WO2022012280A1 - 一种肺癌的外周血tcr标志物及其检测试剂盒和应用 - Google Patents

一种肺癌的外周血tcr标志物及其检测试剂盒和应用 Download PDF

Info

Publication number
WO2022012280A1
WO2022012280A1 PCT/CN2021/101540 CN2021101540W WO2022012280A1 WO 2022012280 A1 WO2022012280 A1 WO 2022012280A1 CN 2021101540 W CN2021101540 W CN 2021101540W WO 2022012280 A1 WO2022012280 A1 WO 2022012280A1
Authority
WO
WIPO (PCT)
Prior art keywords
lung cancer
ser
ala
gly
thr
Prior art date
Application number
PCT/CN2021/101540
Other languages
English (en)
French (fr)
Inventor
张志新
杨鑫
卓越
钟雪梅
治想林
Original Assignee
成都益安博生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 成都益安博生物技术有限公司 filed Critical 成都益安博生物技术有限公司
Publication of WO2022012280A1 publication Critical patent/WO2022012280A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/86—Lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a peripheral blood TCR marker of lung cancer and a detection kit and application thereof.
  • lung cancer is the most common malignant tumor in the world, ranking first in the incidence of various types of cancer (11.6%, tied with female breast cancer), and ranking first in the proportion of cancer deaths. (18.4%).
  • East Asia is one of the regions with the highest incidence of male lung cancer.
  • the incidence of lung cancer is about 40/100,000, which is higher than the world average (31.5/100,000).
  • lung cancer is the malignancy with the highest incidence in men and the malignancy with the highest death rate (both men and women).
  • the actual benefit of patients is very limited. Early diagnosis and early treatment are urgent needs to improve the treatment effect of lung cancer, improve the prognosis of patients, and save social resources.
  • the diagnostic methods for lung cancer are mainly divided into four categories: biochemical methods, molecular biological methods, cytological methods and imaging methods.
  • the patient's tissue samples (blood, serum, pleural effusion, etc.) are checked for tumor markers by methods such as enzyme-linked immunoassay (ELISA).
  • ELISA enzyme-linked immunoassay
  • CEA Carcinoembryonic antigen
  • CEA is a glycoprotein expressed in fetal epithelial cells with a molecular weight of 180kD. Very low expression in normal mucosal epithelium and other tissues of adult colon, high expression in gastrointestinal adenocarcinoma, lung adenocarcinoma and medullary thyroid carcinoma, which is mainly used to detect epithelial tumors, especially those derived from Adenocarcinoma of the glandular epithelium. The increase of CEA level in lung cancer adenocarcinoma is the most obvious, indicating that CEA has an important role in distinguishing benign and malignant lung tumors and their histological types. CEA can be used as a tumor marker for the detection of pleural effusion in patients with lung cancer.
  • SCCA Squamous Cell Carcinoma Antigen
  • Molecular markers commonly used in the diagnosis of lung cancer include carbohydrate antigens (Carbohydrate Antigen, including CA19-9, CA12-5, CA50, CA15-3, etc.), cytokeratin 19 fragment antigen 21-1 (Cytokeratin19 Fragment Antigen21-1, CYFRA21-1), human pleiotrophin (Pleiotrophin, PTN), neuron-specific enolase (Neuron-Specific Enolase, NSE), etc., but the specificity of detecting a single antigen for the diagnosis of lung cancer is low, and the detection of multiple antigens and antibodies Combination tests may improve the diagnostic efficiency of lung cancer.
  • Carbohydrate Antigen including CA19-9, CA12-5, CA50, CA15-3, etc.
  • cytokeratin 19 fragment antigen 21-1 Cytokeratin19 Fragment Antigen21-1, CYFRA21-1
  • human pleiotrophin Pleiotrophin, PTN
  • neuron-specific enolase Neuron-specific
  • Lung cancer tumor markers by biochemical methods are often difficult to be satisfactory in both sensitivity and specificity.
  • multiple markers must be comprehensively evaluated. This will inevitably increase the detection cost, and the comprehensive evaluation of various indicators makes it difficult to interpret the final detection results.
  • Tissue samples (blood, serum, biopsy specimens) of patients are examined for tumor markers by means of gene sequencing.
  • Common molecular biological tumor markers are:
  • EGFR gene is overexpressed in most non-small cell lung cancers (NSCLC) and is an important therapeutic target.
  • NSCLC non-small cell lung cancers
  • EGFR overexpression among which the expression rate of squamous cell carcinoma is about 85%, the expression rate of adenocarcinoma and large cell carcinoma is about 65%, and the expression rate of small cell carcinoma is lower.
  • KRAS is related to the occurrence and prognosis of lung cancer. 20% to 30% of NSCLC patients have KRAS mutation.
  • TTF-1 Thyroid transcription factor 1
  • TTF-1 is a nuclear protein with a molecular weight of 38-40KD, which is present in fetal lung tissue and adult type II alveolar epithelial cells, but not expressed in type I alveolar epithelial cells .
  • the positive expression of TTF-1 is a specific immunohistochemical diagnostic marker for lung adenocarcinoma, which is helpful for the identification of metastatic adenocarcinoma and primary adenocarcinoma.
  • N-CAM/CD56 Neural cell adhesion molecule
  • CD56 is a specific marker for the diagnosis of small cell carcinoma, which is strongly positive in 90% of SCLC cell membranes.
  • the diagnostic sensitivity and specificity of monoclonal antibody for lung cancer were 92.00% and 89.00%, respectively.
  • P53 is an important anti-cancer gene, which is related to biological functions such as cell cycle regulation, repair, cell differentiation, and apoptosis. Abnormal functions can lead to abnormal cell growth.
  • the most common mutation in lung cancer patients is P53.
  • P53 mutations in lung cancer cells predict poor prognosis in EMT, cell migration and tumor metastasis.
  • the molecular biology method of lung cancer tumor markers also has the problem that it is difficult to be satisfactory in both sensitivity and specificity.
  • multiple markers When applied to the diagnosis of lung cancer, multiple markers must be comprehensively evaluated. This will inevitably increase the detection cost, and the comprehensive evaluation of various indicators makes it difficult to interpret the final detection results.
  • the detection of exfoliated cells in sputum, bronchial secretions and pleural effusion of patients has some problems: the specificity of tumor detection is high, but the sensitivity is not ideal; when cancer cells are detected, the tumor cannot be located; in order to ensure Reliability, detection requires a large workload; extraction of pleural effusion is more painful and harmful to patients, and so on.
  • CT X-ray computed tomography
  • Nuclear medicine imaging examination such as Positron Emission Tomography/CT (PET/CT)
  • PET/CT Positron Emission Tomography/CT
  • PET/CT Positron Emission Tomography/CT
  • Staging because PET functional images are not affected by the anatomical structure, it can accurately display the recurrence and metastasis after the anatomical structure changes or the complex anatomical structure
  • the efficacy evaluation for the targeted drugs that inhibit tumor activity, the efficacy evaluation is more sensitive and accurate.
  • the disadvantage is that the equipment is expensive and the detection cost is high.
  • the purpose of the present invention is to provide a peripheral blood TCR marker for lung cancer and a detection kit and application thereof in view of the deficiencies in the prior art.
  • a peripheral blood TCR marker of lung cancer comprising at least one of the proteins shown in the sequence SpecSeq1-100, the specific sequence is shown in Table 1:
  • protein sequence of the marker is the sequence shown in SpecSeq1-100 after one or more bases are substituted, deleted and/or replaced, and a protein with the same function can be expressed.
  • the preparation includes a plasmid, viral vector or nucleic acid fragment of the T cell receptor containing the marker.
  • a kit for detecting lung cancer comprising antibodies that can specifically bind to the above markers.
  • a preparation comprising an antibody that can specifically bind to the above markers; the preparation can be used for diagnosis, prediction, detection or screening of lung cancer.
  • a protein chip for detecting lung cancer includes a substrate and a specific antibody spotted on the substrate.
  • the specific antibody is an antibody that can specifically bind to the above marker.
  • B lymphocytes and T lymphocytes in the human body are two important types of cells in the acquired immune system.
  • B cells recognize antigens through the B cell receptor (BCR) on the cell surface. Later, when B cells differentiate into plasma cells, BCR is expressed as an antibody and secreted outside the cell.
  • T cells recognize antigens through T cell receptors (TCRs) on the cell surface.
  • BCR and TCR The diversity of BCR and TCR is the basis for the establishment of the adaptive immune system.
  • the theoretical value of BCR diversity is 10 18 and the theoretical value of TCR diversity is 10 14 .
  • antigenic determinant 3 CDR3
  • CDR3 antigenic determinant 3
  • BCR and TCR will change with different antigenic stimulation. Therefore, the occurrence and development of diseases can be tracked by using BCR or TCR high-throughput sequencing results.
  • MCHII histocompatibility antigen II
  • Antigen fragments presented by normal cells do not cause an immune response due to immune tolerance.
  • the abnormal protein expressed by the mutated gene and its fragments are presented on the cell surface, which will cause the human immune system to produce a targeted immune response. Therefore, analyzing the changes of BCR or TCR can detect the occurrence and development of tumors.
  • a method for analyzing peripheral blood TCR sequence based on high-throughput sequencing is used to detect whether there is lung cancer, and the specific steps are as follows:
  • CDR3 antigenic determinant 3
  • the CDR3 sequences that appear in two or more samples are sorted from high to low according to "the sum of the repeated occurrences of the sequence C X in all lung cancer samples ⁇ the number of lung cancer samples containing this sequence" , to screen out the sequence of CDR3, a TCR marker for lung cancer.
  • the lung cancer characteristic index is defined as: in a certain sample, the sum of the repeated occurrences C X of all CDR3 sequences belonging to the lung cancer characteristic sequence set in the sample;
  • an artificial intelligence analysis model is first established by using the non-lung cancer control group samples and the TCR high-throughput sequencing data of lung cancer patients. Those with a higher risk of lung cancer;
  • the cost of the high-throughput sequencing instrument used in the present invention is lower than that of large-scale imaging equipment, and it can be outsourced to a third party.
  • the labor cost of sampling and processing is lower than the simultaneous detection of multiple markers, and it is also lower than that of a large number of cytology detection, therefore, the present invention greatly reduces the detection cost;
  • the present invention only needs to take a small amount of peripheral blood, and the sampling is simple and safe;
  • TCR CDR3 sequence described in the present invention can also be used for immunotherapy of lung cancer.
  • Fig. 1 is the CDR3 sequence of the control group and the characteristic sequence of lung cancer in the present invention.
  • the abscissa represents the sequence in which the CDR3 sequence of a specific amino acid combination was added to the control sequence set or the lung cancer characteristic sequence set, and the ordinate represents the logarithm of the number of times the sequence repeats in a sample, C X ; the immune profiles of lung cancer patients have There are many types of lung cancer feature sequences with high repetitions, and healthy people rarely have lung cancer feature sequences, while the lung cancer features of unknown subjects are more obvious, indicating a higher risk of lung cancer.
  • Fig. 2 is the lung cancer characteristic index of healthy people, non-tumor patients, non-lung cancer tumor patients and lung cancer patients, and the lung cancer characteristic index of healthy people, non-tumor patients, and non-lung cancer tumor patients according to the lung cancer characteristic sequence set in the present invention. All were significantly different from those of lung cancer patients, proving the specificity of the lung cancer feature sequence set. Based on this, it can be determined whether the unknown subject suffers from lung cancer.
  • Collected 1301 controls including healthy and non-tumor disease patients, 1300 were used for model establishment, 1 healthy person was used for validation), 41 lung cancer patients (40 were used for model establishment, 1 was used for validation) and The peripheral blood of 1 subject with unknown health status (10 mL per person) was obtained by high-throughput sequencing to obtain the antigenic determinant 3 (CDR3) amino acid sequence of the TCR of the subject and the control group to ensure the functional TCR of each sample.
  • the total number of CDR3 sequences is not less than 30000;
  • the immune profiles of 1 healthy person, 1 lung cancer patient and 1 subject with unknown health status are mapped with reference to the control sequence set and the lung cancer characteristic sequence set, as shown in Figure 1B-D. It can be seen from the figure that the immune profiles of lung cancer patients contain a large number of lung cancer characteristic sequences with a high number of repetitions; the immune profiles of healthy people contain only a very small number of lung cancer characteristic sequences; while subjects with unknown health status have The sequence of lung cancer characteristics is higher than that of healthy people, indicating that this person has a higher risk of developing lung cancer.
  • the peripheral blood (10 mL per person) of 304 non-lung cancer tumor patients and 6 subjects with unknown health status was collected, and the antigenic determinant 3 (CDR3) amino acid sequence of the TCR of the subjects and the control group was obtained by high-throughput sequencing , to ensure that the total number of CDR3 sequences of functional TCRs of each sample is not less than 30,000; the CDR3 sequences of TCRs of each sample are randomly sampled without replacement, so that the total number of CDR3 sequences of each sample is 30,000.
  • CDR3 sequences of TCRs of each sample are randomly sampled without replacement, so that the total number of CDR3 sequences of each sample is 30,000.
  • Example 3 According to the immune profiles of 100 healthy people, 45 non-tumor disease patients, and 40 lung cancer patients from Example 1, as well as 304 non-lung cancer tumor patients and 6 subjects with unknown health conditions newly obtained in Example 2 , to analyze the lung cancer characteristic index.
  • the lung cancer characteristic index is defined as: in a certain sample, the sum of the repeated occurrence times C X of all CDR3 sequences belonging to the lung cancer characteristic sequence set in the sample.
  • the analysis results are shown in Table 2 and Figure 2 below.
  • the lung cancer TCR marker CDR3 sequence of the present invention does have significant lung cancer specificity, and can not only be used for lung cancer to predict the risk of lung cancer in subjects, but also for biological immunotherapy of lung cancer in the future.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Food Science & Technology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种肺癌的外周血TCR标志物及其检测试剂盒和应用，该标志物包括序列SpecSeq1-100所示蛋白中的至少一种。基于高通量测序方法，只需要采取少量外周血，提取RNA，通过对样本的处理建立免疫图谱文库，再经过高通量测序和TCR数据分析，首先确定肺癌外周血中特征性TCR序列，然后将待测样本测试结果与该特征性TCR序列比对，从而确定是否患有肺癌。该方法能够同时比较巨大数量的肺癌特异性TCR序列，相比单独检测一种或几种标记物，具有更高的特异性和准确性，提高了诊断效率。

Description

一种肺癌的外周血TCR标志物及其检测试剂盒和应用 技术领域
本发明属于基因工程技术领域，尤其涉及一种肺癌的外周血TCR标志物及其检测试剂盒和应用。
背景技术
据世界卫生组织2018年最新统计数据显示，肺癌是全球最常见的恶性肿瘤，在各类癌症发病占比排名第一(11.6％，与女性乳腺癌并列)，癌症死亡占比中排名也是第一(18.4％)。东亚是男性肺癌发病率最高的地区之一，在中国肺癌的发病率约为40/10万人，高于世界平均水平(31.5/10万人)。在中国，肺癌是男性发病占比最高的恶性肿瘤，也是死亡占比最高的恶性肿瘤(男女均是)。现有的肺癌治疗手段虽多，但病人的实际获益十分有限。早诊断、早治疗，是提高肺癌治疗效果，改善患者预后，节约社会资源的迫切需求。
目前针对肺癌的诊断手段主要分为生物化学方法、分子生物学方法、细胞学方法和影像学方法四大类。
1、生物化学方法
通过酶联免疫检测(ELISA)等方法，检查患者组织样本(血液、血清、胸腔积液等)中的肿瘤标志物。常见生物化学检测肿瘤标志物有：
癌胚抗原(CEA)：CEA是表达于胎儿上皮细胞的一种糖蛋白，分子量为180kD。在成人结肠的正常黏膜上皮和其他组织当中有极低的表达，在胃肠道腺癌、肺腺癌和甲状腺髓样癌当中高表达，其主要是用于检测上皮性肿瘤，尤其是来源于腺上皮的腺癌。CEA水平在肺癌腺癌中的升高是最为明显的，表明CEA在鉴别肺部的良、恶性肿瘤及其组织学分型中具有重要的作用。CEA可以作为肺癌患者胸腔积液检测的肿瘤标志物。
鳞状细胞癌抗原(Squamous Cell Carcinoma Antigen，SCCA)是丝氨酸蛋白酶抑制剂家族的一员，在肺癌患者组织和血清中SCCA过度表达,可能是诊断肺癌更加有希望的生物标志物。
常用于肺癌诊断的分子标记物还有糖类抗原(Carbohydrate Antigen，包括CA19-9、CA12-5、CA50、CA15-3等)、细胞角蛋白19片段抗原21-1(Cytokeratin19 Fragment Antigen21-1,CYFRA21-1)、人多效蛋白(Pleiotrophin,PTN)、神经元特异性烯醇化酶(Neuron-Specific Enolase,NSE)等，但检测单个抗原对肺癌诊断的特异度较低，多个抗原抗体的组合检测可能会提高肺癌的诊断效率。
生物化学方法的肺癌肿瘤标志物，往往难以在灵敏度和特异性两方面都令人满意，应用于肺癌诊断时，必须综合评估多种标志物。这必然提高检测成本，并且多种指标的综合评估，对最终检测结果的判读造成了困难。
2、分子生物学方法
通过基因测序的方法，检查患者组织样本(血液、血清、活检标本)中的肿瘤标志物。常见的分子生物学肿瘤标志物有：
EGFR基因：EGFR在大多数非小细胞肺癌(NSCLC)中过表达且是重要的治疗靶标。目前已知大部分的NSCLC均存在EGFR过表达，其中鳞癌的表达率约为85％，腺癌和大细胞癌的表达率约为65％，而小细胞癌的表达率较低。
KRAS基因：KRAS与肺癌的发生以及预后有关。有20％～30％的NSCLC患者存在着KRAS的基因突变。
甲状腺转录因子1(TTF-1)：TTF-1是一种分子量为38～40KD的核蛋白，在胎儿的肺组织和成人Ⅱ型肺泡上皮细胞中存在，但在Ⅰ型肺泡上皮细胞中不表达。TTF-1的阳性表达是肺腺癌特异的免疫组化诊断标志物，有助于转移性腺癌和原发性腺癌的鉴别。
神经细胞黏附分子(N-CAM/CD56)等,其中,CD56是诊断小细胞癌的特异性标记物,其在90％SCLC细胞膜强阳性。而早期标志物监测中单克隆抗体对肺癌诊断效果中,诊断敏感性和特异性分别为:92.00％、89.00％。
P53是一个重要的抗癌基因，其与细胞周期的调控、修复、细胞分化、细胞凋亡等生物学功能有关，功能异常可导致细胞生长异常。肺癌患者最常见为P53突变，P53通过影响beta-catenin信号通路发挥对EMT的调控作用，降低P53表达能够促进betacatenin的细胞核内积累及转录活性，抑制beta-catenin信号通路可减弱P53降低所促进的肺癌细胞EMT、细胞迁移和肿瘤转移，其P53突变预示较差的预后。
综上所述，分子生物学方法的肺癌肿瘤标志物，也存在难以在灵敏度和特异性两方面都令人满意的问题。应用于肺癌诊断时，必须综合评估多种标志物。这必然提高检测成本，并且多种指标的综合评估，对最终检测结果的判读造成了困难。
3、细胞学方法
对病人痰液、支气管分泌物、胸腔积液进行脱落细胞检测，但存在一些问题：对肿瘤检测的特异性较高，但灵敏度不理想；检测出癌细胞，也无法对肿瘤进行定位；为保证可靠性，检测需要的工作量大；抽取胸腔积液对病人的痛苦和伤害较大等等。
4、影像学方法
1)X线计算机断层成像(Computed Tomography,CT)，其检出和诊断能力总体略逊于磁共振成像。目前除常见应用于肺癌临床诊断及分期外，更多应用于肺癌局部治疗的疗效评价。但CT存在辐射伤害较大，且难以发现≤1CM肿瘤等缺点。
2)核医学影像检查，如正电子发射计算机断层成像(Positron Emission Tomography/CT, PET/CT)，优势在于：对肿瘤进行分期，通过一次检查能够全面评价淋巴结转移及远处器官的转移；再分期，因PET功能影像不受解剖结构的影响，可准确显示解剖结构发生变化后或者是解剖结构复杂部位的复发转移灶；疗效评价，对于抑制肿瘤活性的靶向药物，疗效评价更加敏感、准确；指导放疗生物靶区的勾画、穿刺活检部位；评价肿瘤的恶性程度和预后。缺点在于设备昂贵，检测费用高。
3)磁共振成像(Magnetic Resonance Imaging,MRI)，具有无辐射影响，组织分辨率高，可以多方位、多序列参数成像，并具有形态结合功能(包括弥散加权成像、灌注加权成像和波谱分析)综合成像技术能力，成为临床肺癌检出、诊断和疗效评价的常用影像技术。但核磁共振检测存在设备昂贵体积巨大，占用空间大，检测费用高等缺点。
发明内容
本发明的目的在于：针对现有技术中存在的不足，提供一种肺癌的外周血TCR标志物及其检测试剂盒和应用。
本发明采用的技术方案如下：
一种肺癌的外周血TCR标志物，包括序列SpecSeq1-100所示蛋白中的至少一种，具体序列见表1：
表1 标志物序列
序列编号 蛋白序列
SpecSeq1 Ala Ser Ser Trp Leu Gly Gln Phe Asn Thr Glu Ala Phe
SpecSeq2 Ala Ser Ser Arg Phe Arg Asp Gly Arg Trp Pro Gln His
SpecSeq3 Ala Ser Ser Leu Ala Pro Asn Ser Tyr Tyr Gly Tyr Thr
SpecSeq4 Ala Ser Ala Ala Leu Gly Glu Gly Arg Tyr Glu Gln Tyr
SpecSeq5 Ala Thr Ser Arg Glu Tyr Thr Gly Thr Ser Thr Asp Thr Gln Tyr
SpecSeq6 Ser Val Gln Ser Ser Gly Thr Gly Glu Gly Pro Leu His
SpecSeq7 Ala Ser Ser Ser Pro Thr Val Leu Asp Thr Gln Tyr
SpecSeq8 Ala Ser Ser Leu Lys Pro Ser Gly Gly Asp Glu Gln Phe
SpecSeq9 Ser Val Leu Pro Gly Gly Arg Glu Lys Leu Phe
SpecSeq10 Ala Ser Ser Ile Ala Gly Asp Ser Ser Tyr Asn Glu Gln Phe
SpecSeq11 Ala Ser Ser Gln Val Val Arg Pro Arg Lys Glu Thr Gln Tyr
SpecSeq12 Ala Ile Ser Glu Ser Thr Ala Trp Ser Glu Glu Thr Gln Tyr
SpecSeq13 Ala Ser Ser Ser Pro Gln Gly Ile Arg Asp Val Glu Gln Phe
SpecSeq14 Ala Ser Ser Asp Pro Asp Ser Thr Ala Thr Asn Glu Lys Leu Phe
SpecSeq15 Ala Ser Ser Tyr Pro Asn Pro Gly Glu Gly Glu Ala Phe
SpecSeq16 Gln Gln Leu Lys Ala Arg Ala Gly Glu Arg Ala Val
SpecSeq17 Ala Thr Gly Tyr Gln Pro Gln His
SpecSeq18 Ala Ser Ser Leu Ala Leu Ala Gly Thr Thr Gln Glu Thr Gln Tyr
SpecSeq19 Ser Ala Arg Glu Val Ala Glu Leu Leu Tyr Glu Gln Tyr
SpecSeq20 Ala Thr Ser Arg Thr Arg Leu Ala Gly Gly Lys Asn Glu Gln Phe
SpecSeq21 Ala Ser Ile Leu Thr Gly Ser Ser Asn Gln Pro Gln His
SpecSeq22 Ala Thr Ser Glu Lys Glu Ser Asn Tyr Gly Tyr Thr
SpecSeq23 Ala Ser Ser Leu Leu Gly Val Gly Asp Phe Tyr Asn Glu Gln Phe
SpecSeq24 Ala Ser Ser Tyr Ile Lys Gly Asn Glu Gln Phe
SpecSeq25 Ala Ser Ser Leu Ser Tyr Ser Ala Asp Asn Glu Gln Phe
SpecSeq26 Ala Thr Ser Arg Val Arg Gly Leu Gly Gln Pro Gln His
SpecSeq27 Ala Ser Ser Phe Glu Glu Gly Gly Thr Asp Thr Gln Tyr
SpecSeq28 Ala Ser Thr Ser Tyr Gln Gly Arg Met Thr Tyr Gly Tyr Thr
SpecSeq29 Ala Ser Ser Ser Ala Gln Gly Gly Lys Asn Ile Gln Tyr
SpecSeq30 Pro Ala Pro Pro Ser Gly Arg Gly Val Thr Ser Ser Thr
SpecSeq31 Ala Ser Ser Tyr Ser Thr Thr Ala Gly Glu Tyr
SpecSeq32 Ala Ser Ser Thr Ser Trp Thr Asn Thr Glu Ala Phe
SpecSeq33 Ala Ser Arg Pro Lys Pro Gly Gln Ala Phe Ser Gly Asn Thr Ile Tyr
SpecSeq34 Ala Ser Ser Leu Pro Gly Leu Leu Ala Glu Gln Tyr
SpecSeq35 Ala Ile Ser Glu Arg Leu Gly Pro Tyr Glu Gln Tyr
SpecSeq36 Gln Arg Arg Pro Arg Gly Gly Ala Leu Arg Ala Val
SpecSeq37 Ser Ala Pro Gly Leu Ala Gly Gly Phe Leu Tyr Thr Gln Tyr
SpecSeq38 Ala Ser Ser Arg Leu Gln Arg Arg Asn Gln Pro Gln His
SpecSeq39 Ala Ser Ser Phe Arg Ser Gly Arg His Thr Thr Gly Glu Leu Phe
SpecSeq40 Gln Gln Leu Asp Arg Leu Gly Leu Trp Leu His
SpecSeq41 Ala Thr Ser Leu Ala Gly Ala Pro Asp Thr Gln Tyr
SpecSeq42 Ser Ala Arg Gly Leu Ala Gly Lys Leu Ser Ser Tyr Asn Glu Gln Phe
SpecSeq43 Ala Ser Ser His Asp Gly Ala Ala Ser Thr Tyr Glu Gln Tyr
SpecSeq44 Ala Ser Ser Val Arg Gln Ser Ser Asn Thr Glu Ala Phe
SpecSeq45 Ala Thr Ser Asp Leu Gln Gly Asp Ser Asn Glu Gln Phe
SpecSeq46 Ala Thr Ser Arg Asp Thr Leu Ala Gly Ala Asp Gly Asp Thr Gln Tyr
SpecSeq47 Ala Ser Lys Met Gly Glu Asn Glu Gln Phe
SpecSeq48 Ser Ala Leu Gly Thr Glu Glu Asn Tyr Gly Tyr Thr
SpecSeq49 Ala Ser Ser Pro Arg Pro Leu Ala Gly Ala Glu Asp Thr Gln Tyr
SpecSeq50 Ala Ser Ser Thr Thr Gln Asp Ser Asp Thr Gln Tyr
SpecSeq51 Ala Ser Ser Glu Asp Leu Ser Asp Arg Ser Arg Glu Thr Gln Tyr
SpecSeq52 Ala Ser Ile Pro Gly Asn Ile Tyr Glu Gln Tyr
SpecSeq53 Ala Thr Ser Glu Gly Gln Gly Glu Glu Glu Gln Tyr
SpecSeq54 Ala Ser Ser Arg Gly Glu Gly Thr Gln Glu Thr Gln Tyr
SpecSeq55 Ala Ser Arg Ser Gly Thr Thr Pro Ser Tyr Glu Gln Tyr
SpecSeq56 Ser Ala Arg Gly Glu Asp Arg Val Ile Asp Tyr Gly Tyr Thr
SpecSeq57 Ser Val Ala Ser Asp Gly Arg Gly Arg Ala Phe
SpecSeq58 Ala Ser Ser Tyr Arg Arg Gly Gln Asn Glu Gln Phe
SpecSeq59 Ser Pro Ser Gly Gly Arg Gly Thr Asp Thr Gln Tyr
SpecSeq60 Ala Ser Ser Phe Gly Gln Arg Gly Ser Asn Thr Gly Glu Leu Phe
SpecSeq61 Ala Ser Ser Gly Thr Gly Met Asn Thr Asp Thr Gln Tyr
SpecSeq62 Ala Ser Thr Ser Arg Asn Glu Ala Phe
SpecSeq63 Ala Ser Ser Asp Lys Pro Ile Glu Ala Phe
SpecSeq64 Gln Gln Leu Gly Pro Ser Gly Asp Asp Pro Arg Asp Pro Val
SpecSeq65 Ala Ser Ser Pro Met Pro Ala Gly Leu Asn Glu Gln Phe
SpecSeq66 Ala Ser Ser Phe Phe Asp Ser Asn Ser Gly Ala Asn Val Leu Thr
SpecSeq67 Ala Ser Ser Leu Lys Thr Gly Thr Ile Tyr Gly Tyr Thr
SpecSeq68 Ala Ser Gly Lys Ser Gln Tyr Thr Glu Ala Phe
SpecSeq69 Ser Val Val Trp Ser Gly Pro Leu Tyr Gly Tyr Thr
SpecSeq70 Ala Ser Ser Glu Gly Gly Phe Pro Leu Asn Glu Gln Phe
SpecSeq71 Ala Thr Ser Asp Leu Val Lys Thr Asp Thr Gln Tyr
SpecSeq72 Ala Ser Ser Gln Asp Phe Ser Val Trp Asp Thr Gln Tyr
SpecSeq73 Ala Ser Met Arg Thr Ala Ala Tyr Asn Glu Gln Phe
SpecSeq74 Ser Val Leu Asn Leu Ala Asp Lys Asn Ile Gln Tyr
SpecSeq75 Ala Ser Ser Ile Ala Gly Asp Ser Ser Tyr Asn Gly Ala Val
SpecSeq76 Ala Ser Ser Met Pro Gly Thr Arg Gly Arg Arg Glu Gln Tyr
SpecSeq77 Ala Ser Ser Tyr Ser Ala Ser Thr Val Gly Glu Thr Gln Tyr
SpecSeq78 Ala Ser Ser Gln Leu Gly Gly Arg Val Thr Thr Asp Thr Gln Tyr
SpecSeq79 Ala Ser Arg Ser Gly Ser Met Tyr Thr Asp Thr Gln Tyr
SpecSeq80 Ala Ser Ser Thr Val Ala Gly Arg Gly Tyr Glu Gln Tyr
SpecSeq81 Ala Thr Ser Ser Val Ala Gly Glu Glu Gln Phe
SpecSeq82 Ala Ile Ser Glu Ser Asn Glu Ala Val Arg Asp Thr Gln Tyr
SpecSeq83 Ala Ser Ser Tyr Gly Gly Gln Met Asn Glu Gln Phe
SpecSeq84 Pro Ala Ala Trp Ser Leu Ala Gly Ala Val Gly Lys Arg Pro Ser Thr
SpecSeq85 Ala Ser Ser Leu Glu Ser Gly Gly Trp Asn Gly Tyr Thr
SpecSeq86 Ala Ser Ser Leu Lys Ile Arg Thr Phe Asn Gln Pro Gln His
SpecSeq87 Ala Ser Gly Arg Leu Phe Gly Thr Asp Thr Glu Ala Phe
SpecSeq88 Ala Ser Ser Leu Pro Gly Gln Ile Leu Tyr Glu Gln Tyr
SpecSeq89 Ala Ser Ser Lys Leu Gly Gln Gly Val Thr Glu Ala Phe
SpecSeq90 Ala Ser Ser Ala Gly Thr Leu Tyr Thr
SpecSeq91 Ala Ser Ser Thr Arg Gln Ala Ala Gln Asn Ser Asn Gln Pro Gln His
SpecSeq92 Ala Ser Ser Tyr Gly Pro Gly Trp Gly Asn Glu Gln Phe
SpecSeq93 Ala Ser Ser Glu Asp Ser Glu Tyr Asn Glu Gln Phe
SpecSeq94 Ser Val Glu Gly Ser Thr Ser Gly Gly Pro Ser Thr Asp Thr Gln Tyr
SpecSeq95 Ala Ser Ser Pro Ser Gly Ser Gly Tyr Gln Glu Thr Gln Tyr
SpecSeq96 Ala Ser Ser Gln Ser Cys Arg Ala Gln Pro Gln His
SpecSeq97 Ala Ser Ser Gln Thr Gly Glu Ser Ile Gln Tyr
SpecSeq98 Ala Ser Ser Tyr Lys Glu Gly Val Ser Pro Leu His
SpecSeq99 Ala Ser Ser Gly Arg Arg Tyr Asn Val Glu Gln Phe
SpecSeq100 Ala Ser Ser Ala Phe Thr Lys Ser Thr Asp Arg Gln Tyr
进一步地，标志物的蛋白序列为SpecSeq1-100所示的序列经取代、缺失和/或替换一个或多个碱基后，能表达相同功能的蛋白。
上述的标志物在制备检测或治疗肺癌的制剂中的应用。
进一步地，制剂包括含有该标志物的T细胞受体的质粒、病毒载体或核酸片段。
一种用于肺癌检测的试剂盒，包括能与上述标志物发生特异性结合的抗体。
一种制剂，包括能与上述标志物发生特异性结合的抗体；制剂可用于对肺癌进行诊断、预测、检测或筛查。
一种检测肺癌的蛋白质芯片，蛋白质芯片包括基片和点样在基片上特异性抗体，特异性抗体为能与上述标志物发生特异性结合的抗体。
本发明的原理为：人体内的B淋巴细胞和T淋巴细胞是获得性免疫系统中重要的两类细胞。B细胞通过细胞表面的B细胞受体(BCR)识别抗原，后期BCR在B细胞分化成浆细胞时，表达成抗体，分泌到细胞外。T细胞通过细胞表面的T细胞受体(TCR)识别抗原。BCR和TCR的多样性是建立获得性免疫系统的基础。BCR多样性的理论值是10 18，TCR多样性的理论值是10 14。BCR与TCR序列中，抗原决定簇3(CDR3)是决定其抗原特异性最重要的部分，因此CDR3的序列被认为可以代表BCR、TCR序列的特性。
在各种疾病中，随着不同的抗原刺激，BCR和TCR的多样性或者表达水平都会发生改变。因此，利用BCR或者TCR高通量测序结果可以追踪疾病的发生、发展。人体内细胞中，衰老蛋白质降解后，其片段会被运输到细胞表面，通过组织相容性抗原II(MCHII)呈递给免疫系统中的T细胞。正常细胞呈递的抗原片段，由于免疫耐受的关系，不会引起免疫反应。一旦当正常细胞出现癌变后，突变的基因表达的异常蛋白，其片段被呈递到细胞表面后，就会引起人体免疫系统产生针对性的免疫反应。因此，分析BCR或TCR的变化，能够检测出肿瘤的发生和发展。
本发明应用时，采用基于高通量测序分析外周血TCR序列方法以检测是否患有肺癌，具体步骤如下：
(1)采集受试者和对照组外周血，通过高通量测序得到受试者和对照组的TCR的抗原决定簇3(CDR3)氨基酸序列，保证每个样本的功能性TCR的CDR3序列总数综合不低于30000；
(2)对每个样本的TCR的CDR3序列进行随机不放回抽样，使每个样本的CDR3序列数量总和均为30000；对任一特定CDR3序列X，在该样本中重复出现次数计为C X；
(3)TCR CDR3数据分析：通过分析确定肺癌TCR标志物CDR3序列：
a)将对照组样本所有CDR3序列归总、去重，设为对照序列集；
b)将肺癌样本所有CDR3序列归总、去重，再去除所有与对照序列集中包含CDR3序列重复的序列，设为肺癌特征序列集；
c)将肺癌特征序列集中，出现于两个及以上样本中的CDR3序列，按“在所有肺癌样本 中该序列重复出现次数C X的总和×包含该序列的肺癌样本数”从高到低排序，以筛选出肺癌TCR标志物CDR3序列。
(4)利用肺癌特征性指数判断未知受试者罹患肺癌风险：
a)根据健康对照组、非肿瘤病人、非肺癌肿瘤患者、肺癌患者以及未知健康状况受试者的免疫图谱，分析其肺癌特征性指数；
其中肺癌特征性指数定义为：某个样本中，属于肺癌特征序列集的所有CDR3序列在该样本内重复出现次数C X的总和；
b)当未知健康状况受试者的肺癌特征性指数高于或接近“其它肿瘤”组的平均值+2×SD时，此人具有较高风险罹患肺癌；如其肺癌特征指数接近健康人或非肿瘤疾病组平均值，则表明其患肺癌风险低。
综上所述，由于采用了上述技术方案，本发明的有益效果是：
1、本发明中，首先利用非肺癌的对照组样本和肺癌病人的TCR高通量测序数据建立了人工智能分析模型，通过和这些肺癌特异性TCR序列对比，可以清楚的判断待测样本中是否有较高肺癌风险者；
2、通过高通量测序分析TCR变化可以发现很早期的肺癌，利用肺癌特有的TCR CDR3序列分析人的免疫系统中的T细胞对肺癌的反应，是一种新型的检测方法；
3、本发明中，由于采用高通量测序技术，同时比较巨大数量的特异性TCR序列，比起单独检测一种或几种标记物，具有更高的特异性和准确性；
4、本发明中使用的高通量测序仪器成本低于大型影像学设备，且可向第三方外包，此外，采样、处理的人力成本低于同时检测多种标记物，也低于大量细胞学检测，因此，本发明大大降低了检测成本；
5、本发明只需要采取少量外周血，采样简便、安全；
6、本发明中所述TCR CDR3序列，也可用于肺癌的免疫治疗。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明中，对照组CDR3序列及肺癌特征序列。横坐标代表某一特定氨基酸组合的CDR3序列被加入对照序列集合或肺癌特征序列集合的先后顺序,纵坐标代表该序列在某一样 本中重复出现次数C X的对数值；肺癌患者的免疫图谱具有多个种类且重复次数较高的肺癌特征序列，健康人极少肺癌特征序列，而未知受试者的肺癌特征比较明显，说明罹患肺癌风险较高。
图2为本发明中，针对肺癌特征序列集，计算健康人、非肿瘤病人、非肺癌肿瘤患者和肺癌患者的肺癌特征性指数，健康人、非肿瘤病人、非肺癌肿瘤患者的肺癌特征性指数均与肺癌患者具有显著差异，证明了肺癌特征序列集的特异性。据此可以判断未知受试者是否罹患肺癌。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
通过免疫图谱分析，获得肺癌TCR标志物CDR3序列集：
1、采样及免疫图谱分析
采集1301名对照组(包括健康人和非肿瘤疾病病人，1300人用于建立模型，1个健康人用于验证)、41名肺癌患者(40人用于建立模型，1人用于验证)及1名未知健康状况受试者的外周血(每人10mL)，通过高通量测序得到受试者和对照组的TCR的抗原决定簇3(CDR3)氨基酸序列，保证每个样本的功能性TCR的CDR3序列总数综合不低于30000；
2、对每个样本的TCR的CDR3序列进行随机不放回抽样，使每个样本的CDR3序列数量总和均为30000。对任一特定CDR3序列X，在单样本测序结果中重复出现次数计为C X；
3、通过分析TCR CDR3数据，确定肺癌TCR标志物CDR3序列：
a)将1300名用于建立模型的对照组样本的所有CDR3序列归总去重，设为对照序列集；
b)将40名用于建立模型的肺癌样本的所有CDR3序列归总去重，再去除所有与对照序列集中包含序列重复的序列，设为肺癌特征序列集。如图1A所示，其中横坐标代表某一特定氨基酸组合的CDR3序列被加入对照序列集合或肺癌特征序列集合的先后顺序,纵坐标代表该序列在某一样本中重复出现次数C X的对数值。
c)按照同样作图方法，将1名健康人、1名肺癌患者和1名健康状况未知受试者的免疫图谱，参照对照序列集合和肺癌特征序列集合进行作图，如图1B-D。从图中可见，肺癌患者的免疫图谱中，含有较多种类且较高重复出现次数的肺癌特征序列；健康人的免疫图谱中，只有极少量肺癌特征序列；而未知健康状况受试者，有高于健康人的肺癌特征序列，说明此人有较高风险罹患肺癌。
d)将肺癌特征序列集中，将所有出现在两个及以上参与建模肺癌样本里的CDR3序列，按“所有参与建模肺癌样本里该序列单样本中重复出现次数C X的总和×包含该序列的参与建模肺癌样本数”从高到低排序，排名前100者即为肺癌TCR标志物CDR3序列，具体序列如SpecSeq1～100所示。
实施例2
验证肺癌TCR标志物CDR3序列集的特异性：
1、采样及免疫图谱分析
采集304名非肺癌的肿瘤患者、6名未知健康状况受试者的外周血(每人10mL)，通过高通量测序得到受试者和对照组的TCR的抗原决定簇3(CDR3)氨基酸序列，保证每个样本的功能性TCR的CDR3序列总数综合不低于30000；对每个样本的TCR的CDR3序列进行随机不放回抽样，使每个样本的CDR3序列数量总和均为30000。
2、从实施例1的对照组中随机挑选100名健康人及45名非肿瘤疾病病人。
3、根据来自实施例1的100名健康人、45名非肿瘤疾病病人、40名肺癌患者，以及实施例2新获取的304名非肺癌肿瘤患者、6名未知健康状况受试者的免疫图谱，分析其肺癌特征性指数。
其中肺癌特征性指数定义为：某个样本中，属于肺癌特征序列集的所有CDR3序列在该样本内重复出现次数C X的总和。
分析结果见下表2及图2。肺癌组与健康人(p＝1.9E-78)、非肿瘤疾病(p＝6.6E-43)、其它肿瘤(p＝9.9E-162)都有显著差异，这证明了肺癌特征序列集的特异性。
表2 不同样本组的肺癌特征性指数
Figure PCTCN2021101540-appb-000001
Figure PCTCN2021101540-appb-000002
Figure PCTCN2021101540-appb-000003
Figure PCTCN2021101540-appb-000004
Figure PCTCN2021101540-appb-000005
Figure PCTCN2021101540-appb-000006
Figure PCTCN2021101540-appb-000007
Figure PCTCN2021101540-appb-000008
Figure PCTCN2021101540-appb-000009
Figure PCTCN2021101540-appb-000010
Figure PCTCN2021101540-appb-000011
4、分析各组的肺癌特征指数(下表3)，未知健康状况受试者(检测样本)中，前4人的肺癌特征指数高于或接近“其它肿瘤”组的平均值+2×SD(347+2×1048＝2443)，此4人具有较高风险罹患肺癌；后2人肺癌特征指数接近健康人或非肿瘤疾病组平均值，罹患肺癌风险低。与临床体检结果对照后，前4人确为肺癌患者，而后2人为健康人。证明了利用肺癌特征序列集及肺癌特征性指数，预测受试者罹患肺癌风险的可行性。
表3 肺癌特征性指数分析表
Figure PCTCN2021101540-appb-000012
综上所述，本发明所述肺癌TCR标志物CDR3序列，确实具有显著的肺癌特异性，不仅可以用于肺癌预测受试者罹患肺癌风险，未来还可用于肺癌的生物免疫治疗。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (7)

  1. 一种肺癌的外周血TCR标志物，其特征在于，包括序列SpecSeq1-100所示蛋白中的至少一种。
  2. 根据权利要求1所述的肺癌的外周血TCR标志物，其特征在于，所述标志物的蛋白序列为SpecSeq1-100所示的序列经取代、缺失和/或替换一个或多个碱基后，能表达相同功能的蛋白。
  3. 权利要求1或2所述的标志物在制备检测或治疗肺癌的制剂中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述制剂包括含有该标志物的T细胞受体的质粒、病毒载体或核酸片段。
  5. 一种用于肺癌检测的试剂盒，其特征在于，包括能与权利要求1所述标志物发生特异性结合的抗体。
  6. 一种制剂，其特征在于，包括能与权利要求1所述标志物发生特异性结合的抗体。
  7. 一种检测肺癌的蛋白质芯片，其特征在于，所述蛋白质芯片包括基片和点样在基片上特异性抗体，所述特异性抗体为能与权利要求1所述标志物发生特异性结合的抗体。
PCT/CN2021/101540 2020-07-11 2021-06-22 一种肺癌的外周血tcr标志物及其检测试剂盒和应用 WO2022012280A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010665298.3 2020-07-11
CN202010665298.3A CN111665359A (zh) 2020-07-11 2020-07-11 一种肺癌的外周血tcr标志物及其检测试剂盒和应用

Publications (1)

Publication Number Publication Date
WO2022012280A1 true WO2022012280A1 (zh) 2022-01-20

Family

ID=72392037

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/101540 WO2022012280A1 (zh) 2020-07-11 2021-06-22 一种肺癌的外周血tcr标志物及其检测试剂盒和应用

Country Status (2)

Country Link
CN (1) CN111665359A (zh)
WO (1) WO2022012280A1 (zh)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111665359A (zh) * 2020-07-11 2020-09-15 成都益安博生物技术有限公司 一种肺癌的外周血tcr标志物及其检测试剂盒和应用
CN112321700A (zh) * 2020-09-21 2021-02-05 四川省人民医院 一种新型冠状病毒感染的外周血tcr标志物及其检测试剂盒和应用
CN113125724A (zh) * 2021-03-15 2021-07-16 成都益安博生物技术有限公司 一种经典霍奇金淋巴瘤的外周血tcr标志物及其检测试剂盒和应用
CN113030473A (zh) * 2021-03-15 2021-06-25 成都益安博生物技术有限公司 一种急性b淋巴细胞白血病的外周血tcr标志物及其检测试剂盒和应用
CN113563454A (zh) * 2021-07-23 2021-10-29 成都益安博生物技术有限公司 一种IgA肾病的外周血TCR标志物及其检测试剂盒和应用
CN113567682A (zh) * 2021-07-23 2021-10-29 成都益安博生物技术有限公司 一种阿尔茨海默病的外周血tcr标志物及其检测试剂盒和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008108257A1 (ja) * 2007-03-05 2008-09-12 International Institute Of Cancer Immunology, Inc. 癌抗原特異的ｔ細胞のレセプター遺伝子およびそれによりコードされるペプチドならびにそれらの使用
CN107488737A (zh) * 2017-10-12 2017-12-19 中国医学科学院肿瘤医院 一种检测外周血tcr可变区编码基因的液体活检试剂盒及其应用
CN110627895A (zh) * 2018-06-25 2019-12-31 北京大学 肺癌特异性tcr及其分析技术和应用
CN111665359A (zh) * 2020-07-11 2020-09-15 成都益安博生物技术有限公司 一种肺癌的外周血tcr标志物及其检测试剂盒和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008108257A1 (ja) * 2007-03-05 2008-09-12 International Institute Of Cancer Immunology, Inc. 癌抗原特異的ｔ細胞のレセプター遺伝子およびそれによりコードされるペプチドならびにそれらの使用
CN107488737A (zh) * 2017-10-12 2017-12-19 中国医学科学院肿瘤医院 一种检测外周血tcr可变区编码基因的液体活检试剂盒及其应用
CN110627895A (zh) * 2018-06-25 2019-12-31 北京大学 肺癌特异性tcr及其分析技术和应用
CN111665359A (zh) * 2020-07-11 2020-09-15 成都益安博生物技术有限公司 一种肺癌的外周血tcr标志物及其检测试剂盒和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
REUBEN ALEXANDRE, ZHANG JIEXIN, CHIOU SHIN-HENG, GITTELMAN RACHEL M., LI JUN, LEE WON-CHUL, FUJIMOTO JUNYA, BEHRENS CARMEN, LIU XI: "Comprehensive T cell repertoire characterization of non-small cell lung cancer", NATURE COMMUNICATIONS, vol. 11, no. 1, 1 December 2020 (2020-12-01), XP055886793, DOI: 10.1038/s41467-019-14273-0 *
WANG XIANG; ZHANG BOTAO; YANG YIKUN; ZHU JIAWEI; CHENG SHUJUN; MAO YOUSHENG; FENG LIN; XIAO TING: "Characterization of Distinct T Cell Receptor Repertoires in Tumor and Distant Non-tumor Tissues from Lung Cancer Patients", GENOMICS PROTEOMICS AND BIOINFORMATICS, BEIJING GENOMICS INSTITUTE, BEIJING, CN, vol. 17, no. 3, 1 June 2019 (2019-06-01), CN , pages 287 - 296, XP085863935, ISSN: 1672-0229, DOI: 10.1016/j.gpb.2018.10.005 *

Also Published As

Publication number Publication date
CN111665359A (zh) 2020-09-15

Similar Documents

Publication Publication Date Title
WO2022012280A1 (zh) 一种肺癌的外周血tcr标志物及其检测试剂盒和应用
WO2022012282A1 (zh) 一种肝癌的外周血tcr标志物及其检测试剂盒和应用
WO2022012291A1 (zh) 一种乳腺癌的外周血tcr标志物及其检测试剂盒和应用
WO2022012284A1 (zh) 一种黑色素瘤的外周血tcr标志物及其检测试剂盒和应用
WO2022012279A1 (zh) 一种大肠癌的外周血tcr标志物序列及其检测试剂盒和应用
WO2022012292A1 (zh) 一种胰腺癌的外周血tcr标志物及其检测试剂盒和应用
WO2022012289A1 (zh) 一种卵巢癌的外周血tcr标志物及其检测试剂盒和应用
CN111172279B (zh) 外周血甲基化基因及idh1联合检测诊断肺癌模型
WO2022012293A1 (zh) 一种子宫内膜癌的外周血tcr标志物及其检测试剂盒和应用
Fujikawa et al. The clinical significance of lymphovascular invasion in gastric cancer
WO2022194039A1 (zh) 一种急性b淋巴细胞白血病的外周血tcr标志物及其检测试剂盒和应用
WO2022012290A1 (zh) 一种前列腺癌的外周血tcr标志物及其检测试剂盒和应用
WO2022194042A1 (zh) 一种急性髓细胞性白血病的外周血tcr标志物及其检测试剂盒和应用
WO2022194033A1 (zh) 一种弥漫大b细胞淋巴瘤的外周血tcr标志物及其检测试剂盒和应用
WO2022012283A1 (zh) 一种宫颈癌的外周血tcr标志物及其检测试剂盒和应用
WO2022194036A1 (zh) 一种经典霍奇金淋巴瘤的外周血tcr标志物及其检测试剂盒和应用
Qiang et al. Carcinoembryonic antigen, α-fetoprotein, and Ki67 as biomarkers and prognostic factors in intrahepatic cholangiocarcinoma: A retrospective cohort study
Cai et al. Nomogram to predict cancer specific survival in patients with pathological stage IA non-small cell lung cancer
CN109971853A (zh) 一种与非小细胞肺癌诊断相关的分子标志物及其应用
Huang et al. Evaluation of HE4 in the diagnosis and follow up of non-small cell lung cancers
Salama et al. Immunohistochemical comparison of CD44 and hypoxia inducible factor-1 alpha (HIF-1α) in oral squamous cell carcinoma and oral epithelial dysplasia: a comparative study
Xie et al. Artificial intelligence scale-invariant feature transform algorithm-based system to improve the calculation accuracy of Ki-67 index in invasive breast cancer: a multicenter retrospective study
Sun et al. Radiomics nomogram for the prediction of Ki-67 index in advanced non-small cell lung cancer based on dual-phase enhanced computed tomography
Zhang et al. Identification of plasma SAA2 as a candidate biomarker for the detection and surveillance of non-small cell lung cancer.
Luo et al. Strategies for five tumour markers in the screening and diagnosis of female breast cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21842619

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21842619

Country of ref document: EP

Kind code of ref document: A1