卵巢癌抗独特型单链抗体6B11ScFv/鼠GMCSF融合蛋白(6B11mGM)的构建、表达和特性研究

抗独特型抗体能够模拟肿瘤抗原,调节机体免疫功能,已成为肿瘤免疫治疗研究中一个十分重要的研究领域.本中心制备的卵巢癌抗独特型抗体6B11能模拟卵巢癌抗原诱导特异性抗肿瘤免疫应答.为降低鼠源性单抗进入体内引起的不良免疫反应,本中心已成功制备了基因工程抗独特型单链抗体(6B11ScFv).为了增加6B11ScFv的免疫原性,又构建了6B11ScFv与人GM-CSF融合蛋白(6B11GM).但由于缺     

卵巢癌基因工程抗独特型疫苗6B11和3D5的基础研究

抗独特型抗体(Ab_2βb)能够模拟肿瘤抗原,调节机体免疫功能,又被称为“抗独特型疫苗”,6B11为本中心制备的鼠单克隆抗独特型抗体,可模拟卵巢癌单抗OC166-9;3D5杂交瘤是用抗卵巢癌单抗OC125免疫小鼠脾细胞与骨髓瘤细胞融合而成,分泌的抗体能模拟卵巢癌相关抗原CA125.两种抗独特型抗体均可在体内外诱导产生特异性抗卵巢癌体液和细胞免疫反应.有可能作为“卵巢癌疫苗”用于卵巢癌的治疗.本研究利用分子克隆技术,对6B11和3D5在基因水平进行改造,构建6B11和3D5卵巢癌基因工程疫苗.     

TCRγ独特型DNA疫苗诱导特异性免疫反应的实验研究

目的：观察TCRγ独特型DNA疫苗诱导BALB／c小鼠的抗人类Jurkat淋巴肿瘤免疫反应的情况。方法：碱裂解法大量提取质粒，制备DNA疫苗。选用16只BALB／c小鼠随机分为pcDNA3组、pcDNA3／TCRγV1组，双侧股四头肌内注射疫苗免疫，于第0、2、4周各免疫1次，共3次。每次免疫前及免疫开始后至第8周取鼠血，用间接免疫荧光法检测小鼠血清中抗体生成情况；RT-PCR法检测重组质粒在小鼠肌肉中的mRNA表达。结果：pcDNA3／TCRγV1组小鼠血清中全部产生了特异性抗独特型抗体，抗体滴度在第4周开始增高，第6周时达到高峰(1：160)。在pcDNA3／TCRγV1组小鼠中用RT—PCR法检测到了重组质粒在肌肉中的mRNA表达。结论：TCRγV1独特型DNA疫苗可以诱导小鼠产生特异性抗淋巴瘤细胞独特型抗体。     

妇科

卵巢癌抗独特型微抗体疫苗诱导BALB／C小鼠体内抗肿瘤免疫应答的实验研究//Survivin反义寡核苷酸对人卵巢癌细胞SKOV3的抑制作用//RhoA、RhoC及其效应分子ROCK-1的表达与卵巢癌细胞系恶性行为相关性的体外研究//封闭ERCC1基因表达对卵巢癌细胞耐药的影响//卵巢上皮性癌组织中环氧合酶2的表达与治疗反应和预后的相关性研究     

卵巢癌抗独特型微抗体刺激树突细胞激活的T细胞对自体卵巢癌细胞杀伤作用的研究

背景与目的：以抗原肽、蛋白、肿瘤细胞冻融物致敏树突细胞的肿瘤免疫治疗已在多种肿瘤展开。6B11抗独特型微抗体能模拟卵巢癌相关抗原OC166-9,本研究用6B11抗独特型做抗体体外刺激树突细胞,以诱导出对卯巢癌患者自体肿瘤细胞抗原特异性的细胞毒作用。方法：取10例卵巢癌患者的外周血,分离并诱导培养树突细胞,在培养过程中用6B11抗独特型微抗体刺激,促成熟后在体外激活自体T细胞。^3H-TdR掺入法测DCs对自体T细胞刺激增殖的作用;^51Cr6h释放实验测激活的T细胞对自体肿瘤细胞特异性的杀伤。结果：9例中有4例(#4,#5,#7,#10)6B11微抗体负载组DC刺激自体T细胞增殖^3H-TdR掺入的cpm值高于对照组;杀伤实验的10例患者中5例(#1,#2,#4,#7,#9)6B11微抗体负载组DC刺激自体T细胞(简称MINI-DC-T)对自体肿瘤细胞有特异性杀伤。在效靶比为20：1时,各组不同的效应细胞对自体肿瘤细胞的杀伤率分别为：MINI-DC-T组25％～100％、F(ab)’2-DC-T组18％～40％、unpulsed-DC-T组13％～43％、T细胞组9％～58％,MINI-DC-T组高于其它各组。在效靶比为20：1时,4例(#l,#2,#4,#9)患者MINI-DC-T对自体肿瘤细胞及SKOV3、HLE、K562细胞的杀伤率分别为25％～100％、5％～51％、2％～38％和2％～25％。MINI-DC-T对自体肿瘤细胞的杀伤高于对照组靶细胞,并且这种杀伤可以部分地被anti-MHC-Ⅰ类抗体阻断。说明不同抗原负载DC诱导的CTL对自体肿瘤细胞的杀伤有抗原特异性。结论：6B11抗独特型微抗体可以模拟卵巢癌相关抗原,用它刺激的树突细胞在体外可以诱导出抗原特异性细胞毒T细胞。     

卵巢癌特异性免疫细胞治疗的体内外实验研究

背景与目的:卵巢癌抗独特型微抗体(6B11mini)是部分人源化的抗独特型卵巢癌疫苗,体内外实验研究证明,它可以诱导出特异的抗卵巢癌体液免疫和细胞免疫.本研究评价将6B11mini作为抗原,采用免疫细胞的方法处理卵巢癌时的安全性和有效性.方法:用纯化的6B11mini负载树突细胞(dendritic cell,DC),与外周血单个核细胞(peripheral blood mononucleocyte,PBMNC)混合培养,在多种细胞因子共同作用下获得效应细胞6B11-OCIK.通过软琼脂克隆形成实验及接种裸鼠皮下瘤实验,观察6B11-OCIK在体内和体外的成瘤能力;将6B11-OCIK静脉注射BALB/c小鼠,观察急性毒性反应:体外51Cr释放实验检测6B11-OCIK对靶细胞的杀伤作用.用人卵巢癌细胞株SKOV3构建严重联合免疫缺陷(severe combined immune deficieney,SCID)小鼠卵巢癌移植瘤模型,注射6B11-OCIK,并以CIK、PBMNC细胞以及生理盐水作为对照组,分别观察各组肿瘤生长情况.结果:软琼脂培养14 d,卵巢癌SKOV3细胞克隆形成良好,克隆形成率50%:皮下接种裸鼠后14 d,阳性对照的宫颈癌HeLa细胞组全部成瘤,6B11-OCIK、CIK、WI-38组和新鲜PBMNC组持续观察13周没有成瘤.6B11-OCIK静脉注射BALB/c小鼠,30 min内各剂量实验组和生理盐水对照组动物都无任何明显的不良反应:输注后第13天处死小鼠,解剖小鼠观察其各主要脏器无明显异常.在体外杀伤实验中6B11-OCIK对抗原阳性的肿瘤细胞具有特异性杀伤作用,并且与MHC限制性相关;在荷瘤SCID小鼠中6B11-OCIK治疗组肿瘤重量与生理盐水对照组相比差异有统计学意义(P=0.023),与CIK组、新鲜单采组相比差异无统计学意义(P=0.540,P=0.285).结论:6B11-OCIK在动物体内应用时符合安全性指标,并对卵巢癌的生长有一定的抑制作用.     

小细胞肺癌抗独特型疫苗功能的试验研究

目的探讨小细胞肺癌(SCLC)抗独特型抗体3F6和其单链抗体(3F6ScFv)诱导体液和细胞免疫应答的能力,以证明其作为抗SCLC疫苗的可行性。方法3F6和3F6ScFv(Ab2)免疫BALB/c小鼠获得抗血清,以ELISA和Westernblot方法分别检测抗血清中Ab3结合SCLC细胞膜表面特异抗原(NCI H128抗原)的能力,用竞争Westernblot检测Ab3与2F7竞争结合NCI H128抗原的能力。以迟发型超敏反应(DTH反应)和小鼠脾脏淋巴细胞增殖实验检测3F6及3F6ScFv诱发细胞免疫应答的潜能。结果ELISA和Westernblot方法均证明,3F6和3F6ScFv免疫同系小鼠所产生的Ab3能特异的与NCI H128抗原相结合,与对照血清相比,差异有统计学意义(P<0.001),且有很强的与2F7(Ab1)竞争结合靶抗原的能力。在DTH反应中,3F6和3F6ScFv所致小鼠足垫肿胀的程度均明显高于对照组(P<0.001)。小鼠脾脏淋巴细胞增殖反应试验证明,用3F6和3F6ScFv免疫的小鼠脾脏淋巴细胞对靶细胞的再次刺激有明显的增殖反应,与阴性肿瘤细胞对照组和阴性抗体对照组相比,差异有统计学意义(P<0.05)。结论抗独特型抗体3F6和3F6ScFv均有模拟SCLC细胞膜表面特异抗原的能力,成功诱导了相应的体液和细胞免疫应答,可作为抗SCLC疫苗进行深入研究。     

接种9724肝癌细胞后不同免疫力小鼠体内T细胞的作用

目的：探讨人类肝癌细胞系9724在不同免疫力小鼠中的生长特点及T细胞对癌细胞的排斥特点,方法：体外培养9724,接种到不同免疫力小鼠（正常BALB／c,B细胞缺陷的CBA／N,T细胞缺陷的BALB／c-nu,T细胞、B细胞缺陷的SICD小鼠及免疫重建的SCID小鼠）中,观察其生长特性;测定小鼠脾细胞杀伤力流式细胞仪测定外周血CD^ ,CD8^ 的百分率。结果：BALB／c小鼠和免疫重建的BALB／c-PBL-SCID不成瘤;CBA／N随接种数量和途径不同而有不同的表现;BALB／c-nu,SCID小鼠和免疫重建的CBA／N－PBL－SCID100％成瘤。接种过癌细胞的BALB／c和CBA／N小鼠脾细胞对癌细胞杀伤力较强,免疫重建的SCID杀伤力较小。不同组别CD4^ 百分率都下降,CD8^ 变化不大,CD4^ /CD8^ 比值下降,。结论：小鼠成癌率与T细胞的作用最为密切;T细胞在异种瘤移植排斥中起主要的特异性的免疫杀伤作用。     

妇科

D卵巢原发癌与转移癌中survivin mRNA和caspase-3 mRNA的临床研究,乙酰肝素酶基因转染SKOV3细胞对细胞增殖、粘附、侵袭能力的影响,Kilon基因在卵巢上皮性肿瘤中的表达状况的探讨,抗HER-2工程抗体chA21和Herceptin对人卵巢癌SKOV3裸小鼠移值瘤的抑制作用,ERK信号转导通路蛋白在上皮性卵巢肿瘤组织中的表达及与肿瘤恶性程度的相关性研究,内皮抑素基因联合重组干扰素-α蛋白对卵巢癌抑制作用的研究,卵巢癌6811抗独特型微抗体诱导抗肿瘤免疫应答的体外实验,端粒酶抑制剂对顺铂不同敏感性卵巢癌细胞株的影响,     

地方株HPV16E7基因疫苗诱发的小鼠免疫反应

目的评价地方株HPV16E7基因疫苗诱导的免疫反应。方法从先期构建的地方株HPV16E7基因重组质粒pGEM-T-E7经限制性核酸内切酶酶切,获得E7基因亚克隆到真核表达载体pcDNA3.1(-)中,构建地方株pcDNA-E7基因疫苗,转染CHO细胞,通过IFA试验验证E7蛋白的表达。用pcDNA-E7肌注方法免疫6周龄BALB/c小鼠,同时设pcDNA空载体为阴性对照,于第1天、第15天和第43天共免疫3次,用IFA和MTT法分析基因免疫在小鼠体内诱导的免疫应答。结果pcDNA-E7免疫小鼠后能检测出特异的抗体,而原载体免疫后不能产生特异抗体;但2组刺激淋巴细胞增殖的能力差异不显著。结论地方株HPV16E7基因疫苗可诱导产生小鼠免疫反应。     

Preparation of humanized ovarian carcinoma anti-idiotypic minibody

Murine anti-idiotypic monoclonal antibody (MAb) 6B11 mimicking the tumor-associated antigen OC166-9 is used as a vaccine for the induction of an anti-tumoral immunity in experiments of in vitro and in vivo animal model with ovarian carcinoma. In this article, we have humanized 6B11 anti-idiotypic minibody using overlap polymerase chain reaction (PCR) and DNA recombinant technique, prokaryotic expression vector was produced by genetic fusion of 6B11V(L)-V(H) to human IgG1 hinge and CH3 region. Transformed E. coli BL21(DE3) were propagated and induced by isopropyl-beta D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein band with molecular weight of 50kD appeared as the expected size after transformation. Molecular weight of 100 kDa may be examined by electrophoresis in nondenaturing systems. The fusion protein was analyzed with enzyme-linked immunosorbant assay (ELISA), inhibition ELISA tests and Western blot, respectively. The humanized anti-idiotype minibody showed capacity of bivalent binding to ovarian cancer MAb COC166-9 and goat anti-human immunoglobulin IgG1. It is useful reagents for clinical use.     

Adoptive chemoimmunotherapy of a moloney lymphoma

Adult BALB/c mice were inoculated with BALB/c Moloney lymphoma cells. On day 6, mice with palpable tumors received sublethal cyclophosphamide (CY) and BALB/c or DBA 2 spleen cells. All untreated mice died with tumor on days 8-12. Spleen cells alone had no effect. All mice treated with CY alone died with tumor by day 30, as did all mice treated with CY plus cells from non-immune donors or from DBA donors immunized against normal BALB/c tissue. By contrast, treatment with CY plus cells from BALB/c or DBA/2 donors immunized with antigenically-related murine sarcoma virus (Moloney) was moderately effective — 30 of 65 mice lived beyond day 60 and 14 of 65 beyond day 100. Lethal X-irradiation of the immune cells abolished their efficacy. Thus, an established antigenic lymphoma was inhibited or eradicated by a combination of chemotherapy and immunotherapy with spleen cells from syngeneic or H-2-compatible donors, but only if the cells were viable and were derived from donors pre-immunized against tumor-associated antigens. Finally, when lethal X-irradiation was substituted for sublethal CY, immunotherapy was not effective, probably because the tumor cells were far less sensitive to X-ray than to CY.     

Native anti-tumor responses elicited by immunization with a low dose of unmodified live tumor cells

The present study demonstrates that immunization with a low dose of unmodified live myeloma tumor cells (FO) elicited tumor-specific immunity. BALB/c mice were vaccinated with 10(4) live dendritic cells (DC)-FO fusion cells or 10(3) live FO cells. 80% of vaccinated mice survived from the later challenge with 1 x 10(6) FO cells, whereas all control mice developed tumors. Additionally, vaccination with live FO cells gave no protection against the growth of Lewis lung carcinoma cells in C57BL/6 mice. Cellular immunity was found to be primarily responsible for anti-tumor responses. In an adoptive immune model, the development of myeloma was greatly reduced by transfusion of lymphocytes but not sera from mice immunized with FO. T cells from immunized mice also induced lysis of FO cells in the cytotoxic T lymphocyte (CTL) assay. After co-culture with FO, IFN-gamma released from immunized T helper cells increased >10-fold, while IL-4 remained unchanged in comparison with control T cells. These findings provided the first evidence that immunization with a low dose of unmodified live FO cells was safe to mice and capable of eliciting specific protective immunity against tumor growth.     

Suppressor T cells in BCG-treated mice interfere with an in vivo specific antitumoral immune response

The interference by BCG in the induction and expression of a specific antitumoral immune reaction was studied in B6 mice, using the in vivo Winn assay and also active immunization. T cells immunized against MCA-induced fibrosarcoma (MC B6-1) transferred together with the tumour cells protected the syngeneic host against tumour take. Pretreatment of normal B6 mice with moderate or high doses of BCG prevented the development of a protective immune response after immunization. Moreover, a single dose of 1 mg, or 2 doses of 0.01 mg BCG, completely eliminated an established antitumour immunity. Suppressor cells are involved in the BCG-induced inhibitory effect; they interfered (1) with the expression of the antitumour response, since their addition to immune T cells in the Winn test resulted in decreased protection and (2) with the induction of the antitumour response, since injection of spleen cells from BCG-treated mice (BCG SpC) into normal mice before immunization inhibited the development of immunity. Treatment of BCG SpC with anti Thy 1.2 and anti Lyt 1.2 antibodies plus complement before injection into normal mice significantly decreased the suppressive activity, showing that the suppressor cells induced by BCG are T cells expressing the Lyt 1+ phenotype. The partial increase in protection obtained after IL-2 administration to BCG-treated mice suggests that the suppressive action of BCG SpC on the IL-2 producing capacity of helper T cells is only one of a number of possible mechanisms of T-cell-mediated suppression.     

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。     

bcr-abl和白介素-7共表达基因疫苗诱导小鼠免疫保护作用

目的探讨小鼠白介素-7（IL-7）基因对bcr-abl融合基因疫苗诱导机体免疫应答的影响。方法采用pVbcr-abl／mIL-7质粒免疫BALB／c小鼠,检测小鼠血清中bcr-abl特异性抗体水平。以转染bcr-abl融合基因片段的SF2／0细胞为靶细胞,用LDH释放实验检测免疫小鼠脾细胞特异性细胞毒活性。结果pVbcr-abl／mIL-7质粒能诱导小鼠产生血清特异性抗bcr-abl抗体,pVbcr-abl／mIL-7免疫组的血清特异性抗体滴度高于pVbcr-abl免疫组,脾细胞杀伤SP2／0／bcr-abl靶细胞的细胞毒活性明显增强。结论共表达bcr-abl和mIL-7的基因疫苗能在小鼠体内诱导较高的特异性体液免疫和细胞免疫水平,为慢性粒细胞白血病基因疫苗的临床前实验研究提供了依据。     

Vaccination with viable human umbilical vein endothelial cells prevents metastatic tumors by attack on tumor vasculature with both cellular and humoral immunity.

PURPOSE: Because tumor endothelium is rarely targeted by immunity but is critically important for tumor growth, the immunity against tumor endothelium is to be developed as a novel antitumor strategy. EXPERIMENTAL DESIGN: First, viable human umbilical vein endothelial cells (HUVEC) were immunized to C57BL/6 and BALB/c mice to evoke specific CTLs as well as antibodies against tumor endothelium. Lewis lung carcinoma or myeloma cells were subsequently inoculated to evaluate the effect on tumor growth by vaccination. Second, the effect on tumor metastasis by vaccination was studied using tumor-resected mice receiving HUVEC immunization 3 days after excision. Third, the immune sera and T lymphocytes from HUVEC-immunized mice were transferred to tumor-bearing mice and added to cultured HUVECs to investigate their antiproliferative effect. RESULTS: Viable HUVEC immunization showed potent antitumor effects in Lewis lung carcinoma and myeloma tumor models. Both immune sera and CTL inhibited tumor growth and specifically suppressed proliferation of HUVECs. Particularly, tumors entirely disappeared on day 90 after tumor inoculation in four of six tumor-bearing mice receiving CTL therapy. In a metastatic tumor model, we found that the HUVEC vaccination prolonged life span from 30.9 to 41.5 days after tumor resection compared with PBS-treated mice without apparent side effects. CONCLUSIONS: Vaccination with viable HUVECs evoked both humoral and cellular immunity against tumor microvasculature, and therefore significantly inhibited tumor growth and prolonged life span of tumor-resected mice. This may provide with a novel treatment for metastatic tumors. Moreover, we have established a convenient method to evoke specific CTL against tumor angiogenesis.