探討放射線與免疫治療的合併療法所引發之腫瘤新生抗原特異T細胞反應

Investigation of Neoantigen-Specific T Cell Response Induced by Combination Therapy of Radiation Immunotherapy

施季韜(THOMAS CHITAO SHIH)

指導教授 ： 陶秘華

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201800331

摘要

部分腫瘤已可由癌症免疫治療治癒，然而並非對所有病人都有療效。文獻證實癌症免疫治療的療效與腫瘤突變數量、腫瘤浸潤的T淋巴細胞相關，而胺基酸突變所形成之腫瘤新生抗原是其理想治療標的。我們先前證實放射線結合IL-12和GM-CSF之局部免疫治療的合併療法能治癒數個小鼠腫瘤模型，且治療引發腫瘤內浸潤CD8+ T細胞數量與活性顯著上升。因此，我們推論合併療法的療效是藉由引發腫瘤新生抗原特異T細胞辨識腫瘤新生抗原所造成。由於文獻曾報告B16F10黑色素瘤之突變，我們挑選其中21個活體試驗中能引發免疫反應突變分析，藉由定序確認了我們的B16F10細胞株具備其中16個突變。接著以程式預測21個突變的免疫性，以Immune Epitope Database and Analysis Resources (IDEB) 和 Bioinformatics and Molecular Analysis Section (BIMAS)工具，預測出其中20個對C57BL/6J 小鼠的major histocompatibility complex (MHC) Class I分子有高結合力。同時我們建立B16F10皮下腫瘤實驗動物模型，單以放射線或免疫治療皆無明顯療效，而合併療法能抑制腫瘤生長，並增加腫瘤浸潤CD8+ T細胞數量、引流淋巴結CD4+ T、CD8+ T細胞活性。我們初步以IFN ELISPOT assay證實了合併療法引發脾臟細胞的腫瘤新生抗原特異T細胞反應。未來需要進一步探討腫瘤新生抗原特異T細胞數量與活性和腫瘤復發時之關係，並以所預測的腫瘤新生抗原，製成疫苗，加強特異T細胞活性，以預防復發，並提供長期保護。

關鍵字

癌症免疫治療 ； 合併療法 ； 腫瘤新生抗原 ； 抗原免疫性預測 ； 抗原特異性T細胞反應

並列摘要

Immunotherapies have led to promising clinical outcomes in certain cancer patients; however, not all patients benefited from immunotherapies. Evidence suggests that the therapeutic efficacy of cancer immunotherapy correlates with tumor mutation burden and the number of tumor-infiltrating T cells. Neoantigens, which are tumor specific nonsynonymous mutations with amino acid substitutions in expressed proteins, have recently been reported as ideal targets for effective cancer immunotherapy. Our previous study has shown that a combination therapy of ionizing radiation and local immunotherapy, achieved by intratumoral injection of adenoviral vectors encoding interleukin 12 and granulocyte-marcophage colony stimulating factor (Ad/IL-12 + GM-CSF), resulted in regression of large established tumors of several murine tumor models. Mechanistic studies revealed that the number of tumor-infiltrating CD8+ T cells was significantly increased. We hypothesized that these tumor-infiltrating CD8+ T cells generated by the combination therapy are neoantigens-specific and correlated with the therapeutic efficacy. We chose B16F10, a highly malignant murine melanoma, as the tumor model to investigate this hypothesis because the neoantigen information of this tumor cell line is available in the public database. Among the many mutations reported in B16F10 tumor cells, we chose 21 of them as the potential neoantigens due to their mutated peptides can induce T cell responses in vivo. To confirm whether these reported mutations are also present in our B16F10 cell line, we synthesized their corresponding primers and carried out PCR for sequencing analysis. Our results confirmed 16 mutations in our cell line but the other 5 remained as wild type sequences. Using the T cell epitope analysis tools provided by Immune Epitope Database and Analysis Resources (IDEB) and Bioinformatics and Molecular Analysis Section (BIMAS), we confirmed that peptides containing 20 of the 21 mutations had a high binding affinity to the major histocompatibility complex (MHC) Class I molecules of C57BL/6J mice. We demonstrated that combination therapy of radiation and Ad/IL-12 + GM-CSF significantly suppressed tumor growth, while the single therapy of radiation or Ad/IL-12 + GM-CSF had much less effect. In addition, the combination therapy resulted in a significant increase of tumor-infiltrating CD8+ T cells. In the combination therapy group, CD4+ T cells and CD8+ T cells from the draining lymph nodes showed activation phenotypes. Our preliminary data showed that combination therapy also increased the number of neoantigens-specific IFN-producing splenocytes by an ELISPOT assay. Further studies are required to examine the relationship between tumor relapse and the dynamics of neoantigens-specific T cells. Moreover, vaccinations with the predicted neoantigens may further strengthen the antitumor T cells and provide long-lasting protection against tumor relapse following the primary cancer therapy.

並列關鍵字

Cancer immunotherapy ； Combination therapy ； Tumor neoantigen ； Immunogenicity prediction ； Neoantigen specific T cell response

參考文獻

1. Cooper GM. 2000. The Cell: A Molecular Approach. ASM Press.

2. Garraway LA, Lander ES. 2013. Lessons from the cancer genome. Cell 153:17-37.

3. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Jr., Kinzler KW. 2013. Cancer genome landscapes. Science 339:1546-58.

4. O'Brien SG. 2003. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 348:994-1004.

5. Sledge GW. 2012. The challenge and promise of the genomic era. J Clin Oncol 30:203-209.