T cells against T2 cells pulsed with HER-2(p369) peptide. HER-2/neu peptide-specific recognition could be demonstrated in six of nine patients after immunization, whereas there was no HER-2/neu peptide-specific recognition before immunization. The peptide-specific CTL lines isolated from two of the patients could also lyse a HER2/neu-transfected cell line. Furthermore, a peptide-specific delayed-type hypersensitivity response occurred in three of nine patients. One of the patients underwent a partial clinical response concurrent with a decrease of tumor marker. Another patient demonstrated a stabilization of disease status for a period of 3 months. CONCLUSIONS: Taken together, tumor vaccination therapy with DCs pulsed with HER-2/neu-peptides may be a potential candidate for the novel treatment of gastric cancer patients.