Development of novel antitumor immunogenetherapy using artificial cancer antigen-secreting tumors.

通过这个科研资助计划，我们检测了分泌型Survivin-Fc嵌合体蛋白诱导Survivin特异性CTL.1的能力。我们已经证明Survivin是肿瘤抗原，并且CD8T细胞识别Survivin衍生的多肽和人类白细胞抗原-A24 MHC-I类复合体。首先，我们将Survivin的编码基因与腺病毒E3衍生的信号序列和免疫球蛋白的Fe部分进行融合。将该基因导入K562细胞，建立了一对稳定的克隆(ssSVN-K562)。测定培养上清液中Survivin蛋白的分泌量。检测到的Survivin蛋白含量小于1/m l。将来自人类白细胞抗原A24阳性的肿瘤患者的CD8T细胞与自体树突状细胞和SSSVN-K562共培养。然而，Survivin特异性CTL不能诱导。我们推测，如此低的分泌量的Survivin不能刺激CTL。接下来，我们建立了肿瘤细胞克隆(CT-26和EG.7)，将Hsp72或Hsc73基因与Adeno E3来源的信号序列融合，构建了肿瘤细胞克隆。众所周知，HSP72和Hsc73与细胞内广泛的肿瘤抗原肽结合。肿瘤攻击实验表明，这些克隆具有免疫原性，部分肿瘤克隆被自发排斥。此外，用这些克隆免疫可产生针对亲本肿瘤株的强大的CTL反应。此外，放射治疗已建立的肿瘤的热休克蛋白分泌肿瘤显示肿瘤生长迟缓，并显示出生存的好处(提交稿件)。我们还证明了HSP72和HSC 73的分泌型结合了抗原肽，例如卵子衍生的多肽。提示将分泌型Hsp72或Hsc73基因导入肿瘤细胞可能是一种很有前途的免疫遗传治疗方法。

By this Grant-in-Aid for Scientific Research, we have examined the ability of secretary form of Survivin-Fc chimera protein for induction of Survivin- specific CTL.1. We have demonstrated that Survivin, inhibitor of apoptosis, is the tumor antigen and CD8+ T cells recognize the Survivin- derived peptide and HLA-A24 MHC class I complex.2. First, we have made the gene encoding Survivin fused with adeno E3-derived signal sequence and Fe portion of IgG. K562 cells were transfected with this gene and established couple of stable clones (ssSVN-K562).3. Quantity of secreted Survivin protein in the culture supernatant was measured. Less thanlong/ml of Survivin protein was detected.4. CD8+ T cells from HLA-A24-positive, tumor-bearing patients were cocultured with autologous dendritic cells and ssSVN-K562. However, Survivin-specific CTLs were unable to induce. We reasoned that such a low amount of secreted Survivin could not stimulate CTLs.5. Next, we have established the tumor cell clones (CT-26 and EG.7), which were transfected with Hsp72 or Hsc73 gene fused with adeno E3-derived signal sequence. It is well established that Hsp72 and Hsc73 bind broad array of tumor antigen peptide within the cells. Tumor challenge assay revealed that these clones become immunogenic and some tumor clones were rejected spontaneously. Furthermore, immunization with these clones elicits powerful CTL responses against parental tumor lines. In addition, treatment with irradiated Hsp secreting tumor for established tumors revealed the growth retardation of tumors and showed the survival benefits (manuscript in submitting). We also demonstrated that secretary form of Hsp72 and Hsc 73 binds an antigenic peptide, such as Ova-derived peptide.6. These data suggest that transfection with secretary form of Hsp72 or Hsc73 gene to tumor cells might bea promising immunogenetherapy.