柔红霉素连接单克隆抗体用于胃癌导向治疗的实验研究

本文采用直接和间接偶连方法将柔红霉素成功连接于胃癌单克隆抗体MGb_2,每一抗体所载的药物分子分别为6个(直接法)和54个(间接法)。ELISA证实所制备结合物中MGb_2活性保存较好,且能与胃癌靶细胞特异地结合。噻唑蓝(MTT)比色法体外细胞毒试验表明结合物对胃癌细胞SGC—7901具有选择性杀伤作用,而对非靶细胞毒性很弱,提示柔红霉素对胃癌细胞杀伤作用是由单抗MGb_2介导的。     

Tumor localization of anti-gastric cancer monoclonal antibody MGb_2-daunomycin conjugate in nude mice bearing human gastric carcinoma SGC-7901

In the present study,an anti-gastric cancer monoclonal antibody MGb_2-daunomycin(DM)conjugate was prepared by cis-aconitic anhydride method.The distribution of the conjugatein nude mice bearing human gastric carcinoma SGC-7901 was investigated.Four to five moleculesof DM were introduced into each molecule of antibody while the antigen-binding capacity ofthe antibody was well-retained.This conjugate could be satisfactorily localized in the tumortissue.Ninety-six hours after intraperitoneal injection of ~(125)I-MGb_2-DM conjugate,the ratio ofradioactivity per milligram of tumor to that per milligram of blood reached 203,the result beingquite similar to that of unmodified MGb_2.     

Targeted anti-tumor agents and their cytotoxicity on gastric cancer cells in vitro.

Several chemotherapeutic drugs including methotrexate, daunomycin, mitomycin C and toxin such as ricin, A chain of ricin were chosen to conjugate with murine monoclonal antibodies MGb2 and MG11. The drugs were linked to antibody directly or through human serum albumin as intermediary. The ELISA results showed that the antibody retained well the antigen-binding capacity during conjugation. These conjugates showed highly selective cytotoxic effect on target cells. In chronic cytotoxicity tests, the cytotoxic effect of these conjugates on human gastric cancer cells KATOIII was quite similar to that of free drugs or ricin, but greater than that of irrelevant conjugates, while they had a little effect on non-target cells, suggesting that the selective cytotoxicity on target cells of the conjugates be mediated by antibodies.

     

单克隆抗体连接表阿霉素导向治疗胃癌的实验研究

作者首次报道了胃癌单克隆抗体MGb_2-表阿霉素结合物的制备及其对胃癌细胞的体外杀伤作用。首先用高碘酸钠氧化葡聚糖,使其成为多醛基葡聚糖,并以此为中间载体将表阿霉素与MGb_2连接。经测定,每一抗体能携带45个药物分子,且其抗体活性保存良好。该结合物对胃癌细胞SGC-7901杀伤作用优于游离药物及无关抗体结合物,且对非靶细胞(Hep-2)毒性作用很弱。说明表阿霉素-葡聚糖-MGb_2对胃癌细胞具有较强选择性杀伤作用,并可能成为胃癌导向治疗的免疫抗癌药物。     

人类肿瘤相关蛋白hyrdC的原核表达、抗体制备及其在胃癌中的初步检测

目的:制备原核表达的hyrdC纯化蛋白及相应单克隆抗体,并以该抗体初步检测胃癌中hyrdC蛋白的表达.方法:用RT-PCR方法获取人hyrdC基因序列,插入原核表达载体pET32a构建重组质粒,纯化得到TRX-hyrdC融合蛋白,将其作为抗原免疫BALB/c小鼠,按常规方法制备抗hyrdC单克隆抗体,以ELISA、Western印迹法筛选及鉴定;采用免疫组织化学法(SP法)比较胃癌组织及瘤旁正常组织中hyrdC抗体水平的差异.结果:测序结果显示克隆的hyrdC ORF与GenBank中登录的序列一致.所获TRX-hyrdC融合蛋白相对分子质量与理论计算值相符,获得2株分泌抗hyrdC单克隆抗体的细胞株;Western印迹法显示该抗体能特异性地结合人hyrdC蛋白;SP法测定显示,hyrdC蛋白在胃癌组织中表达明显高于正常胃组织(P＜0.001).结论:成功制备了抗hyrdC单克隆抗体;SP法测定胃癌组织与瘤旁正常组织中hyrdC抗体水平有显著差异.     

~(125)I-MGB_2-DM在荷胃癌裸鼠体内的定位

作者用碘~(125)标记由顺-乌头酸酐法制备的胃癌单抗(MGB_2)-柔红霉素(DM)结合物,观察其在荷人胃癌SGC-7901裸鼠体内分布状态.在MGB_2—DM中,每个抗体分子引入4～5个分子DM,抗体活性保存良好.结果表明,~(125)Ⅰ—MGB_2—DM能较满意地定位于肿瘤组织,腹腔给予~(125)Ⅰ-MGB_2 DM 96h,瘤/血比值达203,其结果与未修饰MGB_2相近.     

Preparation and cytotoxicity of McAb-HSA-MTX conjugates

In the present study, methotrexate (MTX), was conjugated with a murine monoclonal antibody (79) to human common acute lymphoblastic leukemia antigen (CALLA), with human serum albumin (HSA) as an intermediary. The highest molar ratio of McAb 79:HSA:MTX in the conjugates was 1:2. 63:117. The conjugates obtained retained both antibody binding and drug activities. Although there was some loss of drug activity in binding to antibody, the toxicity of McAb79-HSA-MTX was entirely specific for the target cell, and the cytotoxicity of McAb79-HSA-MTX against CALLA+ cells was greater than that of control S13 (Anti-human urokinase)-HSA-MTX. The ratio of 79-HSA-MTX cytotoxicity to the target and non-target cells was 66:1, whereas there was no cytotoxicity to target cells when McAb79 was used only. There was no cytotoxic difference between 79-HSA-MTX and S13-HSA-MTX against CALLA- SB cells. These results suggest that the cytotoxicity of 79-HSA-MTX against CALLA leukemia cells is specific and this specificity is mediated by McAb79.     

Endocytosis of immuno-ricin A chain specific for gastric cancer and its implications in antibody targeting therapy

In the present study,ricin A chain(RTA)was first biotinylated,and then chemicallyintroduced to a monoclonal antibody against gastric cancer,MGb_2 to generate an immuno-ricin Achain conjugate(I-RTA)specific for gastric cancer.Subsequently,its endocytosis in human gas-tric cancer cell line SGC-7901 was investigated by double dynamic EM labeling technique utilizingstreptavidin-gold and sheep anti-mouse IgG-gold probes.In addition,the effects of verapamil onI-RTA endocytosis process were also observed.Our findings demonstrated that the main entryroute of I-RTA was non-coated microinvagination,followed by coated pits and interiorization ofmicrovilli.Intracellularly,the endocytosed I-RTA was quickly transported from tubulovesicularstrueutres to multivesicular bodies,and finally to lysosomes where it was degraded,while in thenon-membranous structures I-RTA was scanty.In the presence of verapamil,however,I-RTAwas found to stay longer in tubulovesicular structures and to move in a less amount and moreslowly into lysosomes.In the meantime,I-RTA was seen to be increased in the non-membranousstructures of the cytosol,and the in vitro killing efficacy was greatly augmented.     

ANTITUMOR EFFECTS OF MONOCLONAL ANTIBODY FAB’FRAGMENT—CONTAINING IMMUNOCONJUGATES

Objective.Using monoclonal antibody(mAb)Fab‘fragment to develop mAb immunoconjugates for cancer.Methods.Fab‘ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol(DTT),while Fab‘ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β-mercaptoethanol.The conjugation between Fab‘ fragment and pingyangmycin(PYM),an antitumor antibiotic,was mediated by dextran T-40.Immunoreactivity of Fab‘-PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clono-genic assay.Antitumor effects of the Fab‘-PYM conjugates were evaluated by subcutaneously transplanted tumors in mice.Results.The molecular weight of Fab‘ fragment was approximately 53 kD,while the average molecular weight of Fab‘-PYM conjugate was 170kD.The Fab‘-PYM conjugates showed immunoreactivity with anti-gen-relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab‘-PYM conju-gates were more effective aganist the growth of tumors in mice than free PYM and PYM conjugated with intact mAb.Conclusion.Fab‘-PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.     

与人胃癌反应的单克隆抗体GL—013

应用B-细胞杂交瘤技术,以胃癌转移淋巴结中的癌细胞为免疫原,经ABC-免疫组织化学方法筛选,研制出一种鼠单克隆抗体GL-013。免疫亲和层析纯化抗原,Western Blot鉴定抗原分子量约大于200kd的糖蛋白,抗原对1%过碘酸处理敏感,与CEA、NCA和血型前体Lewisy抗原不相关。免疫组化研究表明抗体与胃癌有较高的特异性,抗原的表达与转移相关。     

131I-CD44v6胃癌单抗在荷瘤裸鼠活体内的放射免疫显像

目的：探讨CD44v6单抗与胃癌转移模型的结合能力，了解其应用于胃转移灶定位诊断和异向治疗的可能性。方法：利用荷GC803胃癌细胞株的裸鼠胃癌转移模型腹腔内注射5．5MBq^131I-CD44v6单抗及对照组注入5．5MBq^131I-MMIgG(即正常IgG)后24，48，96h分别作肿瘤放射免疫显像，并测定肿瘤与肿瘤对称部位感兴趣区的放射性计数比值T／NT。结果：发现实验组96h后能清晰显像，且两组之间T／NT差异有显著性（P＜0．05）。结论：CD46v6单抗可与胃癌转移灶特异性结合，未来可作为胃癌转移灶放射免疫显像诊断及治疗的导向载体。     

ANTITUMOR ACTIVITY OF IMMUNOCONJUGATES COMPOSED OF BOANMYCIN AND MONOCLONAL ANTIBODY

ＡＮＴＩＴＵＭＯＲＡＣＴＩＶＩＴＹＯＦＩＭＭＵＮＯＣＯＮＪＵＧＡＴＥＳＣＯＭＰＯＳＥＤＯＦＢＯＡＮＭＹＣＩＮＡＮＤＭＯＮＯＣＬＯＮＡＬＡＮＴＩＢＯＤＹ￥ＺｈｅｎＹｏｎｇｓｕ；（甄永苏）；ＰｅｎｇＺｅ（彭泽）；ＤｅｎｇＹｏｎｇｃｈｕａｎ；（邓甬川）Ｘｕ...     

Anti-FD4 idiotypic antibody mimicking human gastric cancer-associated antigen.

Rabbits were immunized with the Fab fragment of a murine monoclonal antibody (McAb) PD4 against human gastric cancer to produce anti-PD4-idiotypic antibody (alpha PD4-Ab2). The alpha PD4-Ab2 could not only competitively inhibit binding of McAb PD4 to gastric cancer cell MGC803, but also induce delayed-type hypersensitivity (DTH) to MGC803 in mice. Spleen cells of mice immunized with alpha PD4-Ab2 were fused with myeloma cell SP2/0 to form hybridoma secreting Ab3 which could be bound to target cell MGC803. McAb C7-Ab3, one of the Ab3, could selectively react with a 40 kD tumor-associated antigen located on MGC803 cell membrane, as well as McAb PD4. The results indicate that alpha PD4-Ab2 possesses determinants (internal image antigen) similar to those on MGC803, and could mimic human gastric cancer-associated antigen.

     

Boron neutron capture therapy of human gastric cancer by boron-containing immunoliposomes under thermal neutron irradiation]

Boron neutron capture therapy (BNCT) is based on the nuclear reaction yielding high LET Li-7 and alpha particles when boron-10 is irradiated with thermal neutrons. (Et4N)2(10)B10H10 was entrapped in 40 nm liposomes coating the monoclonal antibody, MGb 2, against human gastric cancer. There were 1.4 x 10(4) 10B atoms encapsulated and 20 molecules of MGb 2 incorporated per liposomes ELISA indicated that the immunoreactivity of antibodies on liposomes retained 80%. Preferred binding to human gastric cancer cell line SGC-7901 was observed as many as 15.1 x 10(9) 10B atoms/tumor cell, 38-fold more than that to normal human embryonic lung cell line SL 7. The fluorescent immunoliposome-stained tumor cells showed membrane-fluorescence while SL 7 cells showed no obvious fluorescence. Irradiated with thermal neutrons (0.025 eV, 3.12 x 10(11)n/cm2, gamma-ray 0.84 Gy), 10B-containing immunoliposomes pretreated SGC-7901 cells survived 27%, significantly lower than non-irradiated cells or non-pretreated cells with irradiation (P less than 0.001). The results demonstrated that boron-containing immunoliposomes could bind selectively and deliver sufficient amount of boron-10 to the target tumor cells.     

胃癌单克隆抗体8711C1对人胃癌定位作用的实验研究

用^125I标记胃癌单克隆抗体8711C1在人胃癌小鼠体内进行肿瘤定位实验。结果显示胃癌单克隆抗体8711C1能特异性浓聚于小鼠胃癌组织，使肿瘤组织在体外显影，表明该抗体具有良好的活体内定位作用。     

鼠源性单克隆抗体Met4用于检测胃癌组织Met的表达及临床意义

目的:观察研究肝细胞生长因子受体Met在胃癌组织及相对应的淋巴结转移灶中的表达水平,同时比较分析抗Met单克隆抗体Met4和多克隆抗体C28在检测Met表达水平中的差别?方法:采用免疫组织化学间接法检测胃癌组织芯片中Met表达水平,比较Met4与C28的检测效能?结果:Met在细胞膜及胞浆阳性表达率在不同病理分级组间无统计学意义(P > 0.05)?Met4组Met的阳性染色部位于细胞膜(浆),而C28组Met阳性染色部位在细胞浆中,二者在Met亚细胞染色定位上有差别?Met4的阳性检出率(58.3%)高于C28阳性检出率(30.5%),具有统计学意义(P < 0.05)?Met4组21例胃癌原发灶Met阳性病例中14例淋巴结转移灶Met表达阳性,C28组11例原发灶Met阳性病例中6例淋巴结转移灶Met表达阳性?原发灶中Met阳性表达与转移淋巴结中Met的阳性表达有显著相关性(P < 0.05)?结论:Met阳性表达可能在肿瘤的淋巴转移过程起重要作用?Met4用于肿瘤的诊断及靶向治疗,具有良好的临床应用前景?     

Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA

Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdrl) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdrl cDNA by electroporation. RT-PCR was used to detect mdrl mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method. Results mdrl mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21%-37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3-45.8 times, and that to colchicines to 6.7-11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered. Conclusions CIK cells were successfully transfected with mdrl cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.     

胃癌组织中基质金属蛋白酶-2和组织基质金属蛋白酶抑制剂-2的表达及其与临床病理特征的关系

目的 探讨基质金属蛋白酶-2(MMP-2)和组织基质金属蛋白酶抑制剂-2(TIMP-2)在人胃癌组织中的表达,及其与胃癌临床病理特征的关系.方法 选取100个胃癌手术切除标本中胃癌组织、癌旁组织及正常胃组织,采用免疫组织化学法检测MMP-2和TIMP-2蛋白的表达,并分析两者的表达及其与胃癌临床病理特征之间的相关性.结果 胃癌组织中的MMP-2蛋白阳性率为77.0%,显著高于癌旁组织的33.0%和正常胃组织的8.0%(P值均＜0.01),癌旁组织又显著高于正常胃组织(P＜0.01).胃癌组织中的TIMP-2蛋白阳性率为29.0%,显著低于癌旁组织的62.0%和正常胃组织的76.0%(P值均＜0.01),癌旁组织又显著低于正常胃组织(P＜0.01).随着肿瘤浸润深度的增加(Tis～T4)、周围淋巴结转移的增多(N0～N3)、临床分期的进展(0～Ⅳ期),MMP-2蛋白阳性率逐渐升高,TIMP-2蛋白阳性率逐渐降低,且差异有统计学意义(P值均＜0.01).结论 MMP-2和TIMP-2的表达可能与胃癌的发生、侵袭和转移有关,检测MMP-2和TIMP-2的表达有助于预测胃癌转移.     

趋化因子基质细胞衍生因子-1及其受体对胃癌腹膜转移潜能的影响

目的 探讨趋化因子基质细胞衍生因子-1(SDF-1)及趋化因子受体4(CXCR4)对胃癌腹膜转移潜能的影响.方法 采用逆转录多聚酶链式反应(RT-PCR)检测胃癌细胞NUGC4及间皮细胞HMrSV中CXCR4和SDF-1 mRNA的表达.采用MTT实验检测NUGC4细胞增殖能力的变化,采用间皮细胞黏附实验及间皮细胞迁移实验评价体外培养的NUGC4细胞与间皮细胞发生黏附的能力及其穿越间皮细胞发生迁移的能力.通过建立裸鼠胃癌腹膜种植瘤模型,评价NUGCA细胞腹膜肿瘤生成能力及荷瘤裸鼠生存时间的变化.结果 NUGC4细胞表达较高强度的CXCR4mRNA,而间皮细胞表达高强度的SDF-1 mRNA.抗CXCR4单抗对NUGC4细胞增殖具有显著的抑制作用(P＜0.05);SDF-1可促进NUGC4细胞与间皮细胞黏附及穿越间皮细胞发生迁移,抗CXCR4单抗处理可显著抑制NUGC4细胞的黏附及迁移能力(P＜0.05).体内实验结果显示,CXCR4拮抗剂AMD3100治疗组裸鼠的平均生存时间显著长于对照组[(43.8±2.8)vs(28.2±2.5)d,P＜0.01],瘤结节数目显著低于对照组[(64.6±8.2)vs(103±12.4),P＜0.01].结论 趋化因子SDF-1及其受体CXCR4参与胃癌腹膜转移过程,与肿瘤细胞增殖、肿瘤细胞-问皮细胞问的黏附和肿瘤细胞迁移等步骤密切相关;干扰SDF-1/CXCR4生物学轴可以作为胃癌腹膜转移的潜在治疗策略.     

细胞毒T淋巴细胞杀伤胃癌细胞的超微结构观察

目的 研究特异性细胞毒Ｔ淋巴细胞杀伤胃癌细胞ＢＧＣ－８２３的超微结构变化。方法 用２２例患者的胃腺癌细胞反复冻融后的滤液,抗ＣＤ３单克隆抗体、白细胞介素－２（ＩＬ－２）患者自体外周血淋巴细胞共同培养,体外诱导ＣＴＬ,然后将ＣＴＬ与ＢＧＣ－８２３共同孵育,用电子显微镜观察ＣＴＬ作用于ＢＧＣ－８２３后的超微结构改变。结果 电镜观察可见ＣＴＬ包围并破坏靶细胞,靶细胞凋亡的形态表现为异染色质浓缩呈特征性半