Li Wei, Cui Heng, Chang Xiao-Hong, Cheng Hong-Yan, Cheng Ye-Xia, Feng Jie, Fu Tian-Yun
Department of Gynecologic Oncology Center, Peking University People's Hospital, Beijing 100044, China.
Zhonghua Fu Chan Ke Za Zhi. 2008 Oct;43(10):764-9.
To identify the T cell epitopes from ovarian cancer associated anti-idiotypic antibody 6B11 in order to explore the molecular basis of 6B11 induced cellular immune responses against ovarian cancer.
Potential human leukocyte antigen (HLA) A0201 ligands were predicted by using SYFPEITHI algorithm and tested by the T2 binding assay for screening of HLA-A2 binding peptides from 6B11 complimentary determining region (CDR). Cytotoxic T lymphocytes (CTL) to 6B11 or peptides were generated by 3 rounds of in vitro stimulation with 6B11 or peptide-pulsed dendritic cells (DC), and then tested by (51)Cr-release assay to ascertain the CTL epitope of 6B11. Cell proliferation assay was performed by using 6B11 specific CTL as responder cells and peptide-pulsed DC as stimulators to ascertain the helper T lymphocyte (Th) epitope of 6B11. Cytokine assay and interferon-gamma ELISPOT assay were performed to verify the CTL and Th epitope of 6B11 further.
Light chain CDR3 peptide (VL CDR3) of 6B11 induced specific CTL to kill HLA-A2 and target antigen positive ovarian cancer cells, which could be blocked by anti human major histocompatibility complex (MHC) I antibody. Heavy chain CDR3 peptide (VH CDR3) of 6B11 stimulated the proliferation of 6B11-primed CTL, which could be blocked mainly by anti human MHC-II antibody, and further experiments showed that part of the VH CDR3 peptide-primed CTL killed K562 cells. Peptides in VL CDR3 and VH CDR3 were the CTL and Th epitope mimicking the original antigen of 6B11 respectively. Collaboration of 6B11 CTL and Th epitope, or 6B11 CTL epitope and keyhole limpet hemocyanin (KLH), the former was more powerful in inducing specific cellular immune responses against ovarian cancer. 6B11 or corresponding CTL and Th epitope specific CTL secreted high levels of interleukin-2 (1630, 1503 ng/L) and interferon-gamma (5620, 5421 ng/L), while basal level of interleukin-4 was detected (253, 274 ng/L). ELISPOT assay confirmed the existence of specific interferon-gamma secreting cells in 6B11 or epitopes specific CTL (196/1 x 10(6) T cell, 184/1 x 10(6) T cell).
VL CDR3 and VH CDR3 peptides of 6B11 are the CTL and Th epitopes mimicking original antigen which could duplicate the activity of intact 6B11 to induce the cellular immune responses against ovarian cancer. The results have significant theoretical and practical value in application of anti-idiotypic antibody as anti tumor vaccine. The acquired CTL and Th epitopes as constituents of future multiple peptides against ovarian cancer could be used in peptide vaccine based ovarian cancer therapy.
鉴定卵巢癌相关抗独特型抗体6B11的T细胞表位，以探讨6B11诱导针对卵巢癌的细胞免疫应答的分子基础。
采用SYFPEITHI算法预测潜在的人类白细胞抗原（HLA）A0201配体，并通过T2结合试验进行检测，以从6B11互补决定区（CDR）筛选HLA-A2结合肽。用6B11或肽脉冲树突状细胞（DC）进行3轮体外刺激，产生针对6B11或肽的细胞毒性T淋巴细胞（CTL），然后通过51Cr释放试验检测以确定6B11的CTL表位。以6B11特异性CTL作为反应细胞，肽脉冲DC作为刺激剂进行细胞增殖试验，以确定6B11的辅助性T淋巴细胞（Th）表位。进行细胞因子检测和干扰素-γ ELISPOT试验以进一步验证6B11的CTL和Th表位。
6B11的轻链CDR3肽（VL CDR3）诱导特异性CTL杀伤HLA-A2和靶抗原阳性的卵巢癌细胞，其可被抗人主要组织相容性复合体（MHC）I抗体阻断。6B11的重链CDR3肽（VH CDR3）刺激经6B11致敏的CTL增殖，其主要可被抗人MHC-II抗体阻断，进一步实验表明部分经VH CDR3肽致敏的CTL杀伤K562细胞。VL CDR3和VH CDR3中的肽分别是模拟6B11原始抗原的CTL和Th表位。6B11 CTL和Th表位，或6B11 CTL表位与钥孔戚血蓝蛋白（KLH）协同作用，前者在诱导针对卵巢癌的特异性细胞免疫应答方面更强。6B11或相应的CTL和Th表位特异性CTL分泌高水平的白细胞介素-2（1630、1503 ng/L）和干扰素-γ（5620、5421 ng/L），而白细胞介素-4检测到基础水平（253、274 ng/L）。ELISPOT试验证实6B11或表位特异性CTL中存在特异性分泌干扰素-γ的细胞（196/1×10(6) T细胞，184/1×10(6) T细胞）。
6B11的VL CDR3和VH CDR3肽是模拟原始抗原的CTL和Th表位，其可重现完整6B11诱导针对卵巢癌的细胞免疫应答的活性。该结果在抗独特型抗体作为抗肿瘤疫苗的应用中具有重要的理论和实际价值。所获得的CTL和Th表位作为未来针对卵巢癌的多种肽的组成成分，可用于基于肽疫苗的卵巢癌治疗。
