T cell antigen receptor and its application

Abstract

The present invention relates to the technical field of T cell antigen receptors, and discloses a T cell antigen receptor and its application. The T cell antigen receptor provided by the present invention can specifically recognize and bind to HLA-A*11:01CMVpp65 501‑509 Antigen complex. It has been verified experimentally that the T cell antigen receptor provided by the present invention can be specifically activated by CMV epitope peptides, has good in vitro activity in killing target cells, and can release cytokines such as IFN-γ. In addition, after ALA-SCAN experiments and bioinformatics whole protein group comparisons, the on-target-off-tumor risk of the T cell antigen receptor provided by the present invention is eliminated, so that it has a higher clinical application value and better safety.

Description

Technical Field

[0001] The present invention relates to the technical field of T cell antigen receptors, and in particular to a T cell antigen receptor and applications thereof. Background Art

[0002] Human cytomegalovirus (CMV) is a widespread herpes virus with an infection rate of 50-100% in the adult population. In individuals with a normal immune system, CMV infection usually does not show any symptoms. However, in individuals with suppressed immune systems, such as AIDS patients or those who have received an organ transplant, reactivation of CMV can lead to severe or even fatal disease. Traditional treatment for CMV includes the use of antiviral drugs such as ganciclovir, sodium phosphite, and cidofovir, but these drugs are generally limited and often cause serious side effects.

[0003] Cell receptor therapy is an adoptive cell transfer therapy (ACT) technique that primarily involves collecting a person's own immune cells, introducing them into cell receptors, and culturing them in vitro to expand their numbers and develop targeted killing capabilities. These cells are then infused back into the patient's body to kill pathogens, cancer cells, or mutated cells in the blood and tissues. T cell antigen receptors (TCRs) are surface proteins on T cells that specifically recognize antigens. By introducing specific TCR sequences into T cells, T cells that specifically recognize specific antigens and kill cells containing those antigens can be obtained, thereby treating diseases caused by viral infections such as CMV through cell receptor therapy.

[0004] The Major Histocompatibility Complex (MHC) is a collective term for a group of genes that encode the major histocompatibility antigens of animals. The human MHC is called the human leukocyte antigen (HLA). HLA is highly polymorphic, and HLA typing varies among populations in different countries and regions. The HLA-A3 superfamily (including HLA-A11, HLA-A33, HLA-A68, HLA-A31, etc.) accounts for the largest proportion of HLA typing in the Chinese population, representing approximately 52.7% of the HLA typing in the Chinese population.

[0005] Although researchers have developed a variety of TCRs targeting CMV, there are no reports on TCR sequences based on HLA-A3 superfamily restricted epitopes. Summary of the Invention

[0006] The present invention aims to overcome the existing problems of the lack of TCRs targeting HLA major typing-restricted epitopes in the Chinese population and related efficient CMV treatment methods, and to provide a T cell antigen receptor and its application. The T cell antigen receptor provided by the present invention can specifically recognize and bind to the HLA-A*11:01CMV antigen complex, has good target cell killing activity, can release cytokines such as IFN-γ, and experimentally eliminates the on-target / off-tumor risk of the T cell antigen receptor.

[0007] To achieve the above objectives, the present invention provides a T cell antigen receptor that specifically recognizes and binds to a human cytomegalovirus binding epitope comprising the amino acid sequence shown in SEQ ID NO: 1.

[0008] The second aspect of the present invention provides a nucleic acid molecule encoding the T cell antigen receptor described in the first aspect.

[0009] The third aspect of the present invention provides a nucleic acid expression vector, which comprises a coding gene and a vector sequence, wherein the coding gene is provided by the nucleic acid molecule described in the second aspect.

[0010] The fourth aspect of the present invention provides a vector cell, which comprises the T cell antigen receptor described in the first aspect, and / or the vector cell comprises the nucleic acid molecule described in the second aspect, and / or the vector cell comprises the nucleic acid expression vector described in the third aspect.

[0011] The fifth aspect of the present invention provides the use of the T cell antigen receptor described in the first aspect, and / or the nucleic acid molecule described in the second aspect, and / or the nucleic acid expression vector described in the third aspect, and / or the vector cell described in the fourth aspect in the preparation of drugs and / or vaccines for preventing and / or treating diseases caused by human cytomegalovirus infection.

[0012] The sixth aspect of the present invention provides a pharmaceutical composition for preventing and / or treating diseases caused by human cytomegalovirus infection, wherein the pharmaceutical composition comprises: the T cell antigen receptor described in the first aspect, and / or the nucleic acid molecule described in the second aspect, and / or the nucleic acid expression vector described in the third aspect, and / or the vector cell described in the fourth aspect.

[0013] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0014] (1) The TCR molecules provided by the present invention can recognize and bind to HLA-A*11:01CMV pp65 501-509Antigen complexes are more suitable for use in the Chinese population. After being activated by specific CMV epitope peptides, this TCR molecule has good in vitro activity in killing target cells and can release cytokines such as IFN-γ, thus having the potential to be used in the treatment / prevention of CMV through cell receptor therapy.

[0015] (2) After ALA-SCAN experiments and bioinformatics whole-protein group comparison, the on-target / off-tumor off-target risk of the TCR molecule provided by the present invention was eliminated, and it was confirmed that the TCR molecule has clinical application value. It was also confirmed that the use of the TCR molecule has a smaller off-target toxicity risk, thus having better safety.

[0016] (3) The method for in vitro culture and expansion of specific T cells containing the TCR molecules provided by the present invention is simple and easy to operate. PBMCs can be directly stimulated with CMV pp65 epitope peptides for culture. This enables large-scale clinical application of treatment / prevention methods using the TCR molecules of the present invention and specific T cells containing the TCR molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The CMV was detected by flow cytometry on T cells that were not stimulated and stimulated with CMV epitope peptides in Example 1. + CD8 + T cell percentage results chart.

[0018] Figure 2 This is a graph showing the results of ELISpot detection of IFN-γ secretion by T cells in Example 1.

[0019] Figure 3 Schematic diagram of the construction of the in vitro transcription plasmid vector in Example 2.

[0020] Figure 4 This is a diagram showing the TCR mRNA gel electrophoresis verification results obtained in Example 2.

[0021] Figure 5 This is the map of the HLA lentiviral expression plasmid used in Example 3.

[0022] Figure 6 This is a graph analyzing the HLA expression levels of monoclonal cells used in the flow cytometry experiment in Example 3.

[0023] Figure 7 This is a flow cytometric analysis diagram of the electroporated ZZ1 and ZZ2 NFAT-Luc Jurkat in Example 3.

[0024] Figure 8A and Figure 8BThis is a statistical result diagram of the NFAT-fluorescence values ​​of ZZ1-NFAT-Luc Jurkat and ZZ2-NFAT-Luc Jurkat detected by the microplate reader in Example 3.

[0025] Figure 9 This is a flow cytometric analysis chart of the specific ratio of ZZ1 TCR-T and ZZ2 TCR-T detected by flow cytometry in Example 4.

[0026] Figure 10 This is a graph showing the killing efficiency of ZZ1 TCR-T and ZZ2 TCR-T on target cells detected by EuTDA in Example 4.

[0027] Figure 11A and 11B This is a graph showing the results of ELISA detection of cytokine IFN-γ secretion by ZZ1 TCR-T and ZZ2 TCR-T in Example 5.

[0028] Figure 12 Schematic diagram of the alanine mutation positions of the epitope peptides in the alanine scanning peptide library constructed in Example 6.

[0029] Figure 13 This is a statistical graph of IFN-γ spot secretion by ZZ1 TCR-T detected by ELISPOT in Example 6.

[0030] Figure 14 This is a statistical graph of IFN-γ spot secretion by ZZ2 TCR-T detected by ELISPOT in Example 6.

[0031] Figure 15 This is the flow cytometry detection of ZZ3 TCR-T CD8 in Example 1. + CMV + Positive percentage result graph.

[0032] Figure 16 This is a statistical result diagram of the NFAT-fluorescence value of ZZ3-NFAT-Luc Jurkat detected by microplate reader in Comparative Example 1. DETAILED DESCRIPTION

[0033] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0034] As used herein, the term "T cell antigen receptor (TCR)" refers to the protein molecule that T cells use to recognize specific antigens on the surface of target cells. The variable regions (V regions) of the α and β chains of the TCR contain complementarity-determining regions (CDRs) and the antibody framework region (FR). The amino acid sequence of the CDRs determines the antibody's specificity for binding to the antigen, while the FRs primarily stabilize the spatial configuration of the CDRs to facilitate binding between the CDRs and the antigenic determinants.

[0035] In the present invention, "TCR" is the abbreviation of "T cell antigen receptor", and the two have the same meaning; "TCR-T cell" is the abbreviation of "T cell containing TCR (molecule)", and the two have the same meaning; "HLA" is the abbreviation of "human leukocyte antigen", and the two have the same meaning; "CMV" is the abbreviation of "human cytomegalovirus", and the two have the same meaning; "MHC" is the abbreviation of "major histocompatibility complex", and the two have the same meaning; "fP2A" is the abbreviation of "furin SGSG p2A", and the two have the same meaning. The above groups of terms with the same meaning can be used interchangeably.

[0036] With the development of ACT technology, researchers in this field have developed a variety of different immune cell therapies for the treatment and / or prevention of cancer and viral infections that are difficult to effectively treat with existing drugs. For example, CAR-T therapy and TCR-T cell therapy. Among them, compared with CAR-T therapy, the advantage of TCR-T cell therapy is that TCR-T cells can not only recognize cell membrane antigens, but also recognize intracellular antigens presented by peptide-major histocompatibility complexes. They are not restricted by the expression of antigens on the surface of target cells, thus being able to recognize target antigens more widely and induce more durable immune synapse formation. TCRs can also be activated by single target antigen molecules and are more sensitive to low-copy number antigens than CAR-T. However, TCRs can only recognize peptide-HLA and kill target cells with matching HLA alleles. Moreover, HLA typing is highly polymorphic and exhibits relatively obvious regional distribution characteristics. Therefore, when designing and screening TCR molecules in TCR therapy, the selection of HLA typing epitopes that they can recognize and bind to has a significant impact on the therapeutic effect of the TCR molecule on patients in a specific region.

[0037] The inventors also found in their research that current research on TCR-T therapy is more focused on cancer, especially solid tumor treatment, and there are fewer studies on TCR-T therapy for viral infectious diseases. Although a few researchers have developed some TCR molecules and corresponding TCR-T therapies for CMV, such as the TCR molecules for CMV pp65 epitopes disclosed in CN113121676A and the TCR molecules targeting HCMV disclosed in CN111875698A, these currently developed TCR molecules targeting CMV all specifically recognize and bind to the HLA-A*02 epitope, which belongs to the high-frequency HLA subtype of the European and American population and has a low proportion in the Chinese population, making it unsuitable for CMV treatment / prevention in the Chinese population. After extensive research, the inventors of the present invention found that by screening to obtain specific TCR molecules targeting CMV that can specifically recognize the HLA-A*11:01 subtype epitope (this HLA subtype is a high-frequency subtype in the Chinese population), it is possible to further obtain products and methods that are more suitable for CMV prevention and / or treatment in my country. In addition, after further research, the inventors found that through special design, after the above-mentioned TCR molecules are introduced into T cells in vitro, the expansion of specific T cells can be achieved only through a relatively simple method, which greatly simplifies the operation of the cell expansion step in TCR-T therapy and is more conducive to the large-scale application and promotion of this method.

[0038] Based on the above findings, the present invention provides a T cell antigen receptor in a first aspect, which specifically recognizes and binds to a human cytomegalovirus binding epitope comprising the amino acid sequence shown in SEQ ID NO: 1.

[0039] ATVQGQNLK (SEQ ID NO: 1)

[0040] SEQ ID NO:1 is HLA-A*11:01CMV pp65 501-509 The amino acid sequence of the antigen complex (i.e., the amino acid sequence of positions 501-509 of the HLA-A*11:01-restricted CMV pp65 epitope antigen complex). That is, the TCR provided by the present invention can specifically recognize and bind to the CMV antigen peptide-MHC molecule complex, activate TCR-T cells, and then produce high levels of cytokines and kill target cells. Generally, the TCR provided by the present invention specifically binds to the antigen peptide from CMV through MHC presentation.

[0041] Preferably, the TCR is a soluble TCR.

[0042] The present invention does not particularly limit the specific structure and sequence of the TCR provided, and any TCR that can specifically bind to the amino acid sequence shown in SEQ ID NO: 1 falls within the scope of protection of the present invention. According to a preferred embodiment of the present invention, the TCR includes at least one α chain variable region and / or at least one β chain variable region.

[0043] Preferably, the TCR is a heterodimer comprising at least one α chain variable region and at least one β chain variable region.

[0044] According to some preferred embodiments of the present invention, the α chain variable region includes the complementarity determining region of the α chain, and the β chain variable region includes the complementarity determining region of the β chain.

[0045] A complementarity-determining region (CDR) refers to a sequence segment within a TCR sequence that forms precise complementarity with an HLA-antigen complex, thereby enabling the TCR molecule to specifically recognize the HLA-antigen complex. In the TCR sequences provided herein, the CDR within the α / β chain can be a continuous amino acid sequence or multiple discontinuous amino acid sequences, as long as it enables the TCR sequence of the present invention to specifically recognize and bind to the amino acid sequence set forth in SEQ ID NO: 1.

[0046] The inventors of the present invention cleverly discovered in their research that when the complementary determining regions in the α chain / β chain in the TCR molecule have a specific amino acid sequence, its ability to specifically recognize and bind to the target sequence (such as SEQ ID NO: 1, etc.) is stronger.

[0047] In order to make the TCR have better performance in specific recognition and binding to the target sequence, according to some preferred embodiments of the present invention, the complementarity determining region of the α chain includes an amino acid sequence that has at least 80% homology with at least one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or it can be a range formed by any two of the above values ​​and any intermediate values).

[0048] VSGNPY (SEQ ID NO: 2)

[0049] YITGDNLV (SEQ ID NO: 3)

[0050] CAVRDMEGGNNARLMF (SEQ ID NO: 4)

[0051] DRGSQS (SEQ ID NO: 10)

[0052] IYSNGD (SEQ ID NO: 11)

[0053] CAVKGGYNKLIF (SEQ ID NO: 12)

[0054] Preferably, the complementarity determining region of the α chain comprises the amino acid sequence shown by at least one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0055] According to some preferred embodiments of the present invention, the complementarity determining region of the β chain comprises an amino acid sequence that is at least 80% homologous to at least one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or a range formed by any two of the above values ​​and any intermediate values).

[0056] SNHLY (SEQ ID NO: 5)

[0057] FYNNEI (SEQ ID NO: 6)

[0058] CASSEAYGGYNEQFF (SEQ ID NO: 7)

[0059] SGHRS (SEQ ID NO: 13)

[0060] YFSETQ (SEQ ID NO: 14)

[0061] CASSLGGEGDRPQHF (SEQ ID NO: 15)

[0062] Preferably, the complementarity determining region of the β chain comprises the amino acid sequence shown by at least one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

[0063] According to some preferred embodiments of the present invention, the complementarity determining region of the α chain includes three discontinuous amino acid sequences, which are named CDR1α, CDR2α, and CDR3α, respectively, wherein CDR1α includes an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 10 (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or a range formed by any two of the above values ​​and any intermediate values); CDR2α includes an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 10 (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or a range formed by any two of the above values ​​and any intermediate values); The amino acid sequence of SEQ ID NO: 11 has an amino acid sequence that is at least 80% identical (for example, the identity can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or a range formed by any two of the foregoing values ​​or any intermediate values); and the CDR3α comprises an amino acid sequence that is at least 80% identical (for example, the identity can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or a range formed by any two of the foregoing values ​​or any intermediate values) to the amino acid sequence of SEQ ID NO: 4 and / or SEQ ID NO: 12.

[0064] Preferably, CDR1α comprises the amino acid sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 10; CDR2α comprises the amino acid sequence shown in SEQ ID NO: 3 and / or SEQ ID NO: 11; and CDR3α comprises the amino acid sequence shown in SEQ ID NO: 4 and / or SEQ ID NO: 12.

[0065] More preferably, the amino acid sequence of CDR1α is shown as SEQ ID NO: 2 and / or SEQ ID NO: 10; the amino acid sequence of CDR2α is shown as SEQ ID NO: 3 and / or SEQ ID NO: 11; and the amino acid sequence of CDR3α is shown as SEQ ID NO: 4 and / or SEQ ID NO: 12.

[0066] According to some preferred embodiments of the present invention, the complementarity determining region of the β chain includes three discontinuous amino acid sequences, which are named CDR1β, CDR2β, and CDR3β, respectively, wherein CDR1β includes an amino acid sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 5 and / or SEQ ID NO: 13 (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or a range formed by any two of the above values ​​and any intermediate values); CDR2β includes an amino acid sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 5 and / or SEQ ID NO: 13 (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or a range formed by any two of the above values ​​and any intermediate values); The amino acid sequence of SEQ ID NO: 14 has at least 80% homology (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or a range formed by any two of the foregoing values ​​or any intermediate values); and the CDR3β includes an amino acid sequence having at least 80% homology (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%, or a range formed by any two of the foregoing values ​​or any intermediate values) to the amino acid sequence of SEQ ID NO: 7 and / or SEQ ID NO: 15.

[0067] Preferably, CDR1β comprises the amino acid sequence shown in SEQ ID NO: 5 and / or SEQ ID NO: 13; CDR2β comprises the amino acid sequence shown in SEQ ID NO: 6 and / or SEQ ID NO: 14; and CDR3β comprises the amino acid sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 15.

[0068] More preferably, the amino acid sequence of CDR1β is shown as SEQ ID NO: 5 and / or SEQ ID NO: 13; the amino acid sequence of CDR2β is shown as SEQ ID NO: 6 and / or SEQ ID NO: 14; and the amino acid sequence of CDR3β is shown as SEQ ID NO: 7 and / or SEQ ID NO: 15.

[0069] The α chain variable region / β chain variable region contained in the TCR provided by the present invention preferably includes three complementary determining regions composed of discontinuous amino acid sequences, that is, the complementary determining region of the α chain variable region includes the above CDR1α, CDR2α and CDR3α, and the complementary determining region of the β chain variable region includes the above CDR1β, CDR2β and CDR3β, wherein CDR1α, CDR2α, CDR3α and CDR1β, CDR2β, CDR3β can be arbitrarily combined according to the above sequences, as long as the TCR can specifically recognize and bind to HLA-A*11:01CMV pp65 501-509 Antigen complex.

[0070] According to a preferred embodiment of the present invention, the complementarity determining region of the α chain variable region includes the amino acid sequence shown in SEQ ID NO: 2-4; the complementarity determining region of the β chain variable region includes the amino acid sequence shown in SEQ ID NO: 5-7.

[0071] According to a preferred embodiment of the present invention, the complementarity determining region of the α chain variable region includes the amino acid sequence shown in SEQ ID NOs: 10-12; the complementarity determining region of the β chain variable region includes the amino acid sequence shown in SEQ ID NOs: 13-15.

[0072] According to a particularly preferred embodiment of the present invention, the α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8 and / or SEQ ID NO: 16.

[0073] MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDMEGGNNARLMFGDGTQLVVKPNIQNPDPAV YQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSRAKRSGSG(SEQ ID NO:8)

[0074] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKGGYNKLIFGAGTRLAVHPYIQNPEPAV YQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSRAKRSGSG(SEQ ID NO:16)

[0075] According to a particularly preferred embodiment of the present invention, the β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 and / or SEQ ID NO: 17.

[0076] MDTWLVCWWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEAYGGYNEQFFGPGTRLTVLEDLKNVFPPKVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:9)

[0077] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSLGGEGDRPQHFGDGTRLSILEDLRNVTPPKVSLFEPSKAEIA NKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:17)

[0078] Preferably, in the TCR, the amino acid sequence of the α chain variable region is shown as SEQ ID NO: 8, and the amino acid sequence of the β chain variable region is shown as SEQ ID NO: 9.

[0079] Preferably, in the TCR, the amino acid sequence of the α chain variable region is shown as SEQ ID NO: 16, and the amino acid sequence of the β chain variable region is shown as SEQ ID NO: 17.

[0080] In the TCR provided by the present invention, the α chain variable region and the β chain variable region can be connected by any common connection method of the α chain variable region and the β chain variable region of TCR molecules in the art to form a complete heterodimeric TCR molecule.

[0081] According to some preferred embodiments of the present invention, in the TCR, the α chain variable region is directly or indirectly connected to the β chain variable region. Direct connection refers to a connection method in which the amino acid sequences of the α chain variable region and the β chain variable region are directly connected (for example, connected by a disulfide bond, etc.); indirect connection refers to a connection between the α chain variable region and the β chain variable region through another amino acid sequence (usually referred to as a "linker sequence"), for example, the α chain variable region and the β chain variable region are respectively connected to different segments in the linker sequence through a disulfide bond, etc., or the α chain variable region and the β chain variable region are both connected to the same segment in the linker sequence through a disulfide bond, etc.

[0082] Preferably, in the TCR, the α chain variable region and the β chain variable region are indirectly linked.

[0083] According to some preferred embodiments of the present invention, the α chain variable region can be connected to the β chain variable region via the fp2A sequence (amino acid sequence shown in SEQ ID NO: 55).

[0084] SGSGATNFSLLKQAGDVEENPGP(SEQ ID NO:55)

[0085] The present invention has no particular limitation on the sequences of the α chain and β chain constant regions in the provided TCR molecules, as long as they can combine with the α chain variable region and β chain variable region having the above-mentioned characteristics to form a stable TCR molecule with the desired functions of the present invention.

[0086] In order to improve the efficiency of exogenous TCR expression, according to a preferred embodiment of the present invention, wherein the TCR includes an α chain constant region and a β chain constant region, the α chain constant region includes an amino acid sequence having at least 80% homology to SEQ ID NO: 26 (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or a range formed by any two of the above values ​​and any intermediate value); the β chain constant region includes an amino acid sequence having at least 80% homology to SEQ ID NO: 26; NO:27 has an amino acid sequence with at least 80% homology (for example, the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or a range formed by any two of the above values ​​and any intermediate value).

[0087] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ IDNO:26)

[0088] EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:27)

[0089] According to a particularly preferred embodiment of the present invention, the TCR includes an α chain constant region, an α chain variable region, a β chain constant region, and a β chain variable region, the amino acid sequence of the α chain variable region is shown in SEQ ID NO: 8, and the amino acid sequence of the α chain constant region is shown in SEQ ID NO: 26; the amino acid sequence of the β chain variable region is shown in SEQ ID NO: 9, and the amino acid sequence of the β chain constant region is shown in SEQ ID NO: 27.

[0090] Preferably, the amino acid sequence of the TCR is shown in SEQ ID NO:52.

[0091] According to a particularly preferred embodiment of the present invention, the TCR includes an α chain constant region, an α chain variable region, a β chain constant region, and a β chain variable region, the amino acid sequence of the α chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the α chain constant region is shown in SEQ ID NO: 26; the amino acid sequence of the β chain variable region is shown in SEQ ID NO: 17, and the amino acid sequence of the β chain constant region is shown in SEQ ID NO: 27.

[0092] Preferably, the amino acid sequence of the TCR is shown in SEQ ID NO:53.

[0093] The second aspect of the present invention provides a nucleic acid molecule encoding the T cell antigen receptor described in the first aspect.

[0094] Any nucleic acid molecule (including DNA and RNA) capable of encoding the TCR provided by the present invention is within the scope of protection of the present invention. Based on the degeneracy of codons, those skilled in the art can independently design and synthesize different nucleic acid molecules capable of encoding the TCR provided by the present invention based on the characteristics of the aforementioned TCR sequence.

[0095] According to a preferred embodiment of the present invention, the nucleic acid molecule includes at least one nucleotide sequence shown in SEQ ID NO: 28 (encoding CDR1α shown in SEQ ID NO: 2), SEQ ID NO: 29 (encoding CDR2α shown in SEQ ID NO: 3), SEQ ID NO: 30 (encoding CDR3α shown in SEQ ID NO: 4), SEQ ID NO: 36 (encoding CDR1α shown in SEQ ID NO: 10), SEQ ID NO: 37 (encoding CDR2α shown in SEQ ID NO: 11), and SEQ ID NO: 38 (encoding CDR3α shown in SEQ ID NO: 12).

[0096] GTCTCCGGAAACCCCTAT(SEQ ID NO:28)

[0097] TACATCACTGGTGATAACCTGGTG(SEQ ID NO:29)

[0098] TGTGCGGTGCGTGACATGGAAGGGGGTAATAACGCTCGGCTGATGTTT(SEQ ID NO:30)

[0099] GACCGTGGCAGTCAGAGC(SEQ ID NO:36)

[0100] ATCTACAGCAATGGCGAT(SEQ ID NO:37)

[0101] TGCGCCGTTAAAGGGGGCTACAACAAGCTGATCTTC (SEQ ID NO:38)

[0102] According to a preferred embodiment of the present invention, the nucleic acid molecule further comprises a nucleotide sequence as shown in at least one of SEQ ID NO:31 (encoding CDR1β shown in SEQ ID NO:5), SEQ ID NO:32 (encoding CDR2β shown in SEQ ID NO:6), SEQ ID NO:33 (encoding CDR3β shown in SEQ ID NO:7), SEQ ID NO:39 (encoding CDR1β shown in SEQ ID NO:13), SEQ ID NO:40 (encoding CDR2β shown in SEQ ID NO:14), and SEQ ID NO:41 (encoding CDR3β shown in SEQ ID NO:15).

[0103] AGCAACCACCTCTAC (SEQ ID NO: 31)

[0104] TTTTACAACAACGAGATC(SEQ ID NO:32)

[0105] TGCGCCTCTTCCGAAGCCTACGGTGGCTACAACGAGCAGTTTTTC(SEQ ID NO:33)

[0106] TCTGGCCACCGCTCT (SEQ ID NO: 39)

[0107] TACTTCTCGAGACCCAG(SEQ ID NO:40)

[0108] TGCGCTCCTCCTTGGGCGGGGAGGGCGACAGGCCTCAGCACTTT(SEQ ID NO:41)

[0109] According to a preferred embodiment of the present invention, the nucleic acid molecule includes a nucleotide sequence shown in at least one of SEQ ID NO: 34 (encoding the α chain variable region of SEQ ID NO: 8), SEQ ID NO: 35 (encoding the β chain variable region of SEQ ID NO: 9), SEQ ID NO: 42 (encoding the α chain variable region of SEQ ID NO: 16) and SEQ ID NO: 43 (encoding the β chain variable region of SEQ ID NO: 17).

[0110] ATGGCGTCTGCTCCTATCAGCATGCTAGCTATGTTGTTCACCTTGAGCGGATTGAGGGCTCAGTCCGTAGCGCAGCCGGAGGATCAGGTCAATGTGGCGGAGGGCAATCCCCTGACCGTTAAGTGCACTTACAGCGTCTCCGGAAACCCCTATCTGTTTTGGTACGTGCAGTACCCCAACAGGGGTCTGCAGTTCCTGTTAAAGTACATCACTGGTGATAACCTGGTGAAAGGCAGTTACGGCTTCGAGGCCGAGTTCAACAAGTCCCAGACCTCCTTTCACCTGAAGAAGCCCTCGGCTCTGGTCTCCGACTCTGCGCTGTACTTCTGTGCGGTGCGTGACATGGAAGGGGGTAATAACGCTCGGCTGATGTTTGGCGACGGGACCCAGCTGGTAGTCAAGCCCAACATCCAGAATCCAGATCCAGCCGTGTACCAGCTGCGCGACTCCAAAAGCAGCGACAAATCGGTTTGTCTATTCACCGACTTCGACTCCCAAACTAATGTCAGTCAGTCCAAGGACTCTGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGTCCATGGATTTCAAGTCCAACTCCGCCGTAGCATGGTCTAACAAGAGCGACTTTGCCTGCGCCAACGCGTTCAACAACTCTATCATCCCCGAGGACACGTTCTTTCCCTCTCCGGAGTCCTCCTGCGACGTGAAGCTCGTGGAGAAATCTTTTGAGACCGACACAAATTTGAACTTCCAGAACCTGTCAGTCATCGGCTTCCGCATCCTTCTGTTGAAGGTCGCGGGATTCAACCTCCTGATGACCCTCCGCCTTTGGTCTTCCCGGGCCAAGCGCAGCGGTTCGGGC(SEQ ID NO:34)

[0111] ATGGATACCTGGCTGGTGTGCTGGGCTATTTTCTCCCTGCTCAAGGCCGGTCTCACCGAACCGGAGGTGACCCAGACTCCCTCTCACCAGGTCACCCAGATGGGCCAGGAGGTGATTTTGCGCTGCGTGCCCATCAGCAACCACCTCTACTTCTATTGGTACCGCCAAATCCTAGGCCAGAAAGTGGAGTTCCTGGTGTCCTTTTACAACAACGAGATCAGCGAGAAAAGCGAGATTTTCGATGACCAGTTCAGCGTGGAGCGCCCTGACGGCTCCAACTTTACGCTCAAGATCCGATCCACCAAGCTGGAGGACTCGGCAATGTATTTCTGCGCCTCTTCCGAAGCCTACGGTGGCTACAACGAGCAGTTTTTCGGCCCGGGGACCCGCCTGACTGTGCTGGAGGACCTTAAGAACGTGTTCCCACCGAAGGTGGCGGTTTTCGAGCCCAGCGAGGCCGAGATAAGCCACACTCAGAAGGCGACTCTCGTCTGTCTGGCGACAGGCTTCTACCCTGATCATGTGGAGCTGAGTTGGTGGGTCAACGGTAAGGAGGTGCATTCGGGCGTGTCTACCGATCCTCAGCCACTTAAAGAACAGCCTGCCCTGAACGACTCTCGTTATTGTCTGTCATCCCGCCTGCGCGTCTCTGCCACGTTCTGGCAGAACCCGCGCAACCACTTCCGTTGCCAGGTGCAATTCTACGGATTATCCGAGAATGACGAGTGGACCCAGGACCGCGCCAAGCCTGTGACCCAGATCGTCTCTGCTGAAGCATGGGGCCGAGCAGACTGCGGGTTCACGTCTGAGAGCTATCAGCAGGGAGTGCTGTCCGCTACCATCTTGTACGAGATTCTGCTGGGCAAGGCTACACTGTACGCAGTGCTGGTTTCCGCCCTTGTACTGATGGCCATGGTGAAGCGCAAGGACAGTAGAGGC(SEQ ID NO:35)

[0112] ATGAAATCTCTTCGCGTGCTGCTGGTGATTTTGTGGCTCCAGCTGTCTTGGGTCTGGTCTCAGCAGAAGGAGGTGGAACAGAACTCCGGACCTTTGTCGGTGCCGGAGGGCGCCATTGCTTCCCTCAACTGCACTTATTCTGACCGTGGCAGTCAGAGCTTCTTTTGGTACCGCCAGTACAGTGGCAAGTCGCCGGAGCTGATCATGTTTATCTACAGCAATGGCGATAAGGAGGATGGGCGCTTCACGGCGCAGCTTAACAAAGCTTCCCAGTACGTGAGCCTTCTCATTCGCGACAGCCAGCCGAGTGACTCCGCAACCTACCTGTGCGCCGTTAAAGGGGGCTACAACAAGCTGATCTTCGGTGCCGGCACCCGCCTGGCCGTGCACCCGTATATCCAGAACCCCGAGCCAGCGGTGTACCAGTTGAAGGACCCGCGCTCGCAGGACTCCACCCTGTGCCTGTTCACCGATTTCGACTCCCAAATTAACGTGCCAAAGACTATGGAGAGCGGTACGTTTATCACCGACAAAACCGTGCTGGACATGAAGGCCATGGATTCCAAGAGCAACGGCGCTATCGCTTGGTCCAACCAGACCTCCTTCACCTGCCAGGACATCTTCAAGGAGACCAATGCCACCTATCCTAGTTCCGACGTGCCCTGTGACGCAACACTAACCGAGAAGTCATTCGAGACCGACATGAACCTCAACTTCCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTCAAGGTTGCCGGCTTCAACCTGCTGATGACCCTCCGGCTTTGGTCTTCGCGTGCCAAGCGCAGCGGTTCCGGC(SEQ ID NO:42)

[0113] ATGGGGTCCCGCCTGCTGTGCTGGGTACTCCTGTGCCTGCTGGGAGCTGGACCAGTGAAGGCTGGAGTTACTCAGACTCCCCGTTACCTCATAAAAACGCGTGGTCAGCAGGTCACTCTGTCATGCAGCCCCATCTCTGGCCACCGCTCTGTATCGTGGTACCAGCAGACACCTGGGCAGGGTCTGCAGTTCCTGTTTGAGTACTTCTCCGAGACCCAGCGCAACAAGGGCAACTTCCCGGGTAGATTCTCTGGGCGACAGTTCAGCAATAGCCGGTCTGAGATGAACGTGTCCACCCTCGAGCTTGGTGATTCCGCTCTGTACCTGTGCGCCTCCTCCTTGGGCGGGGAGGGCGACAGGCCTCAGCACTTTGGTGACGGCACTCGACTTAGTATCCTGGAGGACCTGCGCAATGTGACCCCACCCAAGGTGTCGCTGTTCGAGCCCTCCAAGGCAGAGATCGCCAACAAGCAGAAGGCGACTCTGGTCTGTCTGGCGCGTGGCTTTTTCCCCGACCATGTCGAGCTGAGCTGGTGGGTCAACGGTAAAGAGGTCCACTCAGGCGTCTCGACCGACCCCCAGGCCTACAAGGAGAGCAACTATTCCTACTGCTTGTCTTCCAGATTACGGGTGAGCGCAACTTTTTGGCACAACCCCCGCAACCACTTTAGGTGTCAGGTGCAGTTCCATGGACTTTCTGAAGAAGATAAATGGCCTGAAGGGAGCCCTAAGCCCGTCACCCAGAACATCAGCGCCGAGGCCTGGGGCCGCGCGGACTGTGGCATCACCTCCGCTAGCTACCACCAAGGCGTGCTGTCTGCGACCATCCTGTACGAGATTCTGCTAGGCAAAGCTACACTGTACGCCGTGTTGGTGAGCGGCCTGGTCCTGATGGCCATGGTGAAGAAGAAGAACTCC(SEQ ID NO:43)Preferably,The nucleic acid molecule further comprises a nucleotide sequence as shown in at least one of SEQ ID NO: 56 (encoding the α chain constant region shown in SEQ ID NO: 26) and SEQ ID NO: 57 (encoding the β chain constant region shown in SEQ ID NO: 27).

[0114] ATCCAGAACCCCGAGCCCGCCGTGTACCAGCTGAAGGACCCCAGGAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAGAGCGGCACCTTCATCACCGACAAGACCGTGCTGGACATGAAGGCCATGGACAGCAAGAGCAACGGCGCCATCGCCTGGAGCAACCAGACCAG CTTCACCTGCCAGGACATCTTCAAGGAGACCAACGCCACCTACCCCAGCAGCGACGTGCCCTGCGACGCCACCCTGACCGAGAAGAGCTTCGAGACCGACATGAACCTGAACTTCCAGAACCTGAGCGTGATGGGCCTGAGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGGCTGTGGAGCAGC(SEQ ID NO:56)

[0115] GAGGACCTGAGGAACGTGACCCCCCAAGGTGAGCCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAACAAGCAGAAGGCCACCCTGGTGTGCCTGGCCAGGGGCTTCTTCCCCGACCACGTGGAGCTGAG CTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGAGCACCGACCCCCAGGCCTACAAGGAGAGCAACTACAGCTACTGCCTGAGCAGCAGGCTGAGGGTGAGCGCCACCTTCTGGCACAACCCCAGGA ACCACTTCAGGTGCCAGGTGCAGTTCCACGGCCTGAGCGAGGAGGACAAGTGGCCCGAGGGCAGCCCCAAGCCCGTGACCCAGAACATCAGCGCCGAGGCCTGGGGCAGGGCCGACTGCGGCATCACCAGC GCCAGCTACCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACAGC(SEQID NO:57)

[0116] According to a particularly preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO: 58.

[0117]

[0118] According to another particularly preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:59.

[0119]

[0120] The third aspect of the present invention provides a nucleic acid expression vector, which comprises a coding gene and a vector sequence, wherein the coding gene is provided by the nucleic acid molecule described in the second aspect.

[0121] The nucleic acid expression vector provided by the present invention is used to express the TCR provided by the present invention in vitro. That is, by introducing the nucleic acid expression vector into cells in vitro, the expression of the TCR provided by the present invention can be achieved.

[0122] In the nucleic acid expression vector provided by the present invention, the coding gene is used to encode the nucleic acid sequence expressing the TCR molecule provided by the present invention, and its characteristics are as described above and will not be repeated here. The vector sequence is usually provided by the starting vector (usually a plasmid vector) of the nucleic acid expression vector, and its function is to load the coding gene and introduce it into the cell to promote the expression of the TCR molecule in the cell. Any vector commonly used in the art for constructing an in vitro expression system can be applied to the present invention.

[0123] The present invention further provides a method for constructing the nucleic acid expression vector. Any method for constructing an in vitro expression vector commonly used in the art can be applied to the construction of the nucleic acid expression vector provided by the present invention, as long as the method can introduce the above-mentioned encoding gene into the vector sequence and enable it to be expressed in cells in vitro. The specific details will not be repeated here.

[0124] The fourth aspect of the present invention provides a vector cell, which comprises the T cell antigen receptor described in the first aspect, and / or the vector cell comprises the nucleic acid molecule described in the second aspect, and / or the vector cell comprises the nucleic acid expression vector described in the third aspect.

[0125] The vector cells provided by the present invention can be used to express the TCR molecules provided by the present invention in vitro, and can also be used in corresponding CMV prevention / treatment methods, or for preparing related drugs. Any common cells in the art that can achieve the above-mentioned purposes can be applied to the present invention. For example, T cells (especially T cells derived from the patient's own body) can be selected as vector cells, and the above-mentioned TCR, nucleic acid molecules or nucleic acid expression vectors can be introduced therein to prepare products such as cell pharmaceutical compositions for CMV treatment / prevention.

[0126] For example, cells commonly used in the art for in vitro protein expression, such as prokaryotic expression vectors, can be selected as carrier cells, into which the above-mentioned nucleic acid molecules or nucleic acid expression vectors are introduced to produce TCR molecules for introduction into T cells.

[0127] The present invention further provides a method for preparing the vector cell. Any method commonly used in the art for introducing a protein (such as the TCR of the present invention), a nucleic acid molecule, or a nucleic acid expression vector into a cell can be applied to the present invention, such as electroporation, lentiviral transfection, etc.

[0128] The fifth aspect of the present invention provides the use of the T cell antigen receptor described in the first aspect, and / or the nucleic acid molecule described in the second aspect, and / or the nucleic acid expression vector described in the third aspect, and / or the vector cell described in the fourth aspect in the preparation of drugs and / or vaccines for preventing and / or treating diseases caused by human cytomegalovirus infection.

[0129] The present invention has no particular limitation on the specific pharmaceutical form and ingredients of the drug, as long as it contains at least one of the T cell antigen receptor, nucleic acid molecule, nucleic acid expression vector, and carrier cell provided by the present invention.

[0130] According to an exemplary preferred embodiment of the present invention, the drug / vaccine may comprise a fusion protein as a (main) active component, wherein the fusion protein contains the aforementioned TCR.

[0131] Preferably, the fusion protein may also contain an antibody.

[0132] More preferably, the antibody includes single chain antibody (scFv), monoclonal antibody, chimeric antibody, multispecific antibody, preferably single chain antibody.

[0133] More preferably, the antibody is a humanized antibody.

[0134] Preferably, the fusion protein may further contain a CD3 agonist.

[0135] Preferably, the fusion protein may further contain an antibody that specifically targets CD3.

[0136] The sixth aspect of the present invention provides a pharmaceutical composition for preventing and / or treating diseases caused by human cytomegalovirus infection, wherein the pharmaceutical composition comprises: the T cell antigen receptor described in the first aspect, and / or the nucleic acid molecule described in the second aspect, and / or the nucleic acid expression vector described in the third aspect, and / or the vector cell described in the fourth aspect.

[0137] In the pharmaceutical composition provided by the present invention, the TCR, nucleic acid molecule, nucleic acid expression vector, and vector cell can all serve as (one of) the (main) active ingredients of the pharmaceutical composition. According to some preferred embodiments of the present invention, the pharmaceutical composition may further include pharmaceutically acceptable excipients (e.g., buffers, preservatives, stabilizers, etc.).

[0138] According to some preferred embodiments of the present invention, the pharmaceutical composition may further include other active ingredients, such as drugs that have no or substantially no adverse effects on the effects of the TCR, nucleic acid molecules, nucleic acid expression vectors, and vector cells provided by the present invention when used together, and that do not produce or substantially do not produce (serious) side effects on the human body (for example, antiviral drugs, antibodies, vaccine preparations, etc. that can be used to prevent / treat CMV infection).

[0139] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0140] In the following examples, experiments involving human sample collection and testing have been conducted with the informed consent of the relevant personnel. Unless otherwise specified, all reagents and materials used were purchased from reputable chemical and biological reagent or material suppliers and were of analytical grade.

[0141] In the following examples, unless otherwise specified, the operating temperature is room temperature (25±5°C).

[0142] Example 1

[0143] This example is used to illustrate the preparation and expansion of TCR-T cells provided by the present invention and the verification of their killing effect.

[0144] Preparation and expansion of TCR-T cells

[0145] 1. Experimental Materials

[0146]

[0147] 2. Experimental methods

[0148] Peripheral blood mononuclear cells (PBMCs) from HLA-A*11:01 healthy donors and patients with various cancer types were stimulated with chemically synthesized CMV epitope peptides. After two rounds of CMV epitope peptide stimulation, specific T cells were detected using flow cytometry and enzyme-linked immunospot (ELISpot) assays to confirm the generation of CMV-specific T cells. The specific methods are as follows:

[0149] Peripheral blood was collected from HLA-A*11:01 donors, and PBMCs were isolated by density gradient centrifugation. The isolated PBMCs were seeded into well plates at a specific cell density and cultured in X-VIVO15 (LONZA) supplemented with human serum, GM-CSF (Xiamen Tebao Biotechnology Co., Ltd.), and IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.), among other cytokines.

[0150] HLA-A*11:01 CMV epitope peptide (GenScript; specific sequence is shown as SEQ ID NO: 1) was dissolved in X-VIVO15 to prepare a 100 μg / mL CMV epitope peptide solution, which was sterilized by filtration and set aside. Stimulation was performed by adding sterile CMV epitope peptide solution once on day 2 and again on day 4 of cell culture, to a final concentration of 5 μg / mL.

[0151] The total cell culture period was approximately 14 days. During the culture process, cells were observed every 2-3 days, half of the culture medium was replaced, and the viable cell density, cell suspension volume, and cell viability were recorded using a cell counter. On day 14, T cells were stained with mouse anti-human CD8 antibody (Biolegend) and HLA-A*11:01CMV epitope tetramer (MBL) for flow cytometry. The experimental results are shown in Figure 1 Comparison with the control T cell group (not stimulated with CMV epitope peptides) revealed the presence of CMV-specific T cells after CMV peptide stimulation. A flow cytometer was used to isolate the target CD8-positive and tetramer-positive T cell population.

[0152] ELISpot assay was performed using IFN-γ as the cytokine to be tested. A certain amount of cells were taken and washed with X-VIVO15 medium. After resuspending the cells with X-VIVO15 containing 10% FBS, 2×10 cells were added to each well. 4 The cells were plated in Elispot-IFN-γ assay plates (Mabtech) using a cell number of 10 μg / mL CMV epitope peptide (MBL) for 18-24 hours before antibody incubation and color development. The plate was placed in a cool, dark place at room temperature and allowed to dry naturally before imaging and reading the immune spots using a CTL S6. The results are detailed in the table. Figure 2 .

[0153] pass Figure 1 and Figure 2 It can be seen that the specific T cell expansion method provided by the present invention can obtain HLA-A*11:01CMV-specific T cells, and compared with the control T cell group ( Figure 2 Compared with the stimulated CMV pp65 group ( Figure 2Significantly more spots were detected in the middle DF wells, indicating that CD8 + T cells can specifically recognize CMV pp65 epitope peptides and secrete cytokines such as IFN-γ.

[0154] Example 2

[0155] This example is used to illustrate the construction of the in vitro transcription plasmid vector and mRNA preparation of the TCR provided by the present invention.

[0156] 1. Experimental Materials

[0157] name factory Item No. Nuclease-free water (non-DEPC treated) Invitrogen AM9932 QIAGEN OneStep RT-PCR Kit(100) Qiagen 210212 Single Cell Sequence-Specific Amplification Kit Nanjing Novozymes Biotech Co., Ltd. P621-01 HotStarTaq Master Mix Kit(250) Qiagen 203443 Qubit 1X dsDNA HS Assay Kit Invitrogen Q33230 DL500 DNA Marker TaKaRa 3590A 6×Loading Buffer TaKaRa 9156

[0158] 2. Experimental methods

[0159] Reference Figure 3 The plasmid map of the TCR was constructed by inserting the TCR encoding genes (referred to as TCRα / β encoding genes) into the pUC57 plasmid (GenScript) as a vector plasmid. The TCR encoding genes include those for the α and β chains. The α chain encoding gene (the TRA ORF portion in the figure) includes the α chain variable region encoding gene and the α chain constant region encoding gene; the β chain encoding gene (the TRB ORF portion in the figure) includes the β chain variable region encoding gene and the β chain constant region encoding gene; the α and β chain encoding genes are connected by an internal self-cleaving porcine teschovirus 2A sequence (p2A sequence). The specific sequences are shown in Table 1.

[0160] Table 1

[0161]

[0162] In vitro transcription plasmid vector construction : The TCRα / β encoding gene sequence (including BamHI and SacI restriction sites) commissioned to Nanjing GenScript Co., Ltd. was digested with BamHI and SacI restriction endonucleases and cloned into the pUC57 plasmid between the BamHI and SacI restriction sites. This plasmid contains a Kozak sequence (GCCACC) downstream of the BamHI restriction site and a double termination codon sequence (TGATAA) upstream of the SacI restriction site.

[0163] Using the constructed in vitro transcription plasmid vector, mRNA was prepared using the following method:

[0164] The in vitro transcription plasmid vector was linearized using the system in Table 2. After the reaction system was configured, it was placed at 50°C for overnight reaction (no more than 18 hours). After the reaction, the temperature was raised to 80°C and kept for 20 minutes to inactivate the enzyme in the system.

[0165] Table 2

[0166] name Addition amount plasmids 5 μg <![CDATA[NEBuffer TM r3.1]]> 4μL BqI 2μL Nuclease-free water To a final volume of 40 μL

[0167] use Purify the linearized plasmid from the reaction product using DNA Clean Beads. After electrophoresis verification and concentration testing of the purified product, prepare the in vitro transcription system according to Table 3 and incubate at 37°C for 2.5 hours. After incubation, add 4 μL of DNase I to the reaction system and incubate at 37°C for 15 minutes to digest the transcribed DNA template.

[0168] Table 3

[0169] name Addition amount Template (purified linearized plasmid) 1 μg 10×Transcrption Buffer 8μL UTP solution 8μL ATP solution 8μL CTP solution 8μL GTP solution 8μL T7 RNA Polymerase Mix 8μL <![CDATA[RNase-free ddH2O]]> To a final volume of 80 μL

[0170] use Purify the in vitro transcription product using DNA Clean Beads (DNA magnetic beads). After electrophoresis verification and concentration testing, add 100 μg of the purified product to an appropriate volume of nuclease-free water to prepare a 140 μL RNA solution. Heat at 65°C for 5 minutes and then cool on ice for 5 minutes to obtain a heat-denatured RNA solution. Prepare the capping system according to Table 4 and incubate at 37°C for 1.5 hours.

[0171] Table 4

[0172] Components Dosage Final concentration Heat-denatured RNA 140 μL 0.5 μg / μL 10x Capping buffer 20 μL 0.5 U / μL Vaccinia capping enzyme (10 U / μL) 10 μL 2.5 U / μL mRNA Cap 2'-O-methyltransferase (50U / μL) 10 μL 0.5mM GTP (10 mM) 10 μL 0.2mM SAM (4mM) 10 μL 500ng / μL

[0173] use DNA Clean Beads (DNA magnetic beads) were used to purify the reaction product to obtain capped TCR mRNA. After BspQI digestion, electrophoresis verification was performed. The results are shown in the table below. Figure 4 In the figure, samples ZZ1, ZZ2 and ZZ3 are the electrophoresis results of the mRNA digestion products (named ZZ1 mRNA, ZZ2 mRNA and ZZ3 mRNA, respectively) obtained after the nucleic acid expression vectors I, II and III were treated as described above.

[0174] Example 3

[0175] This example is used to illustrate the activation verification results of the TCR provided by the present invention.

[0176] 1. Experimental Materials

[0177]

[0178]

[0179] Preparation of cells for experiments:

[0180] To meet the functional validation requirements for HLA-A*11:01-restricted TCR sequences, we engineered human lymphoma blasts (K562) to construct stable cell lines expressing different HLA subtypes. Since K562 cells lack expression of HLA class I and II molecules on their surface, we artificially constructed single-HLA expressing cell lines and used them as target cells to validate HLA-restricted killing. The specific construction method is as follows:

[0181] HLA overexpression lentiviral packaging:

[0182] (1) Take 293FT cells in good growth state and inoculate them into T175 culture flasks. Add 20 mL of Opti-MEM complete medium (Thermo Fisher Scientific) to each flask. The inoculation volume is about 1.2×10 7 Cells / flask were cultured at 37°C overnight until the cell confluence reached 50-60%. The reagent prepared according to the formula in Table 5 (the amount in the table is for one T175 culture flask) was added for transfection, mixed, and then placed in a 37°C incubator for continued culture.

[0183] Table 5

[0184]

[0185] *For details of the HLA lentiviral expression plasmid map, please see Figure 5 .

[0186] (2) 48 h after transfection, the culture supernatant was collected and stored at 4°C. Then, 20 mL of fresh Opti-MEM complete medium (Thermo Fisher Scientific) was added to each culture flask and the cells were cultured at 37°C.

[0187] (3) After 72 h of culture, the culture supernatant was collected again and combined with the 48 h supernatant collected in step (2). The supernatant was centrifuged at 4500 rpm for 5 min at 4°C to remove the precipitate. The obtained supernatant was filtered through a 0.22 μm vacuum filter bottle. PEG8000 concentration reagent with a volume of 1 / 5 was added to the filtrate, mixed, and concentrated and precipitated at 4°C overnight.

[0188] (4) After the concentration and sedimentation, centrifuge at 4500 rpm for 50 min at 4°C to collect the precipitate, resuspend it in 200 μL of pre-cooled D-PBS, mix well, and dispense 50 μL / tube into 1.5 mL centrifuge tubes. Store at -80°C for later use.

[0189] Infect target cells with lentivirus and obtain positive monoclonal clones:

[0190] (1) Take the cultured K562 wild-type cells and prepare 3×10 5 A cell suspension of 10 cells / mL was inoculated into a 12-well plate at a volume of 1 mL per well. At the same time, 5 μL of polybrene infection reagent and 50 μL of lentivirus were added to each well. After mixing, the cells were cultured at 37°C and passaged.

[0191] (2) After two passages, HLA expression was detected by flow cytometry, with wild-type K562 cells used as a negative control. HLA positivity and expression levels were detected by flow cytometry, and cells were isolated by flow cytometry for single clones. The top 5% of cells with the highest HLA expression intensity were selected and placed in at least one 96-well plate for expansion and culture.

[0192] (3) Using wild-type K562 cells as a negative control, the HLA expression level of monoclonal cells was again detected by flow cytometry. Cell lines with significantly higher expression levels than those of wild-type cells were considered to be HLA overexpressing cell lines. The monoclonal cells with the highest expression levels were selected as target cells for functional verification of HLA-A*11:01-restricted TCR sequences in subsequent experiments (the flow cytometry results are shown in Figure 2). Figure 6 As shown, K562-HLA-A11:01 OE The HLA positivity rate of cells was 98.68%).

[0193] 2. Experimental methods

[0194] Using genetically engineered NFAT-Luc Jurkat cells, the activation status of the TCR pathway was verified by measuring the Luciferase fluorescence value. The specific method is as follows:

[0195] (1) Obtaining NFAT-Luc Jurkat cells expressing TCR:

[0196] NFAT-Luc Jurkat cells were cultured to 2 × 10 7 Cells were collected at a density above 10 cells / mL, washed once with DPBS (Cytiva), and resuspended in R solution (Thermo Fisher Scientific) to prepare 2×10 7 / mL cell suspension was added to 1.5mL EP tubes at a rate of 100μL / tube. Simultaneously, 5μg CD8 mRNA (GenScript), 5μg ZZ1 TCR mRNA, and 5μg ZZ2 TCR mRNA were added to the EP tubes for electroporation of CD8 mRNA or TCR mRNA, respectively, and labeled. 3mL of electroporation E2 solution (Thermo Fisher Scientific) was added to the electroporation cuvette, which was placed in the cuvette tank of an electroporator (Thermo Fisher Scientific). The cell mixture was aspirated using a 100μL pipette tip. Electroporation conditions were: 1400V, 20ms, 2 pulses. The added mRNA was then introduced into NFAT-Luc Jurkat cells to obtain Jurkat cells expressing CD8 or TCR.

[0197] After 24 hours of electroporation, the Jurkat cells expressing CD8 or TCR were stained with HLA-A*11:01CMV tetramer (MBL). The staining results are shown in Figure 7 As can be seen from the figure, cells expressing the corresponding transferred mRNAs were successfully obtained by electroporation, and specific T cell populations expressing the two TCR mRNAs obtained in Example 2 could be detected by HLA-A*11:01CMV tetramerization.

[0198] (2) TCR activation verification:

[0199] NFAT-Luc Jurkat cells expressing ZZ1 TCR and ZZ2 TCR were co-incubated with the HLA-overexpressing K562 cells prepared above at a ratio of 20:1 at 37°C for 4 hours. After incubation, the cells were washed with DPBS (Cytiva) and 100 μL of Bio-Lite detection reagent (Norvozymes) equilibrated to room temperature was added to each well. After standing in the dark for 2-5 minutes, the cells were detected using a microplate reader. The results are shown in Figure 2. Figure 8A and Figure 8B As can be seen from the figure, only HLA-A*11:01K562 cells loaded with CMV pp65 epitope peptide can activate Jurkat Luc fluorescence.

[0200] Example 4

[0201] This example is used to illustrate the verification of the killing effect of TCR-T cells provided by the present invention.

[0202] 1. Experimental Materials

[0203]

[0204] 2. Experimental methods

[0205] Peripheral blood was obtained from healthy donors. PBMCs from the donors were isolated by Ficoll density gradient centrifugation and resuspended in XVIVO15 medium. T cells were purified and isolated using magnetic beads. T cells were activated with CD3 / CD28 magnetic beads and resuspended in XVIVO15 medium containing 2.5% human autologous serum and 30 IU / mL IL-2 for 3 days. Cells were collected, washed once with DPBS, and resuspended in R solution to prepare 2×10 7 / mL cell suspension was added to 1.5mL EP tubes at 100μL per portion, and 5μg of ZZ1 TCR mRNA and ZZ2 TCR mRNA were added respectively. 3mL of E2 solution was added to the electroporation cuvette, which was placed in the cuvette slot of the electroporator. The cell mixture was aspirated using a 100μL pipette tip. Electroporation conditions were: 1400V, 10ms, 3 pulses. TCR mRNA was introduced into activated T cells to obtain TCR-expressing T cells.

[0206] 24 hours after electroporation, the T cells expressing TCR were stained with HLA-A*11:01CMV tetramer (MBL). Figure 9 .

[0207] K562 cells were cultured to a cell density of 1×10 7 Cells were collected, washed once with 1640 medium and resuspended in 2×10 6 / mL cell suspension, pipette 200 μL / well of the cell suspension into a 48-well cell culture plate, add 60 μL / well of 200 μM polypeptide (SEQ ID NO: 1) and then add 1640 medium to make up to 400 μL / well, and incubate at 37°C overnight.

[0208] K562 cells were collected at 8×10 5 1 μL of EuTDA dye was added to each well, incubated at 37°C for 20 minutes, centrifuged, and washed three times with 1 mL of 1× PBS. After the final wash, the cells were resuspended in T cell culture medium.

[0209] TCR-expressing T cells (TCR-T cells, containing the ZZ1 TCR and ZZ2 TCR obtained in Example 2, respectively, and flow cytometry verification was performed to confirm that TCR mRNA was successfully transferred into activated T cells) were incubated with the above-mentioned K562 cells at a ratio of 20:1, 10:1, 5:1, 2.5:1, and 1.25:1 at 37°C for 4 hours. After incubation, centrifuge at 500g for 5 minutes at room temperature. 20 μL of supernatant was transferred to a flat-bottomed 96-well plate of the EuTDA Cytotoxicity Reagents kit, 200 μL of Eu solution was added to each well, and the plates were shaken and incubated at room temperature for 10 minutes at 250 rpm. The fluorescence values ​​at the wavelengths of excitation light 330 / 80 and emission light 620 / 10 were recorded with a microplate reader within 5 hours. The window value and killing efficiency were calculated based on the fluorescence values ​​of each group. The calculation formula is as follows, and the results are shown in detail. Figure 10 ((A) is ZZ1 TCR-T; (B) is ZZ2 TCR-T).

[0210]

[0211]

[0212] Example 5

[0213] This example is used to illustrate the results of the T cell peptide sensitivity evaluation of the TCR provided by the present invention.

[0214] 1. Experimental Materials

[0215] name factory Item No. D-PBS Cytiva SH30028.02 Human IFN-γPrecoated ELISA Kit Shenzhen Dakoway Bioengineering Co., Ltd. 1110003 RPMI-1640 Thermo Fisher Scientific R5886 Fetal bovine serum Thermo Fisher Scientific SV30087.02 Penicillin-streptomycin Thermo Fisher Scientific 15140-122 X-VIVO 15 Lonza 04-418Q

[0216] 2. Experimental methods

[0217] The method in Example 3 was used to obtain T cells expressing ZZ1 TCR and ZZ2 TCR. Then the two TCR-T cells were respectively loaded with 10 -5 M to 10 -12 The cells were incubated with K562, a CMV epitope peptide from M, at a 1:1 ratio for 18-24 hours. After incubation, the cells and supernatant were collected from each group. The CD3+4-1BB+ / CD3+ ratio (%) was determined by flow cytometry, and a fitting curve was drawn to estimate the EC50. The IFN-γ concentration in the supernatant of each cell group was determined by ELISA, and a fitting curve was drawn to estimate the EC50.

[0218] K562 cells were collected and counted using a cell counter to record the viable cell density and cell viability. The cells were washed once and resuspended in RPMI 1640 (Thermo) medium. 5K562 cells were seeded in 24-well plates, with a total of 9 groups. Different concentrations of CMV epitope peptides were added to different groups to make the final peptide concentration 10 -12 M, 10 - 11 M, 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 M, loading time was 4 hours. The electroporated TCR-T cells were collected and counted using a cell counter to record the viable cell density and cell viability. The cells were resuspended in X-VIVO 15 containing 10% FBS (Cytiva) and 2×10 5 Tetramer detection was performed on a certain number of cells.

[0219] K562 cells loaded with epitope peptides were collected and the cell density was adjusted to 1 × 10 cells using XVIVO 15 medium containing 10% FBS. 6 After adjustment, 100 μL of cell suspension was added to a 96U well plate, with 3 replicates per group. The TCR-T cell density was adjusted to 1×10 using XVIVO15 containing 10% FBS. 6 Cells / mL were added to a 96-well plate seeded with K562 target cell suspension (100 μL). Mix thoroughly to achieve a 1:1 ratio of effector cells to target cells. After incubating TCR-T cells with K562 at 37°C overnight (approximately 18-24 hours), the 96-well plate samples were centrifuged and the supernatant was analyzed by ELISA-IFN-γ (Dakoway).

[0220] Dissolve the lyophilized powder of ELISA standards according to the label instructions, let it stand for 5-10 minutes, and then dilute it with 1× diluent to 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.3 pg / mL, respectively.

[0221] Dilute the collected cell supernatant 10- and 100-fold with diluent. Add 100 μL of the diluted sample to each well of a 96-well ELISA plate. Add 50 μL / well of the diluted antibody to the standard and sample wells, mix thoroughly, and cover with a sealing film. Incubate at 37°C for 2 hours. After incubation, discard the liquid from the wells and add 300 μL / well of 1x washing buffer. Allow to stand for 1 minute, then discard the liquid. Repeat this step four times. Add 100 μL / well of diluted avidin to the sample wells, cover with a sealing film, and incubate at 37°C for 30 minutes. After incubation, wash the plate again and add 100 μL / well of TMB. Incubate at 37°C in the dark for 5-30 minutes. After color development is complete, add 100 μL / well of stop solution. Within 10 minutes after stopping, read the plate simultaneously using a detection wavelength of 450 nm and a calibration wavelength of 630 nm. The IFN-γ concentration of each cell supernatant was calculated based on the standard curve. The results were statistically analyzed using GraphPad Prism 9 and fitted curves were drawn. The experimental results are detailed in Figures 11A-11B As can be seen from the figure, TCR-T cells expressing the two TCR mRNAs obtained in Example 2 can recognize HLA-A*11:01K652 cells loaded with different concentrations of CMV pp65 antigen peptides and release cytokines such as IFN-γ, indicating that both TCR-T cells can specifically recognize and kill cells expressing CMV pp65. The EC of ZZ1 TCR-T cells was estimated by the fitting curve. 50 About 10 -5 Although ZZ2TCR-T cells did not reach the peak of IFN-γ secretion, EC could not be calculated. 50 However, the experimental results showed that it also has a good ability to recognize and kill HLA-A*11:01K652 cells loaded with CMV pp65 antigen peptide.

[0222] Example 6

[0223] This example is used to illustrate the TCR motif evaluation results provided by the present invention.

[0224] 1. Experimental Materials

[0225]

[0226] 2. Experimental methods

[0227] The ALA-SCAN method was used to identify specific amino acid sites in the TCR provided by the present invention that are closely related to its function, stability and conformation. The alanine scanning peptide library constructed was as follows: Figure 12 As shown (the specific sequence of the epitope peptide can be found in Table 6).

[0228] Table 6

[0229] Epitope peptide number ID sequence 1 SEQ ID NO:61 AAVQGQNLK 2 SEQ ID NO:62 ATAQGQNLK 3 SEQ ID NO:63 ATVAGQNLK 4 SEQ ID NO:64 ATVQAQNLK 5 SEQ ID NO:65 ATVQGANLK 6 SEQ ID NO:66 ATVQGQALK 7 SEQ ID NO:67 ATVQGQNAK 8 SEQ ID NO:68 ATVQGQNLA

[0230] T cells expressing ZZ1 TCR and ZZ2 TCR were obtained using the method in Example 4.

[0231] K562 cells were cultured until the cell density reached 1×10 7 Cells were collected, washed once with 1640 medium and resuspended to obtain 2×10 6 / mL cell suspension, 200 μL / well of the cell suspension was pipetted into a 48-well cell culture plate, and 60 μL of epitope peptide (SEQ ID NO: 58-65) was added to each well, and 1640 medium was added to 400 μL / well, and incubated at 37°C overnight.

[0232] The two TCR-expressing T cells were co-incubated with the above K562 cells at a ratio of 1:2 at 37°C for 24 h. Figure 13-14 The figure shows that the motif recognized by the electroporated ZZ2 TCR is -TVQGQNLK, while the motif recognized by the ZZ1 TCR is ---QGQ—K. This result indicates that the motif recognized by ZZ2 is more conserved.

[0233] Comparative Example 1

[0234] Using the method in Example 3, NFAT-Luc Jurkat cells were electroporated with the ZZ3 TCR mRNA obtained in Example 2 to obtain Jurkat cells expressing ZZ3 TCR. The flow cytometry results were as follows: Figure 15 As shown in the experimental results, it can be seen that there is no CMV-A*11:01 tetramer-positive population in ZZ3Jurkat cells.

[0235] The ZZ3 TCR sequence (SEQ ID NO: 54) contains an α chain variable region with an amino acid sequence as set forth in SEQ ID NO: 24, a β chain variable region with an amino acid sequence as set forth in SEQ ID NO: 25, and an α chain constant region and a β chain constant region with amino acid sequences as set forth in SEQ ID NOs: 26-27, respectively. The α chain variable region includes CDR1α, CDR2α, and CDR3α with amino acid sequences as set forth in SEQ ID NOs: 18-20, respectively (the genes encoding them are set forth in SEQ ID NOs: 44-46, respectively); the β chain variable region includes CDR1β, CDR2β, and CDR3β with amino acid sequences as set forth in SEQ ID NOs: 21-23, respectively (the genes encoding them are set forth in SEQ ID NOs: 47-49, respectively).

[0236] The experimental results of the NFAT-Luc reporter system are as follows Figure 16As shown in the figure, the fluorescence value of the control group is in the range of 12 to 14, and the fluorescence value of ZZ3 Jurkat is 18, which is close to the control group, indicating that this TCR cannot be activated by K562-A*11:01-pep.

[0237] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A T cell antigen receptor, characterized in that The T cell antigen receptor specifically recognizes and binds to a human cytomegalovirus binding epitope comprising an amino acid sequence as shown in SEQ ID NO: 1, the T cell antigen receptor comprising at least one α chain variable region and at least one β chain variable region, the α chain variable region comprising a complementarity determining region of the α chain, and the β chain variable region comprising a complementarity determining region of the β chain; wherein the amino acid sequences of the complementary determining regions 1-3 of the α chain variable region are shown in SEQ ID NOs: 2-4, respectively, and the amino acid sequences of the complementary determining regions 1-3 of the β chain variable region are shown in SEQ ID NOs: 5-7, respectively; or The amino acid sequences of the complementarity determining regions 1-3 of the α chain variable region are shown in SEQ ID NOs: 10-12, respectively, and the amino acid sequences of the complementarity determining regions 1-3 of the β chain variable region are shown in SEQ ID NOs: 13-15, respectively.

2. The T cell antigen receptor according to claim 1, wherein The α chain variable region includes the amino acid sequence shown in SEQ ID NO:8, and the β chain variable region includes the amino acid sequence shown in SEQ ID NO:9; Alternatively, the α chain variable region comprises the amino acid sequence shown in SEQ ID NO:16, and the β chain variable region comprises the amino acid sequence shown in SEQ ID NO:

17.

3. The T cell antigen receptor according to claim 1 or 2, wherein In the T cell antigen receptor, the α chain variable region is directly or indirectly linked to the β chain variable region.

4. The T cell antigen receptor according to claim 3, wherein In the T cell antigen receptor, the α chain variable region is indirectly linked to the β chain variable region.

5. The T cell antigen receptor according to claim 4, wherein The α chain variable region is indirectly linked to the β chain variable region via the furin SGSG p2A sequence.

6. A nucleic acid molecule encoding the T cell antigen receptor according to any one of claims 1 to 5.

7. A nucleic acid expression vector, characterized in that: The nucleic acid expression vector comprises a coding gene and a vector sequence, wherein the coding gene is provided by the nucleic acid molecule according to claim 6.

8. A vector cell, characterized in that: The vector cell comprises the T cell antigen receptor according to any one of claims 1 to 5, and / or the vector cell comprises the nucleic acid molecule according to claim 6, and / or the vector cell comprises the nucleic acid expression vector according to claim 7.

9. Use of the T cell antigen receptor according to any one of claims 1 to 5, and / or the nucleic acid molecule according to claim 6, and / or the nucleic acid expression vector according to claim 7, and / or the vector cell according to claim 8 in the preparation of a medicament for preventing and / or treating diseases caused by human cytomegalovirus infection.

10. Use of the T cell antigen receptor according to any one of claims 1 to 5, and / or the nucleic acid molecule according to claim 6, and / or the nucleic acid expression vector according to claim 7, and / or the vector cell according to claim 8 in the preparation of a medicament for preventing and / or treating diseases caused by human cytomegalovirus infection using TCR-T therapy.

11. A pharmaceutical composition for preventing and / or treating diseases caused by human cytomegalovirus infection, characterized in that: The pharmaceutical composition comprises: the T cell antigen receptor according to any one of claims 1 to 5, and / or the nucleic acid molecule according to claim 6, and / or the nucleic acid expression vector according to claim 7, and / or the vector cell according to claim 8.

Images