CN113150111A

CN113150111A - HLA-A0201 restrictive CMVpp65 specific T cell receptor and application thereof

Info

Publication number: CN113150111A
Application number: CN202110133157.1A
Authority: CN
Inventors: 王亚栋; 卫莹; 李啸扬
Original assignee: Shanghai Muxi Biotechnology Co ltd
Current assignee: Shanghai Muxi Biotechnology Co ltd
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2021-07-23

Abstract

The invention provides an HLA-A0201 restrictive CMVpp65 specific T cell receptor and application thereof. The T cell receptor includes an alpha chain and a beta chain; the amino acid sequence of the alpha chain CDR3 region is SEQ ID NO: 8 or to the nucleotide sequence as set forth in SEQ ID NO: 8 and retains the function thereof, and the amino acid sequence of the beta chain CDR3 region is the amino acid sequence shown in SEQ ID NO: 11 or to the nucleotide sequence as set forth in SEQ ID NO: 11 and the amino acid sequence shown in the formula 11 is subjected to chemical group modification and retains the function of the amino acid sequenceAnd (4) sequencing. The HLA-A0201 restriction CMVpp65 specific T cell receptor provided by the invention can specifically recognize CMVpp65 source, presents NLV epitope in HLA-A0201 expression cell and ectopically expresses CD8 of the T cell receptor⁺The T lymphocyte has higher killing property to LCL021 target cells incubated with NLV, and the cell can be applied to more people due to the high frequency of HLA-A0201 expression, so that the cell has high medicinal value.

Description

HLA-A0201 restrictive CMVpp65 specific T cell receptor and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an HLA-A0201 restrictive CMVpp65 specific T cell receptor and application thereof.

Background

Human Cytomegalovirus (HCMV) is a herpes virus with an infection rate of 50-100% in healthy people. HCMV is already infected shortly after birth or in young children and rarely causes significant disease conditions under the supervision and control of a healthy immune system. However, in the case where lymphocytes are removed by hematopoietic stem cells due to a pretreatment process, or in the case where a recipient inhibits T cell activity by long-term use of drugs such as ATG, cyclosporin a, etc. after solid organ transplantation, T lymphocytes in the body are decreased and disabled, and the ability to fight HCMV infection is lost, and viruses are activated and replicated in this case, causing a series of serious life-threatening diseases, especially HCMV infection in about 50% of recipients after solid organ transplantation. Of the infected patients, about 10-50% of patients develop clinical symptoms, mainly manifested by inflammation of various organs, such as pneumonia, hepatitis, enteritis, encephalitis, nephritis, etc. Similar conditions occur after hematopoietic stem cell transplantation, with the most common symptoms being pneumonia and enteritis caused by HCMV, with the most common pneumonia resulting in a mortality rate of 30-60%. Currently, the prevention and treatment drugs for CMV infection include ganciclovir and the like, but the drugs have poor treatment effects, adverse reactions such as bone marrow suppression and the like, and influence the transplantation effect.

The idea that HCMV-infected diseases are caused by the reduction and incapacitation of T cell number, and that the T cell immune response of patients to CMV can be recovered by infusing lymphocytes capable of specifically recognizing and killing CMV-infected cells, namely CMV-CTLs (CMV specific cytoxic T lymphocytes), can effectively treat and prevent the diseases, has been verified in a series of clinical researches. However, the culture method of CMV-CTLs is time-consuming and labor-consuming, inefficient, and due to HLA (human leukocyte differentiation antigen) restriction, such cell preparations are essentially only applicable to themselves and cannot cope with urgent and complicated clinical needs. If common HLA restrictive CMV antigen-specific T lymphocytes are isolated, T cell receptor genes encoding genes mainly responsible for antigen recognition are obtained, and the genes are further introduced into allogeneic lymphocytes to be efficiently expressed, a novel lymphocyte with the same antigen recognition and killing functions as those of original CMV-CTLs is produced. Such cells can be stored for a long period of time after culture and used immediately when needed, possess similar shelf-life product characteristics to traditional drugs, and are one of the most promising approaches to solving such cell products. HLA-a 0201 is an HLA allele that is expressed at high frequency in all ethnic groups of the world. After HLA-A0201 restrictive HCMV antigen specific T Cell Receptor (TCR) is discovered and screened, and the encoding gene is obtained and allogeneic lymphocytes are transduced, the cells are endowed with HLA-A0201 restrictive mode, and CMV-infected cells are recognized and killed. Due to the high frequency of the expression of the HLA-A0201, the method can be applied to more people and has high application value.

Disclosure of Invention

The invention provides an HLA-A0201 restriction CMVpp65 specific T cell receptor and application thereof aiming at the defects of the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an HLA-a x 0201 restricted CMVpp65 specific T cell receptor comprising an alpha chain and a beta chain; the amino acid sequence of the alpha chain CDR3 region is SEQ ID NO: 8 or to the nucleotide sequence as set forth in SEQ ID NO: 8 and retains the function thereof, and the amino acid sequence of the beta chain CDR3 region is the amino acid sequence shown in SEQ ID NO: 11 or to the nucleotide sequence as set forth in SEQ ID NO: 11, and the amino acid sequence shown in the figure 11 is subjected to chemical group modification and retains the function of the amino acid sequence.

Further, the above T cell receptor is composed of an α chain and a β chain; the amino acid sequence of the alpha chain CDR3 region is SEQ ID NO: 8, the amino acid sequence of the beta chain CDR3 region is SEQ ID NO: 11; wherein the beta chain CDR3 region sequences play a key role in epitope recognition.

In a second aspect, the invention provides a nucleotide sequence encoding the above T cell receptor, comprising a nucleotide sequence encoding the alpha chain, a nucleotide sequence encoding the beta chain and a T2A polypeptide coding sequence; the T2A polypeptide coding sequence links the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain.

In a third aspect, the present invention provides an HLA-a x 0201 restriction epitope peptide derived from CMVpp65 antigen recognized by the above-mentioned T cell receptor, the amino acid sequence of the epitope peptide is as set forth in SEQ ID NO: shown at 7.

In a fourth aspect, the present invention provides a T lymphocyte expressing the above-mentioned T cell receptor, having the above-mentioned nucleotide sequence or recognizing the above-mentioned HLA-a 0201-restricted epitope peptide.

Further, the T lymphocyte is CD8⁺T lymphocytes.

In a fifth aspect, the present invention provides a method for preparing the above T lymphocyte, comprising the steps of:

step one, constructing a nucleotide sequence: the T2A polypeptide coding sequence is connected with a nucleotide sequence coding for an alpha chain and a nucleotide sequence coding for a beta chain;

step two, constructing an expression vector: inserting the nucleotide sequence into a lentivirus expression vector, and packaging into virus particles;

and step three, infecting the receptor cells to prepare the T lymphocytes.

Further, the receptor cell in step three is CD8⁺T lymphocytes.

In a sixth aspect, the present invention provides a biological agent comprising the above-described lymphocytes.

In a seventh aspect, the invention provides an application of the T cell receptor, the nucleotide sequence, the T lymphocyte or the biological agent in preparing a medicament for treating human cytomegalovirus infectious diseases.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the HLA-A0201 restriction CMVpp65 specific T cell receptor provided by the invention can specifically recognize CMVpp65 source, presents NLV epitope in HLA-A0201 expression cell and ectopically expresses CD8 of the T cell receptor⁺T lymphocyte has higher killing property to LCL021 target cell incubated with NLV, and the cell can be applied due to high frequency of expression of HLA-A0201Has high medicinal value for more people.

Drawings

FIG. 1 is a graph showing the flow-through comparison of the reactivity of CMV-CTLs against different epitope peptides, which were cultured in one example of the present invention;

FIG. 2 shows a graphical representation of a cloned snail sequence of a TCR sequencing result in accordance with an embodiment of the invention; wherein, the graph A is a cloned snail graph of an alpha chain sequencing result, and the graph B is a cloned snail graph of a beta chain sequencing result;

FIG. 3 shows the results of TCR binding capacity measurements after co-transfection of T lymphocytes with different combinations of alpha and beta chains according to one embodiment of the invention; wherein, the graph A is the detection result of alpha 1/beta 1 binding capacity, and the graph B is the detection result of alpha 3/beta 2 binding capacity;

FIG. 4 is a schematic diagram of the B1A1-TCR expression structure design according to an embodiment of the invention;

FIG. 5 shows the killing activity of B1A1-TCR-T cell ratios and different effective target ratios on LCL021 target cells incubated with NLV and QYD peptides in accordance with one embodiment of the invention; in panel A, CFSE labeled LCL021 cells were loaded with NLV and QYD epitope peptides, respectively, and cultured in mixed culture with B1A1-TCR-T cells at different ratios overnight; addition of PI dye labels dead cells, thus, the CFSE that appears in the right quadrant of the flow chart⁺The cells are target cells (LCL021), and the cells are shown in two quadrants on the right side in the flow chart; if the target cells are killed by the B1A1-TCR-T cells, the target cells are stained by PI, appear in the upper right quadrant, and the cells which are not killed are still in the lower right quadrant, so that the killing rate can be obtained by dividing the ratio of the cells in the upper right quadrant by the ratio of the cells in the two right quadrants; panel a selects representative three effective target ratio results, as well as flow results for target cells only to demonstrate this method and the manner of calculation; FIG. 5B shows the B1A1-TCR-T cell ratio, which varies from experiment to experiment due to the inability to infect all T cells simultaneously by means of lentiviral infection;

FIG. 6 shows the killing rate of LCL021 target cells incubated with NLV and QYD peptides at different effective target ratios in one embodiment of the invention;

FIG. 7 showsIn one embodiment of the invention, the B1A1-TCR on CD8⁺And CD8^-Expression in a subpopulation;

FIG. 8 shows a CD8 according to an embodiment of the invention⁺And CD8^-Killing rate of B1a1-TCR-T against LCL021 target cells incubated with NLV and QYD peptides;

FIG. 9 shows a CD8 according to an embodiment of the invention⁺And CD8^-B1A1-TCR-T IFN-gamma secretion following antigen exposure.

Detailed Description

The invention provides an HLA-A x 0201 restrictive CMVpp65 specific T cell receptor, which can specifically recognize an amino acid sequence derived from CMVpp65 antigen as shown in SEQ ID NO: 7, an HLA-a x 0201 restricted epitope peptide; the receptor includes an alpha chain and a beta chain; the amino acid sequence of the alpha chain CDR3 region is SEQ ID NO: 8 or to the nucleotide sequence as set forth in SEQ ID NO: 8 and retains the function thereof, and the amino acid sequence of the beta chain CDR3 region is the amino acid sequence shown in SEQ ID NO: 11 or to the nucleotide sequence as set forth in SEQ ID NO: 11, and the amino acid sequence shown in the figure 11 is subjected to chemical group modification and retains the function of the amino acid sequence.

In a preferred embodiment of the invention, the T cell receptor consists of an alpha chain and a beta chain; the amino acid sequence of the alpha chain CDR3 region is SEQ ID NO: 8, the amino acid sequence of the beta chain CDR3 region is SEQ ID NO: 11.

the invention provides a nucleotide sequence for coding the T cell receptor, which comprises a nucleotide sequence for coding an alpha chain, a nucleotide sequence for coding a beta chain and a T2A polypeptide coding sequence; the T2A polypeptide coding sequence links the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain.

The invention also provides a T lymphocyte B1A1-TCR-T expressing the T cell receptor, having the nucleotide sequence or recognizing the HLA-A0201 restriction epitope peptide.

In a preferred embodiment of the present invention, the T lymphocyte is CD8⁺T lymphocyte CD8⁺B1A1-TCR-T。

The invention also provides a preparation method of the T lymphocyte B1A1-TCR-T, which comprises the following steps:

and step three, infecting the receptor cells to prepare the T lymphocytes.

In a preferred embodiment of the present invention, the recipient cell in step three is CD8⁺T lymphocytes.

The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

This example provides a method of constructing an HLA-a x 0201 restricted CMVpp65 specific T cell receptor comprising the steps of:

cultivation of CMV-CTLs

A series of HLA-A0201 restriction epitope peptides derived from the HCMV important antigen pp65 (epitope peptide means that the cell can hydrolyze the antigen intracellularly into oligopeptide fragments with certain length, the peptides are combined with HLA molecules under the assistance of molecular chaperones and presented on the surface of the cell for recognition by TCR, and one HLA molecule can only bind one epitope peptide) are synthesized, see Table 1.

Table 1 some HLA-a x 0201 restricted epitope peptides derived from CMVpp65 antigen

After dissolving these epitope peptides in the corresponding solvents, peripheral blood mononuclear cells of healthy HLA-A0201 positive patients are stimulated, and the culture medium is replaced and cytokines are supplemented every 2-3 days. By day 14, the epitope peptides are used for stimulating cultured cells respectively in the presence of Brefeldin A, and after surface staining, fixing, membrane rupture and intracellular staining, flow cytometry is used for detecting whether lymphocytes capable of secreting IFN-gamma after being stimulated by the epitope peptides exist, and the result is shown in figure 1, and the sequence is shown as SEQ ID NO: the epitope peptide of 7 can stimulate part of cultured cells to secrete IFN-gamma, namely the cultured lymphocytes can specifically recognize NLV epitope, and other epitope peptides do not stimulate the corresponding effector cells.

Sequencing of specific TCR genes 2, NLV

NLV epitope peptide reactive lymphocytes are separated by adopting IFN-g differentiation assay cell enriche detect Kit of America whirlpool, and after the lymphocytes are amplified to a corresponding quantity in vitro, total RNA is extracted by using TaKaRaMiniBEST Universal RNA Extraction Kit, and TCR transcripts are sequenced. After obtaining relatively complete transcript complementary DNA sequence by 5' RACE method, in Illumina

And carrying out deep sequencing by an X10 system, carrying out sequencing correction on a sequencing result by a PCR method, carrying out V, D, J, C genotyping by IMGT, and finally determining the TCR alpha/beta protein and DNA sequence. Table 2 below lists the CDR3 amino acid sequences and corresponding ratios of TCR α and β chain components relative to the first three. The alpha chain and beta chain with the first occupation ratio are similar, and each occupation ratio is about 35 percent; the sum of the alpha chain ratios of the second and third ranks is similar to the beta chain ratio of the second rank, and accounts for about 17 percent. This result is more evident in the snail graph (fig. 2).

TABLE 2TCR sequencing results and corresponding ratios

3. Combined validation of potential NLV-specific TCR alpha and beta chains

The complete TCR consists of one α chain and one β chain. Based on the ratios of different α and β chains in the sequencing results, the following are suspected of possible TCR compositions: α 1/β 1, α 2/β 2 and α 3/β 2. By means of gene synthesis, alpha 1, alpha 2, alpha 3, beta 1 and beta 2 chain DNA sequences are synthesized and inserted into corresponding slow virus expression vector separately to be packed into corresponding slow virus particle. The five lentivirus particles were infected with activated T lymphocytes according to the α 1/β 1, α 2/β 2 and α 3/β 2 combinations, respectively, and after 5-7 days of culture, they were stained with the Tetramer T-Select HLA-A02: 01CMV pp65 Tetramer-NLVPMVATV-PE from MBL, and then examined whether the Tetramer could be stained after expression of the different combinations. As shown in FIG. 3, it was found that only the α 1/β 1 combination could obtain staining.

Example 2

In this example, a B1a1-TCR-T cell expressing the T cell receptor provided in example 1 and functional verification thereof were constructed, and the specific preparation process was as follows:

the expression construct is redesigned, and after the α 1 and β 1 chain encoding genes are ligated with the T2A polypeptide coding sequence, the construct is inserted into a lentiviral expression vector and packaged into a viral particle. This structure allows the α 1 and β 1 chains to be separated into two separate protein sequences by automatic cleavage at a specific site in T2A after translation into a protein, and further allows the α 1 and β 1 chains to be cleaved at a stringency of 1: 1 ratio, see FIG. 4.

After 24 hours of activating and culturing PBMC of healthy patients by adopting a 24-well plate coated by an OKT3 monoclonal antibody, adding a proper amount of virus particles, infecting T lymphocytes to prepare NLV epitope specific TCR-T (hereinafter referred to as B1A1-TCR-T), and respectively detecting the TCR structure expression condition and the corresponding function when the B1A1-TCR-T cells are amplified to a corresponding amount.

Killing experiment: LCL021 is an immortal B lymphocyte cell strain, which expresses HLA-A0201 and HLA-A2402 alleles at the same time, LCL021 is marked with CFSE dye, and after incubation with HLA-A0201 limiting epitope peptide NLV and HLA-A2402 limiting epitope peptide QYDPVAALF (SEQ ID NO: 14), respectively, the cell is mixed with cultured B1A1-TCR-T cells according to different effective target ratios and cultured overnight, dye PI is added, and the killing of TCR-T cells to target cells is detected by a flow cytometer. As shown in FIG. 5, this killing effect is specific, and B1A1-TCR-T cells have killing activity only on target cells incubated with NLV peptide, but have no significant effect on QYD incubated cells. In addition, as shown in fig. 6, the killing effect is dose-dependent, and higher killing rate can be obtained by the higher target ratio of the experimental group.

Validation of TCR transduction to CD8 only⁺Cells can exert their killing function to a greater extent:

(1) since the killing function of the obtained B1A1-TCR-T cells is limited by HLA-I molecules, and the recognition of the restricted epitope is always carried by CD8 cells. Thus, CD8 was isolated using magnetic beads⁺Cells and CD8^-Cells, B1A1-TCR-T cells were prepared separately. The results are shown in FIG. 7, which is a non-CD 8⁺Cell comparison, CD8^-Cells appear to be more susceptible to infection by lentiviruses.

(2) After these two cells were again co-cultured overnight with LCL021 cells incubated with HLA-A02: 01 restriction epitope peptide NLV and HLA-A24: 02 restriction epitope peptide QYD, the results of the killing rate obtained are shown in FIG. 8.

(3) The function of B1A1-TCR-T was further verified. Secretion of cytokines is an important aspect of the function of lymphocytes. When lymphocytes recognize the corresponding antigens, they secrete a series of cytokines, such as IFN-gamma, TNF-alpha, GM-CSF, etc., of which the most important one is IFN-gamma. The cytokine can induce inflammatory reaction, improve lymphocyte function, enhance antigen presentation of peripheral target cells, and promote recognition and killing of target cells by lymphocytes.

As can be seen from FIG. 9, only CD8⁺The B1A1-TCR-T can secrete IFN-gamma after contacting NLV epitope peptide, and can secrete a large amount of IFN-gamma under the condition of very low antigen concentration (IC50 is about 0.01 mu M). In response, CD8⁺B1A1-TCR-T of (1-B-T) has no effect on the QYD epitope peptideReactive, CD8^-B1A1-TCR-T of (1) was not responsive to either of the NLV and QYD epitope peptides. This function is seen to be highly specific and requires the involvement of CD 8.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

<110> Shanghai Wood sunset Biotech Ltd

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Claims

1. An HLA-a x 0201 restricted CMVpp65 specific T cell receptor comprising an alpha chain and a beta chain; the amino acid sequence of the alpha chain CDR3 region is SEQ ID NO: 8 or to the nucleotide sequence as set forth in SEQ ID NO: 8 and the amino acid sequence of the beta chain CDR3 region is the amino acid sequence shown in SEQ ID NO: 11 or to the nucleotide sequence as set forth in SEQ ID NO: 11, and the amino acid sequence shown in the figure 11 is subjected to chemical group modification and retains the function of the amino acid sequence.

2. The T cell receptor of claim 1, wherein the T cell receptor consists of an alpha chain and a beta chain; the amino acid sequence of the alpha chain CDR3 region is SEQ ID NO: 8, the amino acid sequence of the beta chain CDR3 region is SEQ ID NO: 11.

3. a nucleotide sequence encoding a T cell receptor according to claim 1 or 2 comprising a nucleotide sequence encoding the α chain, a nucleotide sequence encoding the β chain and a T2A polypeptide coding sequence; the T2A polypeptide coding sequence links the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain.

4. An HLA-a 0201 restriction epitope peptide derived from the CMVpp65 antigen recognized by the T cell receptor according to claim 1 or 2, wherein the amino acid sequence of said epitope peptide is as set forth in SEQ ID NO: shown at 7.

5. A T lymphocyte expressing a T cell receptor according to claim 1 or 2, having a nucleotide sequence according to claim 3 or recognizing an HLA-a 0201 restricted epitope peptide according to claim 4.

6. Root of herbaceous plantThe T lymphocyte of claim 5, wherein the T lymphocyte is CD8⁺T lymphocytes.

7. A method for producing T lymphocytes according to claim 5 or 6, comprising the steps of:

step one, constructing a nucleotide sequence: linking a T2A polypeptide coding sequence to a nucleotide sequence encoding an alpha chain and a nucleotide sequence encoding a beta chain;

step two, constructing an expression vector: inserting the nucleotide sequence into a lentiviral expression vector and packaging into a viral particle;

and step three, infecting receptor cells to prepare the T lymphocytes.

8. The method of claim 7, wherein the recipient cell is CD8 in step three⁺T lymphocytes.

9. A biological agent comprising the T lymphocyte of claim 5 or 6.

10. Use of the T cell receptor according to claim 1 or 2, the nucleotide sequence according to claim 3, the T lymphocyte according to claim 5 or 6 or the biological agent according to claim 9 for the preparation of a medicament for the treatment of human cytomegalovirus infectious diseases.