TCR targeting cytomegalovirus antigens and T cells expressing same and uses

Abstract

The present invention provides a TCR targeting a cytomegalovirus antigen and T cells expressing the same, as well as uses thereof. Specifically, the present invention discloses specific T cells that target the cytomegalovirus pp65 antigen epitope with HLA-A*0201 restriction, and uses thereof, in which the T cell receptor of the T cell specifically targets CMV-pp65 and can accurately and rapidly eliminate the CMV virus.

Description

[Technical field]

[0001] The present invention relates to TCRs capable of recognizing cytomegalovirus-derived antigenic peptides, cytomegalovirus-specific T cells expressing said TCRs, and their use in the prevention and treatment of cytomegalovirus-associated diseases. [Background technology]

[0002] Cytomegalovirus (CMV) belongs to the β subfamily of the human herpesviridae family, has clear host species specificity, and is the largest and most structurally complex virus in the human herpesviridae family. CMV is highly contagious among humans, with an infection rate of over 95% in adults. It is usually asymptomatic, and many infected individuals have no clinical symptoms, but under certain conditions, it can affect multiple organs and systems and lead to severe disease. In recent years, with the great progress in the diagnosis and treatment of CMV infection, the incidence of CMV infection after transplantation has obviously decreased, but the incidence of CMV infection remains high in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients, and the indirect consequences, such as secondary graft dysfunction, GVHD, and all-cause mortality, still have a significant impact on patient prognosis. Meanwhile, existing anti-CMV drugs also have many shortcomings, such as monotonous formulations, similar targets, and granulocytopenia / kidney damage. Current antiviral drugs, such as acyclovir and ganciclovir, often cause severe side effects, such as bone marrow suppression.

[0003] Human cytomegalovirus (HCMV) contains approximately 20-25 proteins, of which three proteins play the most important role in mediating HCMV infection and viral replication: pp65 protein encoded by ORF UL83, phosphorylated protein pp71 encoded by ORF UL82, and pUL69 protein encoded by ORF UL69. The common feature of the above three proteins is that they initiate HCMV infection of host cells and viral replication, and at the same time play an important role in evading T cell-mediated cytotoxicity. Research has shown that of the above three proteins, the pp65 protein has the most severe effect on vascular lesions and rejection of grafts. Immune damage caused by allo-HSCT reactivates latent HCMV, leading to severe clinical complications.

[0004] In recent years, cellular immunotherapy, including immune checkpoint inhibitors, CAR-T (Chimeric Antibody Receptor Engineered T Cells) and TCR-T (T cell Receptor Engineered T Cells), has made great progress in the field of tumor treatment. TCR-T therapy involves modifying T cells by genetic engineering to acquire specific TCRs against tumor antigens or specific viral antigens, and then injecting them into the body to treat tumors or eliminate viral infections.

[0005] Due to the weakened immune function of allo-HSCT patients, they are unable to effectively eliminate CMV and are prone to primary CMV infection or reactivation of latent CMV. CMV infection / reactivation in HSCT patients leads to CMV-related fever and a series of related diseases that extend to organs, which are closely related to disease recurrence and subject survival, and are less effective with current conventional drug treatments. Therefore, if CMV-TCR-T cell therapy can be applied in the field of treatment and prevention of CMV infection after HSCT or other organ transplantation, it will have great clinical value and future use. Summary of the Invention

[0006] An object of the present invention is to provide a specific TCR that targets the cytomegalovirus antigen epitope pp65 in an HLA-A*0201 restricted manner, a TCR-T cell expressing said TCR, and uses thereof.

[0007] In a first aspect of the invention there is provided a T cell receptor (TCR), comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, the amino acid sequence of the CDR3 of the TCR alpha chain variable domain being CAFPYNNNDMRF (SEQ ID NO: 13); and / or The TCR β chain variable domain has an amino acid sequence of CDR3 CASSLEGYTEAFF (sequence number 21). In another preferred embodiment, the TCR specifically binds to the NLVPMVATV-HLA-A*0201 complex. In another preferred embodiment, the three complementarity determining regions (CDRs) of the TCR α chain variable domain are: α-CDR1: SSNFYA (SEQ ID NO: 9), α-CDR2: MTLNGDE (SEQ ID NO: 11), α-CDR3: CAFPYNNNDMRF (sequence number 13). In another preferred embodiment, the three complementarity determining regions of the TCR β chain variable domain are: β-CDR 1: MNHEY (SEQ ID NO: 17), β-CDR 2: SMNVEV (SEQ ID NO: 19), β-CDR 3: CASSLEGYTEAFF (SEQ ID NO: 21). In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the TCR alpha chain variable domain has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7, and / or the TCR beta chain variable domain has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. In another preferred embodiment, the TCR comprises the amino acid sequence of the alpha chain variable domain SEQ ID NO:7. In another preferred embodiment, the TCR comprises the amino acid sequence of the β chain variable domain SEQ ID NO:15. In another preferred embodiment, the TCR is an αβ heterodimer and comprises a TCR α chain constant region TRAC*01 and a TCR β chain constant region TRBC1*01 or TRBC2*01. In another preferred embodiment, the amino acid sequence of the alpha chain of the TCR is SEQ ID NO:3. In another preferred embodiment, the amino acid sequence of the β chain of the TCR is SEQ ID NO:5. In another preferred embodiment, the TCR contains an artificial interchain disulfide bond between the α and β chains. In another preferred embodiment, the amino acid sequence of the TCR is shown in SEQ ID NO:1.

[0008] In a second aspect of the invention there is provided a multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR according to the first aspect of the invention.

[0009] In a third aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention, or a complementary sequence thereof. In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:8 encoding the TCR alpha chain variable domain. In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 16 encoding the TCR β chain variable domain. In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:4 encoding the TCR alpha chain. In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:6 encoding the TCR β chain. In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:2.

[0010] In a fourth aspect of the present invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the present invention. Preferably, the vector is a viral vector. More preferably, the vector is a lentiviral vector.

[0011] In a fifth aspect of the invention there is provided an isolated host cell which contains a vector according to the fourth aspect of the invention or has incorporated into its genome an exogenous nucleic acid molecule according to the third aspect of the invention.

[0012] In a sixth aspect of the invention there is provided a cell transfected with a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention. Suitably the cell is a T cell or a stem cell.

[0013] In a seventh aspect of the invention there is provided a pharmaceutical composition comprising a pharma- ceutically acceptable vehicle and a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention.

[0014] In an eighth aspect of the invention there is provided the use of a T cell receptor as defined in the first aspect of the invention, or a TCR complex as defined in the second aspect of the invention, a nucleic acid molecule as defined in the third aspect of the invention, a vector as defined in the fourth aspect of the invention, or a cell as defined in the sixth aspect of the invention, for the manufacture of a medicament for treating a cytomegalovirus associated disease.

[0015] In a ninth aspect of the invention there is provided a method of treating a disease which comprises administering to a subject in need of treatment a suitable amount of a T cell receptor as described in the first aspect of the invention, or a TCR complex as described in the second aspect of the invention, a nucleic acid molecule as described in the third aspect of the invention, a vector as described in the fourth aspect of the invention, or a cell as described in the sixth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention.

[0016] Preferably, the disease is a disease associated with cytomegalovirus infection (cytomegalovirus infection), such as CMV retinitis, CMV pneumonia, CMV gastroenteritis and CMV encephalitis.

[0017] Of course, it is understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (for example, in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1: Flow chart of CMV-pp65-TCR-T specificity stimulation and screening. [Diagram 2] FIG. 2 shows the results of in vitro specific expansion of CMV-pp65-TCR-T cells. [Diagram 3] FIG. 3 is information on the CMV-pp65-TCR-T vector. [Figure 4] FIG. 4 shows the in vitro binding of CMV-pp65-TCR-T. [Diagram 5] FIG. 5: Verification of in vitro activation of CMV-pp65-TCR-T. [Figure 6] FIG. 6: Verification of in vitro killing of CMV-pp65-TCR-T. [Figure 7] FIG. 7 shows the validation of in vivo killing in a subcutaneous tumor transplant model of CMV-pp65-TCR-T. [Figure 8] FIG. 8 shows validation of in vivo killing in a CMV-pp65-TCR-T metastasis model.

[0019] Specific embodiments The present invention discloses specific T cells targeting the cytomegalovirus (CMV) pp65 antigen epitope NLVPMVATV with HLA-A*0201 restriction and their use, the T cell receptor (TCR) of the T cells specifically targets CMV-pp65 and can accurately and rapidly eliminate the CMV virus. When a TCR gene with high affinity for CMV-pp65 is infected into T cells via lentivirus, CMV-pp65-specific TCR-T cells can be produced. The CMV-pp65-TCR-T cells can directly and rapidly eliminate the CMV virus through precise targeting, and indirectly, can thoroughly eliminate the virus through immune reconstitution mediated by TCR-T cells, prevent reinfection, and exert a long-term protective effect. Therefore, the pp65 antigen epitope-specific pp65-TCR-T cells of the present invention can bind to and kill target cells expressing CMV pp65 antigen specifically in a HLA-A*0201 restricted manner, providing a new clinical protocol for TCR-T treatment of CMV-related diseases, and has great clinical value and future use in the field of treatment and prevention of CMV infection after HSCT or other organ transplantation.

[0020] term MHC molecules are proteins of the immunoglobulin superfamily and can be MHC class I or class II molecules. Therefore, there is specificity in antigen presentation, and different individuals have different MHCs and can present different short peptides of protein antigens on the surface of their APC cells. The human MHC is usually called the HLA gene or HLA complex. The T cell receptor (TCR) is the sole receptor for specific antigenic peptides presented by the major histocompatibility complex (MHC). In the immune system, T cells specific for different antigens exert an immune response against their target cells by direct physical contact between the T cell and the antigen-presenting cell (APC) through the binding of the antigen-specific TCR to the pMHC complex, and by interaction with other cell membrane surface molecules of both the T cell and the APC, which results in a series of subsequent cell signaling events and other physiological responses. TCR is a glycoprotein present on the surface of the cell membrane in the form of an α / β or γ / δ heterodimer. In 95% of T cells, the TCR heterodimer consists of an α and a β chain, while 5% of T cells have a TCR consisting of a γ and a δ chain. Naturally occurring αβ heterodimeric TCRs have α and β chains, which constitute the subunits of the αβ heterodimeric TCR. In a broad sense, the α and β chains each contain a variable region, a joining region, and a constant region, and the β chain usually further contains a short polyvariable region between the variable region and the joining region, but this polyvariable region is usually considered to be part of the joining region. Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, which are fitted into framework regions. The CDR regions determine the binding of the TCR to the pMHC complex, and CDR3 is called the hypervariable region because it is composed of the variable region and the joining region. The TCR α and β chains are generally considered to have two "domains" each, namely a variable domain and a constant domain, with the variable domain being composed of a linked variable region and a linking region. Sequences of TCR constant domains can be found in the International Immunogenetic Information System (IMGT) public database, e.g., the sequence of the constant domain of the TCR molecule α chain is "TRAC*01", and the sequence of the constant domain of the TCR molecule β chain is "TRBC1*01" or "TRBC2*01". The TCR α and β chains further include a transmembrane domain and an intracellular domain. In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention" and "T cell receptor of the present invention" can be used interchangeably.

[0021] Natural and artificial interchain disulfide bonds A pair of disulfide bonds exists between the Cα chain and the Cβ chain in the membrane proximal region of a native TCR, and is referred to as a "native interchain disulfide bond" in the present invention. In the present invention, an artificially introduced interchain conjugated disulfide bond whose position is different from that of the native interchain disulfide bond is referred to as an "artificial interchain disulfide bond."

[0022] Detailed Description of the Invention TCR molecule In the process of antigen processing, antigens are degraded within cells and then delivered to the cell surface via MHC molecules. T cell receptors can recognize peptide-MHC complexes on the surface of antigen-presenting cells. Thus, in a first aspect of the present invention, a TCR molecule capable of binding to the NLVPMVATV-HLA-A*0201 complex is provided. Preferably, the TCR molecule is isolated or purified. Each of the α and β chains of the TCR has three complementarity determining regions (CDRs). In one preferred embodiment of the invention, the alpha chain of the TCR comprises a CDR having the following amino acid sequence: α-CDR1: SSNFYA (SEQ ID NO: 9), α-CDR2: MTLNGDE (SEQ ID NO: 11), α-CDR3: CAFPYNNNDMRF (sequence number 13). In one preferred embodiment of the invention, the β chain of the TCR comprises a CDR having the following amino acid sequence: β-CDR 1: MNHEY (SEQ ID NO: 17), β-CDR 2: SMNVEV (SEQ ID NO: 19), β-CDR 3: CASSLEGYTEAFF (SEQ ID NO: 21). The amino acid sequence of the CDR region of the present invention can be inserted into any suitable framework region to produce a chimeric TCR. If the framework region is compatible with the CDR region of the TCR of the present invention, a person skilled in the art can design or synthesize a TCR molecule having a corresponding function with the CDR region disclosed in the present invention. Therefore, the TCR molecule of the present invention is a TCR molecule comprising the sequence of the CDR region of the above α and / or β chain and any suitable framework region. The TCR alpha chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID NO: 7, and / or the TCR beta chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID NO: 15. In one preferred embodiment of the present invention, the TCR molecule of the present invention is a heterodimer composed of an α chain and a β chain. Specifically, on the one hand, the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the α chain variable domain comprises CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 13) of the α chain. Preferably, the TCR molecule comprises the amino acid sequence of the α chain variable domain SEQ ID NO: 7. More preferably, the amino acid sequence of the variable domain of the α chain of the TCR molecule is SEQ ID NO: 7. On the other hand, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the β chain variable domain comprises CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 19), and CDR3 (SEQ ID NO: 21) of the β chain. Preferably, the TCR molecule comprises the amino acid sequence of the β chain variable domain SEQ ID NO: 15. More preferably, the amino acid sequence of the variable domain of the β chain of the TCR molecule is SEQ ID NO: 15. In one preferred embodiment of the present invention, the TCR molecule of the present invention is a single-chain TCR molecule composed of a part or all of the α chain and / or a part or all of the β chain. For a description of single-chain TCR molecules, see Chung et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. According to the description in the literature, a person skilled in the art can easily construct a single-chain TCR molecule comprising the CDR region of the present invention. Specifically, the single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably linked in the order from the N-terminus to the C-terminus. The amino acid sequence of the α chain variable domain of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 9), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 13) of said α chain. Preferably, said single chain TCR molecule comprises the amino acid sequence of the α chain variable domain SEQ ID NO: 7. More preferably, the amino acid sequence of the variable domain of the α chain of the single chain TCR molecule is SEQ ID NO: 7. The amino acid sequence of the β chain variable domain of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 19) and CDR3 (SEQ ID NO: 21) of said β chain. Preferably, said single chain TCR molecule comprises the amino acid sequence of the β chain variable domain SEQ ID NO: 15. More preferably, the amino acid sequence of the variable domain of the β chain of the single chain TCR molecule is SEQ ID NO: 15. In one preferred embodiment of the present invention, the constant domain of the TCR molecule of the present invention is a human or murine constant domain. The skilled artisan knows the amino acid sequence of the human constant domain or can obtain it by browsing and searching relevant books or the public database of the IMGT (International Immunogenetic Information System). For example, the sequence of the constant domain of the TCR molecule α chain of the present invention may be "TRAC*01", and the sequence of the constant domain of the TCR molecule β chain of the present invention may be "TRBC1*01" or "TRBC2*01". Preferably, the amino acid sequence of the TCR molecule α chain of the present invention is SEQ ID NO: 3, and / or the amino acid sequence of the β chain is SEQ ID NO: 5. The TCRs of the present invention may contain an artificial disulfide bond introduced between residues of the α and β chain constant domains. It should be noted that any TCR of the present invention may contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, whether or not it contains an artificial disulfide bond introduced between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked via a natural disulfide bond present in the TCR. The constant regions of the TCRs of the invention can be engineered to prevent mismatches with endogenous TCR chains by introducing artificial disulfide bonds between residues of the α and β chain constant domains in the TCRs of the invention. The TCR of the present invention may also comprise a chimeric TCR derived from sequences from more than one species. For example, studies have shown that murine TCRs can be expressed more effectively in human T cells than human TCRs. Thus, the TCR of the present invention may comprise a human variable domain and a murine constant domain. The drawback of this approach is that it may induce an immune response. Therefore, when used in adoptive T cell therapy, a regulatory plan for immunosuppression is required to allow the transfer of murine T cells. In this specification, the names of amino acids are represented by one-letter or three-letter alphabets that are universally used around the world, and the correspondence between the one-letter and three-letter alphabets of the amino acid names is as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0023] nucleic acid molecule In a second aspect of the invention there is provided a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a part thereof, which part may be one or more of the CDRs, the variable domains of the α and / or β chains, and the α chain and / or the β chain. The nucleotide sequence encoding the CDR regions of the α chain of the TCR molecule according to the first aspect of the invention is as follows: α CDR1- TCCAGCAATTTCTACGCC (SEQ ID NO: 10) α CDR2- ATGACCCTCAACGGCGATGAA (SEQ ID NO: 12) α CDR3- TGTGCTTTTCCTTATAACAACAACGATATGAGGTTC (SEQ ID NO: 14) The nucleotide sequence encoding the CDR regions of the β chain of the TCR molecule according to the first aspect of the invention is as follows: β CDR1- ATGAACCATGAATAC (SEQ ID NO: 18) β CDR2- TCTATGAATGTGGAGGTG (SEQ ID NO: 20) β CDR3- TGCGCTTCCTCCCTCGAGGGGTACACCGAGGCATTTTTT (SEQ ID NO: 22) Thus, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the alpha chain of the TCR of the present invention includes SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14, and / or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the beta chain of the TCR of the present invention includes SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22. The nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but can encode a polypeptide of the present invention, for example, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the α chain variable domain of the TCR of the present invention comprises SEQ ID NO:8, and / or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the β chain variable domain of the TCR of the present invention comprises SEQ ID NO:16. More preferably, the nucleotide sequence of the nucleic acid molecule of the present invention comprises SEQ ID NO:4 and / or SEQ ID NO:6. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO:2. It should be noted that due to the degeneracy of genetic codons, different nucleotide sequences may code for similar polypeptides. Thus, the nucleic acid sequences encoding the TCRs of the present invention may be similar to the nucleic acid sequences shown in the figures of the present invention, or may be degenerate variants. In one embodiment of the present invention, a "degenerate variant" is a nucleic acid sequence that codes for the protein sequence of SEQ ID NO:1, but which differs from the sequence of SEQ ID NO:2. The nucleotide sequence may be codon optimized. Different cells vary in their specific codon usage, but depending on the cell type, expression can be increased by altering the codons in the sequence. Codon preference tables for mammalian cells and several other organisms are known to those of skill in the art. The full length sequence of the nucleic acid molecule of the present invention or a fragment thereof can usually be obtained by, but is not limited to, PCR amplification, recombinant techniques, or artificial synthesis. Currently, the DNA sequence encoding the TCR of the present invention (or a fragment thereof, or a derivative thereof) can already be obtained completely by chemical synthesis. Furthermore, this DNA sequence can be introduced into various known DNA molecules (or vectors, etc.) and cells well known in the art. The DNA can be the coding strand or the non-coding strand.

[0024] vector The present invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, phages and animal and plant viruses. Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Advantageously, the vector is capable of transferring a nucleotide of the invention into a cell, such as a T cell, to induce expression of an antigen-specific TCR in the cell, and ideally, the vector is capable of sustaining high levels of expression in the T cell.

[0025] cell The present invention also relates to host cells genetically engineered with the vectors or coding sequences of the present invention, said host cells containing the vectors of the present invention or chromosomally integrating the nucleic acid molecules of the present invention, the host cells being selected from prokaryotic and eukaryotic cells, such as E. coli, yeast cells, CHO cells, 293T cells, etc. The invention also includes isolated cells, particularly T cells, expressing a TCR of the invention. The T cells may be derived from T cells isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells may be isolated from peripheral blood mononuclear cells (PBMCs) and may be CD4 + Helper T cells or CD8 + The cells may be cytotoxic T cells. + Helper T cells / CD8 + They may be present in a mixed population of cytotoxic T cells. Alternatively, the cells of the present invention may be derived from stem cells, such as hematopoietic stem cells (HSCs). Since the CD3 molecule is not expressed on the surface of the stem cells, the transfer of genes to the HSCs does not result in the expression of the TCR on the surface of the cells. However, when the stem cells differentiate into lymphoid precursors in the thymus, the expression of the CD3 molecule initiates the expression of the introduced TCR molecule on the surface of the thymocytes. Many methods are suitable for transfecting T cells with DNA or RNA encoding the TCR of the present invention (e.g., Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art are aware of many suitable methods for performing adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0026] Cytomegalovirus (CMV)-related diseases The present invention also relates to a method for treating and / or preventing a CMV-associated disease in a subject, comprising adoptively transferring CMV-specific T cells into the subject, the CMV-specific T cells being capable of primarily recognizing the CMV matrix phosphoprotein pp65, the CMV-specific T cells being capable of recognizing the epitope NLVPMVATV. CMV is a widespread human herpesvirus that infects approximately 50% of normal individuals. In many cases, the immune response is able to suppress acute infection by recognizing CMV-derived antigens. The virus then persists in a latent state throughout the host's life. Outward replication is prevented by immune system effector factor mechanisms, including neutralizing antibodies against viral membrane proteins, HLA-restricted CMV-specific helper and cytotoxic T cells, and MHC-restricted effector factors. CMV infection is a major concern for some high-risk populations. The main areas of risk for infection include fetuses or newborns, and immunocompromised individuals, such as organ transplant recipients, leukemia patients, or those infected with the human immunodeficiency virus (HIV). In general, there are three clinical manifestations of CMV infection, including: (1) Neonatal CMV inclusion disease, which can range from asymptomatic to severe and disabling, affecting the liver, spleen, and central nervous system; (2) acute acquired CMV infection, which resembles infectious mononucleosis and presents with symptoms such as fever, malaise, and skeletal muscle pain; (3) CMV infection in immunocompromised individuals (e.g., organ transplant recipients or individuals with HIV) who are at risk for CMV retinitis, CMV pneumonia, CMV gastroenteritis, and CMV encephalitis. The TCRs of the present invention may be used to treat and / or prevent reactivation of latent CMV following allogeneic hematopoietic stem cells. CMV disease in allo-HSCT recipients is believed to be primarily due to reactivation of latent virus. Viral spread can occur via donor bone marrow transfusions or allogeneic blood products. In immunocompromised bone marrow transplant recipients, viral reactivation usually leads to progressive CMV infection, which contributes to infectious morbidity and mortality in these patients. Progressive CMV infection is a consequence of both post-transplant immunosuppression and slow immune recovery in these patients. In the methods of the invention, for example, Allo-HSCT recipients are adoptively immunotherapyed with T cells expressing a CMV-specific T cell receptor of the invention.

[0027] Prevention and Treatment Methods The term "prevention" refers to avoiding, slowing, resisting or inhibiting the progression of a disease. For example, the likelihood of CMV infection and / or CMV reactivation can be prevented or reduced. As used herein, "treatment" refers to alleviating, curing or reducing the symptoms of a disease or reducing or preventing the progression of a disease. The relevant disease can be prevented or treated by isolating T cells from a patient or volunteer suffering from the disease and introducing a TCR of the invention into the T cells, and then delivering these genetically modified cells into the patient. Thus, the present invention provides a method of treating a CMV-associated disease comprising delivering isolated T cells expressing a TCR of the present invention, preferably autologous T cells, into a patient, which generally comprises: (1) Isolate the patient's T cells; (2) transducing T cells ex vivo with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention; (3) Delivering genetically modified T cells into the patient’s body. The number of isolations, transfections and forwardings may be determined by a physician.

[0028] The main advantages of the present invention are: (1) The TCR of the present invention can rapidly eliminate the CMV virus through direct and precise targeting. (2) The TCR of the present invention has a high protein expression level in the body and does not mismatch with endogenous TCR chains. (3) T cells expressing the TCR of the present invention have strong killing ability and high specificity, and therefore can be effectively used to treat CMV infection in hematopoietic stem cell transplant (HSCT) patients or other transplant patients. The present invention will be further described by the following specific examples. It is understood that these examples are only used to explain the present invention and do not limit the scope of the present invention. Experimental methods for which specific conditions are not shown in the following examples are usually performed under standard conditions, such as those described in Sambrook and Russell et al., Molecular Cloning-A Laboratory Manual (3rd Edition) (2001), or in accordance with the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise specified, percentages and parts are calculated by weight. All experimental materials and reagents used in the following examples are commercially available unless otherwise specified.

[0029] Example 1 Induction of differentiation of monocytes into dendritic cells (DC cells) in vitro (1) PBMCs (or regenerated cryopreserved PBMCs) from healthy donors / patients' blood were obtained by density gradient centrifugation with lymphocyte separation medium (Lymphoprep), resuspended in cytotoxic T lymphocyte medium, centrifuged at 350 g for 5 min to remove the supernatant, and resuspended in a 6-well plate with an appropriate amount of DC cell medium. After culturing in an incubator for 2 h, the medium and clumped non-adherent cells were aspirated and discarded. (2) 3 ml of DC medium containing DC Differentiation Supplement (GM-CSF and IL-4) at a final concentration of 1x was gently poured along the sidewall of each well, and the 6-well plate was cultured in a 37°C, 5% CO2 incubator for 5 d. (3) On day 5, without changing the medium, 30 μl of 100×DC Maturation Supplement was added to each well (final concentration of DC Maturation Supplement was 1×). The 6-well plate was induced to mature for 2 d in a 37° C., 5% CO2 incubator. (4) On day 7, mature DC cells were harvested by mixing vigorously with a pipette and resuspending the cells at the bottom of the well plate. The mature DC cells were transferred to a 15 mL centrifuge tube and centrifuged at 350 g for 5 min. The supernatant was removed, and the density of the mature DC cells was 2 × 10 6 DCs were resuspended in DC medium to give cells / ml and used in the subsequent experiments.

[0030] Example 2 In vitro stimulation and expansion of human CMV-pp65 antigen-specific T cells (1) 5 μg / ml of CMV-PP65 peptide of interest was added to the mature DC cells. After sealing with a Parafilm, the cells were placed in a rotator (MACSmix) and rotated in an incubator for 4 h. After incubation of mDC with CMV-PP65 peptide of interest was completed, the CMV-PP65 peptide-loaded DC cells were irradiated with an irradiation dose of 40 Gy using an irradiation device. After irradiation, the CMV-PP65 peptide-loaded DC cells were collected in a 15 ml centrifuge tube by centrifugation. (2) PBMCs were obtained from healthy donor blood by density gradient centrifugation using lymphocyte separation fluid (Lymphoprep) and then isolated using a kit (EasySep TM CD8+ T cells were isolated from PBMCs using a Human CD8 Positive Selection Kit, and CMV-PP65 peptide-loaded DCs and CD8+ T cells were cultured in a 12-well plate at a DC / T ratio of 1:2.5. The culture system was supplemented with IL-21 at a final concentration of 30 ng / ml and CMV-PP65 peptide at a final concentration of 5 μg / ml. (3) The 12-well plate was incubated overnight in an incubator at 37°C and 5% CO2. On the first day, 10 ng / ml of IL-2, IL-7, and IL-15 were added to each well, and the plates were further cultured in an incubator at 37°C and 5% CO2 for 10 days. Half the volume of the liquid was replaced every 2-3 days during the culture process. Then, on the 5th and 10th days, the CMV-PP65 peptide was added to each culture system at a final concentration of 5 μg / ml. (4) On the 11th day, referring to the above steps, irradiated CMV-PP65 target peptide-loaded DC cells (irradiated CMV-peptide-loaded DCs) were prepared and subjected to a second in vitro stimulation with the cells in each culture system, and the stimulation was continued for 7 days. (5) On the 18th day, 500 μl of cells were aspirated from each well of the well plate and washed twice with buffer (FACS buffer). Then, CMV-PP65-HLA:A:0201 Dextramer-FITC and CD8a-PE were added and mixed uniformly. The cells were then incubated at 4°C for 20 min away from light. After the incubation was completed, the cells were washed three times with FACS buffer and resuspended in 400 μl of a buffer containing 1×DAPI working solution (FACS buffer), and the CD8+ / Dextramer+ (%) ratio was detected.

[0031] Example 3 Obtaining HLA:A:0201-restricted CMV-pp65 antigen-specific TCR by single cell sequencing (1) CD8+ / Dextramer+ T cells were sorted by flow cytometry, and the sequences of TCRα and TCRβ chains of CD8+ / Dextramer+ T cells were obtained using 10X Genomics single-cell sequencing technology. (2) After obtaining the TCR sequence by single-cell sequencing, the sequence of the most frequently occurring TCRα / β chain was optimized, and its constant region was modified so that the exogenous TCR would not mismatch with the endogenous TCR of the T cell. Codon optimization was performed on the expression sequence to improve the protein expression level. TCRα and TCRβ were simultaneously expressed in one expression vector, and enzymatic cleavage was performed with P2A and Furin protease to prevent the generation of an extra proline tail (Furin-cleavage). (3) A lentiviral expression vector was constructed, and the above-mentioned TCRα and TCRβ sequences were inserted into the same lentiviral expression vector. The virus was packaged in 293T cells to produce specific viral particles of the CMV-pp65 antigen-specific TCR.

[0032] Example 4 Generation of CMV-pp65-TCR-T cells by T cell activation following infection with CMV-pp65-TCR lentivirus (1) Kit (EasySep TM CD8+ T cells were isolated from PBMCs using a Human CD8 Positive Selection Kit (Human CD8 Positive Selection Kit), and activated in vitro with CD3 / 28 immunobeads (Dynabeads) at a ratio of 1:3 CD8+ T cells:CD3 / 28 immunobeads (Dynabeads). CD8+ T cells were stimulated and activated at 37°C for 2 d. (2) Activated CD8+ T cells were infected with CMV-pp65-TCR lentivirus particles, and 8 μg / ml polybrene was added, followed by centrifugation at 500×g, 90 min, and 30° C. After centrifugation, the cells were cultured in an incubator at 37° C. and 5% CO2 for 3 days, and then a second round of infection with CMV-pp65-TCR lentivirus was performed. (3) Three days after the second infection with CMV-pp65-TCR lentivirus, CMV-pp65-TCR-T cells were aspirated and washed twice with buffer (FACS buffer), then stained with CMV-PP65-HLA*A:0201 Dextramer-FITC and CD8a-PE antibodies, and the ratio of CD8+ / Dextramer+ (%) was detected by FACS. CD8a+ / Dextramer+ T cells were further separated by flow cytometry for subsequent in vitro killing experiments.

[0033] Example 5 HLA:A*0201 restricted CMV-pp65-TCR-T in vitro activation experiment A. Detection of the expression levels of CD137, IFNγ, and TNF-α in TCR-T cells by FACS (1) K562 cells were infected with lentivirus to prepare K562-A:0201-GFP, K562-A:0201-GFP-mCherry-vector, and K562-A:0201-GFP-pp65-mCherry cells, respectively. (2) The CMV-pp65-TCR-T / Reported-TCR-T cells prepared in Example 4 were inoculated into round-bottom 96-well plates and cultured with TCR-T cells and K562 cells at an effector-target ratio of 1:1. (3) After 24 h of co-culture, extracellular staining was performed, followed by fixation to disrupt the membrane, and intracellular staining was performed to detect the expression levels of CD137, TNFα, and IFNγ in CD8+ T cells in the co-culture system by FACS to evaluate whether K562-A:0201-GFP-pp65-mCherry target cells could specifically activate CMV-pp65-TCR-T cells in vitro.

[0034] B. Elispot detection of IFNγ release levels from TCR-T cells (1) Coating antibody (Mabtech IFNγ, 1-D1K: 1 mg / ml) was diluted to 15 μg / ml (1.5 μg / 100 μl) with sterile calcium- and magnesium-ion-free 1xDPBS (pH 7.4). 100 μl of the diluted 1-D1K antibody solution was added to each well and incubated overnight at 4-8°C. (2) After removing the antibody from the plate and washing with sterile DPBS, cells were plated at an effector-target ratio of 1:1, and each well was filled with 100 μl of 1640 complete medium, 3 × 10 4 CMV-pp65-TCR-T cells or K562 cells were added, the edge of the plate was gently tapped to uniformly distribute the cells, and the plate was placed in an incubator and cultured at 37°C and 5% CO2 for 20 hours. (3) After 20 h, the cells were removed and washed 4-6 times with DPBS to ensure complete cell removal. 100 μl of biotin-labeled detection antibody 7-B6-1-biotin was added to each well and incubated at 37°C for 2 h. After incubation, the plate was washed 5 times with DPBS, discarding the liquid the last time, and then tapped lightly on sterile paper to dry. Then, 200 μl of Streptavidin-HRP was added to each well and incubated at 37°C for 1 h. (4) After incubation, the plate was washed five times with DPBS, and 100 μl of the substrate TMB was added to each well and reacted until clear spots appeared. Finally, deionized water was added to stop the color reaction. The plate was scanned by an Elispots-reader device and statistical analysis was performed.

[0035] Example 6 Specific killing of pp65-HLA*A0201-K562 cells by HLA:A*0201-restricted CMV-pp65-TCR-T in vitro (1) K562 cells were infected with lentivirus to prepare K562-A:0201-GFP control cells and K562-A:0201-GFP-pp65-mCherry target cells, respectively. (2) The CMV-pp65-TCR-T / Reported-TCR-T cells prepared in Example 4 were seeded and cultured in round-bottom 96-well plates with T cells and K562 cells at effector-target ratios of 1:1 and 5:1, respectively. (3) After 24 h of co-culture, we evaluated whether CMV-pp65-TCR-T and Reported-TCR-T specifically killed K562-A:0201-GFP-pp65-mCherry cells by detecting the ratio of K562-A:0201-GFP or K562-A:0201-GFP-pp65-mCherry in the co-culture system by FACS, and compared the killing efficiency of the two TCR-Ts.

[0036] Example 7 Specific killing of pp65-HLA*A0201-K562 cells by HLA:A*0201-restricted CMV-pp65-TCR-T in vivo (1) Infect K562 cells with lentivirus and infect K562-A:0201-GFP-pp65-luciferase target cells with lentivirus to induce CD8 + T cells were infected to prepare CMV-pp65-TCR-T effector cells. (2) Mouse subcutaneous tumor transplantation model: On day 0, 1 × 10 6 On day 3, 5 × 10 target cells were injected subcutaneously into the tail of each mouse. 6 The first CD8 + T cells (CD8 + T day 3 group) and CMV-pp65-TCR-T cells (pp65-TCR-T day 3 group) were intravenously injected, and on day 7, 5 × 10 6 Mice were intravenously injected with CMV-pp65-TCR-T cells (pp65-TCR-T day 7 group) at d0 (+4h), d7, d14, d21, d28, and d35 after tumor cell injection. Bioluminescence imaging was performed on the mice to detect the expression of luciferase in tumor cells, and the photon intensity of the luciferase was quantified to evaluate whether CMV-pp65-TCR-T inhibited the growth of K562-A:0201-GFP-pp65-luciferase target cells. (3) Mouse tail vein metastasis model: On day 0, 1 × 10 6 On day 3, 1 × 10 target cells were injected intravenously into the tail of each mouse. 7 The first CD8 + T cells and CMV-pp65-TCR-T cells were intravenously injected. Bioluminescence imaging was performed on the mice on d0(+4h), d28, d35, d42, d49, and d56 after tumor cell injection to detect the expression of luciferase in tumor cells and quantify the photon intensity of the luciferase to evaluate the inhibitory effect of CMV-pp65-TCR-T on target cells K562-A:0201-GFP-pp65-luciferase.

[0037] Test Results: CMV-pp65-TCR-T screening procedure: FIG. 1 shows the flow of specific stimulation and screening of CMV-pp65-TCR-T of the present invention. The entire test consisted of CD8+ T cells from healthy HLA-A:0201 donors being stimulated twice with autologous mDCs loaded with pp65 polypeptide, and then the specificity of CMV-pp65 antigen-specific TCR-T cells was analyzed with CMV-PP65-HLA*A:0201 Dextramer. Flow cytometry results (Figure 2) showed that the CD8a + / Dextramer + It was shown that the ratio of T increased from 0.16% to 81.3%, indicating that CMV-pp65-specific TCR-T cells were specifically expanded in vitro.

[0038] Obtaining CMV-pp65-TCR sequence by single cell sequencing: After loading pp65 polypeptide onto autologous mDCs and stimulating them twice, CMV-pp65 antigen-specific TCR-T cells were sorted by flow cytometry using CMV-PP65-HLA*A:0201 Dextramer and CD8a antibody, and the resulting CMV-pp65-TCR-T cells were subjected to single-cell sequencing using the 10×Genomics method to obtain the α and β chain sequences of CMV-pp65-TCR-T cells. After single-cell sequencing, the top 10 frequencies obtained were ranked. + / Dextramer + It was shown that 99.64% of the T cell clones in T cells were composed of one TCR-T, proving the good specificity of this experiment (antigen-specific T cell priming). The T cell clone contained two α chains, one of which was an extra α chain, which was subsequently eliminated by the CMV-PP65-HLA*A:0201 Dextramer in vitro binding experiment. The final TCR sequence is as follows: CMV-pp65-TCR: MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLEGYTEAFFGQGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGV CTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRAKRGSGATNFSLLKQAGDVEENPGPMEKNPLAAPLLILWFHLDC VSSILNVEQSPQSLHVQEGDSTNFTCFSSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFPYNNNDMRFGAGTRLTVEPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDK C VLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 1), in the sequence C is the cysteine ​​residue introduced by artificial mutation. The codon-optimized TCR coding sequence is as follows:

[0039] Among them, The alpha chain and its coding sequence are as follows: MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFSSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFPYNNNDMRGAGTRLTVEPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 3) ATGGAAAAGAACCCTCTGGCTGCCCCCCTGTTGATACTGTGGTTCCACCTGGACTGCGTGAGCAGCATTCTGAACGTCGAACAGTCCCCTCAGTCTCTGCACGTCCAAGAGGGAGATTCCACCAATTTTACTTGTAGTTTTAGCTCCAGCAATTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGTCTCCCGAAGCCCTCTTTGTGATGACCCTCAACGGCGATGAAAAAAAAAAAGGGAGAATTAGCGCCACCCTGAACACTAAAGAGGGCTATTCTTACTTGTATATAAAAGGGTCTCAGCCTGAGGACAGCGCTACATACTTGTGTGCTTTTCCTTATAACAACAACGATATGAGGTTCGGGGCCGGAACAAGGTTGACCGTTGAGCCTGACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAATGCGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACCGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCGGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGT(SEQ ID NO: 4) The β-chain and its coding sequence are as follows. MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLEGYTEAFFGQGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 5) ATGGGGCCCCAGCTTTTGGGTTATGTAGTTCTTTGTCTCCTGGGCGCTGGACCGCTGGAAGCACAAGTCACTCAAAACCCTAGGTACCTGATCACCGTGACCGGAAAGAAGCTGACTGTGACCTGCTCTCAGAACATGAACCATGAATACATGTCATGGTACAGGCAGGATCCTGGGCTCGGACTTAGACAGATTTACTACTCTATGAATGTGGAGGTGACAGATAAAGGGGATGTGCCAGAAGGCTATAAGGTCTCCAGGAAGGAGAAGAGAAACTTTCCCTTGATCTTGGAGTCTCCGTCCCCAAACCAGACCTCTCTGTACTTCTGCGCTTCCTCCCTCGAGGGGTACACCGAGGCATTTTTTGGACAGGGGACTAGACTGACCGTGGTGGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCTGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCA(SEQ ID NO: 6)

[0040] The variable region of the α chain and its coding sequence are as follows. MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFSSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFPYNNNDMRFGAGTRLTVEP (SEQ ID NO: 7) ATGGAAAAGAACCCTCTGGCTGCCCCCCTGTTGATACTGTGGTTCCACCTGGACTGCGTGAGCAGCATTCTGAACGTCGAACAGTCCCCTCAGTCTCTGCACGTCCAAGAGGGAGATTCCACCAATTTTACTTGTAGTTTTAGCTCCAGCAATTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGTCTCCCGAAGC CCTCTTTGTGATGACCCTCAACGGCGATGAAAAAAAAAAAAGGGAGAATTAGCGCCACCCTGAACACTAAAGAGGGCTATTCTTACTTGTATATAAAAGGGTCTCAGCCTGAGGACAGCGCTACATACTTGTGTGCTTTTCCTTAACAACAACGATATGAGGTTCGGGGCCGGAACAAGGTTGACCGTTGAGCCT (SEQ ID NO: 8) The three CDR sequences contained therein are as follows: α-CDR1: SSNFYA (SEQ ID NO: 9), coding sequence: TCCAGCAATTTTCTACGCC (SEQ ID NO: 10); α-CDR2: MTLNGDE (SEQ ID NO: 11), coding sequence: ATGACCCTCAACGGCGATGAA (SEQ ID NO: 12); α-CDR3: CAFPYNNNDMRF (SEQ ID NO: 13), coding sequence: TGTGCTTTTCCTTATAACAACAACGATATGAGGTTC (SEQ ID NO: 14)

[0041] The beta chain variable region and its coding sequence are as follows: MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLEGYTEAFFGQGTRLTVV (SEQ ID NO: 15) ATGGGGCCCCAGCTTTTGGGTTATGTAGTTCTTTGTCTCCTGGGCGCTGGACCGCTGGAAGCACAAGTCACTCAAAACCCTAGGTACCTGATCACCGTGACCGGAAAGAAGCTGACTGTGACCTGCTCTCAGAACATGAACCATGAATACATGTCATGGTACAGGCAGGATCCTGGGCTCGGACTTAGACAGATTTACTA CTCTATGAATGTGGAGGTGACAGATAAAGGGGATGTGCCAGAAGGCTATAAGGTCTCCAGGAAGGAGAAGAGAAACTTTCCCTTGATCTTGGAGTCTCCGTCCCCAAACCAGACCTCTCTGTACTTCTGCGCTTCCTCCCTCGAGGGGTACACCGAGGCATTTTTTGGACAGGGGACTAGACTGACCGTGGTG (SEQ ID NO: 16)

[0042] The three CDR sequences contained therein are as follows: β-CDR 1: MNHEY (SEQ ID NO: 17), coding sequence: ATGAACCATGAATAC (SEQ ID NO: 18); β-CDR 2: SMNVEV (SEQ ID NO: 19), coding sequence: TCTATGAATGTGGAGGTG (SEQ ID NO: 20); β-CDR 3: CASSLEGYTEAFF (SEQ ID NO: 21), coding sequence: TGCGCTTCCTCCCTCGAGGGGTACACCGAGGCATTTTTT (SEQ ID NO: 22).

[0043] Construction and sequence optimization of CMV-pp65-TCR-T vector: After obtaining the sequence of pp65-TCR (SEQ ID NO: 1) by sequencing, the sequence is optimized, specifically including: (1) Their constant regions were modified to prevent mismatches with endogenous TCR chains; (2) Human-derived codon optimization was performed on the expression sequence to improve the expression level of TCR protein; (3) The α- and β-chains were expressed simultaneously on one plasmid by P2A and furin cleavage, avoiding the terminal proline residues that would result from the use of P2A alone. The optimized pp65-TCR sequence (Figure 3) was then cloned into a lentiviral expression vector.

[0044] CMV-pp65-TCR-T in vitro binding experiment: We packaged TCR lentivirus in 293T cells, infected healthy HLA-A:0201 donor PBMCs or jurkat T cells twice with CMV-pp65-TCR lentivirus, and then stained CMV-pp65-HLA*A:0201 Dextramer. Flow cytometry revealed that Tetramer + / mTCRβC + JurkatT (Figure 4A) and Dextramer + / First CD8 + The results showed that the CMV-pp65-TCR-T cells screened and identified in this project were distinct from the jurkatT and primary CD8 T cells from healthy donors. + It was shown to be normally expressed in T cells and capable of binding CMV-PP65-HLA*A:0201 Tetramer / Dextramer.

[0045] CMV-pp65-TCR-T in vitro activation experiment: Control cells K562-A:0201-GFP, vector control cells K562-A:0201-GFP-mCherry-vector, and target cells K562-A:0201-GFP-pp65-mCherry were prepared in vitro, and CMV-pp65-TCR-T effector cells were co-cultured with target cells or controls (vector control and blank control) in vitro. After intracellular and extracellular staining, the expression levels of CD137 and IFNγ in CMV-pp65-TCR-T cells were detected by flow cytometry. The results of flow cytometry experiments (Figure 5) showed that K562-A:0201-GFP-pp65-mCherry target cells specifically activated CMV-pp65-TCR-T cells in vitro, and after activation, the expression levels of CD137 and IFNγ of CMV-pp65-TCR-T were significantly enhanced, while the vector control K562 cells and control K562 cells had no obvious activation effect on CMV-pp65-TCR-T cells. Similarly, the results of IFNγ-Elispot experiments showed that K562-A:0201-GFP-pp65-mCherry target cells specifically activated CMV-pp65-TCR-T cells in vitro, and after activation, the IFNγ expression level of CMV-pp65-TCR-T was significantly increased, whereas the control cells K562-A:0201-GFP had no activating effect on CMV-pp65-TCR-T.

[0046] CMV-pp65-TCR-T in vitro killing experiment: The target cells K562-A:0201-GFP-pp65-mCherry, the Reported TCR-T published in CN102656188A, and the CMV-pp65-TCR-T of this project were prepared in vitro. CMV-pp65-TCR-T or Reported TCR-T effector cells and target cells were co-cultured in vitro for 24 h at effector-target ratios of 1:1 and 5:1, and then the ratio of GFP+ target cells was detected by flow cytometry. The flow cytometry experiment results (Figure 6A and B) showed that both CMV-pp65-TCR-T and Reported TCR-T cells killed K562-A:0201-GFP-pp65-mCherry cells expressing pp65 in vitro, but the CMV-pp65-TCR-T published in this project had a better killing effect than Reported TCR-T. Then, CMV-pp65-TCR-T and Reported TCR-T effector cells were co-cultured with target cells in vitro at an effector-target ratio of 1:1, and after 24 h, the cytokine release status of CMV-pp65-TCR-T and Reported TCR-T was evaluated by intracellular staining and flow cytometry to detect the expression of cytokine TNF-α. The results of the flow cytometry experiment showed that the CMV-pp65-TCR-T cells screened and identified in this project released higher levels of TNF-α in vitro than Reported TCR-T and specifically killed K562-A:0201-GFP-pp65-mCherry cells expressing pp65 (Figure 6C), but there was no cytokine release of TNF-α and IFN-γ after co-culture of CMV-pp65-TCR-T with K562-A0201 control cells, indicating its good specificity.

[0047] In vivo killing experiment of CMV-pp65-TCR-T in a subcutaneous tumor transplant model K562-A:0201-GFP-pp65-luciferase target cells and CMV-pp65-TCR-T effector cells were prepared in vitro, and 1 × 10 6 On day 3, 5 × 10 target cells were injected subcutaneously into the tail of each mouse. 6 The first CD8 + T cells (CD8 +T day 3 group) and CMV-pp65-TCR-T cells (pp65-TCR-T day 3 group) were intravenously injected, and on day 7, 5 × 10 6 CMV-pp65-TCR-T cells (pp65-TCR-T day7 group) were intravenously injected into the mice (Figure 7A). Bioluminescence imaging detection was performed on the mice 0-5 weeks after tumor cell injection (Figure 7B), and the luciferase photon intensity of the mice was quantified. The results of the in vivo experiment showed that the CMV-pp65-TCR-T cells of this project could specifically kill K562-A:0201-GFP-pp65-luciferase target cells in vivo (Figure 7B, C). Injection of CMV-pp65-TCR-T on day 3 or day 7 could significantly inhibit the growth of K562-A:0201-GFP-pp65-luciferase tumor cells and significantly extend the survival time of the mice (Figure 7D). The killing effect on target cells was significantly higher than that of CD8 cells that were not transduced with pp65-TCR. + This was shown to be better than the T control cells.

[0048] In vivo killing experiments in a CMV-pp65-TCR-T metastatic model: K562-A:0201-GFP-pp65-luciferase target cells and CMV-pp65-TCR-T effector cells were prepared in vitro. On day 0, 1 × 10 6 On day 3, 5 × 10 target cells were injected intravenously into the tail of each mouse. 7 The first CD8 + T cells and CMV-pp65-TCR-T cells were intravenously injected (Figure 8A). Bioluminescence imaging detection was performed on the mice 4-8 weeks after tumor cell injection (Figure 8B), and the luciferase photon intensity of the mice was quantified. The results of in vivo experiments showed that the CMV-pp65-TCR-T cells of this project could specifically kill K562-A:0201-GFP-pp65-luciferase target cells in the body (Figure 8, B and C), and injection of CMV-pp65-TCR-T on day 3 significantly inhibited the growth of K562-A:0201-GFP-pp65-luciferase tumor cells, and the inhibitory effect was significantly better than that of the CD8+T control cells that were not transduced with pp65-TCR.

[0049] Technical Effects CMV has a very wide range of human infections, with an infection rate of more than 95% among adults in China. It is usually asymptomatic, and many infected people have no clinical symptoms, but under certain conditions, it can affect multiple organs and systems and lead to severe disease. CMV activation is common in hematopoietic stem cell transplant (HSCT) patients, and some patients have poor drug treatment effects or low drug resistance and cannot complete the drug treatment course. Patients have a weakened immune function and cannot effectively eliminate CMV, which may lead to a series of related diseases ranging from CMV-related fever to organs, with a high mortality rate. Existing anti-CMV drugs also have many shortcomings, such as monotonous dosage forms, similar targets, and granulocytopenia / kidney damage. Therefore, a new clinical treatment protocol for the treatment of CMV infection in HSCT patients is urgently needed. The CMV-TCR-T cells of this project can precisely target and eliminate target cells infected with the CMV virus, and at the same time, indirectly eliminate the virus through immune reconstruction mediated by TCR-T cells, prevent reinfection, and exert a long-term protective effect. The CMV-pp65-TCR-T invention of this project has the following features: In vitro and in vivo experiments showed that CMV-pp65-TCR-T can accurately target and rapidly eliminate target cells expressing CMV antigens, has high protein expression in the body, no mismatch with endogenous TCR chains, strong killing ability, high specificity, etc. In vitro experiments showed that the CMV-pp65-TCR-T of this project has a more pronounced killing effect on target cells than the Reported-TCR-T published in CN102656188A, and can be effectively used to treat CMV infection in HSCT patients or other transplant patients. In addition, compared with the existing related disclosed inventions, this project provides the evidence of ELISPOT (enzyme-linked immunospot) in vitro experiment, which is the gold standard recognized in the TCR-T field, and shows that the TCR obtained by screening in this project can specifically recognize CMV antigen. At the same time, in addition to demonstrating that CMV-specific TCR-equipped T cells can specifically eliminate target cells expressing CMV antigen in the tail vein model, the invention of this project also demonstrates that CMV-specific TCR-equipped T cells obtained by screening in this project can specifically eliminate target cells expressing CMV antigen in a subcutaneous tumor transplant model, which is closer to the clinical situation of CMV infection. In other words, the invention of this project is complete, meets the standards of the field, and provides in vivo and in vivo data that are closer to the clinical CMV infection situation, and the specificity and efficacy of the CMV antigen-specific TCR obtained by screening have been fully demonstrated.

[0050] All documents related to the present invention are incorporated herein by reference as if each document were incorporated individually. After reading the above content of the present invention, it will be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalent forms thereof are within the scope of the claims of the present invention.

Claims

1. a T cell receptor (TCR), comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, the amino acid sequence of the CDR3 of the TCR alpha chain variable domain being CAFPYNNNDMRF (SEQ ID NO: 13); and / or A TCR characterized in that the amino acid sequence of CDR3 of the TCR β chain variable domain is CASSLEGYTEAFF (sequence number 21).

2. The three complementarity determining regions (CDRs) of the TCRα variable domain are: α-CDR1: SSNFYA (SEQ ID NO: 9), α-CDR2: MTLNGDE (SEQ ID NO: 11), α-CDR3: CAFPYNNNDMRF (SEQ ID NO: 13); and / or The three complementarity determining regions of the TCR β chain variable domain are: β-CDR1: MNHEY (SEQ ID NO: 17), β-CDR2: SMNVEV (SEQ ID NO: 19), β-CDR3: CASSLEGYTEAFF (SEQ ID NO:21) The T cell receptor (TCR) of claim 1.

3. The TCR described in claim 1, characterized in that it comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the TCR alpha chain variable domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7, and / or the TCR beta chain variable domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

15.

4. The TCR of claim 1, characterized in that the amino acid sequence of the TCR is set forth in SEQ ID NO:

1.

5. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR according to any of the preceding claims.

6. A nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of claim 1 or its complementary sequence.

7. The nucleic acid molecule comprises a nucleotide sequence encoding a TCR alpha chain variable domain, SEQ ID NO:8; and / or The nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 16, which encodes the TCR β chain variable domain. The nucleic acid molecule according to claim 6.

8. A vector comprising the nucleic acid molecule according to claim 6, preferably a viral vector, more preferably a lentiviral vector.

9. 10. An isolated host cell, characterized in that it contains a vector according to claim 8 or has an exogenous nucleic acid molecule according to claim 6 integrated into its chromosome.

10. A cell transduced with a nucleic acid molecule according to claim 6 or a vector according to claim 8, preferably a T cell or a stem cell.

11. A pharmaceutical composition comprising a pharma- ceutical acceptable vehicle and a TCR according to any one of claims 1-4, a TCR complex according to claim 5, a nucleic acid molecule according to claim 6, or a cell according to claim 10.

12. Use of a T cell receptor according to any one of claims 1 to 4, or a TCR complex according to claim 5, or a cell according to claim 10, for the manufacture of a drug for treating a cytomegalovirus infection.

13. A method for treating a disease, comprising administering to a subject in need of treatment a TCR according to any one of claims 1-4, a TCR complex according to claim 5, a nucleic acid molecule according to claim 6, or a cell according to claim 10 or a pharmaceutical composition according to claim 11.

14. The method according to claim 13, characterized in that the disease is a disease associated with cytomegalovirus infection (cytomegalovirus infection), such as CMV retinitis, CMV pneumonia, CMV gastroenteritis and CMV encephalitis.

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