Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4 T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction.

The breaking of immune tolerance of "selfantigens" associated with angiogenesis is an attractive approach to cancer therapy by active immunity. We used vascular endothelial growth factor receptor-2 (VEGFR-2) as a model antigen to explore the feasibility of the immunotherapy with a vaccine based on a xenogeneic homologous protein. To test this concept, we prepared a quail homologous VEGFR-2 protein vaccine (qVEGFR) based on quail VEGFR-2. At the same time, a protein vaccine based on the corresponding ligand-binding domain of mouse self-VEGFR-2 (mVEGFR) was also prepared and used as a control. We found that immunotherapy with qVEGFR was effective at protective and therapeutic antitumor immunity in several solid and hematopoietic tumor models in mice. Autoantibodies against mouse VEGFR-2 (Flk-1) were identified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Anti-VEGFR antibodyproducing B cells were detectable by ELISPOT. Endothelial deposition of immunoglobulins developed within tumor. VEGF-mediated endothelial cell proliferation was inhibited in vitro by immunoglobulins from qVEGFR-immunized mice. Antitumor activity was caused by the adoptive transfer of the purified immunoglobulins. Antitumor activity and production of autoantibodies against Flk-1 could be abrogated by the depletion of CD4 T lymphocytes. Angiogenesis was apparently inhibited within the tumors, and the vascularization of alginate beads was also reduced. No marked toxicity was found in the immunized mice. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factor receptor associated with angiogenesis in a crossreaction with single xenogeneic homologous protein.

Purpose: CXC chemokine ligand 10 (CXCL10) is a potent inhibitor of angiogenesis.We wonder whether the combination of CXCL10 with cisplatin would improve the therapeutic antitumor efficacy. Experiment Design:We evaluated the antitumor activity of the combination therapy in the immunocompetent C57BL/6 and BALB/c mice bearing LL/2 Lewis lung cancer and CT26 colon adenocarcinoma, respectively. Mice were treated with either CXCL10 s.c. at 25Mg per kg per day once daily for 30 days, cisplatin cycled twice (5 mg/kg i.p. on days 14 and 21 after the initiation of CXCL10), or both agents together. Tumor volume and survival time were observed. Antiangiogenesis of CXCL10 in vivo were determined by alginate capsule models and CD31 immunohistochemistry. Histologic analysis and assessment of apoptotic cells were also conducted in tumor tissues. Results: CXCL10 + cisplatin reduced tumor growthin LL/2 and CT26 tumormodel, respectively, more effectively, although cisplatin or CXCL10 individually resulted in suppression of tumor growth and improved survival time of tumor-bearing mice. CXCL10 successfully inhibited angiogenesis as assessed by alginate model and CD31 (P<0.05). Histologic analysis of tumors exhibited that CXCL10 in combination with cisplatin led to the increased rate of apoptosis, tumor necrosis, and elevated lymphocyte infiltration. Conclusions: Our data suggest that the combination of CXCL10, a well-tolerated angiogenesis inhibitor, with cisplatin can enhance the antitumor activity.The present findings may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung and colon carcinoma.

The breaking of immune tolerance against self epidermal growth factor receptor (EGFr) should be a useful approach for the treatment of receptor-positive tumors with active immunization. To test this concept, we constructed a plasmid DNA encoding extracellular domain of xenogeneic (human) EGFr (hEe-p) or corresponding control mouse EGFr (mEe-p) and empty vector (c-p). Mice immunized with hEe-p showed both protective and therapeutic antitumor activity against EGFr-positive tumor. Sera isolated from the hEe-p-immunized mice exhibited positive staining for EGFr-ositive tumor cells in flow cytometric analysis and recognized a single 170-kDa band in Western blot analysis. Ig subclasses responded to rEGFr proteins were elevated in IgG1, Ig2a, and Ig2b. There was the deposition of IgG on the tumor cells. Adoptive transfer of the purified Igs showed the antitumor activity. The increased killing activity of CTL against EGFr-positive tumor cells could be blocked by anti-CD8 or anti-MHC class I mAb. In vivo depletion of CD4 T lymphocytes could completely abrogate the antitumor activity, whereas the depletion of CD8 cells showed partial abrogation. The adoptive transfer of CD4-depleted (CD8) or CD8-depleted (CD4) T lymphocytes isolated from mice immunized with hEe-p vaccine showed the antitumor activity. In addition, the increase in level of both IFN-and IL-4 was found. Taken together, these findings may provide a new vaccine strategy for the treatment of EGFr-positive tumors through the induction of the autoimmune response against EGFr in a cross-reaction between the xenogeneic homologous and self EGFr. The Journal of Immunology, 2003, 170: 3162-3170.

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors and is the second most common cause of cancer death in China. Therefore, it is very important to detect this disease and the recurrence at its earlier period. Serum tumor markers, as the effective method for detecting hepatocellular carcinoma for a long time, could be divided into 4 categories: oncofetal antigens and glycoprotein antigens; enzymes and isoenzymes; genes; and cytokines. Serum alpha fetoprotein (AFP) is the most widely used tumor marker in detecting patients with hepatocellular carcinoma, and has been proven to have capability of prefiguring the prognosis. However, it has been indicated that AFP-L3 and DCP excel AFP in differentiating hepatocellular carcinoma from nonmalignant hepatopathy and detecting small hepatocellular carcinoma. Some tumor markers, such as human cervical cancer oncogene and human telomerase reverse transcriptase mRNA, have also been indicated to have higher accuracies than AFP. Furthermore, some other tumor markers, such as glypican-3, gamma-glutamyl transferase II, alpha-lfucosidase, transforming growth factor-beta1, tumorspecific growth factor, have been indicated to be available supplementaries to AFP in the detection. AFP mRNA has been shown to correlate with the metastasis and recurrence of HCC, and it may be the most useful marker to prefi gure the prognosis. Some other markers, such as gamma-glutamyl transferase mRNA, vascular endothelial growth factor, and interleukin-8, could also be used as available prognostic indicators, and the simultaneous determination of AFP and these markers may detect the recurrence of HCC at its earlier period.

本文综述了有关癌蛋白免疫原性及分子疫苗研究新进展。恶性肿瘤形成是癌蛋白表达的结果，癌蛋白具有免疫、原性经、细胞抗原受体认别后可激发机体特异性免疫反应；p53、MARGE-1突变基因编码癌蛋白，其抗原肽分子疫苗可诱导特异性CTL产生发挥抗癌作用；DPC4新的抑癌基因作为TGF-β信号旁路的一部分参与细胞增殖与生长。分子疫苗主动免疫治疗结合回输体外扩增的CTL是一种增强机体抗癌效应的新方法。

目的：研究回输自体PHA LAK细胞对恶性实体瘤患者淋巴细胞表型及功能状态的影响。方法：入组204例实体瘤患者，并以10例健康成人作为对照，采集与分离外周血单个核细胞，体外扩增PHA LAK细胞，采用流式细胞术分析淋巴细胞表型，MTT法检测淋巴细胞杀伤活性。结果：实体瘤患者较正常人淋巴细胞亚群CD1+/CD4+细胞、CD2+/CD2+细胞、B细胞、NK细胞减少，淋巴细胞杀伤活性下降。扩增的PHA LAK是以激活的淋巴细胞亚群CD3+/CD2+口胞、CD3+/CD56+细胞、CD4+/CD28+细胞为主的异质性群体，其数量及杀伤活性明显增加，激活淋巴细胞标记性抗原分子表达亦增加。PHA LAK细胞回输治疗后，患者外周血上述激活淋巴细胞亚群数量增加，淋巴细胞杀伤活性增强。结论：实体瘤患者淋巴细胞亚群数量及功能低下，PHA LAK细胞过继性免疫治疗使患者体内激活淋巴细胞亚群数量增加，杀伤活性增强。

融合细胞株Eahy926是人肺腺癌细胞株A549和人脐静脉内皮细胞杂交而成的永生化细胞株，具有血管内皮细胞的特性，已广泛用于内皮细胞相关研究。本研究应用蛋白质组学技术分析融合细胞株Eahv926与亲本人肺腺癌细胞株A549的蛋白质差异表达，探讨融合细胞生物学特性变化及其机制。对Eahy926和亲本A549的细胞总蛋白质进行双向凝胶电泳，在PDQuest。软件辅助下找出差异表达蛋白质点，经肽质量指纹图谱（peptide mass fingerprinting，PMF）和串级质谱（tandem mass spectrometry，TMS）分析，SWISS-PROT数据库检索，成功鉴定出28个差异蛋白，如CATB、CK8、CK18、annexin A2、GRP78、HsP90、HSP60、vimentin等一些与分子伴侣、氧化应激、能量代谢、信号转导等有关，并与肿瘤细胞分裂增殖、分化凋亡、侵袭转移、免疫逃逸以及肿瘤血管生长密切关联的蛋白质。研究发现，融合细胞株Eahy926和人肺腺癌细胞株A549的蛋白质组表达谱存在明显差异，这将有助于今后进一步探讨肿瘤细胞与内皮细胞的相互作用机制及其融合细胞的特性，筛选肿瘤增殖和转移相关蛋白质及分子标志物，亦可为肿瘤的抗血管生成治疗提供新思路。