WO2022012198A1 - 一种用于杀伤肿瘤的tcr-t细胞及其制备方法和应用 - Google Patents

一种用于杀伤肿瘤的tcr-t细胞及其制备方法和应用 Download PDF

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WO2022012198A1
WO2022012198A1 PCT/CN2021/097813 CN2021097813W WO2022012198A1 WO 2022012198 A1 WO2022012198 A1 WO 2022012198A1 CN 2021097813 W CN2021097813 W CN 2021097813W WO 2022012198 A1 WO2022012198 A1 WO 2022012198A1
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cells
tumor
tcr
cell
cancer
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刘海平
李翔
李游
卞超超
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广州泛恩生物科技有限公司
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Definitions

  • the invention relates to the technical field of immunotherapy, in particular to a TCR-T cell used for killing tumors and a preparation method and application thereof.
  • T cells recognize antigens presented by Human Leukocyte Antigen (HLA) on the surface of target cells through the T Cell Receptor (TCR) on their surface to directly attack and kill target cells.
  • HLA Human Leukocyte Antigen
  • TCR T Cell Receptor
  • Traditional TCR-T cell therapy that recognizes tumor antigens is directed against known tumor antigens.
  • TCRs that can recognize these known antigens are cloned and their corresponding HLA subtypes are identified. Then, patients with simultaneous expression of the tumor antigen and HLA subtype were selected to prepare TCR-T cells for reinfusion therapy.
  • few tumor antigens are currently known, and these antigens are only expressed in a small subset of patients. Coupled with the simultaneous expression of the corresponding HLA molecules, only very few patients can meet these two requirements at the same time. Most patients cannot be treated with such TCR-T cells, which recognize known tumor antigens.
  • tumor cells the amino acid sequence variation caused by gene mutation is the main source of tumor antigens.
  • tumor mutations are random, these tumor antigens generated by random mutation are usually unique to each patient. That is, very few tumor antigens are shared among patients. Therefore, it is necessary to establish corresponding TCR-T cells that recognize the tumor antigens unique to each patient in order to achieve effective treatment for most patients.
  • T cells that recognize tumor antigens in the body will be activated by tumor antigens and enter tumor tissues to expand and kill tumor cells in tumors. Therefore, if T cells that recognize tumor antigens can be identified and isolated from the patient's tumor, the TCR sequences carried by them can be obtained, and expressed in the patient's peripheral blood T cells, the patient's own, that is, individualized tumor can be established. Antigen-recognizing TCR-T cells for personalized tumor therapy.
  • tumor tissue also contains a large number of T cells that do not recognize tumor antigens from adjacent tissues and blood, which is a mixture of various T cells.
  • TCR-T cell therapy To obtain TCRs that can recognize tumor antigens from these mixed T cells, it is first necessary to stimulate tumor antigens to determine which T cells can recognize tumor antigens, and then obtain the TCR sequences carried by these T cells for individualization. TCR-T cell therapy.
  • T cells that recognize tumor antigens analyze the gene expression characteristics of these T cells, compare the molecular characteristics differences between T cells that recognize tumor antigens and T cells that do not recognize tumor antigens, and find the T cells that recognize tumor antigens.
  • Cell-specific molecular markers through which T cells that can recognize tumor antigens can be isolated and obtained for subsequent treatment.
  • T cells need to be isolated from tumor tissue and cultured in vitro.
  • the tumor antigens in the tumor tissue must be identified, and then the isolated T cells are stimulated with the identified tumor antigens in vitro.
  • T cells that can be stimulated by tumor antigens can be proved to be able to recognize tumor antigens. Due to the extremely complex experimental process and high technical difficulty of the entire identification, it is still impossible to systematically identify and analyze the tumor antigens of T cells in a tumor tissue. It also makes it impossible to quickly obtain T cells and TCRs that recognize tumor antigens from tumor tissues for treatment.
  • the purpose of the present invention is to provide a technical solution that can accurately and rapidly analyze the tumor antigen recognition ability of T cells in view of the above technical problems to be solved, and compare T cells that recognize tumor antigens with T cells that do not recognize tumor antigens.
  • the differences in gene expression between the two groups the molecular markers unique to T cells that recognize tumor antigens in tumor tissues are found, and these molecular markers are used to realize the rapid separation of T cells that recognize tumor antigens, so as to quickly obtain TCRs that recognize tumor antigens. , established an individualized TCR-T cell therapy technology that recognizes tumor antigens.
  • the present invention provides a TCR-T cell for killing tumors
  • the TCR-T cell is a T cell carrying a TCR that recognizes the tumor antigen
  • the TCR in the TCR-T cell is derived from the following Any one or more of the T cells in:
  • CD4 T cells expressing one or more of these genes TNFRSF18, CXCL13, TNFRSF4, TNFSF8, ENTPD1, ACP5, LAYN, TNFRSF9, CTLA4, CD200, TIGIT in the tumor;
  • CD8 T cells expressing one or more of the genes TNFRSF18, CXCL13, CXCR6, GALNT2, ENTPD1, ACP5, HAVCR2, LAYN, TNFRSF9, CTLA4, CD109 in the tumor.
  • the present invention also provides the application of the TCR-T cells in the preparation of medicaments for treating tumors.
  • the tumor includes but is not limited to any one or more of lung cancer, melanoma, intestinal cancer, liver cancer, gastric cancer, breast cancer, cervical cancer, ovarian cancer, kidney cancer, bladder cancer, and esophageal cancer.
  • the present invention also provides the TCR-T cell preparation method, which comprises: introducing one or more nucleotide sequences of TCRs that recognize tumor antigens into T cells for expression, and constructing the TCR-T cells .
  • the steps of identifying TCRs that recognize tumor antigens are as follows: by flow sorting, magnetic bead separation or in situ sequencing of tumor tissue, the markers TNFRSF18 and CXCL13 expressing CD4 T cells that recognize tumor antigens are obtained from tumor tissue.
  • the present invention also provides a method for identifying T cells and TCRs that recognize tumor antigens, which includes the following steps: by cloning high-frequency TCRs in tumor tissue, establishing TCR-expressing TCR-T cells, using tandem expressing tumor antigens The antigen-presenting cells of the gene are stimulated with tumor antigens in vitro, and the TCR carried by the stimulated TCR-T cells is the TCR that recognizes the tumor antigen, and the T cell that carries the TCR is the T cell that recognizes the tumor antigen;
  • the TCR in the TCR-T cells is derived from any one or more of the following T cells:
  • CD4 T cells expressing one or more of these genes TNFRSF18, CXCL13, TNFRSF4, TNFSF8, ENTPD1, ACP5, LAYN, TNFRSF9, CTLA4, CD200, TIGIT in the tumor;
  • CD8 T cells expressing one or more of the genes TNFRSF18, CXCL13, CXCR6, GALNT2, ENTPD1, ACP5, HAVCR2, LAYN, TNFRSF9, CTLA4, CD109 in the tumor.
  • the TCR-T cells of the present invention can be effectively used in the treatment of tumors, especially immunotherapy.
  • Figure 1 shows the stimulatory response of high frequency TCRs in tumor CD4 and CD8 T cells to tumor antigens.
  • FIG. 2 shows that TCR-T cells constructed from TCRs of tumor antigen-recognizing T cell subsets can effectively treat tumors (*P ⁇ 0.05; **P ⁇ 0.01).
  • Figure 3 shows molecular marker genes of tumor antigen-recognizing T cell subsets, wherein a shows specific gene expression differences in CD4 T cells, and b shows specific gene expression differences in CD8 T cells.
  • Figure 4 shows that T cells sorted by molecular markers of tumor antigen-recognizing T cells can effectively recognize tumor antigens, where a represents CD4 T cells and b represents CD8 T cells (*P ⁇ 0.05; **P ⁇ 0.01) .
  • DNA of tumor tissue was extracted with Qiagen's DNeasy Blood&Tissue Kit (Cat. No. 69506), and RNA of tumor tissue was extracted with Qiagen's miRNeasy-Mini Kit (Cat. No. 217004).
  • the obtained DNA and RNA samples are then sent to a sequencing service company for exon and RNA sequencing.
  • the DNA extracted from peripheral blood leukocytes is sent to a sequencing service company for exon sequencing, which is used as a reference gene sequence for detecting gene mutations in tumors. By comparing the exon sequences of tumor tissue and peripheral blood leukocytes, the mutated sites and corresponding genes in tumor tissue were found.
  • the expression abundance of mutant gene in tumor was sorted, and the mutation with higher expression in tumor tissue was selected as tumor antigen. Then, the 25-amino acid epitope peptide centered on the mutation site and the new polypeptide generated by the frameshift mutation are connected in series as tumor antigens to synthesize the genes of these tumor mutation antigens in series, and express them after EBV virus infection. immortalized patient B cells, thereby establishing antigen-presenting cells that can be used in vitro for a long time and cover most of the tumor mutated antigens, which solves the problem that traditional methods cannot effectively obtain tumor antigens for antigen presentation.
  • RNA sequencing of each T cell was performed.
  • the gene sequences of each TCR were synthesized and constructed into lentiviral expression vectors respectively.
  • the virus was then packaged using 293T cells, and the viral supernatant was collected and concentrated by high-speed centrifugation. After resuspending the virus, it was mixed with peripheral blood lymphocytes activated by CD3/CD28 antibody for 3 days, and polybrene (Polybrene) with a final concentration of 8 ⁇ g/ml was added, and then added to a 24-well plate at 30°C. T cell infection was performed by centrifugation at 2000 rpm for 50 minutes. The supernatant was then removed, and RPMI medium containing 10% FBS and 200 ng/ml IL2 was added for cultivation. Through the lentivirus centrifugal infection method, a TCR-T cell line was established for each TCR, which achieved long-term use in vitro and solved the problem of in vitro culture of tumor T cells in traditional identification methods.
  • the present invention carries out the identification and analysis of T cell recognition of tumor antigens on multiple tumor tissues of different tumor types, including lung cancer, melanoma, intestinal cancer, liver cancer, gastric cancer, breast cancer , cervical cancer, ovarian cancer, kidney cancer, bladder cancer, esophageal cancer and other tumor types.
  • tumor-1, Tumor-2, Tumor-3 After obtaining fresh tumor (lung cancer) tissue (Tumor-1, Tumor-2, Tumor-3), it was divided into three parts.
  • the first part is used to construct PDX (Patient-Derived Xenograft) tumor model in immunodeficient NSG mice.
  • PDX Principal-Derived Xenograft
  • the second part of the tumor tissue is sent to a sequencing service company for exon and RNA sequencing after DNA and RNA are extracted.
  • the DNA extracted from peripheral blood leukocytes is sent to a sequencing service company for exon sequencing, which is used as a reference gene sequence for detecting gene mutations in tumors.
  • the third part of the tumor tissue was lysed into a single-cell suspension using the above-mentioned experimental protocol, and the single CD4 and CD8 T cells were sorted into 96-well plates by flow sorting, and then RNA sequencing of these T cells was performed respectively. , to obtain TCR sequences per cell and RNA expression levels of most genes. From exon and RNA sequencing results, the genes mutated in each tumor tissue and the expression levels of these mutated genes were obtained. Then, these expressed gene mutation sequences are synthesized in the manner of antigen tandem, and then introduced into immortalized patient B cells after EBV virus infection for expression to establish a B cell line that presents tumor mutation antigens.
  • the selected mutant antigens and synthetic tandem antigen gene sequences are shown in Tables 1 to 3.
  • 10 TCRs with the highest frequency were selected from the CD4 and CD8 T cells of each tumor, that is, the most amplified TCRs, and cloned. , constructed into a lentiviral expression vector.
  • the selected TCR information is shown in Table 4.
  • lentivirus is introduced into the corresponding patient peripheral blood T cells to prepare TCR-T cells, and each TCR establishes a TCR-T cell line.
  • Table 1 Tumor-1 tumor mutations and tandem antigen gene sequences
  • the nucleotide sequences of the Tumor-1 tandem antigen gene are shown in SEQ ID NO.1 and SEQ ID NO.2.
  • Table 2 Tumor-2 tumor mutations and tandem antigen gene sequences
  • the nucleotide sequences of the Tumor-2 tandem antigen gene are shown in SEQ ID NO.3 and SEQ ID NO.4.
  • Table 3 Tumor-3 tumor mutations and tandem antigen gene sequences
  • the nucleotide sequences of Tumor-3 tandem antigen genes are shown in SEQ ID NO.5 and SEQ ID NO.6.
  • Table 4 TCR a, b chain amino acid sequence information table
  • B cells expressing tumor antigens were mixed with each TCR-T cell line to conduct antigen stimulation experiments, while B cells that did not express tumor antigens were used as controls.
  • the above-mentioned B cells and T cells were mixed at a volume ratio of 1:1, inoculated in a 96-well plate, and after co-cultivation for 48 hours, the IFNG level in the supernatant was detected by ELISA. If tumor antigens expressed by B cells can be recognized by TCRs expressed by T cells, then T cells will be activated and secrete the effector cytokine IFNG. The higher the degree of activation, the more IFNG is produced. By comparing the levels of IFNG in the supernatant, it is determined whether these TCR-T cells can be activated by tumor antigens, so as to determine whether these TCRs recognize tumor antigens.
  • TCRs that recognize tumor antigens can be accurately identified.
  • 4 TCRs derived from CD4 T cells and 7 TCRs derived from CD8 T cells could be activated by B cells expressing tumor antigens.
  • the number of TCRs identified in the second tumor tissue that could recognize tumor antigens was 4 and 5 in CD4 and CD8, respectively.
  • the third tumor tissue was 3 and 4, respectively.
  • the same-derived tumor PDX model was treated with TCR-T cells that recognize tumor antigens. From the TCRs that have been verified to recognize tumor antigens, two TCRs with the highest frequency in CD8 T cells and one TCR with the highest frequency in CD4 T cells were selected to prepare TCR-T cells for treatment. At the same time, the three TCR-T cells were mixed in a volume ratio of 1:1:1 for multi-TCR treatment.
  • mice When PDX tumors transplanted subcutaneously into NSG mice grew to a size of approximately 50 mm 2 , mice were injected with 6x10 6 TCR-T cells via the tail vein.
  • TCRs that recognize tumor antigens identified by the method of the present invention can not only recognize tumor antigens, but also can effectively treat tumors, and the simultaneous use of multiple TCRs has better therapeutic effects.
  • the CD4 and CD8 T cells carrying these TCRs were divided into two groups, respectively, according to whether the identified TCRs recognized the tumor: positive for tumor antigen recognition and negative for tumor antigen recognition. Then, using the RNA sequencing results of each cell, the RNA expression abundance of each gene between the two groups of T cells was compared, and genes with significant expression differences were found. As a result, a batch of T cells that were positive for tumor antigen recognition was found. specifically expressed genes.
  • CD4 tumor antigen-recognizing T cells were specifically expressed: TNFRSF18, CXCL13, TNFRSF4, TNFSF8, ENTPD1, ACP5, LAYN, TNFRSF9, CTLA4, CD200 and TIGIT (Fig. 3, a).
  • CD8 tumor antigen-recognizing T cells were specifically expressed: TNFRSF18, CXCL13, CXCR6, GALNT2, ENTPD1, ACP5, HAVCR2, LAYN, TNFRSF9, CTLA4 and CD109 (Fig. 3, b).
  • TNFRSF18, CXCL13, ENTPD1, ACP5, LAYN, TNFRSF9 and CTLA4 were all highly expressed in CD4 and CD8 tumor antigen-recognizing T cells.
  • T cells that recognize tumor antigens can significantly distinguish T cells that recognize tumor antigens from T cells that do not recognize tumor antigens.
  • these specifically expressed genes can significantly distinguish T cells that recognize tumor antigens from T cells that do not recognize tumor antigens in different tumor types, with a good broad spectrum. Therefore, T cells that recognize tumor antigens can be isolated and identified from tumors by using these genes as molecular markers, and TCRs carried by them can be obtained for tumor treatment.
  • the molecular markers expressed on the surface of T cells were selected as molecular markers for flow sorting, among which tumor antigen-recognizing CD4 T cells were selected.
  • TNFRSF18, TNFRSF4, ENTPD1, TNFRSF9, CTLA4, CD200 and TIGIT, TNFRSF18, CXCR6, ENTPD1, TNFRSF9, HAVCR2, CTLA4 and CD109 were selected among tumor antigen-recognizing CD8 T cells.
  • 5 lung cancer tissues were processed into single cell suspension, and then a part of the single cell suspension was centrifuged and resuspended in RPMI medium containing 10% FBS at a density of 1 ⁇ 10 6 / ml, and then cryopreserved at -80°C as tumor antigen for subsequent antigen stimulation experiments.
  • the other single-cell suspensions were divided into 7 fractions, and flow-through antibody staining was performed respectively.
  • the antibody combination is 1) CD4, CD8, TNFRSF18; 2) CD4, CD8, ENTPD1; 3) CD4, CD8, TNFRSF9; 4) CD4, CD8, CTLA4; 5) CD4, CD8, TNFRSF4, CXCR6; 6) CD4, CD8, CD200, HAVCR2; 7) CD4, CD8, TIGIT, CD109. Then, the CD4 and CD8 T cells in the tumor tissue were stained with these molecular marker antibodies, and two groups of cells with positive and negative molecular markers were sorted, and a total of 14 groups of CD4 T cells and 14 groups of CD8 T cells were obtained.
  • the sorted T cells of each group were resuspended in RPMI medium containing 10% FBS and 200ng/ml IL2, and seeded into U-bottom 96-well plates coated with CD3/CD28 antibody for non-specific activation. After one week of culture, T cells in each group were taken out, and after two washes with PBS, they were resuspended in RPMI medium containing 10% FBS at a density of 2x10 6 /ml, and 200ul were seeded into a U-bottom 96-well plate. , 3 replicates for no group of cells.
  • T cells after resuscitating the cryopreserved tumor tissue single cell suspension to 37 degrees in a water bath, 50ul was added to the inoculated T cells for specific activation as tumor antigens. After 20 hours, the IFNG content in the medium was detected by ELISA to detect the response of T cells in each group to tumor antigen stimulation.
  • the inventors found that among CD4 T cells, TNFRSF18+, TNFRSF4+, ENTPD1+, TNFRSF9+, CTLA4+, CD200+ and TIGIT+ T cells can all be activated by tumor antigens, while T cells negative for corresponding molecular markers showed very low levels. stimulation effect (Fig. 4, a).
  • TNFRSF18+, CXCR6+, ENTPD1+, TNFRSF9+, HAVCR2+, CTLA4+ and CD109+ T cells were all activated by tumor antigens, while T cells negative for corresponding molecular markers could not be activated (Fig. 4, b ).
  • T cells isolated by these molecular markers and the TCRs they carry can be used for tumor treatment of patients.

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Abstract

本发明提供了一种用于杀伤肿瘤的TCR-T细胞，其特征在于，所述TCR-T细胞为携带有识别肿瘤抗原TCR的T细胞，所述TCR-T细胞中的TCR来源于以下中的任意一个或多个T细胞：1)在肿瘤中表达TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200、TIGIT这些基因中的一个或多个的CD4T细胞；2)在肿瘤中表达TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、TNFRSF9、CTLA4、CD109这些基因中的一个或多个的CD8T细胞。本发明的TCR-T细胞能够有效应用于治疗肿瘤，特别是免疫治疗。

Description

一种用于杀伤肿瘤的TCR-T细胞及其制备方法和应用 技术领域
本发明涉及免疫治疗技术领域，特别是涉及一种用于杀伤肿瘤的TCR-T细胞及其制备方法和应用。
背景技术
T细胞通过其表面的T细胞受体(T Cell Receptor，TCR)识别靶细胞表面的组织相融性分子(Human Leukocyte Antigen，HLA)所呈递的抗原，实现对靶细胞的直接攻击和杀伤。传统的识别肿瘤抗原的TCR-T细胞治疗，是针对已知的肿瘤抗原，克隆能够识别这些已知抗原的TCR，并明确其对应的HLA亚型。然后选择该肿瘤抗原和HLA亚型同时表达的患者，制备TCR-T细胞，进行回输治疗。然而，目前已知的肿瘤抗原较少，并且这些抗原只在一小部分患者中表达。加上同时要表达相对应的HLA分子，导致只有非常少的患者能够同时满足这两个要求。大部分患者无法通过这种识别已知肿瘤抗原的TCR-T细胞进行治疗。
在肿瘤细胞中，基因突变所产生的氨基酸序列变异，是肿瘤抗原的主要来源。然而，由于肿瘤的突变是随机的，这些通过随机突变产生的肿瘤抗原，通常是每个患者所特有的。也就是说，在患者中间共享的肿瘤抗原非常少。因此，必须针对每个患者自身所特有的肿瘤抗原建立相应的识别这些肿瘤抗原的TCR-T细胞，才能够实现对大部分患者的有效治疗。
在肿瘤的发生发展过程中，机体内识别肿瘤抗原的T细胞会被肿瘤抗原所激活，并进入到肿瘤组织，在肿瘤中扩增并杀伤肿瘤细胞。因此如果能够从患者的肿瘤中鉴定和分离出识别肿瘤抗原的T细胞，获得其携带的TCR序列，表达到患者的外周血T细胞中，将能够建立来源于患者自身的，即个体化的肿瘤抗原识别TCR-T细胞，用于个体化的肿瘤治疗。
然而，肿瘤与正常组织以及血液之间的淋巴与血液系统是相互连接的，大量不识别肿瘤抗原的T细胞通过血液和淋巴系统的循环进入到肿瘤。因此肿瘤组织中除了识别肿瘤抗原的T细胞，还含有大量来自于旁边组织和血液的不识别肿瘤抗原的T细胞，是一个各种T细胞的混合体。要从这些混合的T细胞中获得能够识别肿瘤抗原的TCR，首先需要通过肿瘤抗原刺激，确定哪些是能够识别肿瘤抗原的T细胞，然后再获得这些T细胞所携带的TCR序列，用于个体化的TCR-T细胞治疗。同时，在明确这些T细胞是否识别肿瘤抗原后，分析这些T细胞的基因表达特征，比较识别肿瘤抗原的T细胞和不识别肿瘤抗原的T细胞之间的分子特征差异，找到识别肿瘤抗原的T细胞所特有的分子标志物，通过这些特异的分子标志物来分离和获取肿瘤中能够识别肿瘤抗原的T细胞，用于后续的治疗。
鉴定一个T细胞是否识别肿瘤抗原是一个过程非常复杂，且技术要求很高的系统工程。首先需要将T细胞从肿瘤组织中分离出来，在体外进行培养。同时还必须将肿瘤组织中的肿瘤抗原鉴定出来，然后在体外用鉴定出来的肿瘤抗原来刺激分离出来的T细胞。能够被肿瘤抗原刺激起来，才能证明是能够识别肿瘤抗原的T细胞。由于整个鉴定的实验流程极为复杂，技术难度也非常高，导致目前仍然无法对一个肿瘤组织中的T细胞进行系统的肿瘤抗原识别鉴定与分析。也使得目前无法从肿瘤组织中快速获得识别肿瘤抗原的T细胞和TCR，用于治疗。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种能够准确并快速地分析T细胞的肿瘤抗原识别能力的技术方案，并通过比较识别肿瘤抗原的T细胞和不识别肿瘤抗原的T细胞之间的基因表达差异，找到了肿瘤组织中识别肿瘤抗原的T细胞所特有的分子标志物，并通过这些分子标志物实现对识别肿瘤抗原T细胞的快速分离，从而快速获得识别肿瘤抗原的TCR，建立了个体化的识别肿瘤抗原的TCR-T细胞治疗技术。
为了实现以上目的，本发明提供了一种用于杀伤肿瘤的TCR-T细胞，所述TCR-T细胞为携带有识别肿瘤抗原TCR的T细胞，所述TCR-T细胞中的TCR来源于以下中的任意一个或多个T细胞：
1)在肿瘤中表达TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200、TIGIT这些基因中的一个或多个的CD4 T细胞；
2)在肿瘤中表达TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、TNFRSF9、CTLA4、CD109这些基因中的一个或多个的CD8 T细胞。
本发明还提供了所述的TCR-T细胞在制备用于治疗肿瘤的药物中的应用。
优选地，所述肿瘤包括但不限于肺癌、黑色素瘤、肠癌、肝癌、胃癌，乳腺癌、宫颈癌、卵巢癌、肾癌、膀胱癌、食管癌中的任意一种或多种。
本发明还提供了所述的TCR-T细胞的制备方法，其包括：将识别肿瘤抗原的TCR的核苷酸序列的一个或多个导入到T细胞中进行表达，构建所述TCR-T细胞。
优选地，所述识别肿瘤抗原的TCR的鉴定步骤如下：通过流式分选、磁珠分离或肿瘤组织原位测序方法，由肿瘤组织获得表达了识别肿瘤抗原CD4 T细胞的标志物TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200、TIGIT中的任意一种或多种的T细胞及其携带的TCR序列，以及表达了识别肿瘤抗原CD8 T细胞的标志物TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、 TNFRSF9、CTLA4、CD109中的任意一种或多种的T细胞及其携带的TCR序列。
本发明还提供了一种用于鉴定识别肿瘤抗原的T细胞和TCR的方法，其包括以下步骤：通过克隆肿瘤组织中的高频TCR，建立表达TCR的TCR-T细胞，利用表达肿瘤抗原串联基因的抗原呈递细胞进行体外肿瘤抗原刺激，能够被刺激起来的TCR-T细胞所携带的TCR即为识别肿瘤抗原的TCR，携带该TCR的T细胞即为识别肿瘤抗原的T细胞；
其中，所述TCR-T细胞中的TCR来源于以下中的任意一个或多个T细胞：
1)在肿瘤中表达TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200、TIGIT这些基因中的一个或多个的CD4 T细胞；
2)在肿瘤中表达TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、TNFRSF9、CTLA4、CD109这些基因中的一个或多个的CD8 T细胞。
本发明的TCR-T细胞能够有效应用于治疗肿瘤，特别是免疫治疗。
附图说明
图1示出了肿瘤CD4和CD8 T细胞中的高频TCR对肿瘤抗原的刺激应答情况。
图2示出了肿瘤抗原识别T细胞亚群的TCR所构建的TCR-T细胞能够有效治疗肿瘤(*P<0.05；**P<0.01)。
图3示出了肿瘤抗原识别T细胞亚群的分子标志基因，其中a示出了CD4 T细胞中的特异基因表达差异，b示出了CD8 T细胞中的特异基因表达差异。
图4示出了通过肿瘤抗原识别T细胞的分子标志物分选的T细胞能够有效识别肿瘤抗原，其中a表示CD4 T细胞，b表示CD8 T细胞(*P<0.05；**P<0.01)。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。值得注意的是，出于简要清楚的目的，以下实施例中对一些常规的技术操作步骤、试剂、仪器并未进行细致的描述，但应理解，如未特别说明，这些常规技术操作步骤、试剂、仪器对本领域普通技术人员而言是显而易见的。
下面结合具体实例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
首先，用Qiagen的DNeasy Blood&Tissue试剂盒(货号：69506)提取肿瘤组织的DNA， 使用Qiagen的miRNeasy-Mini试剂盒(货号：217004)提取肿瘤组织的RNA。然后将获得的DNA和RNA样本送测序服务公司进行外显子和RNA测序。同时，提取外周血白细胞的DNA送测序服务公司进行外显子测序，作为检测肿瘤中基因突变的参考基因序列。通过比较肿瘤组织和外周血白细胞的外显子序列，找到在肿瘤组织中发生了突变的位点和对应的基因。并根据每个突变基因在肿瘤组织中的RNA表达量，对突变基因在肿瘤中的表达丰度进行排序，选择在肿瘤组织中表达较高的突变作为肿瘤抗原。然后将以突变位点为中心的25氨基酸表位肽，以及移码突变产生的新的多肽，作为肿瘤抗原串联起来，合成串联了这些肿瘤突变抗原的基因，并表达在经过EBV病毒感染后获得永生化的患者B细胞中，从而建立可以在体外长期使用，并且覆盖大部分肿瘤突变抗原的抗原呈递细胞，解决了传统方法中无法有效获得肿瘤抗原进行抗原呈递的难题。
其次，通过流式细胞分选技术，分选出肿瘤中的T细胞后，对每个T细胞进行RNA测序。首先用手术刀将肿瘤组织切成小于1mm 3的碎块后，按10倍体积加入组织消化液(配方：150ml RIPA 1640培养基、3ml胎牛血清、27mg胶原酶、7.5mg DNA酶)，于37℃消化2小时。消化完成后，经70μM筛网过滤，用PBS洗涤并重悬，得到单细胞悬液。在样本中加入终浓度为1:200的荧光流式抗体(配色方案：CD45-APC-Cy7、CD3-APC、CD19-PE)，于4℃染色30分钟后，用PBS洗涤两次，然后用流式细胞分选仪(型号：BD Bioscience Arial III)分选出单个的T淋巴细胞(CD45 +CD3 +)到96孔板。然后在96孔板中进行单细胞RNA反转录和和cDNA扩增，构建文库，送测序服务公司进行测序，测序平台为Illumina HiSeq X Ten。测序结果用CellRanger软件进行分析，获得每个T细胞中的基因表达量，以及其所携带的TCR序列。
然后，合成每个TCR的基因序列，并分别构建到慢病毒表达载体中。然后使用293T细胞包装病毒，收集病毒上清，通过高速离心浓缩病毒。重悬病毒后，与经过CD3/CD28抗体激活了3天的外周血淋巴细胞混匀，并加入终浓度为8μg/ml的聚凝胺(Polybrene)，然后加入到24孔板中，于30℃2000转离心50分钟，进行T细胞感染。然后去除上清，加入含10％FBS和200ng/ml IL2的RPMI培养基进行培养。通过慢病毒离心感染法，为每个TCR建立了一个TCR-T细胞系，实现了在体外的长期使用，解决了传统鉴定方法中的肿瘤T细胞体外培养难题。
在完成了以上两个技术方法的建立后，本发明对不同肿瘤类型的多个肿瘤组织进行了T细胞识别肿瘤抗原的鉴定与分析，包括肺癌、黑色素瘤、肠癌、肝癌、胃癌，乳腺癌、宫颈癌、卵巢癌、肾癌、膀胱癌、食管癌等多种肿瘤类型。
以下是来源于3个肺癌组织的代表性结果。
获取新鲜的肿瘤(肺癌)组织(Tumor-1、Tumor-2、Tumor-3)后，将其分为三部分。其中第一部分用于在免疫缺陷的NSG小鼠中进行PDX(Patient-Derived Xenograft)肿瘤模型的构建。首先用无菌剪刀将肿瘤组织剪成约1mm 3的碎块，用套针接种到免疫缺陷的NSG小鼠左侧腹股沟皮下，成瘤以后再剪成小块，继续接种到NSG小鼠，经过三次成瘤后，将肿瘤切成小块冻存于液氮，用于后续的肿瘤治疗实验。第二部分肿瘤组织在提取DNA和RNA后，送测序服务公司进行外显子和RNA测序。同时，提取外周血白细胞的DNA送测序服务公司进行外显子测序，作为检测肿瘤中基因突变的参考基因序列。第三部分肿瘤组织用上述的实验方案裂解成单细胞悬液后，通过流式分选，将其中单个的CD4和CD8 T细胞分选到96孔板中，然后对这些T细胞分别进行RNA测序，获得每个细胞的TCR序列和大部分基因的RNA表达水平。通过外显子和RNA测序结果，获得在每个肿瘤组织中发生了突变的基因，以及这些突变基因的表达水平。然后，将这些表达的基因突变序列按照抗原串联的方式进行基因合成，并导入到经过EBV病毒感染后获得永生化的患者B细胞中表达，建立呈递肿瘤突变抗原的B细胞系。
所选择的突变抗原与合成的串联抗原基因序列如表1至表3所示。同时，通过分析每个CD4和CD8 T细胞所携带的TCR序列，从每个肿瘤的CD4和CD8 T细胞中分别挑选出频率最高的10个TCR，也就是扩增最多的TCR，将其克隆出来，构建到慢病毒表达载体中。所选择的TCR信息如表4所示。然后通过慢病毒导入到相应的患者外周血T细胞中，制备出TCR-T细胞，每个TCR建立一个TCR-T细胞系。
表1：Tumor-1肿瘤突变与串联抗原基因序列
Figure PCTCN2021097813-appb-000001
Figure PCTCN2021097813-appb-000002
Tumor-1串联抗原基因的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
表2：Tumor-2肿瘤突变与串联抗原基因序列
Figure PCTCN2021097813-appb-000003
Figure PCTCN2021097813-appb-000004
Tumor-2串联抗原基因的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
表3：Tumor-3肿瘤突变与串联抗原基因序列
Figure PCTCN2021097813-appb-000005
Figure PCTCN2021097813-appb-000006
Tumor-3串联抗原基因的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示。
表4：TCR a、b链氨基酸序列信息表
Figure PCTCN2021097813-appb-000007
Figure PCTCN2021097813-appb-000008
为了明确这些TCR是否识别肿瘤抗原，将表达了肿瘤抗原的B细胞与每个TCR-T细胞系混合培养，进行抗原刺激实验，同时以不表达肿瘤抗原的B细胞作为对照。
首先将上述B细胞与T细胞按体积比1:1混合，接种于96孔板，共同培养48小时后，通过ELISA检测上清中的IFNG水平。若B细胞所表达的肿瘤抗原能够被T细胞所表达的TCR所识别，那么T细胞则会被激活并分泌效应性细胞因子IFNG。激活程度越高，产生的IFNG越多。通过比较上清中的IFNG水平，来确定这些TCR-T细胞是否能够被肿瘤抗原所激活，从而明确这些TCR是否识别肿瘤抗原。
结果如图1所示，利用建立的技术体系，能够准确地将识别肿瘤抗原的TCR鉴定出来。在第一个肿瘤组织的高频TCR中，4个来源于CD4 T细胞的TCR和7个来源于CD8 T细胞的TCR能够被表达肿瘤抗原的B细胞所激活。第二个肿瘤组织中所鉴定出来的能够识别肿瘤抗原的TCR，在CD4和CD8中分别为4个和5个。第三个肿瘤组织则分别为3个和4个。这些结果表明，肿瘤中确实存在即能够识别肿瘤抗原的T细胞，也存在不识别肿瘤抗原的T细胞。
接下来，为了验证这些肿瘤特异的TCR是否能够用于肿瘤治疗，用识别肿瘤抗原的TCR-T细胞，对相同来源的肿瘤PDX模型进行治疗。从已经验证了能够识别肿瘤抗原的TCR中，选择了两个在CD8 T细胞中频率最高的TCR和1个在CD4 T细胞中频率最高的TCR，制备成TCR-T细胞进行治疗。同时还将这三个TCR-T细胞进行1:1:1体积比混合，进行多TCR的治疗。当移植到NSG小鼠皮下的PDX肿瘤生长到大小约为50mm 2时，通过尾静脉给小鼠注射6x10 6TCR-T细胞。
结果如图2所示，在对三个肿瘤PDX的治疗中，3个TCR混合的治疗效果最佳，而单个TCR也均表现出了明显的治疗效果。
这些结果表明，通过本表明的方法所鉴定出来的识别肿瘤抗原的TCR不仅能够识别肿瘤抗原，还能够对肿瘤进行有效的治疗，并且同时使用多个TCR有更好的治疗效果。
为了鉴定肿瘤抗原特异T细胞亚群的分子标志物，根据所鉴定的TCR是否识别肿瘤，将携带这些TCR的CD4和CD8 T细胞分别分为两组：肿瘤抗原识别阳性和肿瘤抗原识别阴性。然后利用每个细胞的RNA测序结果，比较每个基因在将这两组T细胞之间的RNA表达丰度，找到具有明显表达差异的基因，结果发现了一批在肿瘤抗原识别阳性的T细胞中特异表达的基因。其中CD4肿瘤抗原识别T细胞特异表达：TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200和TIGIT(图3，a)。CD8肿瘤抗原识别T细胞特异表达：TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、TNFRSF9、CTLA4和CD109(图3，b)。其中TNFRSF18、CXCL13、ENTPD1、 ACP5、LAYN、TNFRSF9和CTLA4在CD4和CD8肿瘤抗原识别T细胞中都高表达。这些特异表达的基因，能够显著区分识别肿瘤抗原的T细胞和不识别肿瘤抗原的T细胞。同时，这些特异表达的基因在不同肿瘤类型中均能够显著区分识别肿瘤抗原的T细胞和不识别肿瘤抗原的T细胞，具有很好的广谱性。因此可以通过这些基因作为分子标志物从肿瘤中分离和鉴定出识别肿瘤抗原的T细胞，获得其携带的TCR，用于肿瘤治疗。
为了进一步验证通过这些分子标志物是否能够鉴定和分离出识别肿瘤抗原的T细胞，选择在T细胞表面表达的分子标志物作为流式分选的分子标记，其中肿瘤抗原识别CD4 T细胞中选择了TNFRSF18、TNFRSF4、ENTPD1、TNFRSF9，CTLA4、CD200和TIGIT，肿瘤抗原识别CD8 T细胞中则选择了TNFRSF18、CXCR6、ENTPD1、TNFRSF9、HAVCR2、CTLA4和CD109。首先按照上述的肿瘤组织消化方法，将5个肺癌组织处理成单细胞悬液后，然后取一部分单细胞悬液离心后，重悬到含有10％FBS的RPMI培养基中，密度为1x10 6/ml，然后冻存到-80℃，作为肿瘤抗原用于后续的抗原刺激实验。其他的单细胞悬液分成7个组分，分别进行流式抗体染色。抗体组合为为1)CD4、CD8、TNFRSF18；2)CD4、CD8、ENTPD1；3)CD4、CD8、TNFRSF9；4)CD4、CD8、CTLA4；5)CD4、CD8、TNFRSF4、CXCR6；6)CD4、CD8、CD200、HAVCR2；7)CD4、CD8、TIGIT、CD109。然后将肿瘤组织中的CD4和CD8T细胞根据这些分子标志物抗体的染色，分选出分子标志物阳性与阴性的两组细胞，共获得14组CD4 T细胞和14组CD8 T细胞。
将分选获得的各组T细胞重悬在含有10％FBS和200ng/ml IL2的RPMI培养基中，接种到包被了CD3/CD28抗体的U型底96孔板中，进行非特异性激活。培养一周后，将各组T细胞取出，结果两次PBS洗涤后，重悬到含有10％FBS的RPMI培养基中，密度为2x10 6/ml，取200ul接种到U型底的96孔板中，没组细胞3个重复。同时将冻存的肿瘤组织单细胞悬液通过水浴复苏到37度后，取50ul加入到接种的T细胞中，作为肿瘤抗原进行特异性激活。20小时后，通过ELISA检测培养基中的IFNG含量来检测各组T细胞对肿瘤抗原刺激的反应。发明人发现，在CD4 T细胞中，TNFRSF18+、TNFRSF4+、ENTPD1+、TNFRSF9+，CTLA4+、CD200+和TIGIT+的T细胞，均能够被肿瘤抗原激活起来，而相应分子标记阴性的T细胞则表现出了很低的刺激效果(图4，a)。同样，在CD8 T细胞中TNFRSF18+、CXCR6+、ENTPD1+、TNFRSF9+、HAVCR2+、CTLA4+和CD109+的T细胞，均能够被肿瘤抗原激活起来，而相应分子标记阴性的T细胞则不能够被激活(图4，b)。
以上结果说明，这些分子标志物能够用于分离肿瘤中识别肿瘤抗原的T细胞。通过这些分子标志物分离出来的T细胞以及其携带的TCR，能够用于患者的肿瘤治疗。

Claims (6)

  1. 一种用于杀伤肿瘤的TCR-T细胞，其特征在于，所述TCR-T细胞为携带有识别肿瘤抗原TCR的T细胞，所述TCR-T细胞中的TCR来源于以下中的任意一个或多个T细胞：
    1)在肿瘤中表达TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200、TIGIT这些基因中的一个或多个的CD4 T细胞；
    2)在肿瘤中表达TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、TNFRSF9、CTLA4、CD109这些基因中的一个或多个的CD8 T细胞。
  2. 根据权利要求1所述的TCR-T细胞在制备用于治疗肿瘤的药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述肿瘤是肺癌、黑色素瘤、肠癌、肝癌、胃癌，乳腺癌、宫颈癌、卵巢癌、肾癌、膀胱癌、食管癌中的任意一种或多种。
  4. 根据权利要求1所述的TCR-T细胞的制备方法，其特征在于包括：将识别肿瘤抗原的TCR的核苷酸序列的一个或多个导入到T细胞中进行表达，构建所述TCR-T细胞。
  5. 根据权利要求4所述的方法，其特征在于，所述识别肿瘤抗原的TCR的鉴定步骤如下：通过流式分选、磁珠分离或肿瘤组织原位测序方法，由肿瘤组织获得表达了识别肿瘤抗原CD4 T细胞的标志物TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200、TIGIT中的任意一种或多种的T细胞及其携带的TCR序列，以及表达了识别肿瘤抗原CD8 T细胞的标志物TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、TNFRSF9、CTLA4、CD109中的任意一种或多种的T细胞及其携带的TCR序列。
  6. 一种用于鉴定识别肿瘤抗原的T细胞和TCR的方法，其特征在于包括以下步骤：通过克隆肿瘤组织中的高频TCR，建立表达TCR的TCR-T细胞，利用表达肿瘤抗原串联基因的抗原呈递细胞进行体外肿瘤抗原刺激，能够被刺激起来的TCR-T细胞所携带的TCR即为识别肿瘤抗原的TCR，携带该TCR的T细胞即为识别肿瘤抗原的T细胞；
    其中，所述TCR-T细胞中的TCR来源于以下中的任意一个或多个T细胞：
    1)在肿瘤中表达TNFRSF18、CXCL13、TNFRSF4、TNFSF8、ENTPD1、ACP5、LAYN、TNFRSF9、CTLA4、CD200、TIGIT这些基因中的一个或多个的CD4 T细胞；
    2)在肿瘤中表达TNFRSF18、CXCL13、CXCR6、GALNT2、ENTPD1、ACP5、HAVCR2、LAYN、TNFRSF9、CTLA4、CD109这些基因中的一个或多个的CD8 T细胞。
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