TIL识别的HLA-A2限制性人黑色素瘤特异抗原肽的研究

The study of human melanoma-specific antigen peptides by HLA-A2 restricted tumor-infiltrating lymphocytes

目的探讨TIL识别的人黑色素瘤HLA-A2限制性的肿瘤抗原肽特性。方法通过亲和层析柱从三种肿瘤细胞系(624-Mel、Chap-Mel和JY)中纯化HLA-A2蛋白和结合的多肽分子,经弱酸高温处理和离心超滤后,应用反相-高效液相色谱层析(RT-HPLC)分离各多肽组份,并将其各组份与T2细胞共同孵育、进行重建TIL识别的人黑色素瘤特异表位试验,鉴定肽分子生物活性,通过质谱分析所获活性肽分子特性,参照活性肽序列,制备人工合成肽加以进一步证实。结果从RT-HPLC层析的624-Mel的肽组份,经特异表位鉴定发现3个活性峰(P19、P25和P31)。其中P31,TIL杀伤率达67%,质谱分析表明肽分子量为948,氨基酸序列为H+Ala Lue Trp Lue Phe Phe Gly Val Lue OH-的9肽分子。经特异表位测定表明人工合成肽具有纯化活性肽的生物活性特征。结论分离鉴定的这一9肽分子是一种TIL识别的HLA-A2限制性肿瘤抗原肽,为肿瘤防治、合成肽疫苗的研究奠定了可靠的实验基础。

Objective： To explore the characteristics of human melanoma-specific antigen peptides by HLA-A2 restricted tumor-infiltrat- ing lyrnphoeytes.Methods：The HLA-A2 protein and polypeptides molecules were purified from the three tumor cell lines（624-Mel, Chap-Mel and JY） by immunoaffinity chromatography, after the peptides bound to HLA-A2 protein solution were acidified with acetic acid and boiled by high temperature, and centrifuged through an Ultra-CL filter, then the pepfides extracts were fraetionated by revered phase high pressure liquid ehromatography（RP-HPLC）. Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by adding to the HLA-A2 Ag-proeeeing mutant cell, T2. The biological feature of one of three active pepfides from RT-HPLC samples was performed by mass spectrometric analysis. The synthetic pepfides identical to active pepfide sequences were determined in the reconstitute test. Results： Three prominent peaks（P19, P25 and P31） of the fraction from 624-Mel were observed in the reconstitute test, TIL killing rate was 67% for （P31） pepfide fraction. The mass speetrometric analysis of one of active pepfides （P31） showed that at mass-to-charge ratio（m/z） 948 has been usually nine residues. The sequence is H^＋ Ala line Trp line Phe Phe Gly Val Luae OH^- . The pepfide synthesized comprising epitopes were veilfied. Conclusion： These results showed the peptides derived from active fractions were related to human melanoma-specific tumor antigen peptides recognized by HLA-A2-restriced TIL. These peptides could devdop novel peptide-based an anti-tumor vaccine for immunotherapy of CTL.