CN113121676B

CN113121676B - Specific T cell receptor targeting cytomegalovirus antigen and application thereof

Info

Publication number: CN113121676B
Application number: CN202010040290.8A
Authority: CN
Inventors: 徐曲苗; 朱琳楠; 王飞; 赵正琦; 林玉琪; 李子怡; 顾颖; 侯勇
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2022-05-31
Anticipated expiration: 2040-01-15
Also published as: CN113121676A

Abstract

The application discloses a specific T cell receptor targeting cytomegalovirus antigen and application thereof. The specific T cell receptor of the targeted cytomegalovirus antigen can be specifically targeted and combined with cytomegalovirus phosphoprotein pp65, an alpha chain and a beta chain of the specific T cell receptor respectively have three complementarity determining regions, and the three complementarity determining regions of the alpha chain are sequences shown in SEQ ID Nos. 4, 5 and 2 in sequence; the three complementarity determining regions of the beta chain are shown in SEQ ID No.6, 7 and 3 in sequence. The specific T cell receptor can specifically target and combine the epitope polypeptide of cytomegalovirus phosphoprotein pp65, and provides a new scheme for TCR-T treatment of cytomegalovirus related diseases; in addition, the preparation method of the specific T cell receptor is simple and easy to operate, and lays a foundation for popularization and application of TCR-T treatment based on the specific T cell receptor.

Description

Specific T cell receptor targeting cytomegalovirus antigen and application thereof

Technical Field

The application relates to the field of cytomegalovirus, in particular to a specific T cell receptor targeting cytomegalovirus antigen and application thereof.

Background

Cytomegalovirus (CMV) belongs to a member of the herpes family of viruses. After cytomegalovirus infection, symptoms are greatly different, and no symptoms exist, and the patients with discomfort of the whole body, fever, low immunity and fetuses can have serious symptoms, such as blindness, brain and viscera damage. The virus can be transmitted through sexual or non-sexual body fluids. Meanwhile, CMV infection is also common in organ transplantation and bone marrow transplantation, and is an important factor causing diseases and deaths after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, current antiviral drugs, such as acyclovir, valganciclovir, ganciclovir, often cause serious side effects, and with the development of resistant viral strains, treatment approaches for such antiviral drugs tend to be ineffective.

In general, immune effector mechanisms inhibit the replication and spread of a large number of viruses. However, immune impairment due to allogeneic stem cell transplantation (allo-HSCT) can reactivate latent human cytomegalovirus leading to serious clinical complications. In recent years, researchers have effectively restored virus-specific T cell responses by culturing enriched CMV-specific T cell clones and T cell lines in vitro and adoptively transplanting patients with CMV-positive stem cell transplants, potentially reducing the risk of Graft Versus Host (GVHD). However, the application of CMV-specific T cells is severely hampered by the complex technical limitations of their in vitro expansion and by the excessive cost requirements. Furthermore, since the specific T reconstitution of transplanted patients from CMV-negative donors is difficult and lack of adoptive therapy, these donors do not obtain CMV-specific T cells and thus still present a high risk. After allogeneic hematopoietic stem cell transplantation, a high affinity T cell response to CMV has not been achieved in the patient's blood, i.e. their immune system is not protected against CMV infection. A lentivirus-engineered T cell receptor-engineered T cell (TCR-T) therapy carrying a CMV high affinity TCR gene can effectively control acute CMV disease and induce an antiviral immune response. This therapy holds the promise of being one of the most effective therapies for CMV infection.

Although TCR-T holds promise for the treatment of diseases caused by CMV infection, it is a great challenge to construct specific TCR-transduced T cells that are functional by obtaining effective T cells from cytotoxic T lymphocytes stimulated in vivo or in vitro in patients and rapidly obtaining their TCR sequences. Previous studies have performed TCR sequence identification by various methods such as 5' Race, multiplex PCR, and the like. However, the screening process is complicated, and the TCR pairing information is lost, which becomes resistance to the wide application. With the development of single cell sequencing technology, high-throughput determination of TCR pairing sequences and evaluation of important gene expression levels of immune cells can be realized at the single cell level.

Disclosure of Invention

The purpose of the application is to provide a novel specific T cell receptor targeting cytomegalovirus antigen and application thereof.

The application specifically adopts the following technical scheme:

the first aspect of the application discloses a specific T cell receptor targeting cytomegalovirus antigens, which can specifically target and combine cytomegalovirus phosphoprotein pp 65; the alpha chain and the beta chain of the specific T cell receptor are respectively provided with three complementarity determining regions, wherein the three complementarity determining regions of the alpha chain are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.2 in sequence; the three complementarity determining regions of the beta chain are shown as SEQ ID No.6, SEQ ID No.7 and SEQ ID No.3 in sequence;

SEQ ID No.2：CARNTGNQFYF

SEQ ID No.3：CASSYVTGTGSYGYTF

SEQ ID No.4：SSNFYA

SEQ ID No.5：MTLNGD

SEQ ID No.6：MNHEY

SEQ ID No.7：SVGAGI。

it should be noted that the complementarity determining regions are key to determining the specific targeting properties and antibody titers of the specific T cell receptors of the present application, as long as the specific T cell receptors comprising the three complementarity determining regions of the α -chain and β -chain as defined herein are capable of having the effect of targeting cytomegalovirus antigens of the present application, and in particular, targeting binding to cytomegalovirus phosphoprotein pp 65. It is understood that the specific T cell receptor of the present application may comprise other non-complementarity determining region sequences in addition to the three complementarity determining region sequences of the α and β chains, and is not particularly limited herein.

Preferably, the amino acid sequence of the variable region of the alpha chain is the sequence shown in SEQ ID No. 9;

SEQ ID No.9：

MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIP。

it should be noted that the α chain of the sequence shown in SEQ ID No.9 is only an α chain sequence of a specific T cell receptor specifically screened in an implementation manner of the present application, and it can be understood that as long as three complementarity determining regions of the α chain are sequentially the sequences shown in SEQ ID No.4, SEQ ID No.5, and SEQ ID No.2, all of them can meet the use requirement of the present application, and are not limited to the α chain of the sequence shown in SEQ ID No. 9.

Preferably, the amino acid sequence of the variable region of the beta chain is the sequence shown in SEQ ID No. 11;

SEQ ID No.11：

MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVTGTGSYGYTFGSGTRLTVV。

it should be noted that the beta-strand of the sequence shown in SEQ ID No.11 is also only a beta-strand sequence of a specific T cell receptor specifically screened in an implementation manner of the present application, and it can be understood that as long as the three complementarity determining regions of the beta-strand are the sequences shown in SEQ ID No.6, SEQ ID No.7, and SEQ ID No.3 in sequence, they can all meet the usage requirements of the present application, and are not limited to the beta-strand of the sequence shown in SEQ ID No. 11.

Preferably, the specific T cell receptor is a sequence shown as SEQ ID No. 13;

SEQ ID No.13：

MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVTGTGSYGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSRAKRSGSGATNFSLLKQAGDVEENPGPMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS。

it is noted that specific T cell receptors may contain other non-variable region amino acid sequences in addition to their critical alpha and beta chains; similarly, the specific T cell receptor of the sequence shown in SEQ ID No.13 is only one specific T cell receptor sequence selected in the present application, and it is understood that the three complementarity determining region sequences of the α chain and the β chain are the sequences defined in the present application to satisfy the requirements of the present application, and are not limited to the specific T cell receptor of the sequence shown in SEQ ID No. 13.

Preferably, the specific T cell receptor of the application is targeted to bind to cytomegalovirus phosphoprotein pp65, and specifically comprises that the specific T cell receptor specifically binds to NLV epitope polypeptide of cytomegalovirus phosphoprotein pp65 when presented by Major Histocompatibility Complex (MHC), wherein the NLV epitope polypeptide is a sequence shown as SEQ ID No. 1; SEQ ID No. 1: NLVPMVATV.

It is noted that the CMV pp65 antigen NLV epitope polypeptide is a known epitope polypeptide, and the key of the present application is to develop a new specific T cell receptor based on the epitope polypeptide.

In a second aspect of the present application, there is disclosed a nucleotide encoding the alpha chain of a specific T cell receptor of the present application.

Preferably, the nucleotide encoding the alpha chain is the sequence shown in SEQ ID No. 8.

It is noted that the α chain-encoding nucleotides of the present application can be used as raw materials or gene editing materials for the preparation of the specific T cell receptors of the present application; wherein, the sequence shown in SEQ ID No.8 is only a nucleotide sequence specifically adopted in one implementation mode of the application; it is understood that synonymous mutations may also be made on the basis of the sequence shown in SEQ ID No.8, thereby obtaining other nucleotides encoding the alpha chain of the application, which are not specifically limited herein.

In a third aspect of the present application, there is disclosed a nucleotide encoding the beta chain of the specific T cell receptor of the present application.

Preferably, the nucleotide encoding the beta-strand is the sequence shown in SEQ ID No. 10.

It should be noted that the nucleotide encoding the β chain of the present application can also be used as a raw material or gene editing material for preparing the specific T cell receptor of the present application; wherein, the sequence shown in SEQ ID No.10 is only a nucleotide sequence specifically adopted in one implementation mode of the application; it is understood that synonymous mutations may also be made on the basis of the sequence shown in SEQ ID No.10, so as to obtain other nucleotides encoding the beta-strand of the application, which is not specifically limited herein.

In a fourth aspect of the present application, there is disclosed a nucleotide encoding a specific T cell receptor of the present application.

Preferably, the nucleotide encoding the specific T cell receptor is the sequence shown in SEQ ID No. 12.

It is noted that the nucleotide encoding the specific T cell receptor of the present application is a raw material for preparing the specific T cell receptor of the present application; wherein, the sequence shown in SEQ ID No.12 is only a nucleotide sequence specifically adopted in one implementation mode of the application; it will be appreciated that synonymous mutations may also be made on the basis of the sequence shown in SEQ ID No.12 to obtain other nucleotides encoding a T cell receptor specific for the present application, and are not specifically limited herein.

In a fifth aspect of the present application, there is disclosed a vector comprising an alpha chain-encoding nucleotide, a beta chain-encoding nucleotide, or a specific T cell receptor-encoding nucleotide of the present application.

It is noted that vectors containing the α chain-encoding nucleotides and/or β chain-encoding nucleotides of the present application can be used as gene editing material for the preparation of vectors encoding specific T cell receptor nucleotides of the present application and thus for the preparation of specific T cell receptors of the present application.

A sixth aspect of the present application discloses a cell comprising a specific T cell receptor of the present application, or a nucleotide encoding an alpha chain, a nucleotide encoding a beta chain or a nucleotide encoding a specific T cell receptor of the present application, or a vector of the present application.

It is contemplated that, among other things, cells containing the alpha chain-encoding nucleotides and/or beta chain-encoding nucleotides of the present application can be further used to prepare the specific T cell receptors of the present application.

Preferably, the cells of the present application are T lymphocytes.

In a seventh aspect of the present application, there is disclosed the use of a specific T cell receptor of the present application, or a nucleotide encoding an alpha chain, a nucleotide encoding a beta chain or a nucleotide encoding a specific T cell receptor of the present application, or a vector of the present application, or a cell of the present application, in the manufacture of a medicament for the treatment and/or prevention of a disease associated with cytomegalovirus.

A seventh aspect of the present application discloses a pharmaceutical composition for the treatment and/or prevention of a cytomegalovirus-associated disease comprising a specific T cell receptor of the present application, or a vector of the present application, or a cell of the present application; the cells of the present application are especially cells containing the specific T cell receptor or vector of the present application, especially vectors containing nucleotides encoding the specific T cell receptor.

It is noted that the specific T cell receptor of the present application is capable of specifically targeting CMV, and thus, corresponding agents can be prepared based on TCR-T therapy. It will be appreciated that when directed to a particular subject, the T lymphocytes employed are preferably T lymphocytes isolated from the subject; alternatively, the T lymphocytes are derived from the same donor as the hematopoietic stem cells, organs, tissues or stem cells at the time of allogeneic transplantation.

It is also noted that the diseases related to cytomegalovirus of the present application include all of the diseases related to cytomegalovirus currently recognized, such as general malaise, fever, blindness, brain and internal organ damage caused by cytomegalovirus infection; and diseases or clinical complications caused by cytomegalovirus infection after organ transplantation, bone marrow transplantation or allogeneic hematopoietic stem cell transplantation, and the like.

The beneficial effect of this application lies in:

the specific T cell receptor can specifically target and combine the epitope polypeptide of cytomegalovirus phosphoprotein pp65, and provides a new scheme for TCR-T treatment of cytomegalovirus related diseases; in addition, the preparation method of the specific T cell receptor is simple and easy to operate, and lays a foundation for popularization and application of TCR-T treatment based on the specific T cell receptor.

Drawings

FIG. 1 is a diagram of the CMV-specific cytotoxic T lymphocyte function test EliSpot in the examples of the present application;

FIG. 2 is a diagram of flow analysis of CMV-specific cytotoxic T lymphocyte sorting in the examples of the present application;

FIG. 3 is a schematic diagram of the assembly structure of the coding nucleotides of the alpha and beta chains of specific T cell receptors in the examples of the present application;

FIG. 4 is a graph of flow analysis of anti-murine TCR β constant region antibodies and tetramer staining of TCR transduced T cells specific for CMV pp65 in the examples of the present application;

FIG. 5 is an ELISpot assay for IFN- γ cytokine secretion from TCR-T in an embodiment of the present application;

FIG. 6 is a diagram of flow analysis for detecting TCR-T cytokine secretion by flow analysis in an embodiment of the present application;

FIG. 7 is a graph of flow analysis of the detection of TCR-T on target cell killing in an example of the present application;

FIG. 8 is a graph of the statistics of the cell killing ratio of the flow assay to detect the killing effect of TCR-T on target cells in the examples of the present application.

Detailed Description

TCR-T therapy is effective in controlling acute CMV disease, causing an antiviral immune response; however, how to obtain effective T cells from cytotoxic T lymphocytes stimulated in vivo or in vitro of a patient and rapidly obtain the TCR sequence thereof, so as to construct specific T cells transduced by functional TCR is a hindrance to TCR-T application. Based on a single cell sequencing technology, the method realizes determination of a TCR pairing sequence through high-throughput sequencing, evaluates the expression level of important genes through immune cells, and researches specific T cell receptors. Specifically, in an implementation manner of the application, antigen-specific cytotoxic T lymphocytes are amplified in vitro by inducing CMV pp65 antigen NLV epitope polypeptide NLVPMVATV based on HLA-A0201 typing, TCR pairing information of positive T cells is screened according to clone sequence frequency through single cell sequencing and MHC tetramer sorting, TCR is constructed, TCR-T function is verified in vitro, TCR-T function is evaluated, and finally the specific T cell receptor is obtained through research and development.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

In the embodiment, antigen CMV pp65 antigen NLV epitope polypeptide NLVPMVATV based on HLA-A0201 typing induces and amplifies cytotoxic T lymphocytes with antigen specificity in vitro, TCR pairing information of positive T cells is obtained after single cell sequencing and MHC tetramer sorting, TCR is screened according to clone sequence frequency, TCR-T is constructed, in-vitro function verification is carried out, the function of the TCR-T is evaluated, and finally the specific T cell receptor of the embodiment is determined. The details are as follows:

CMV pp65 NLV specific cytotoxic T lymphocyte induction and functional assay

1.1 CMV pp65 NLV-specific cytotoxic T lymphocyte Induction

50mL of HLA-A0201-typed healthy human peripheral blood was collected and subjected to Ficoll density gradient centrifugation to obtain Peripheral Blood Mononuclear Cells (PBMC). From the separated PBMCs, CD14 was sorted using anti-CD 14 antibody-coated magnetic beads (Miltenyi) and anti-CD 8 antibody-coated magnetic beads (Miltenyi), respectively⁺Monocytes (DC precursor cells) and CD8⁺T cells, the specific sorting steps refer to magnetic bead instructions. Separating the obtained CD8⁺And (5) freezing and storing the T cells for later use. Separating the obtained CD14⁺Monocyte dose of 1 × 10⁶Cell density of/mL was added to 6-well plates and cultured with X-VIVO containing 2% FBS, 800U/mL GM-CSF and 1000U/mL IL-4 at 37 ℃ with 5% CO₂Culturing in incubator for 3 days to induce CD14⁺The monocytes are differentiated into immature DC cells, and then 20ng/mL IL-1 beta, 20ng/mL IL-6, 40ng/mL TNF-alpha, 100ng/mL PGE-2 and 5 mu g/mL polyIC are added to induce for 48 hours, thereby promoting the maturation of the DC cells.

2mg of CMV pp65 NLV epitope polypeptide having a sequence shown in SEQ ID No.1 was dissolved in 200uL of DMSO solvent at a concentration of 10 mg/mL. Mature DC cells were harvested by pipetting from well plates into 15mL centrifuge tubes, centrifuging at 300g, resuspending in 2mL serum-free X-VIVO medium, and adding CMV pp65 NLV epitope polypeptide at a dilution of 1:1000 to a final concentration of 10. mu.g/mL. Cells were plated at 37 ℃ with 5% CO₂After 2 hours of incubator incubation, 10mL of X-VIVO medium was added and centrifuged at 300 g. The precipitated cells were resuspended in HIPP-T009 medium containing 2% FBS and 30ng/mL IL-21, prepared into mature DC cells loaded with CMV pp65 NLV epitope polypeptide, and prepared for CD8⁺T cell co-culture.

SEQ ID No.1：NLVPMVATV。

Resuscitated CD8 1 day before co-culture⁺T cells. In co-culture, mature DC cells loaded with CMV pp65 NLV epitope polypeptide and CD8⁺The T cells were counted separately and plated at a density of 1:4 to 1:8 for DC: CD8+ T cells in 48-well cell culture plates with a total cell count of 1.3 to 2.8X 10 per well⁶Culture with 30ng/mL IL-21 in HIPP-T009 at 37 deg.C, 5%CO₂The incubator is used for 3 days. 1/2 volumes of supernatant were then carefully discarded, cells were blown out and transferred to 12-well plates, and HIPP-T009 medium (T cell expansion medium) containing 2% FBS, 10ng/mL IL-2, 10ng/mL IL-7 and 10ng/mL IL-15 was used at 37 ℃ with 5% CO₂And culturing and amplifying for 2 days in an incubator. 1/2 volumes of supernatant were then carefully discarded, and the cells were blown out and transferred to 6-well plates and expanded in culture with the above medium for 6 days. In the culture process, the cell density is observed every 2 days, the cells are timely distributed into new holes, and a T cell amplification culture medium is added to ensure enough space and nutrition to amplify the cells. When the cells expanded to 2X 10⁷In the above, IFN-. gamma.secretion was examined by EliSpot.

In contrast, this example set unloaded polypeptide DC to CD8⁺T cell Co-culture group, i.e., mature DC cells were directly added with an equal amount of DMSO solvent and then incubated with CD8 in the same manner⁺T cell co-culture was inoculated in T009 medium, and the remaining culture steps and treatments were identical, for the flow sort control "2. CMV pp65 NLV specific cytotoxic T lymphocyte sorting".

1.2 detection of CMV pp65 NLV-specific cytotoxic T lymphocyte function induced

Enzyme linked immunospot assay (ELISpot) is a novel immunoenzyme technique for quantitative detection of single secretory cells in 96-well plates. The cells are cultured in a 96-well plate coated with specific antibodies, and under the condition of stimulation, the cell secreted cytokine is captured by the coated antibodies, and the cytokine detected in the example is IFN-gamma. After removal of the cells, the captured cytokines can be further labeled with a secondary antibody labeled with alkaline phosphatase, which is colored with 5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium (BCIP/NBT) substrate, and the reacting cells will leave purple spots of varying diameters. In this example, 2X 10 wells per well were added to a 96-well ELISpot plate (Mabtech)⁴T cells and the use of a 5X 10-loaded CMV pp65 NLV epitope polypeptide³Individual T2 cells were co-cultured and labeled as CMV pp65 group. As a control, T cells were co-cultured with T2 cells not loaded with the polypeptide but added with an equal amount of DMSO solventNurse, marked as DMSO group. Co-culture was performed in HIPP-T009 medium containing 2% FBS at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 20 hours. The immunospot was imaged and read using an ELISPOT reader AID isspot (AID-autoimum diagnostic GmbH, stelas burg, germany).

The results of the EliSpot assay are shown in figure 1 and show that significantly more spots were detected in the CMV pp65 group compared to the control DMSO group, demonstrating the in vitro stimulation of amplified CD8⁺The T cells can specifically recognize the CMV pp65 NLV epitope polypeptide-loaded T2 cells and secrete IFN-gamma cytokines.

CMV pp65 NLV specific cytotoxic T lymphocyte sorting

2.1 preparation of tetramers loaded with CMV pp65 NLV epitope polypeptide

For the marker CMV pp65 NLV specific T cells, HLA-a 0201 typing tetramers loaded with CMV pp65 NLV epitope polypeptide were prepared. Specifically, 5. mu.L of 10mM target polypeptide mother liquor is added into 120. mu.L PBS to prepare 125. mu.L of 400. mu.M polypeptide solution, and the solution is placed on ice; mu.L of a diluted 400. mu.M solution of the polypeptide of interest and 20. mu.L of a monomer (Flex-T) were added to a 96-well plate with a U-shaped bottom^TMmonomer, BioLegend,200 μ g/ml), mixed up and down using a pipettor; sealing with sealing membrane, centrifuging at 3000rpm for 1min to get the reaction solution to the bottom of the plate; the sealing film is carefully uncovered, so that liquid is prevented from splashing; placing the flat plate on ice, and placing the flat plate in a dark box of an ultraviolet crosslinking instrument to crosslink for 30min under 365nm ultraviolet light; sealing with sealing film plate, and incubating in 37 deg.C incubator for 30min in dark; after the incubation is finished, placing the plate on ice, adding 4.4 mu L of fluorescence coupled streptavidin (BioLegent, for other brands of fluorescence coupled streptavidin, the molar ratio of the monomers to the streptavidin needs to be ensured to be 6:1), mixing the mixture up and down by a pipettor, and placing the mixture on ice in a dark place for 30 min; during the incubation on ice, stop solutions were prepared: 2.56. mu.L of 500. mu. M D-Biotin, 0.093. mu.L of 10% (w/v) NaN was taken₃And 0.54. mu.L PBS, vortex and mix; after the incubation is finished, adding 3.2 mu L of stop solution into the reaction solution, and uniformly mixing the stop solution and the reaction solution up and down by using a pipettor to stop the reaction; incubating overnight at 4 deg.C with sealing membrane plate, or standing on ice in dark place for 30min for dyeing。

2.2 CMV pp65 NLV specific cytotoxic T lymphocyte sorting

Taking 1 × 10 CMV pp65 specific T cells prepared by 1 CMV pp65 NLV specific cytotoxic T lymphocyte induction and function detection⁷Cells are placed in a 5mL centrifuge tube, centrifuged for 5min at 400g, and then 200 mu L of flow buffer solution is added for resuspension; adding 10 mu L of tetramer loaded with CMV pp65 NLV epitope polypeptide prepared in '2.1', mixing uniformly, and reacting for 30min at 4 ℃; after the reaction time is over, adding 3mL of PBSA, centrifuging for 400g and 5min, carefully discarding the supernatant, adding 4mL of PBSA for resuspension and centrifuging, carefully discarding the supernatant, resuspending with 500 mu L of PBS, and placing on ice for flow detection; after flow-loading, positive populations were selected for sorting of single cells. Wherein the flow buffer is prepared by adding 0.5% FBS into PBS.

At the same time, mature DC cells not loaded with CMV pp65 NLV epitope polypeptide and CD8 were used in this example⁺The same experiment was performed on the co-cultured T cell products for comparison. Wherein the mature DC cells are associated with CD8⁺T cell co-culture was the same as "1. CMV pp65 NLV specific cytotoxic T lymphocyte induction and function test".

The results of flow cytometry after stimulation with CMV antigenic polypeptide are shown in fig. 2, in which the left panel is a flow cytometry sorting plot of the mature DC cells not loaded with CMV pp65 NLV epitope polypeptide co-cultured with CD8+ T cells and the right panel is a flow cytometry sorting plot of the mature DC cells loaded with CMV pp65 NLV epitope polypeptide co-cultured with CD8+ T cells and stained. The results in fig. 2 show that the control group not loaded with CMV pp65 NLV epitope polypeptide stained 0.58% positive with tetramer, possibly non-specific staining of tetramer; whereas T cells stimulated with CMV antigen polypeptide antigen can detect 6.1% of positive CMV-specific T cells with tetramers, the portion indicated by the box in the right panel.

3. 10 XGenomics-based high-throughput single-cell TCRV (D) J full-length sequencing

The droplet microfluidic technology based on the GemCode platform of 10 XGenomics leads magnetic beads, single cell suspension cells, a reverse transcription reaction system and oil to pass through a very fine channel together to generate water-in-oil micro droplets. Separate oil droplets containing one magnetic bead, one cell and a reverse transcription reaction system, i.e., GEMs (Gel Beads-In-Emulsions), can be separated. The GEMS are themselves a single space, each containing a unique barcoded beads. Each of the Beads carries a different Barcode primer for labeling the transcriptome of each single cell, and the source cells of each of the Beads can be tracked after the later analysis. Collecting hundreds of thousands of generated liquid drops in a single tube to carry out reverse transcription reaction, generating a full-length cDNA product, carrying out twice enrichment of a target segment, carrying out TCR segment amplification by using a TCR specific primer, enriching V (D) J segments from the cDNA amplification product, and carrying out V (D) J library construction and sequencing to obtain a paired TCR alpha/beta variable region sequence of a single cell.

In this example, 6.1% of positive CMV-specific T cells obtained by sorting "2. CMV pp65 NLV-specific cytotoxic T lymphocytes" were counted, the cell concentration was adjusted to 800 cells/. mu.L at 700-^TM Single Cell 5'Library&Gel Bead Kit) for GEM generation, reverse transcription, cDNA purification, amplification, TCRV (D) J and transcriptome banking.

Sequencing results show that from 830 single cells successfully used for library construction, 539 cells obtained the full-length TCR sequence, and the ten-first-frequency clonotype (clonotype) and CDR3 sequences thereof are shown in Table 1. Wherein the amino acid sequence of the TCR alpha/beta variable region of the clonotype 1 with the highest frequency is shown as SEQ ID No.9 and SEQ ID No. 11.

TABLE 1 shows the TCR clonotypes with the first ten frequencies in the single cell sequencing results and the CDR3 sequences thereof

CMV pp 65-specific TCR Lentiviral shuttle plasmid vector construction

The exogenous TCR is transferred into the T cell and has the problem of mismatching with the endogenous TCR, and the expression of the exogenous TCR on the T cell is influenced. Research reports that the expression efficiency of exogenous TCR can be effectively improved by connecting the variable region of the transferred TCR with the constant region of a mouse (Foley KC et al (2017) Mol Ther: Oncolytics 5: 105-115). Based on the highest frequency CMV pp65 specific TCR obtained from the sequencing, the α and β variable region sequences were fused with the mouse α constant region and β 2 constant region, respectively, to obtain TCR α and TCR β chains, and TCR β and TCR α chains were linked using the 2A sequence of internal self-splitting Porcine teschovirus (Porcine teschovirus), as shown in FIG. 3. The example of placing TCR β before the 2A sequence is based on literature reports that placing the TCR β gene before the 2A sequence can facilitate TCR expression and assembly due to the greater length of TCR β than TCR α. In this example, TCR β was cloned between the BamHI and EcoRI cleavage sites of prrlsin. cppt. pgk-gfp. wpre lentiviral shuttle plasmid vector (purchased from addge) by gene synthesis and BamHI and EcoRI restriction endonuclease cleavage of codon optimized exogenous TCR gene, and the EGFP gene in the vector was replaced with TCR gene.

Specifically, the TCR sequence of the clone type 1 is taken from '3' high-throughput unicell TCRV (D) J full-length sequencing based on 10 XGenomics and is constructed into a lentiviral vector, and the nucleic acid sequence is the sequence shown in SEQ ID No. 12. Methods for lentiviral shuttle plasmid vector construction reference the instructions for use.

SEQ ID No.12：

5’-ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACGTTACCGGGACAGGGTCCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGATCTGAGAAACGTGACCCCCCCTAAGGTGTCTCTGTTTGAGCCTAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCAGATCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACTCCGGCGTGTCTACAGACCCCCAGGCCTATAAGGAGAGCAATTACTCCTATTGCCTGTCTAGCCGGCTGAGAGTGTCCGCCACCTTCTGGCACAACCCCCGGAATCACTTCAGATGTCAGGTGCAGTTTCACGGCCTGTCCGAGGAGGATAAGTGGCCTGAGGGCTCTCCAAAGCCCGTGACACAGAACATCAGCGCCGAGGCATGGGGAAGAGCAGACTGTGGCATCACCTCTGCCAGCTACCACCAGGGCGTGCTGAGCGCCACAATCCTGTATGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGGACTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCTAGGGCAAAGCGGAGCGGCTCTGGAGCAACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAACCCTGGACCAATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAGAAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACAGCTATTTGTACATCAAAGGATCCCAGCCTGAAGACTCAGCCACATACCTCTGTGCCCGGAACACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAGACATCCAGAACCCCGAGCCTGCCGTGTACCAGCTGAAGGACCCAAGATCCCAGGATAGCACCCTGTGCCTGTTCACCGACTTTGATTCTCAGATCAATGTGCCCAAGACCATGGAGAGCGGCACCTTTATCACAGACAAGACCGTGCTGGATATGAAGGCCATGGACAGCAAGTCCAACGGCGCCATCGCCTGGAGCAATCAGACATCCTTCACCTGCCAGGATATCTTTAAGGAGACAAACGCCACCTACCCTTCTAGCGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGACTGTGGTCCTCT-3’。

Preparation of CMV pp 65-specific TCR-transduced T cells (TCR-T)

TCR transduction into T cells is achieved by infecting human T cells with a lentiviral vector carrying the TCR gene of interest. Briefly, lentiviral shuttle plasmids bearing the TCR gene of interest and a second generation lentiviral packaging plasmid (PsPAX2, pmd2.g, purchased from Addgene) were transferred into 293T cells by pei (polyethylenimine) transfection, resulting in the release of replication-deficient lentiviruses into the supernatant, which was collected 72 hours after transfection and the lentiviruses concentrated by ultracentrifugation. The concentrated lentiviruses were titered and healthy human 0201-typed T cells isolated from PBMC were infected at a certain MOI (Multiplicity of infection). The transduced cells were expanded 24 hours after infection by culture in medium containing IL-2, IL-7, IL-15 cytokines. TCR expression positive rates were detected 5 days post infection with anti-mouse TCR β constant region antibody and tetramer staining.

Specifically, 1X10 TCR-T cells were prepared⁶Dividing the cells into two 5mL centrifuge tubes, centrifuging at 400g for 5min, and adding 100 mu L flow buffer solution for resuspension; one of the tubes was added 1. mu.L of the tetramer loaded with the CMV pp65 NLV epitope polypeptide prepared in "2.1Adding 2 mu L of anti-mouse TCR beta constant region antibody (Biolegend) into the other tube, mixing uniformly and reacting for 30min at 4 ℃; after the reaction time was over, 4mL of PBSA was added, centrifuged at 400g for 5min, the supernatant was carefully discarded, 4mL of PBSA was added again, resuspended and centrifuged, the supernatant was carefully discarded, resuspended in 200. mu.L of PBS, and placed on ice for flow assay. The positive rate of TCR expression was determined 5 days after infection using anti-mouse TCR β constant region antibody and tetramer staining as shown in FIG. 4. In FIG. 4, the left panel shows the staining results of the anti-mouse TCR β constant region antibody, and the right panel shows the staining results of the tetramer. The results in fig. 4 show that the TCR-T cells produced expressed a TCR β constant region at a rate of 10.8% and the tetramer loaded with the CMV pp65 NLV epitope polypeptide stained positive at 12.6%, indicating that the transduced CMV pp 65-specific TCR was expressed more efficiently and without mismatch to the endogenous TCR.

TCR transduced T cell function assay

To confirm that the TCR-T cells prepared in this example have antigen-specific activity, T cell function experiments under antigen stimulation were performed.

1) Detection of IFN-gamma cytokine secretion by TCR-T by ELISpot

Following the protocol shown in 1.2, 2X 10 additions per well were made in 96-well ELISpot plates (Mabtech)⁴TCR-T cells and the use of a 5X 10-loaded CMV pp65 NLV epitope polypeptide³Individual T2 cells were co-cultured, i.e. CMV pp65 group. As a control, T cells were co-cultured with T2 cells not loaded with polypeptide, but added with an equal amount of DMSO solvent, i.e., DMSO group. Co-culture was performed in HIPP-T009 medium containing 2% FBS at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 20 hours. The immunospot was imaged and read using an ELISPOT reader AID isspot (AID-autoimum diagnostic GmbH, stelas burg, germany). EliSpot assay results As shown in FIG. 5, significantly more spots were detected in the CMV pp65 group compared to the control DMSO group, demonstrating that the prepared TCR-T cells specifically recognize T2 cells loaded with CMV pp65 NLV epitope polypeptide and secrete IFN-gamma cytokines.

2) Detection of TCR-T cytokine secretion by flow assay

The intracellular flow analysis method is to combine the anti-cytokine antibody with the cell surface or intracellular specific subgroup mark, which can detect the secretion of different cell subgroup cytokines, and simultaneously adopts special chemistry and antibody selection to ensure the minimum fluorescence background of the resting and cytokine-free secretory cells.

The specific experimental process is as follows:

the first day of rest "5. CMV pp 65-specific TCR transduced T cells (TCR-T)" preparation of the obtained TCR-T cells: take out about 1X10⁷Cells were centrifuged at 400g for 5min at room temperature, the supernatant removed, resuspended in T009 medium containing 2% FBS, plated in 6-well plates and rested for 24 hours.

Next day the target cells were loaded with antigenic polypeptides: t2 cells (purchased from ATCC) were counted and approximately 2X 10 cells were removed⁶Cells, 300g at room temperature, were centrifuged for 5min and resuspended in 2mL of 10% bovine serum IMDM medium and aliquoted into 2 wells of a 24-well plate. Dissolving CMV pp65 NLV epitope polypeptide with sequence shown in SEQ ID No.1 in DMSO to prepare 10 μ g/mL, adding appropriate amount of CMV pp65 NLV epitope polypeptide into one of the wells (CMV pp65 group) according to a dilution ratio of 1:1000, resuspending and mixing, and 5% CO at 37 deg.C to obtain mixture with final concentration of 10ug/mL₂Incubate overnight. An equal amount of DMSO was added to another well for the same treatment and incubation, i.e., DMSO group.

And (3) co-culturing effector cells and target cells on the third day: after the antigen loading time, the T2 cells in the well plate were separately purged with 2% bovine serum T009 medium, centrifuged at room temperature for 300g and 5min, resuspended in 500. mu.L of 2% bovine serum T009 medium, counted, and adjusted to 1.5X 10⁶Concentration in mL. The resting TCR-T effector cells were purged from the well plate, centrifuged at 400g for 5min at room temperature, resuspended in 500. mu.L of medium containing 2% bovine serum T009, counted and adjusted to 6X 10⁶and/mL. Mixing 500 μ L effector cells and 500 μ L CMV pp65 group T2 cells, adding into 12-well plate, mixing 500 μ L effector cells and 500 μ L DMSO group T2 cells, adding into 12-well plate, adding into 37 deg.C, 5% CO₂Incubating in an incubator for 6 h.

Flow dyeing: co-incubated cells were collected from well plates to 5mL aliquotsIn the heart tube, after centrifugation at 300g for 5min at room temperature, 200. mu.L of PBS containing 0.5% FBS was resuspended, and 5. mu.L of the "tetramer loaded with CMV pp65 NLV epitope polypeptide" prepared in 2.1 was added for tetramer surface staining, respectively. Then, the cells were fixed and punched using a cell fixing/punching kit (BD), and the cells co-cultured with the CMV pp65 group and the DMSO group were divided into 7 tubes each of which was about 5X 10⁵Cells, 3 tubes stained with anti-IFN-. gamma.antibody, 3 tubes stained with anti-TNF-. alpha.antibody, and 1 tube stained with isotype control for both antibodies. Then the detection is carried out by using a flow cytometer.

The flow cytometry analysis and detection results are shown in FIG. 6, wherein the left row in FIG. 6 is listed as a DMSO control group, the right row is listed as a CMV pp65 test group, the first row is the analysis result of IFN-gamma-PE-Cy 7, and the second row is the analysis result of TNF-alpha-APC. The results in FIG. 6 show that CMV TCR-T cells can efficiently recognize pp65 NLV epitope-loaded T2 cells, shown as CMV pp65, and secrete IFN-. gamma.and TNF-. alpha.as compared to a control group, shown as DMSO-only, in which CMV TCR-T cells reacted with DMSO-only T2 cells, shown as DMSO in the figure, demonstrating that this group of cells is functional.

3) Detection of killing effect of TCR-T on target cells by flow analysis

The detection of the cell killing effect by flow analysis is simpler, more convenient and faster than the traditional isotope labeling method. The principle is that CFSE is used for fluorescence labeling of target cells, and after the CFSE and effector cells are incubated together, the survival rate of the target cells is detected, so that the killing function of the effector cells is reflected.

The specific experimental process is as follows:

first day the target cells were loaded with antigenic polypeptides: t2 cells (purchased from ATCC) were counted and about 1X10 cells were removed⁶Cells, centrifuged at 300g for 5min at room temperature, resuspended in 2mL 10% bovine serum IMDM medium, and averaged into 2 wells of a 24-well plate. Dissolving CMV pp65 NLV epitope polypeptide with a sequence shown in SEQ ID No.1 in DMSO to prepare 10 mu g/mL, and adding a proper amount of CMV pp65 NLV epitope polypeptide into one hole according to a dilution ratio of 1:1000, namely a CMV pp65 group; the final concentration of the polypeptide is 10ug/mL, the mixture is re-suspended and mixed evenly, and the mixture is treated at 37 ℃ and 5 percent of CO₂Incubate overnight. Add an equal amount of DMSO to another well for the same treatment andculture, i.e. DMSO group.

The next day effector cells were co-cultured with target cells: after the antigen loading time is over, respectively adding CFSE fluorescent dye into the target cells of the two holes, the final concentration is 1 mu g/mL, and placing back the target cells at 37 ℃ and 5% CO₂Incubators were stained for 30 minutes. After staining, the target cells were purged from the well plate, washed by adding 10mL IMDM medium containing 10% bovine serum, centrifuged at 300g for 5min at room temperature, the supernatant was discarded, the washing was repeated again, and after centrifugation, resuspended in 1mL T009 medium containing 2% bovine serum, counted, and adjusted to 2X 10⁵Concentration in mL. Preparation of "5. CMV pp 65-specific TCR-transduced T cells (TCR-T)" TCR-T Effector cells⁶After centrifugation at 400g for 5min at room temperature, the cells were counted in suspension with 1mL of 2% bovine serum T009 medium and adjusted to 4X 10⁶The volume is/mL. After 50. mu.L/well of effector cells were added to a 96-well plate, 50. mu.L/well of the target cell suspension was added and mixed well. Three holes are used for co-incubation of effector cells and CMV pp65 group T2 cells, three holes are used for co-incubation of effector cells and DMSO group T2 cells, and meanwhile, a single target cell group without effector cells is arranged, and each group of three holes are repeated. Placing the hole plate in 5% CO at 37 deg.C₂Incubating in an incubator for 6 h.

Flow analysis: after incubation, the pore plate is placed on ice, 1mg/mLPI dye is added according to the dilution ratio of 1:20 before flow-type sample loading, flow-type sample loading analysis is immediately carried out after the mixture is fully blown away and uniformly mixed, and fluorescent signals are collected in FITC and PE-TexasRed channels.

The flow cytometry analysis and detection results are shown in FIGS. 7 and 8, and FIG. 7 is a flow analysis chart, wherein the upper chart is a DMSO control group, and the lower chart is a CMV pp65 test group; FIG. 8 is the statistical cell killing ratio. The results in FIGS. 7 and 8 show that CMV TCR-T cells can efficiently recognize T2 cells loaded with a pp65 NLV epitope polypeptide, i.e., CMV pp65 in the figure, and have a specific killing effect, compared to a control group in which CMV TCR-T cells react with T2 cells to which DMSO is added only, i.e., DMSO group in the figure.

7. Conclusion

Combining the above test results, the specific T cell receptor of the epitope polypeptide of SEQ ID No.1 targeting cytomegalovirus phosphoprotein pp65 is finally prepared, and the three complementarity determining regions CDR1, CDR2, and CDR3 of the α chain of the specific T cell receptor are sequentially SEQ ID No.4, SEQ ID No.5, and SEQ ID No. 2; the three complementarity determining regions CDR1, CDR2 and CDR3 of the beta chain are shown as SEQ ID No.6, SEQ ID No.7 and SEQ ID No.3 in sequence;

SEQ ID No.1：NLVPMVATV

SEQ ID No.2：CARNTGNQFYF

SEQ ID No.3：CASSYVTGTGSYGYTF

SEQ ID No.4：SSNFYA

SEQ ID No.5：MTLNGD

SEQ ID No.6：MNHEY

SEQ ID No.7：SVGAGI

more specifically, the variable region amino acid sequence of the alpha chain is shown as SEQ ID No.9, and the variable region amino acid sequence of the beta chain is shown as SEQ ID No. 11. Wherein the alpha chain of the sequence shown as SEQ ID No.9 is encoded by the nucleic acid of the sequence shown as SEQ ID No. 8; the beta strand of the sequence shown as SEQ ID No.11 is encoded by a nucleic acid of the sequence shown as SEQ ID No. 10.

SEQ ID No.8：

5’-ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCACTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAGAAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACAGCTATTTGTACATCAAAGGATCCCAGCCTGAAGACTCAGCCACATACCTCTGTGCCCGGAACACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCA-3’

SEQ ID No.9：

MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIP

SEQ ID No.10：

5’-ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACGTTACCGGGACAGGGTCCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTA-3’

SEQ ID No.11：

MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVTGTGSYGYTFGSGTRLTVV

The nucleic acid sequence contained in the lentivirus vector constructed by the TCR is shown as SEQ ID No. 12; accordingly, the amino acid sequence of the TCR expression element is the sequence shown in SEQ ID No. 13.

Based on the specific T cell receptor of the present example, functional CMV TCR-T cells prepared in the present example can be used for treating or preventing cytomegalovirus-related diseases.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> Shenzhen Huashengshengsciences institute

<120> specific T cell receptor targeting cytomegalovirus antigen and application thereof

<130> 19I28984

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 9

<212> PRT

<213> CMV pp65 antigen NLV epitope polypeptide

<400> 1

Asn Leu Val Pro Met Val Ala Thr Val

1 5

<210> 2

<211> 11

<212> PRT

<213> specific T cell receptor alpha chain CDR3

<400> 2

Cys Ala Arg Asn Thr Gly Asn Gln Phe Tyr Phe

1 5 10

<210> 3

<211> 16

<212> PRT

<213> specific T cell receptor beta chain CDR3

<400> 3

Cys Ala Ser Ser Tyr Val Thr Gly Thr Gly Ser Tyr Gly Tyr Thr Phe

1 5 10 15

<210> 4

<211> 6

<212> PRT

<213> specific T cell receptor alpha chain CDR1

<400> 4

Ser Ser Asn Phe Tyr Ala

1 5

<210> 5

<211> 6

<212> PRT

<213> specific T cell receptor alpha chain CDR2

<400> 5

Met Thr Leu Asn Gly Asp

1 5

<210> 6

<211> 5

<212> PRT

<213> specific T cell receptor beta chain CDR1

<400> 6

Met Asn His Glu Tyr

1 5

<210> 7

<211> 6

<212> PRT

<213> specific T cell receptor beta chain CDR2

<400> 7

Ser Val Gly Ala Gly Ile

1 5

<210> 8

<211> 396

<212> DNA

<213> nucleic acid sequence of alpha chain variable region of specific T cell receptor

<400> 8

atggagaaga atcctttggc agccccatta ctaatcctct ggtttcatct tgactgcgtg 60

agcagcatac tgaacgtgga acaaagtcct cagtcactgc atgttcagga gggagacagc 120

accaatttca cctgcagctt cccttccagc aatttttatg ccttacactg gtacagatgg 180

gaaactgcaa aaagccccga ggccttgttt gtaatgactt taaatgggga tgaaaagaag 240

aaaggacgaa taagtgccac tcttaatacc aaggagggtt acagctattt gtacatcaaa 300

ggatcccagc ctgaagactc agccacatac ctctgtgccc ggaacaccgg taaccagttc 360

tattttggga cagggacaag tttgacggtc attcca 396

<210> 9

<211> 132

<212> PRT

<213> amino acid sequence of alpha chain variable region of specific T cell receptor

<400> 9

Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His

1 5 10 15

Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser

20 25 30

Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro

35 40 45

Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys

50 55 60

Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys

65 70 75 80

Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr

85 90 95

Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys

100 105 110

Ala Arg Asn Thr Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu

115 120 125

Thr Val Ile Pro

130

<210> 10

<211> 402

<212> DNA

<213> nucleic acid sequence of beta chain variable region of specific T cell receptor

<400> 10

atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 240

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300

gctccctccc agacatctgt gtacttctgt gccagcagtt acgttaccgg gacagggtcc 360

tatggctaca ccttcggttc ggggaccagg ttaaccgttg ta 402

<210> 11

<211> 134

<212> PRT

<213> amino acid sequence of beta chain variable region of specific T cell receptor

<400> 11

Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala

1 5 10 15

Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu

20 25 30

Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His

35 40 45

Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu

50 55 60

Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro

65 70 75 80

Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg

85 90 95

Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

100 105 110

Ser Tyr Val Thr Gly Thr Gly Ser Tyr Gly Tyr Thr Phe Gly Ser Gly

115 120 125

Thr Arg Leu Thr Val Val

130

<210> 12

<211> 1809

<212> DNA

<213> specific T cell receptor nucleic acid sequences

<400> 12

atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 180

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 240

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 300

gctccctccc agacatctgt gtacttctgt gccagcagtt acgttaccgg gacagggtcc 360

tatggctaca ccttcggttc ggggaccagg ttaaccgttg tagaggatct gagaaacgtg 420

acccccccta aggtgtctct gtttgagcct agcaaggccg agatcgccaa taagcagaag 480

gccaccctgg tgtgcctggc aaggggcttc tttccagatc acgtggagct gagctggtgg 540

gtgaacggca aggaggtgca ctccggcgtg tctacagacc cccaggccta taaggagagc 600

aattactcct attgcctgtc tagccggctg agagtgtccg ccaccttctg gcacaacccc 660

cggaatcact tcagatgtca ggtgcagttt cacggcctgt ccgaggagga taagtggcct 720

gagggctctc caaagcccgt gacacagaac atcagcgccg aggcatgggg aagagcagac 780

tgtggcatca cctctgccag ctaccaccag ggcgtgctga gcgccacaat cctgtatgag 840

atcctgctgg gcaaggccac cctgtacgcc gtgctggtgt ccggactggt gctgatggcc 900

atggtgaaga agaagaactc tagggcaaag cggagcggct ctggagcaac caacttcagc 960

ctgctgaagc aggcaggcga tgtggaggag aaccctggac caatggagaa gaatcctttg 1020

gcagccccat tactaatcct ctggtttcat cttgactgcg tgagcagcat actgaacgtg 1080

gaacaaagtc ctcagtcact gcatgttcag gagggagaca gcaccaattt cacctgcagc 1140

ttcccttcca gcaattttta tgccttacac tggtacagat gggaaactgc aaaaagcccc 1200

gaggccttgt ttgtaatgac tttaaatggg gatgaaaaga agaaaggacg aataagtgcc 1260

actcttaata ccaaggaggg ttacagctat ttgtacatca aaggatccca gcctgaagac 1320

tcagccacat acctctgtgc ccggaacacc ggtaaccagt tctattttgg gacagggaca 1380

agtttgacgg tcattccaga catccagaac cccgagcctg ccgtgtacca gctgaaggac 1440

ccaagatccc aggatagcac cctgtgcctg ttcaccgact ttgattctca gatcaatgtg 1500

cccaagacca tggagagcgg cacctttatc acagacaaga ccgtgctgga tatgaaggcc 1560

atggacagca agtccaacgg cgccatcgcc tggagcaatc agacatcctt cacctgccag 1620

gatatcttta aggagacaaa cgccacctac ccttctagcg acgtgccatg tgatgccacc 1680

ctgacagaga agtccttcga gacagacatg aacctgaatt ttcagaacct gtctgtgatg 1740

ggcctgagaa tcctgctgct gaaggtggcc ggcttcaatc tgctgatgac cctgagactg 1800

tggtcctct 1809

<210> 13

<211> 603

<212> PRT

<213> specific T cell receptor amino acid sequence

<400> 13

Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala

1 5 10 15

Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu

20 25 30

Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His

35 40 45

Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu

50 55 60

Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro

65 70 75 80

Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg

85 90 95

Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser

100 105 110

Ser Tyr Val Thr Gly Thr Gly Ser Tyr Gly Tyr Thr Phe Gly Ser Gly

115 120 125

Thr Arg Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys

130 135 140

Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser

195 200 205

Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe

210 215 220

Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro

225 230 235 240

Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp

245 250 255

Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val

260 265 270

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

275 280 285

Tyr Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys

290 295 300

Lys Asn Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser

305 310 315 320

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu

325 330 335

Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His Leu Asp

340 345 350

Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser Leu His

355 360 365

Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro Ser Ser

370 375 380

Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys Ser Pro

385 390 395 400

Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys Lys Gly

405 410 415

Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr Leu Tyr

420 425 430

Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Arg

435 440 445

Asn Thr Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val

450 455 460

Ile Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp

465 470 475 480

Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser

485 490 495

Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp

500 505 510

Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala

515 520 525

Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys

530 535 540

Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr

545 550 555 560

Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn

565 570 575

Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe

580 585 590

Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600

Claims

1. A specific T cell receptor targeting cytomegalovirus antigens characterized by: the specific T cell receptor can specifically target and combine cytomegalovirus phosphoprotein pp 65; the alpha chain and the beta chain of the specific T cell receptor are respectively provided with three complementarity determining regions, and the three complementarity determining regions of the alpha chain are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.2 in sequence; the three complementarity determining regions of the beta chain are shown as SEQ ID No.6, SEQ ID No.7 and SEQ ID No.3 in sequence;

SEQ ID No.2：CARNTGNQFYF

SEQ ID No.3：CASSYVTGTGSYGYTF

SEQ ID No.4：SSNFYA

SEQ ID No.5：MTLNGD

SEQ ID No.6：MNHEY

SEQ ID No.7：SVGAGI。

2. a specific T-cell receptor according to claim 1, characterized in that: the variable region amino acid sequence of the alpha chain is shown as SEQ ID No. 9.

3. The specific T cell receptor of claim 2, wherein: the amino acid sequence of the variable region of the beta chain is shown as SEQ ID No. 11.

4. The specific T cell receptor according to claim 1 or 2 or 3, characterized in that: the specific T cell receptor is a sequence shown in SEQ ID No. 13.

5. The specific T cell receptor of claim 4, wherein: the targeting binding cytomegalovirus phosphoprotein pp65 specifically comprises that when the targeting binding cytomegalovirus phosphoprotein pp65 is presented by a major histocompatibility complex, the specific T cell receptor specifically binds to NLV epitope polypeptide of cytomegalovirus phosphoprotein pp65, and the NLV epitope polypeptide is a sequence shown as SEQ ID No. 1;

SEQ ID No.1：NLVPMVATV。

6. a nucleotide encoding a specific T-cell receptor according to any one of claims 1 to 5, said nucleotide comprising a nucleotide encoding the alpha chain and a nucleotide encoding the beta chain.

7. The nucleotide of claim 6, characterized in that: the nucleotide for coding the alpha chain is a sequence shown in SEQ ID No. 8.

8. The nucleotide of claim 6, wherein: the nucleotide for coding the beta chain is a sequence shown in SEQ ID No. 10.

9. A nucleotide according to any one of claims 6-8, characterized in that: the nucleotide for coding the specific T cell receptor is a sequence shown in SEQ ID No. 12.

10. A vector comprising the nucleotide of any one of claims 6-9.

11. A cell comprising a specific T cell receptor according to any one of claims 1 to 5 or a nucleotide according to any one of claims 6 to 9 or a vector according to claim 10.

12. The cell of claim 11, wherein: the cells are T lymphocytes.

13. Use of a specific T-cell receptor according to any one of claims 1-5 or a nucleotide according to any one of claims 6-9 or a vector according to claim 10 or a cell according to claim 11 or 12 for the preparation of a medicament for the treatment and/or prevention of a disease associated with cytomegalovirus.

14. A pharmaceutical composition for the treatment and/or prevention of diseases associated with cytomegalovirus, comprising: the pharmaceutical composition comprising a specific T cell receptor according to any one of claims 1 to 5 or a vector according to claim 10 or a cell according to claim 11 or 12.