Establishment of tumor immunotherapy using class II

在第一年，我们试图建立将重组hCIITA分子递送到癌细胞中的系统。我们首次使用HSV的VP 22标签作为候选递送子，VP 22干扰hCIITA的功能。因此，我们接下来使用来源于HIV的达特标签，并获得了Tat-hCIITA具有足够的功能来诱导癌细胞中的MHC抗原和纯化的重组Tat-hCIITA蛋白在培养基中移动到癌细胞中并可以使MHC诱导的事实。第二年，我们尝试建立了结核分枝杆菌α抗原的MHC单倍型匹配寡肽（Tb（k）和Tb（km）= I-A^k-单倍型匹配寡肽，Tb（B）= I-A^b-单倍型匹配寡肽）与hCIITA的共表达系统。我们构建了表达载体pZeo-hCIITA-HA-furin-Tb（k）-His、pZeo-hCIITA-HA-furin-Tb（km）-His和pZeo-hCIITA-HA-furin-Tb（B）-His，并转染入C1300细胞（源自A/J小鼠的小鼠神经母细胞瘤细胞系（I-A^k单倍型），MM 102（源自C3 H/HeN小鼠的小鼠乳腺癌细胞系（I-A^k单倍型）和刘易斯（源自C57 BL/6小鼠的小鼠肺癌细胞系（I-A^B单倍型））。我们建立了稳定表达hCIITA和Tb（B）的共表达质粒（Lewis-pZeo-hCIITA-HA-furin-Tb（B）-His和MM 102-pZeo-hCIITA-HA furin-Tb（B）-His），并证明Lewis-pZeo-hCIITA-HA furin-Tb（B）-His对荷瘤宿主产生了良好的肿瘤免疫。

In the first year, we tried to establish the system to delivery the recombinant hCIITA molecules into cancer cells. We first used VP22 tag derived from HSV as a candidate of deliverers, the VP22 interfered the function of hCIITA. Thus, we next used Tat tag derived from HIV and obtained the facts that Tat-hCIITA has enough function to induce MHC antigens in cancer cells and purified recombinant Tat-hCIITA protein in the culture media move into cancer cells and could make MHC induce. However, it was impossible to get enough amounts of recombinant Tat-hCIITA protein, since the protein formed inclusion body in E.coli.In the second year, we tried to establish the co-expression system of hCIITA and MHC haplotype-matched antigenicoligopeptides (Tb (k) and Tb (km) = I-A^k-haplotype matched oligopeptides, Tb (b) = I-A^b-haplotype matched oligopeptides) derived from Mycobacterium tuberculosis alpha antigen. We constructed the expression vectors, pZeo-hCIITA-HA-furin-Tb (k) -His, pZeo-hCIITA-HA furin-Tb (km) -His, and pZeo-hCIITA-HA-furin-Tb (b)-His and transfected into he C1300 (mouse neuroblastoma cell line derived from A/J mouse (I-A^k haplotype), MM102 (mouse breast cancer cell line derived from C3H/HeN mouse (I-A^k haptotype), and Lewis (mouse lung cancer cell line derived from C57BL/6 mouse (I-A^b haplotype). We have established stably hCIITA-and Tb (b)-coexpressed transfectants (Lewis-pZeo-hCIITA-HA-furin-Tb(b)-His and MM102-pZeo-hCIITA-HA furin-Tb (b)-His) and got the evidence that Lewis-pZeo-hCIITA-HA furin-Tb (b)-His generates well tumor immunity against tumor-laden hosts.