制备并鉴定靶向肺癌抗原KK-LC-1 HLA A*02:01 限制性表位的特异性CTL

  背景及目的：肺癌在中国及世界的恶性肿瘤新发病率和致死率都位居榜 首，肿瘤免疫治疗作为癌症治疗的第三次革命最有希望能治愈癌症，针对肺癌 的免疫治疗研究亟需进展。基于T细胞免疫反应中TCR识别肽-HLA复合物的 重要性，我们想筛选并鉴定靶向肺癌相关抗原的T细胞表位肽，并制备高纯度 的抗原特异性CTL，为肺癌在以TCR为基础的肿瘤免疫疗法及癌症治疗疫苗上 提供基础。

  方法：通过237例非小细胞肺癌（NSCLC）组织切片验证KK-LC-1表达水 平，KK-LC-1作为癌症种系基因的一员，是免疫治疗的理想靶点。通过4种T 细胞表位预测软件及肽-HLA亲和力实验筛选KK-LC-1上HLA-A*02:01限制性 T 细胞表位肽，与HLA-A*02:01+ PBMC共孵育获得特异性CTL并验证杀伤肿 瘤细胞的免疫功能，使用特异性p-HLA四聚体分选得到特异性CTL，扩增培养 后再次验证杀伤肿瘤细胞的免疫功能。

  结果：KK-LC-1在NSCLC中异常高表达，以此作为标靶通过T细胞表位 预测工具筛选得到6个HLA-A*02:01限制性T细胞表位肽，通过肽-HLA亲和 力验证得知LP4表现出最高亲和力。LP4和HLA-A*02:01+ PBMC共孵育可以刺 激特异性CD8 T细胞活化增殖，通过ELISpot方法检测LP4刺激的CTL能分 泌IFN-γ，并特异性对KK-LC-1+ HLA-A*02:01+ NSCLC细胞产生杀伤作用， 用流式细胞术分选和LP4-HLA-A*02:01四聚体结合的CD8 T细胞后进行扩增培 养。扩增培养的CTL仍能特异性杀伤KK-LC-1+ HLA-A*02:01+ NSCLC细胞， 但通过流式细胞术发现和LP4-HLA-A*02:01四聚体结合的CD8 T细胞数量及占 比均极低。进一步验证LP4是特异性刺激LP4-HLA-A*02:01 CD8 T细胞分泌 IFN-γ，但LP4-HLA-A*02:01 复合物与对应TCR的亲和力极低。

  结论：针对LP4能特异性激活HLA-A*02:01+ PBMC中的CD8 T细胞产生 具有特异性杀伤NSCLC细胞的免疫功能，但仍未能确定LP4-HLA-A*02:01复 合物与对应TCR的亲和力强弱，需要进一步的深入探究。虽然以LP4为切入点 研究基于HLA-A*02:01限制性TCR的肺癌免疫疗法就此中断，但是仍可给治 疗性肺癌疫苗提供一定的基础。

[1] HAN B, ZHENG R, ZENG H, et al. Cancer incidence and mortality in China, 2022[J]. Journal of the National Cancer Center, 2024
[2] SUNG H, FERLAY J, SIEGEL R L, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249.
[3] LIU Y, YAN X, ZHANG F, et al. TCR-T Immunotherapy: The Challenges and Solutions[J]. Front Oncol, 2021, 11: 794183.
[4] ZHAO Q, JIANG Y, XIANG S, et al. Engineered TCR-T Cell Immunotherapy in Anticancer Precision Medicine: Pros and Cons[J]. Front Immunol, 2021, 12: 658753.
[5] SHEN Y, WEI X, JIN S, et al. TCR-mimic antibody-drug conjugates targeting intracellular tumor-specific mutant antigen KRAS G12V mutation[J]. Asian Journal of Pharmaceutical Sciences, 2020, 15(6): 777-785.
[6] NATHAN P, HASSEL J C, RUTKOWSKI P, et al. Overall Survival Benefit with Tebentafusp in Metastatic Uveal Melanoma[J]. New England Journal of Medicine, 2021, 385(13): 1196-1206.
[7] FAN T, ZHANG M, YANG J, et al. Therapeutic cancer vaccines: advancements, challenges, and prospects[J]. Signal Transduction and Targeted Therapy, 2023, 8(1)
[8] MENG X, SUN X, LIU Z, et al. A novel era of cancer/testis antigen in cancer immunotherapy[J]. Int Immunopharmacol, 2021, 98: 107889.
[9] YANG P, MENG M, ZHOU Q. Oncogenic cancer/testis antigens are a hallmarker of cancer and a sensible target for cancer immunotherapy[J]. Biochim Biophys Acta Rev Cancer, 2021, 1876(1): 188558.
[10] JIN S, CAO S, LI J, et al. Cancer/testis antigens (CTAs) expression in resected lung cancer[J]. OncoTargets and Therapy, 2018, Volume 11: 4491-4499.
[11] LI Q, HU W, LIAO B, et al. Natural high-avidity T-cell receptor efficiently mediates regression of cancer/testis antigen 83 positive common solid cancers[J]. J Immunother Cancer, 2022, 10(7)
[12] LEKO V, ROSENBERG S A. Identifying and Targeting Human Tumor Antigens for T CellBased Immunotherapy of Solid Tumors[J]. Cancer Cell, 2020, 38(4): 454-472.
[13] PERI A, SALOMON N, WOLF Y, et al. The landscape of T cell antigens for cancer immunotherapy[J]. Nature Cancer, 2023, 4(7): 937-954.
[14] CHEN L, FLIES D B. Molecular mechanisms of T cell co-stimulation and co-inhibition[J]. Nature Reviews Immunology, 2013, 13(4): 227-242.
[15] ARMSTRONG KATHRYN M, PIEPENBRINK KURT H, BAKER BRIAN M. Conformational changes and flexibility in T-cell receptor recognition of peptide–MHC complexes[J]. Biochemical Journal, 2008, 415(2): 183-196.
[16] DENG L, LANGLEY R J, WANG Q, et al. Structural insights into the editing of germ-lineencoded interactions between T-cell receptor and MHC class II by Vα CDR3[J]. Proceedings of the National Academy of Sciences, 2012, 109(37): 14960-14965.
[17] COLE D K, MILES K M, MADURA F, et al. T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions[J]. Journal of Biological Chemistry, 2014, 289(2): 628-638.
[18] XIE N, SHEN G, GAO W, et al. Neoantigens: promising targets for cancer therapy[J]. Signal Transduction and Targeted Therapy, 2023, 8(1)
[19] ABD HAMID M, PENG Y, DONG T. Human cancer germline antigen-specific cytotoxic T cell—what can we learn from patient[J]. Cellular & Molecular Immunology, 2020, 17(7): 684692.
[20] TALEBIAN YAZDI M, LOOF N M, FRANKEN K L, et al. Local and systemic XAGE-1bspecific immunity in patients with lung adenocarcinoma[J]. Cancer Immunol Immunother, 2015, 64(9): 1109-1121.
[21] TAGUCHI A, TAYLOR A D, RODRIGUEZ J, et al. A search for novel cancer/testis antigens in lung cancer identifies VCX/Y genes, expanding the repertoire of potential immunotherapeutic targets[J]. Cancer Res, 2014, 74(17): 4694-4705.
[22] SCHOOTEN E, DI MAGGIO A, VAN BERGEN EN HENEGOUWEN P M P, et al. MAGEA antigens as targets for cancer immunotherapy[J]. Cancer Treat Rev, 2018, 67: 54-62.
[23] FUKUYAMA T, HANAGIRI T, TAKENOYAMA M, et al. Identification of a new cancer/germline gene, KK-LC-1, encoding an antigen recognized by autologous CTL induced on human lung adenocarcinoma[J]. Cancer Res, 2006, 66(9): 4922-4928.
[24] GURE A O, CHUA R, WILLIAMSON B, et al. Cancer-testis genes are coordinately expressed and are markers of poor outcome in non-small cell lung cancer[J]. Clin Cancer Res, 2005, 11(22): 8055-8062.
[25] NAKAMURA Y, NOGUCHI Y, SATOH E, et al. Spontaneous remission of a non-small cell lung cancer possibly caused by anti-NY-ESO-1 immunity[J]. Lung Cancer, 2009, 65(1): 119122.
[26] FUTAWATARI N. Early gastric cancer frequently has high expression of KK-LC-1, a cancertestis antigen. World J Gastroenterol.[J]. World J Gastroenterol, 2017: 23(46):8200-8206.
[27] FUKUYAMA T, FUTAWATARI N, YAMAMURA R, et al. Expression of KK-LC-1, a cancer/testis antigen, at non-tumour sites of the stomach carrying a tumour[J]. Scientific Reports, 2018, 8(1)
[28] TAKAHASHI Y, FUKUYAMA T, FUTAWATARI N, et al. Expression of Kita-Kyushu Lung Cancer Antigen-1 as Detected by a Novel Monoclonal Antibody in Gastric Cancer[J]. Anticancer research, 2019, 39(11): 6259-6263.
[29] CHEN Z, ZUO X, PU L, et al. Hypomethylation‐mediated activation of cancer/testis antigen KK‐LC‐1 facilitates hepatocellular carcinoma progression through activating the Notch1/Hes1 signalling[J]. Cell Proliferation, 2019, 52(3)
[30] KONDO Y, FUKUYAMA T, YAMAMURA R U I, et al. Detection of KK-LC-1 Protein, a Cancer/Testis Antigen, in Patients with Breast Cancer[J]. Anticancer research, 2018, 38(10): 5923-5928.
[31] BU J, ZHANG Y, WU S, et al. KK-LC-1 as a therapeutic target to eliminate ALDH+ stem cells in triple negative breast cancer[J]. Nature Communications, 2023, 14(1)
[32] JIN S, CAO S, GRIGOREV A, et al. Establishment of cancer/testis antigen profiling based on clinicopathological characteristics in resected pathological stage III non-small cell lung cancer[J]. Cancer Management and Research, 2018, Volume 10: 2031-2046.
[33] TAKESHI HANAGIRI, YOSHIKI SHIGEMATSU S S M T. Clinical Significance of Expression of Cancer/testis Antigen and Down-regulation of HLA Class-I in Patients with Stage I Non-small Cell Lung Cancer[J]. Anticancer research, 2013: 33(35):2123-2128.
[34] BAI R, YUAN C. Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1): A Promising Cancer Testis Antigen[J]. Aging Dis, 2022, 13(4): 1267-1277.
[35] HERRERA L R. Reverse Vaccinology Approach in Constructing a Multi-Epitope Vaccine Against Cancer-Testis Antigens Expressed in Non-Small Cell Lung Cancer[J]. Asian Pacific Journal of Cancer Prevention, 2021, 22(5): 1495-1506.
[36] YE Z, LIANG Y, MA Y, et al. Targeted photodynamic therapy of cancer using a novel gallium (III) tris (ethoxycarbonyl) corrole conjugated‐mAb directed against cancer/testis antigens 83[J]. Cancer Medicine, 2018, 7(7): 3057-3065.
[37] MARCINKOWSKI B, STEVANOVIC S, HELMAN S R, et al. Cancer targeting by TCR geneengineered T cells directed against Kita-Kyushu Lung Cancer Antigen-1[J]. J Immunother Cancer, 2019, 7(1): 229.
[38] ICHIKI Y, SHIGEMATSU Y, BABA T, et al. Development of adoptive immunotherapy with KK-LC-1-specific TCR-transduced gammadeltaT cells against lung cancer cells[J]. Cancer Sci, 2020, 111(11): 4021-4030.
[39] SAXENA M, VAN DER BURG S H, MELIEF C J M, et al. Therapeutic cancer vaccines[J]. Nat Rev Cancer, 2021, 21(6): 360-378.
[40] MA M, LIU J, JIN S, et al. Development of tumour peptide vaccines: From universalization to personalization[J]. Scandinavian Journal of Immunology, 2020, 91(6)
[41] HIGASHIHARA Y, KATO J, NAGAHARA A, et al. Phase I clinical trial of peptide vaccination with URLC10 and VEGFR1 epitope peptides in patients with advanced gastric cancer[J]. International Journal of Oncology, 2014, 44(3): 662-668.
[42] ZHOU Y. HER2/neu-based vaccination with li-Key hybrid, GM-CSF immunoadjuvant and trastuzumab as a potent triple-negative breast cancer treatment[J]. Journal of Cancer Research and Clinical Oncology, 2023, 149(9): 6711-6718.
[43] KLEBANOFF C A, CHANDRAN S S, BAKER B M, et al. T cell receptor therapeutics: immunological targeting of the intracellular cancer proteome[J]. Nature Reviews Drug Discovery, 2023, 22(12): 996-1017.
[44] XU R, DU S, ZHU J, et al. Neoantigen-targeted TCR-T cell therapy for solid tumors: How far from clinical application[J]. Cancer Lett, 2022, 546: 215840.
[45] ROBBINS P F, LI Y F, EL-GAMIL M, et al. Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions[J]. The Journal of Immunology, 2008, 180(9): 6116-6131.
[46] FOY S P, JACOBY K, BOTA D A, et al. Non-viral precision T cell receptor replacement for personalized cell therapy[J]. Nature, 2022, 615(7953): 687-696.
[47] PETERS B, NIELSEN M, SETTE A. T Cell Epitope Predictions[J]. Annu Rev Immunol, 2020, 38: 123-145.
[48] MEI S, LI F, LEIER A, et al. A comprehensive review and performance evaluation of bioinformatics tools for HLA class I peptide-binding prediction[J]. Briefings in Bioinformatics, 2020, 21(4): 1119-1135.
[49] NI L, LU J. Interferon gamma in cancer immunotherapy[J]. Cancer Medicine, 2018, 7(9): 45094516.
[50] GOCHER A M, WORKMAN C J, VIGNALI D A A. Interferon-γ: teammate or opponent in the tumour microenvironment?[J]. Nature Reviews Immunology, 2021, 22(3): 158-172.
[51] ZENG X, NONG W X, ZOU X Q, et al. Prediction and identification of HLA-A*0201restricted epitopes from cancer testis antigen CT23[J]. Hum Vaccin Immunother, 2023, 19(3): 2293299.
[52] YANG Z, ZHANG H, XIA X, et al. Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from TC2N[J]. Eur J Microbiol Immunol (Bp), 2024, 14(1): 59-65.
[53] MA N, LIU H, ZHANG Y, et al. Identification of CD8(+) T-cell epitope from multiple myeloma-specific antigen AKAP4[J]. Front Immunol, 2022, 13: 927804.