CN114920822B - T cell receptor for recognizing C7orf50 mutant antigen short peptide and application thereof - Google Patents

T cell receptor for recognizing C7orf50 mutant antigen short peptide and application thereof Download PDF

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CN114920822B
CN114920822B CN202110140898.2A CN202110140898A CN114920822B CN 114920822 B CN114920822 B CN 114920822B CN 202110140898 A CN202110140898 A CN 202110140898A CN 114920822 B CN114920822 B CN 114920822B
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黄晓武
毛丽
李佳
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Abstract

The invention discloses a T cell receptor for recognizing a C7orf50 mutant antigen short peptide and application thereof, wherein the T cell receptor comprises a TCR alpha chain and a TCR beta chain, the full-length amino acid sequence of the TCR alpha chain is shown as SEQ ID NO. 1, and the full-length amino acid sequence of the TCR beta chain is shown as SEQ ID NO. 4 or SEQ ID NO. 7. According to the invention, the TCR sequence of the T cell is obtained by analyzing the existence of the C7orf50 mutant in the tumor cell of the liver cancer patient, separating the T cell for recognizing the antigen short peptide of the C7orf50 mutant from the tumor-infiltrating lymphocyte of the liver cancer patient and further sequencing.

Description

T cell receptor for recognizing C7orf50 mutant antigen short peptide and application thereof
Technical Field
The invention discloses a T cell receptor for recognizing a C7orf50 mutant antigen short peptide and application thereof, and belongs to the technical field of biochemistry.
Background
Liver cancer is a common malignant tumor in China, and the current treatment of liver cancer mainly comprises liver resection operation, but the recurrence rate of liver cancer after radical resection for 5 years exceeds 60%. The liver cancer patients have high fatality rate and poor survival state, and the main reasons are lack of effective treatment means, so that development of new effective treatment methods is urgently needed. The C7orf50 gene is positioned in an open reading zone of chromosome 7, the function is unknown, and the early research result reveals that the C7orf50 gene encoding protein is highly expressed in liver cancer patients and is closely related to clinical prognosis, which suggests that C7orf50 can become a novel therapeutic target for liver cancer.
The tumor cells are enriched with natural mutation which is favorable for cell growth, and along with subsequent rapid proliferation, the false bases in the DNA replication process cannot be repaired in time, so that more and more new mutations, namely tumor specific new antigens, are generated. T cells recognize specific proteins through the surface receptor TCR and clear target cells, but due to changes in tumor microenvironment, etc., the function of T cells is often inhibited and tumor cells cannot be cleared effectively. After in vitro amplification, tumor specific antigen T cells can be identified, cytotoxic substances are released, and the tumor cells are directly killed by direct action, so that separation and enrichment of tumor specific antigen T cells are an important research direction of tumor immune cell treatment. TCR is receptor protein expressed on T cell membrane to recognize antigen short peptide presented on cell surface by main histocompatibility complex, and the derived TCR-T cell therapy is to modify T cell receptor to recognize specific antigen on tumor cell, and to eliminate tumor cell after in vitro culture and amplification to a certain amount. Accordingly, the study of TCR-T cell therapies has focused on the discovery of T cell receptors that recognize tumor-specific antigens.
Disclosure of Invention
The invention solves the technical problems that: how to identify the C7orf50 mutant antigen short peptide in tumor cells.
In order to solve the technical problems, the invention provides a T cell receptor for recognizing a C7orf50 mutant antigen short peptide, which comprises a TCR alpha chain and a TCR beta chain, wherein the full-length amino acid sequence of the TCR alpha chain is shown as SEQ ID NO. 1, and the full-length amino acid sequence of the TCR beta chain is shown as SEQ ID NO. 4 or SEQ ID NO. 7.
Preferably, the amino acid sequence of the variable region of the TCR alpha chain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 2 and the amino acid sequence of the variable region of the TCR beta chain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 5 or SEQ ID NO. 8.
Preferably, the amino acid sequence of the complementarity determining region CDR3 of the TCR alpha chain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 3 and the amino acid sequence of the complementarity determining region CDR3 of the TCR beta chain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 6 or SEQ ID NO. 9.
The invention also provides application of the T cell receptor for recognizing the C7orf50 mutant antigen short peptide, wherein the application is applied to diagnosis and treatment.
Preferably, the application is the application in preparing a kit for diagnosing liver cancer.
Preferably, the use is in the preparation of TCR-T cells specific for C7orf50 mutants.
Preferably, the application is the application in preparing medicines for treating liver cancer.
Compared with the prior art, the invention has the following beneficial effects:
1. the T cell receptor for recognizing the C7orf50 protein mutant antigen short peptide can be used for transducing T cells to obtain the T cells with specificity to the C7orf50 protein mutant, plays a role in cellular immunotherapy, brings a new treatment method for liver cancer patients, and has important clinical significance;
2. the T Cell Receptor (TCR) for recognizing the C7orf50 protein mutant antigen short peptide disclosed by the invention is used for transducing T cells, can specifically target tumor cells, and cannot damage normal cells of the T cells.
Drawings
FIG. 1 is a graph showing the results of a component analysis of liver tumor infiltrating lymphocytes;
FIG. 2A is a schematic diagram showing the results of detecting IFN gamma spot numbers after 24 hours when T2 cells loaded with different antigen short peptides were co-cultured with liver tumor infiltrating lymphocytes, respectively; FIG. 2B is a schematic diagram showing the results of detecting IFN gamma spot numbers after 24 hours, in which T2 cells loaded with the selected mutant antigen peptide and the corresponding wild-type antigen peptide were co-cultured with tumor-infiltrating lymphocytes, respectively;
FIG. 3 is an antigenic peptide/HLA-A 02 x 01 tetramer complex labeling tumor infiltrating lymphocytes;
FIG. 4A is a schematic representation of the results of flow cytometry detection of endocytosis of TCR after co-culturing T2 cells loaded with wild-type and mutant antigenic peptides, respectively, with tumor-infiltrating lymphocytes; FIG. 4B is a schematic representation of the results of flow cytometry detection of 41BB expression in TCR after co-culturing T2 cells loaded with wild-type and mutant antigenic peptides, respectively, with tumor-infiltrating lymphocytes; FIG. 4C is a schematic representation of the results of flow cytometry detection of IFNγ expression in TCR after co-culturing T2 cells loaded with wild-type and mutant antigenic peptides, respectively, with tumor-infiltrating lymphocytes;
FIG. 5 is a graph of single cell TCR sequencing CDR3 results.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
The experimental materials and general experimental methods used in the following examples are as follows:
1. cell culture: t2 cells (174 XCEM.T2) were cultured with IMDM (Invitrogen) containing 10% fetal bovine serum (Gibco), 37℃CO 2 The concentration was 5%.
2. Preparation of single cell suspension of tissue sample: the tissue was minced into a paste, digested with a digestion solution containing collagenase and nuclease for 1 hour, and the cell suspension was passed through a 70 μm plug to remove insufficiently digested tissue. And (3) adding the cell sediment after centrifugation into the erythrocyte lysate for 5 minutes, adding an equal volume of PBS for neutralization, centrifuging, washing once by the PBS, and re-suspending to obtain single cell suspension. After counting, the cells were cultured with a medium (STEMCELL, 10981) containing 6000U/mL of IL2, and T cells were expanded.
3. ELISA spot test: placing T2 cells and T cells loaded with antigen peptide into a culture plate coated with IFNgamma, co-culturing for 24 hours, taking out, removing cells and culture solution, washing the culture plate, adding IFNgamma antibody, incubating for 2 hours at room temperature, adding a substrate for color development after washing, and washing with distilled water to terminate the reaction after spots are formed clearly enough. Plate reading quantitative analysis was performed using a plate reader.
Example 1
An acquisition method of T cell receptor for recognizing C7orf50 mutant antigen short peptide:
1.1 in vitro isolation and expansion of liver tumor infiltrating lymphocytes:
the partially infiltrated lymphocytes in the tumor tissue can identify and kill the tumor cells, but can not normally perform the tumor killing function due to the inhibition effect of the tumor microenvironment, and can be activated again through in vitro amplification. The cancer tissue samples of the liver cancer patients subjected to surgical excision are prepared into single cell suspension, the single cell suspension is cultured by using a culture medium (STEMCELL, 10981) containing 6000U/mL IL2, and the liver tumor-infiltrating lymphocytes are subjected to in vitro expansion, and after 10 days of expansion, the main components of the liver tumor-infiltrating lymphocytes are CD3 positive T cells, as shown in figure 1.
1.2 construction of a liver tumor mutant antigen peptide library:
analyzing tumor mutant neoantigen short peptide of a patient, taking tumor specimens of a liver cancer patient subjected to surgical excision, performing exon sequencing and transcriptome sequencing, and taking peripheral blood of the patient for performing exon sequencing control. After quality evaluation of error rate, data amount, comparison rate and the like is carried out on the original sequencing data, the gene with sense mutation of exons in tumors and the measurement index FPKM (Fragments per Kilobase Million) of gene expression amount being more than 1 is selected for tumor new antigen screening. HLA (human leukocyte antigen) affinity to the mutant polypeptide, wild-type polypeptide, was calculated using NetMHCpan and the affinity results were compared, and if the mutated polypeptide had an affinity to the HLA molecule of less than 500nM and less than the affinity between the corresponding wild-type polypeptide and the HLA molecule (the smaller the affinity, the stronger the affinity, and therefore the greater the affinity of the mutant polypeptide than the wild-type), the mutant polypeptide was defined as a neoantigenic peptide. By this analysis, a library of liver tumor mutant antigen peptides was obtained, including 26 candidate neoantigen peptides, as shown in table 1.
TABLE 1 sequencing analysis and screening of the 26 mutant antigenic peptides of liver cancer patients
Figure BDA0002928786300000041
Figure BDA0002928786300000051
1.3 screening for liver tumor mutant antigenic peptides:
the T2 cells loaded with the antigen peptide are used as antigen presenting cells, and the antigen presenting cells and the tumor-infiltrating lymphocytes are co-cultured to detect whether the tumor-infiltrating lymphocytes can recognize candidate neoantigen peptides, so that the tumor neoantigens can be determined. The T2 cell line is HLA-A 02 x 01 cell deficient in antigen processing related transporter (Transporter associated with antigen processing, TAP), and is a common tool cell for presenting free antigen peptides. 25 polypeptides (except P11) in Table 1 were synthesized, the labels P1-P26 (P11 was not successfully synthesized due to the problem of polypeptide amino acid composition) were loaded on the surface of T2 cells, and then placed together with liver tumor-infiltrating lymphocytes into an ELISA spot plate coated with IFNgamma antibody, after 24 hours of incubation, it was detected that 4 neoantigen peptides (P1, P3, P5 and P21) screened by mutation at one site of the C7orf50 gene were presented and stimulated tumor-infiltrating lymphocytes to release IFNgamma, as shown in FIG. 2A, suggesting that T cells capable of recognizing the neoantigen peptides may be present in tumor-infiltrating lymphocytes. And then synthesizing wild-type peptide fragments corresponding to P1, P3, P5 and P21, and simultaneously taking P8 and the wild-type peptide fragments thereof as a control, and detecting whether tumor-infiltrating lymphocytes specifically recognize mutant neoantigen peptides or not through an ELISA (enzyme-linked immunosorbent assay) and do not recognize the wild-type peptide fragments, wherein as shown in a result of FIG. 2B, the amount of IFNgamma spots generated by a group presenting the wild-type peptide fragments is obviously less than that of the mutant group, so that the tumor-infiltrating lymphocytes can specifically recognize the mutant neoantigen peptides and do not recognize the wild-type peptide fragments.
1.4 antigenic peptide/HLA-A 02 x 01 tetramer labeled liver tumor specific T cells:
QuickSwitch Using MBL company TM Kit, according to the instructions procedure, the wild-type and mutant polypeptides/HLA-A 02 x 01 complex tetramer reacted as described in FIG. 2 above was prepared. The tumor infiltrating lymphocytes were de-labeled with the prepared antigen peptide/HLA-A: 02 x 01 tetramer, and as a result, it was found that the P1 and P5 mutant neoantigen peptide HLA-A:02 x 01 complex tetramer could recognize specific T cells in tumor infiltrating lymphocytes, whereas their corresponding wild-type tetramer could not be successfully recognized, which indicates that the antigen peptide/HLA-A: 02 x 01 tetramer could label T cells recognizing neoantigen peptide, and further indicates that there were T cells in liver tumor infiltrating lymphocytes that specifically recognized neoantigen peptide but not wild-type antigen peptide, as shown in FIG. 3. Negative results were marked because of failure of the preparation of the P3/HLA-A:02 x 01 complex tetramer. However, we also found that the successfully prepared P21/HLA-A:02 x 01 complex tetramer was not recognized, and that loading of P21 this polypeptide on antigen presenting cells could activate T cells to release IFNγ, presumably because both the P21 polypeptide and the P1, P3, and P5 polypeptide sequences contained mutated sites, the difference in the number of amino acids before and after the mutated sites could still activate T cells, and that the P21/HLA-A:02 x 01 complex tetramer could not recognize TCR due to steric hindrance and the like.
1.5 functional validation of liver tumor-specific T cells:
the function of tetramer-labeled tumor-specific T cells was verified, on the one hand to further rule out non-specific recognition of tetramers, and on the other hand to detect the activity of tumor-specific T cells. Tumor infiltrating T cells activate upon contact with target cells, a series of events occur, such as endocytosis of TCR recognition target antigen, up-regulation of surface receptor 41BB expression, and release of ifnγ, etc. Detecting endocytosis of the TCR, expression of 41BB and expression of IFNγ by flow cytometry after co-culturing tumor infiltrating lymphocytes and antigen presenting cells; it was found that T2 cells loaded with mutant P1 and P5 activated tetramer-positive T cells in tumor-infiltrating lymphocytes compared to wild-type peptide, as shown in fig. 4A, tetramer-positive T cells underwent TCR endocytosis; as shown in FIG. 4B, the 41BB expression level in tetramer-positive T cells was up-regulated; the amount of ifnγ expression in tetramer-positive T cells was up-regulated as shown in fig. 4C, corresponding to the results of the enzyme-linked immunosorbent assay in fig. 2.
1.6 tumor specific T cell TCR sequencing:
the tumor infiltrating lymphocytes marked by the antigen peptide/HLA-A: 02 x 01 tetramer complex are separated, single cell TCR sequencing is carried out, and as a result, the obtained TCR sequences of 9906T cells are found that the TCR alpha chains of 9863 (99.6%) T cells are TRAV17-01-TRAJ43-01 and the beta chains are TRBV5-1-TRBD1-01. Wherein, the alpha chain of 8663 TCRs is TRAV17-01-TRAJ43-01-TRAC, the full-length amino acid sequence is SEQ ID NO. 1, the variable region amino acid sequence is SEQ ID NO. 2, and the amino acid sequence of the complementarity determining region CDR3 is: CATPPGNDMRF (SEQ ID NO: 3); beta chain is TRBV5-1-TRBD1-01-TRBJ1-1-TRBC1, full-length amino acid sequence is SEQ ID NO. 4, variable region amino acid sequence is SEQ ID NO. 5, and amino acid sequence of complementarity determining region CDR3 is: CASSLGGVTFF (SEQ ID NO: 6). 1200 TCR alpha chains are TRAV17-01-TRAJ43-01-TRAC1, the full-length amino acid sequence is SEQ ID NO:1, the variable region amino acid sequence is SEQ ID NO:2, and the amino acid sequence of the complementarity determining region CDR3 is: CATPPGNDMRF (SEQ ID NO: 3), the beta chain is TRBV5-1-TRBD1-01-TRBJ2-1-TRBC2, the full-length amino acid sequence is SEQ ID NO:7, the variable region amino acid sequence is SEQ ID NO:8, the amino acid sequence of the complementarity determining region CDR3 is: CASSLAGLQFF (SEQ ID NO: 9). The corresponding CDR3 amino acid sequence of the TCR sequence is shown in figure 5.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Figure BDA0002928786300000081
Figure BDA0002928786300000091
Figure BDA0002928786300000101
Figure BDA0002928786300000111
Figure BDA0002928786300000121
Figure BDA0002928786300000131
Figure BDA0002928786300000141
Figure BDA0002928786300000151
Figure BDA0002928786300000161
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Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr
275 280 285
Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys
290 295 300
Asp Ser Arg Gly
305
<210> 8
<211> 130
<212> PRT
<213> person (Homo sapiens)
<400> 8
Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala
1 5 10 15
Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys
20 25 30
Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His
35 40 45
Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe
50 55 60
Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro
65 70 75 80
Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn
85 90 95
Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Leu Ala Gly Leu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val
115 120 125
Leu Glu
130
<210> 9
<211> 11
<212> PRT
<213> person (Homo sapiens)
<400> 9
Cys Ala Ser Ser Leu Ala Gly Leu Gln Phe Phe
1 5 10

Claims (4)

1. The T cell receptor for recognizing the C7orf50 mutant antigen short peptide is characterized by comprising a TCR alpha chain and a TCR beta chain, wherein the full-length amino acid sequence of the TCR alpha chain is shown as SEQ ID NO. 1, and the full-length amino acid sequence of the TCR beta chain is shown as SEQ ID NO. 4 or SEQ ID NO. 7;
the variable region amino acid sequence of the TCR alpha chain is shown in SEQ ID NO:2, wherein the amino acid sequence of the variable region of the TCR beta chain is shown as SEQ ID NO. 5 or SEQ ID NO. 8;
the amino acid sequence of the CDR3 of the TCR alpha chain is shown in SEQ ID NO. 3, and the amino acid sequence of the CDR3 of the TCR beta chain is shown in SEQ ID NO. 6 or SEQ ID NO. 9.
2. The use of the T cell receptor recognizing the C7orf50 mutant antigen oligopeptide of claim 1 in preparing a kit for diagnosing liver cancer.
3. Use of a T cell receptor recognizing a C7orf50 mutant antigen oligopeptide according to claim 1 for the preparation of a TCR-T cell specific for a C7orf50 mutant.
4. The use of the T cell receptor recognizing the C7orf50 mutant antigen oligopeptide of claim 1 in the preparation of a medicament for treating liver cancer.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998055133A1 (en) * 1997-06-06 1998-12-10 The Regents Of The University Of California A melanoma associated antigen, t cell epitopes thereof and methods of using same
CN110857319A (en) * 2018-08-24 2020-03-03 杭州康万达医药科技有限公司 Isolated T cell receptor, modified cell thereof, encoding nucleic acid and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998055133A1 (en) * 1997-06-06 1998-12-10 The Regents Of The University Of California A melanoma associated antigen, t cell epitopes thereof and methods of using same
CN110857319A (en) * 2018-08-24 2020-03-03 杭州康万达医药科技有限公司 Isolated T cell receptor, modified cell thereof, encoding nucleic acid and application thereof

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