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

通过这项科学研究资助，我们研究了Survivin- fc嵌合体蛋白秘书形式诱导Survivin特异性ctl - 1的能力。我们已经证明，凋亡抑制剂Survivin是肿瘤抗原，CD8+ T细胞识别Survivin衍生肽和HLA-A24 MHC I类复合物。首先，我们将编码Survivin的基因与腺e3衍生的信号序列和IgG的Fe部分融合。用该基因转染K562细胞，建立了两个稳定克隆（ssSVN-K562）。测定培养上清液中Survivin蛋白分泌量。Survivin蛋白含量小于长/ml。来自hla - a24阳性荷瘤患者的CD8+ T细胞与自体树突状细胞和ssSVN-K562共培养。然而，survivin特异性ctl无法诱导。我们推断，如此低的Survivin分泌量不能刺激ctl。接下来，我们建立了肿瘤细胞克隆（CT-26和EG.7），将Hsp72或Hsc73基因与腺e3衍生的信号序列融合转染。已经证实Hsp72和Hsc73在细胞内结合多种肿瘤抗原肽。肿瘤激发试验显示这些克隆具有免疫原性，部分肿瘤克隆自发排斥。此外，用这些克隆免疫可引起针对亲本肿瘤系的强大CTL反应。此外，用放射热休克蛋白分泌肿瘤治疗已建立的肿瘤显示出肿瘤生长迟缓，并显示出生存益处（已提交手稿）。我们还证明Hsp72和Hsc 73的秘书形式结合抗原肽，如ova衍生肽。这些数据表明，将秘书型Hsp72或Hsc73基因转染肿瘤细胞可能是一种有希望的免疫遗传学治疗方法。

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.