基于体细胞突变数据库筛选免疫原性结直肠癌高频新抗原的方法

doi:10.16288/j.yczz.20-032

遗传 ›› 2020, Vol. 42 ›› Issue (6): 599-612.doi: 10.16288/j.yczz.20-032

• 技术与方法 • 上一篇

基于体细胞突变数据库筛选免疫原性结直肠癌高频新抗原的方法

秦丽丽^1,², 李毅坚¹, 梁兆端¹, 陈蕾¹, 李文慧¹, 陈超^1,^3,⁴, 黄亚灵¹, 张乐^1,^3,⁴, 刘松明^1,^3,⁴, 邱思^1,⁴, 葛玉萍¹, 彭文婷^1,³, 林欣欣^1,⁴, 张秀清^1,⁴, 董旋¹(), 李波^1,⁴()

^1. 深圳华大生命科学研究院，深圳 518083
^2. 大理大学基础医学院，大理 671000
^3. 中国科学院大学华大教育中心，深圳 518083
^4. 武汉华大吉诺因生物科技有限公司，武汉 430079

收稿日期:2020-04-10 修回日期:2020-05-15 出版日期:2020-06-20 发布日期:2020-05-19
作者简介:秦丽丽, 在读硕士研究生,专业方向：病理学和病理生理学。E-mail: veritasdoct168@163.com|李毅坚,硕士,助理研究员,研究方向：肿瘤免疫治疗。E-mail:liyijian@genomics.cn; 秦丽丽和李毅坚为并列第一作者。
基金资助:
国家自然科学基金项目编号(81702826);深圳市科技创新委员会项目资助编号(JCYJ20170303151334808);深圳市科技创新委员会项目资助编号(JCYJ20170817150015170)

A method of screening highly common neoantigens with immunogenicity in colorectal cancer based on public somatic mutation library

Qin Lili^1,², Li Yijian¹, Liang Zhaorui¹, Dai Lei¹, Li Wenhui¹, Chen Chao^1,^3,⁴, Huang Yaling¹, Zhang Le^1,^3,⁴, Liu Songming^1,^3,⁴, Qiu Si^1,⁴, Ge Yuping¹, Peng Wenting^1,³, Lin Xinxin^1,⁴, Zhang Xiuqing^1,⁴, Dong Xuan¹(), Li Bo^1,⁴()

^1. BGI-Shenzhen, Shenzhen 518083, China
^2. College of Basic Medicine, Dali University, Dali 671000, China
^3. BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China
^4. BGI-GenoImmune, BGI-Shenzhen, Wuhan 430079, China

Received:2020-04-10 Revised:2020-05-15 Published:2020-06-20 Online:2020-05-19
Supported by:
Supported by the National Natural Science Foundation of China No(81702826);Science, Technology and Innovation Commission of Shenzhen Municipality Nos(JCYJ20170303151334808);Science, Technology and Innovation Commission of Shenzhen Municipality Nos(JCYJ20170817150015170)

摘要/Abstract

摘要：

结直肠癌是世界高发和高致死率的恶性肿瘤。靶向新抗原的免疫治疗已被证实可以诱导癌症患者肿瘤持续消退,但这些特异性新抗原,仅适用于个体精准治疗。随着大量的高频肿瘤基因突变被发现,这些与突变相关的高频新抗原可覆盖更多人群,具有较强的临床意义。然而目前结直肠癌中是否也存在高频新抗原仍不清楚。本研究利用来源于321个结直肠癌患者的体细胞突变数据库,联合1种标准过滤和7种预测算法,筛选并获得了25个基于中国人高频分型HLA-A*1101限制性的高频新抗原,它们均具有高亲和力(IC₅₀<50 nmol/L)和高呈递分值(>0.90);其中,除了阳性对照多肽KRAS_G12V_8-16外,11个高频新抗原能够在体外诱导细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)分泌γ干扰素(interferon gamma, IFN-γ),证实具有免疫原性。选取免疫原性最强的新抗原C1orf170_S418G_413-421及阳性对照多肽KRAS_G12V_8-16体外刺激T细胞,利用流式细胞分选及单细胞转录组测序技术,获得其特异性CTL的免疫组库信息,所构建的TCR-T（T-cell receptor engineered T cell）能够识别新抗原并分泌细胞因子。以上结果表明,本研究开发了一种利用体细胞数据库预测并体外筛选验证具有免疫原性高频新抗原的方法,为结直肠癌及其他癌种的多肽、DC（dendritic cells）疫苗、TCR-like抗体、TCR-T等免疫治疗提供了重要的多肽靶点和TCR信息,具有实际的临床应用价值。

关键词: 结直肠癌, 高频, 新抗原, 呈递

Abstract:

Colorectal cancer (CRC) is a malignant cancer with high incidence and mortality in the world. Immunotherapy targeting neoantigens can induce durable tumor regression in cancer patients, but is almost limited to personalized precision therapy, due to the individual differences of unique neoantigens. With the discovery of many common oncogenic mutations, and such mutation-associated neoantigens could cover more patients, and hence are valuable in clinical field. However, whether the common neoantigens can be identified in CRC is unknown. Combining the somatic mutations data from 321 CRC patients with a filter standard and 7 predicted algorithms, we screened and obtained 25 HLA-A*1101-restricted common neoantigens with a high binding affinity (IC₅₀<50 nmol/L) and presentation score (>0.90). Besides the positive epitope KRAS_G12V_8-16, 11 out of 25 common neoantigens specifically induced in vitro pre- stimulated cytotoxic lymphocyte (CTL) to secrete interferon gamma (IFN-γ). Moreover, combining cell-sorting technology and single-cell RNA sequencing, the immune repertoire profiles of C1orf170_S418G_413-421 and KRAS_G12V_8-16-specific CTL were analyzed and validated. Their related T-cell receptor engineered T cell (TCR-T) cells could also recognize the neoantigens and secrete IFN-γ. Hence, we have established a method to screen for common neoantigens with immunogenicity in CRC based on the public somatic mutation library. It can provide essential peptide and TCR information for immunotherapies, such as peptides, dendritic cells (DC) vaccines, TCR-like antibodies, TCR-T, etc., for the CRC and other cancers, which has practical application value in the clinics.

Key words: colorectal cancer, common, neoantigen, presentation

秦丽丽, 李毅坚, 梁兆端, 陈蕾, 李文慧, 陈超, 黄亚灵, 张乐, 刘松明, 邱思, 葛玉萍, 彭文婷, 林欣欣, 张秀清, 董旋, 李波. 基于体细胞突变数据库筛选免疫原性结直肠癌高频新抗原的方法[J]. 遗传, 2020, 42(6): 599-612.

Qin Lili, Li Yijian, Liang Zhaorui, Dai Lei, Li Wenhui, Chen Chao, Huang Yaling, Zhang Le, Liu Songming, Qiu Si, Ge Yuping, Peng Wenting, Lin Xinxin, Zhang Xiuqing, Dong Xuan, Li Bo. A method of screening highly common neoantigens with immunogenicity in colorectal cancer based on public somatic mutation library[J]. Hereditas(Beijing), 2020, 42(6): 599-612.

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预测的新抗原表位	基因	突变位点	突变频率	新抗原突变位置	亲和力 (nmol/L)	EPIP分数	肿瘤中的作用
GLCE_V533I_526-535	GLCE	V533I	6/321	STIDESPIFK	2.7	0.989077	?
C1orf170_S418G_413-421	C1orf170	S418G	5/321	SVAGPGPNK	21.7	0.974471	?
MUC3A_I29T_28-36	MUC3A	I29T	6/321	STSQVPFPR	13.8	0.970609	?
CCRL2_F179Y_174-183	CCRL2	F179Y	5/321	ATLPEYVVYK	4.7	0.968889	?-
KIAA1683_M313T_311-319	KIAA1683	M313T	6/321	STTTTTPPK	7.9	0.964324	?
KLHL40_N345S_341-349	KLHL40	N345S	5/321	ASLSSQVPK	9.4	0.956861	?
MUC3A_S175P_172-181	MUC3A	S175P	6/321	STYPMTTTEK	5.8	0.952998	?
RNF43_I47V_46-54	RNF43	I47V	7/321	AVIRVIPLK	7.8	0.949232	TSG
SYNE2_A2395T_2389-2398	SYNE2	A2395T	5/321	STQESATVEK	28.9	0.946256	?
TLR10_I369L_367-375	TLR10	I369L	5/321	RTLQLPHLK	13.2	0.945037	?
ANKRD36C_N1571S_1571-1579	ANKRD36C	N1571S	5/321	STMEKCIEK	6.6	0.94484	?
IYD_F231I_229-237	IYD	F231I	6/321	QVIGKIILK	21.4	0.940062	?
SSX5_E19Q_12-20	SSX5	E19Q	5/321	RVGSQIPQK	35.7	0.936871	?
MUC3A_S326T_319-327	MUC3A	S326T	7/321	TTLPTTITR	16.1	0.936033	?
ARHGEF11_H1427R_1427-1435	ARHGEF11	H1427R	5/321	RTIEQLTLK	10.8	0.933929	?
CTNNB1_T41A_41-49	CTNNB1	T41A	6/321	ATAPSLSGK	6.9	0.932574	癌基因
LILRB5_L605F_603-611	LILRB5	L605F	5/321	RSFPLTLPR	6.9	0.931333	?
EIF2A_T92S_86-95	EIF2A	T92S	5/321	ATWQPYSTSK	12.9	0.931289	?
TMPRSS15_P732S_732-740	TMPRSS15	P732S	5/321	STDGGPFVK	12	0.930946	?
MUC6_P2049L_2044-2053	MUC6	P2049L	9/321	GTVPPLTTLK	16.5	0.917963	?
TMEM185B_A42G_42-51	TMEM185B	A42G	5/321	GVFAPIWLWK	5.7	0.904248	?
UNC93A_M403T_402-410	UNC93A	M403T	6/321	STFLCVHVK	4.5	0.903415	?
MUC3A_Q31H_28-36	MUC3A	Q31H	5/321	SISHVPFPR	14.1	0.901598	?
FSIP2_R1288Q_1285-1293	FSIP2	R1288Q	5/321	SSLQSQLSK	13.8	0.900572	?
FSIP2_T184NX	FSIP2	T184NX	2/321	TTLPKFNKK	10.2	0.961067	?

抗原肽	TCR克隆	CDR3 序列	TRV 基因	J 基因	比例(%)
C1orf170_S418G_413-421	Clonotype1	TRA: CAASGGAQKLVF	TRAV29DV5	TRAJ54	53.2
		TRA: CAGLLYNSGNTPLVF	TRAV35	TRAJ29
		TRB: CASSRDRGSNQPQHF	TRBV27	TRBJ1-5
	Clonotype2	TRA: CAASGGAQKLVF	TRAV29DV5	TRAJ54	31.6
	Clonotype2	TRB: CASSRDRGSNQPQHF	TRBV27	TRBJ1-5	31.6
KRAS_G12V_8-16	Clonotype1	TRA: CASNDYKLSF	TRAV8-3	TRAJ20	80.5
	Clonotype1	TRB: CASSLDGVSYEQYF	TRBV11-2	TRBJ2-7	80.5
	Clonotype2	TRA: ?	?	?	14.3
	Clonotype2	TRB: CASSLDGVSYEQYF	TRBV11-2	TRBJ2-7	14.3

基于体细胞突变数据库筛选免疫原性结直肠癌高频新抗原的方法

A method of screening highly common neoantigens with immunogenicity in colorectal cancer based on public somatic mutation library

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