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北京大学学报（医学版） ›› 2022, Vol. 54 ›› Issue (5): 884-895. doi: 10.19723/j.issn.1671-167X.2022.05.016

• 论著 • 上一篇    下一篇

KRAS G12V特异性T细胞受体治疗恶性肿瘤的临床前研究

程晓静1,蒋栋2,张连海1,王江华2,李雅真2,翟佳慧2,闫宝琪2,张露露2,谢兴旺2,*(),李子禹1,*(),季加孚1,*()   

  1. 1. 北京大学肿瘤医院暨北京市肿瘤防治研究所胃肠肿瘤中心，恶性肿瘤发病机制及转化研究教育部重点实验室，北京 100142
    2. 北京可瑞生物科技有限公司，北京 100094
  • 收稿日期:2022-06-27 出版日期:2022-10-18 发布日期:2022-10-14
  • 通讯作者: 谢兴旺,李子禹,季加孚 E-mail:xiexingwang@corregene.com;ziyu_li@hsc.pku.edu.cn;jijiafu@hsc.pku.edu.cn
  • 作者简介:季加孚，主任医师、教授、博士生导师，北京大学肿瘤医院大外科主任、胃肠肿瘤中心主任，教育部恶性肿瘤发病机制及转化重点实验室主任，北京大学肿瘤研究中心主任，国务院政府特殊津贴专家，中国人民政治协商会议第十三届全国委员会委员，中国民主同盟中央委员会常务委员。现任中国抗癌协会副理事长，健康中国行动推进委员会专家咨询委员会委员，中国医疗保健国际交流促进会副会长，中华医学会外科学分会常务委员兼秘书长，美国外科学会会员(Fellow of American College of Surgeons, FACS)，英国皇家外科学院院士(Fellow of Royal Colleges of Surgeons, FRCS)，亚洲外科学会(Asian Surgical Association, ASA)常务委员，国际胃癌学会(International Gastric Cancer Association, IGCA)前任主席。
    以第一作者或通信作者在Lancet、Science、Cell和JAMA等期刊发表论文400余篇，主编国内首部胃癌英文专著及首个SCI肿瘤学期刊(现为高质量科技期刊分级目录T1级期刊)，培养研究生及博士后120余人。以第一完成人获国家科技进步二等奖1项、省部级一等奖3项，获何梁何利科技进步奖、吴阶平·保罗杨森医学医药奖、英国文化教育协会职业成就奖及中菲亚洲国际和平奖。获评国家卫生健康委员会突出贡献专家、北京学者、约翰·霍普金斯大学医学院兼职教授
  • 基金资助:
    北京市科委、中关村管委会医药创新品种及平台培育项目(Z211100002521027);北京大学肿瘤医院科学基金(2021-8);北京市属医学科研院所公益发展改革试点项目(第三批)(京医研2019-01)

Preclinical study of T cell receptor specifically reactive with KRAS G12V mutation in the treatment of malignant tumors

Xiao-jing CHENG1,Dong JIANG2,Lian-hai ZHANG1,Jiang-hua WANG2,Ya-zhen LI2,Jia-hui ZHAI2,Bao-qi YAN2,Lu-lu ZHANG2,Xing-wang XIE2,*(),Zi-yu LI1,*(),Jia-fu JI1,*()   

  1. 1. Department of Gastrointestinal Cancer Center, Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Laboratory of Genetics, Peking University Cancer Hospital & Institute, Beijing 100142, China
    2. Beijing CorreGene Biotechnology Co., Ltd., Beijing 100094, China
  • Received:2022-06-27 Online:2022-10-18 Published:2022-10-14
  • Contact: Xing-wang XIE,Zi-yu LI,Jia-fu JI E-mail:xiexingwang@corregene.com;ziyu_li@hsc.pku.edu.cn;jijiafu@hsc.pku.edu.cn
  • Supported by:
    the Cultivation of Pharmaceutical Innovation Varieties and Platforms of Beijing Municipal Science and Technology Commission and Zhongguancun Administrative Committee(Z211100002521027);the Peking University Cancer Hospital Science Foundation(2021-8);the Pilot Project (3rd Round) to Reform Public Development of Beijing Municipal Medical Research Institute(京医研2019-01)

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摘要：

目的: KRAS G12V是最为常见的KRAS突变类型之一，是一个T细胞表位抗原，目前尚无针对该位点的靶向药物，本研究旨在克隆能够特异性识别该表位抗原的T细胞受体(T cell receptor, TCR)，通过体内外实验对该TCR基因修饰T细胞(TCR engineered T cells, TCR-T)靶向KRAS G12V突变肿瘤的安全性和有效性进行评估。方法: 从1例结直肠癌患者的肿瘤浸润淋巴细胞中获得靶向KRAS G12V8-16表位的高亲和力TCR序列，构建该TCR慢病毒载体并感染人源T细胞，获得TCR-T。采用抗原肽激活、γ-干扰素(interferon-γ, IFN-γ) 酶联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)、T细胞体外增殖等实验，体外评价该TCR-T的免疫杀伤活性和脱靶-交叉反应性；通过体内实验评价该TCR-T的抑瘤效果、安全性等指标。结果: 获得了能特异性识别HLA-A*11：01限制性KRAS G12V8-16表位的高亲和力TCR序列KVA11-01。KVA11-01 TCR-T能够显著杀伤体外过表达HLA-A*11:01和KRAS G12V的多种肿瘤细胞。非特异杀伤实验显示，KVA11-01仅杀伤同时表达HLA-A*11:01和KRAS G12V的肿瘤细胞。体内抑瘤实验显示，KVA11-01 TCR-T可以显著抑制PANC-1和HeLa(体外过表达HLA-A*11:01和KRAS G12V)细胞裸鼠皮下移植瘤的生长。TCR-T细胞可以显著浸润至肿瘤组织内部，有良好的实体肿瘤归巢能力。结论: KVA11-01 TCR-T能够在体内外有效靶向并杀伤携带KRAS G12V突变的多种恶性肿瘤细胞，具有良好的实体瘤组织归巢能力，KVA11-01 TCR-T有望成为携带KRAS G12V突变的实体恶性肿瘤患者的有效治疗手段。

关键词: 肿瘤, 肿瘤浸润淋巴细胞, DNA突变分析, T细胞受体

Abstract:

Objective: KRAS gene is one of the most common mutations of proto-oncogenes in human tumors, G12V is one of the most common mutation types for KRAS. It's challenging to chemically acquire the targeted drug for this mutation. Recent studies reported that this mutation peptides can form a neoepitope for T cell recognition. Our study aims to clone the T cell receptor (TCR) which specifically recognizes the neoepitope for KRAS G12V mutation and constructs TCR engineered T cells (TCR-T), and to investigate if TCR-Ts have strong antitumor response ability. Methods: In this study, tumor infiltrating lymphocytes were obtained from one colorectal cancer patient carrying KRAS G12V mutation. Tumor-reactive TCR was obtained by single-cell RT-5′ rapid-amplification of cDNA ends PCR analysis and introduced into peripheral blood lymphocytes to generate TCR-Ts. Results: We obtained a high-affinity TCR sequence that specifically recognized the HLA-A*11:01-restricted KRAS G12V8-16 epitope: KVA11-01. KVA11-01 TCR-T could significantly kill various tumor cells such as PANC-1, SW480 and HeLa (overexpressing HLA-A*11:01 and KRAS G12V), and secreting high levels of interferon-γ (IFN-γ). Non-specific killing experiments suggested KVA11-01 specifically recognized tumor cells expressing both mutant KRAS G12V and HLA-A*11:01. In vivo assay, tumor inhibition experiments demonstrated that infusion of approximately 1E7 KVA11-01 TCR-T could significantly inhibit the growth of subcuta-neously transplanted tumors of PANC-1 and HeLa (overexpressing HLA-A*11:01 and KRAS G12V) cells in nude mice. No destruction of the morphologies of the liver, spleen and brain were observed. We also found that KVA11-01 TCR-T could significantly infiltrate into tumor tissue and had a better homing ability. Conclusion: KVA11-01 TCR-T cells can effectively target a variety of malignant tumor cells carrying KRAS G12V mutation through in vitro and in vivo assay. KVA11-01 TCR-T cells have excellent biological activity, high specificity of target antigen and homing ability into solid tumor tissue. KVA11-01 TCR-T is expected to be an effective treatment for patients with KRAS G12V mutant solid malignancies.

Key words: Neoplasms, Tumor-infiltrating lymphocytes, DNA mutational analysis, T-cell receptor

中图分类号: 

  • R730.51

图1

从肿瘤浸润淋巴细胞中克隆KVA11-01并构建其对应表达载体"

表1

KVA11-01与TK34 TCR的信息对比"

TCR KVA11-01 TK34
HLA HLA-A*11:01 HLA-A*11:01
KRAS G12V specificity VVGAVGVGK VVGAVGVGK
TCR V alpha TRAV8-3*01/TRAJ4*01 TRAV3-3*01/TRAJ7*01
TCR V beta TRBV7-8*01/TRBD1*01/TRBJ1-3*01 TRBV4*01/TRBD2*01/TRBJ2-1*01

图2

KAV11-01和TK34 TCR亲和力及表达模式的比较"

图3

KVA11-01 TCR-T对抗原阳性肿瘤细胞的特异性杀伤"

图4

评估KVA11-01的脱靶-交叉反应与同种异体反应"

图5

KVA11-01 TCR-T的体内抗肿瘤活性"

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ISSN 1671-167X
CN 11-4691/R
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