T cell receptor and application thereof

Abstract

Relates to the field of biological medicines, and particularly provides a T cell receptor (TCR) and application thereof. The TCR can be used for transforming cells so as to improve or promote the multiplication capacity, killing capacity and / or directional differentiation capacity of the cells expressing the TCR.

Description

T cell receptor and use thereof Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a T cell receptor and uses thereof. Background Art

[0002] Natural killer cells (NKT) are a special type of T lymphocyte subset that has the dual properties of T cells and NK cells. NKT cells can express two receptors, TCR of T cells and NKR-P1 of NK cells. Under the mediation of TCR and NKR, NKT cells can produce a large number of cytokines. Among them, the T cell receptor (TCR) mediates the recognition of peptide epitopes bound to major histocompatibility complex (MHC) molecules to exert adaptive cellular immunity. Normally, the half-life of infused NKT cells in the patient's body is about 2 weeks, the effective period is short, and repeated infusions are required. In addition, NKT cells themselves lack specific antibodies and are not enough to be enriched around tumors or in tumor nests, which restricts the targeted effect of NKT cell treatment. On the other hand, NKTs derived from peripheral blood are relatively rare, accounting for only 0.1-0.5%, and are not easy to use for cell therapy.

[0003] Therefore, this field is in urgent need of a class of TCR molecules that, after being transduced into stem cells (including HSC, iPSC, ESC), can enable the stem cells to better differentiate in the NKT direction, thereby obtaining a large number of NKT cells that can be used for cell therapy.

[0004] Summary of the Invention

[0005] The present invention provides a T cell receptor (TCR) and its use, which can have one or more of the following advantages: the ability to be expressed in cells, the ability to increase the proliferation of cells expressing the TCR, the ability to increase the killing ability of cells expressing the TCR, and / or the ability to promote the directed differentiation of cells expressing the TCR.

[0006] In one aspect, the present invention provides a T cell receptor (TCR) or an antigen-binding fragment thereof, wherein the TCR comprises a β chain variable region (Vβ), the Vβ comprises a CDR3, and the CDR3 of the Vβ comprises the CDR3 sequence of the Vβ provided by the present invention.

[0007] On the other hand, the present invention provides a fusion protein comprising the TCR or antigen-binding fragment thereof according to the present invention.

[0008] In another aspect, the present invention provides a nucleic acid molecule encoding the TCR or antigen-binding fragment thereof and / or the fusion protein of the present invention.

[0009] In another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention.

[0010] On the other hand, the present invention provides a composition comprising the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, and / or the vector described in the present invention, and optionally a pharmaceutically acceptable adjuvant.

[0011] On the other hand, the present invention provides a cell comprising the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the vector described in the present invention and / or the composition described in the present invention.

[0012] On the other hand, the present invention provides a kit comprising the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the vector described in the present invention, the composition described in the present invention and / or the cell described in the present invention.

[0013] In another aspect, the present invention provides a method for influencing cell growth, comprising administering the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the vector described in the present invention, the composition described in the present invention, the cell described in the present invention and / or the kit described in the present invention.

[0014] On the other hand, the present invention provides the use of the TCR or antigen-binding fragment thereof, the fusion protein, the nucleic acid molecule, the vector, the composition, the cell and / or the kit of the present invention in the preparation of a medicament for preventing and / or treating a disease and / or symptom.

[0015] On the other hand, the present invention provides the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the vector described in the present invention, the composition described in the present invention, the cell described in the present invention and / or the kit described in the present invention, which are used to prevent and / or treat diseases and / or symptoms.

[0016] On the other hand, the present invention provides a method for preventing and / or treating a disease and / or symptom, comprising administering the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the vector described in the present invention, the composition described in the present invention, the cell described in the present invention and / or the kit described in the present invention.

[0017] Those skilled in the art will readily appreciate other aspects and advantages of the present invention from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present invention. As will be appreciated by those skilled in the art, the disclosure of the present invention enables those skilled in the art to modify the disclosed embodiments without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the descriptions in the drawings and specification of the present invention are intended to be exemplary only and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The features and advantages of the present invention can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. The accompanying drawings are briefly described as follows:

[0019] FIG1 shows the results of sorting CD45-positive TCRVα24-positive cell populations.

[0020] Figure 2 shows the single-cell transcriptome sequencing results of cells corresponding to each group of TCR (001-007).

[0021] FIG3 shows the results of tetramer-positive cell population sorting.

[0022] FIG4 shows the single-cell transcriptome sequencing results of cells corresponding to each group of TCR (008-015).

[0023] FIG5 shows the detection results of TCR expression in each group.

[0024] 6A-6B show the cell phenotype results in cells expressing the TCR of the present invention.

[0025] FIG7 shows the target cell killing effect of cells expressing the TCR of the present invention.

[0026] FIG8 shows the target cell killing effect of peripheral blood-derived cells expressing the TCR of the present invention. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0028] Definition of terms

[0029] In the present invention, the term "T cell receptor" or "TCR" generally refers to an endogenous or engineered T cell receptor. For example, a TCR may include an extracellular antigen binding domain that binds to a specific antigen epitope in an MHC molecule. A TCR may include a TCRα polypeptide chain and a TCRβ polypeptide chain. A "tumor-specific TCR" refers to a TCR that specifically recognizes a tumor antigen expressed by a tumor cell. A "TCR cell" refers to a cell that expresses a recombinant TCR. The "antigen binding domain" of a TCR refers to a polypeptide that binds to an antigen with a higher degree of specificity due to its primary, secondary or tertiary sequence and / or post-translational modification and / or charge. The antigen binding domain may be derived from any part or fragment of a TCR that, as part of a TCR, retains the antigen binding activity of the TCR. The antigen binding portion encompasses a portion that has the ability to detect, treat or prevent cancer. The TCR antigen binding domain may comprise about 10-95% or more, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95% or more of the full-length TCR. The antigen binding domain may comprise the antigen recognition portion of either or both of the α chain and β chain of the TCR, for example, it may comprise one or more portions of the complementary determining regions CDR1, CDR2, and CDR3 of the variable region of the α chain and / or β chain of the TCR. The antigen binding domain may comprise the following amino acid sequence: CDR1 of the α chain, CDR2 of the α chain, CDR3 of the α chain, CDR1 of the β chain, CDR2 of the β chain, CDR3 of the β chain, or any combination thereof. Preferably, the antigen binding domain comprises the amino acid sequence of the α chain CDR1, CDR2 and CDR3 of TCR or the amino acid sequence of the CDR1, CDR2 and CDR3 of the β chain; or the amino acid sequence of CDR1, CDR2, CDR3 of all α chains and β chains. In one embodiment of the invention, the antigen binding portion may include, for example, the variable region of TCR, which includes a combination of the above-mentioned CDR regions. In this regard, the antigen binding portion may include the amino acid sequence of the variable region (Vα) of the α chain of TCR, the amino acid sequence (Vβ) of the variable region of the β chain, or the amino acid sequence of both Vα and Vβ. In one embodiment of the invention, the antigen binding portion may include a combination of a variable region and a constant region. In this regard, the antigen binding portion may include the full length α or β chain of TCR, or both α and β chains.

[0030] For example, the α and β chains of a TCR are generally considered to each have two "domains" or "regions" referred to as variable and constant domains / regions. The terms "domain" and "region" are used interchangeably herein. The variable domain consists of a concatenation of a variable region and a connecting region. In the specification and claims of the present invention, the term "TCRα variable domain" therefore refers to the concatenation of the TRAV and TRAJ regions, and the term TCRα constant domain refers to the extracellular TRAC region or to the TRAC sequence with a C-terminal truncation. Similarly, the term "TCRβ variable domain" refers to the concatenation of the TRBV and TRBD / TRBJ regions, and the term TCRβ constant domain refers to the extracellular TRBC region or to the TRBC sequence with a C-terminal truncation. Similarly, the connecting region of a TCR is defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant region is defined by the IMGT TRAC and TRBC nomenclature. The β chain variable region is referred to by the abbreviation TRBD in the IMGT nomenclature, and the concatenated TRBD / TRBJ regions are generally considered together to be the connecting region.

[0031] The example of TCR includes but is not limited to full-length TCR, TCR antigen binding fragment, lack of transmembrane region and cytoplasmic region soluble TCR, containing TCR by flexible joint attached variable region single chain TCR, by the TCR chain of disulfide bond connected through transformation, monospecific TCR, multispecific TCR (including bispecific TCR), TCR fusion, human TCR, humanized TCR, chimeric TCR, recombinant TCR and synthetic TCR.The term covers wild-type TCR and genetically modified TCR (for example, comprising chimeric TCR chain, the chimeric TCR chain comprising the first part of the TCR from the first species and the second part of the TCR from the second species).In certain embodiments, TCR includes transmembrane region.In certain embodiments, TCR includes costimulatory signaling region.

[0032] In the present invention, the term "vector" generally refers to a vector by which a polynucleotide sequence (such as a foreign gene) can be introduced into a host cell to obtain the desired gene expression of the introduced nucleotide sequence. Cloning vectors can include, for example, plasmids, phages, viruses, etc. Another type of vector is a viral vector that is connected to the nucleic acid construct to be transported in the viral genome. Viral vectors can replicate autonomously in the host cell into which they are introduced, or can integrate themselves into the genome of the host cell, thereby replicating together with the host genome. In addition, some vectors can instruct the expression of genes operably connected to them. Such vectors are referred to as "recombinant expression vectors" or simply "expression vectors" in this article. In some embodiments, the vector is a viral vector (such as a replication-defective retrovirus, adenovirus, and adeno-associated virus).

[0033] In the present invention, the term "cancer" or "cancer cell" generally refers to a cell that divides in an uncontrolled manner. Examples of such cells include cells with an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include cancerous growths (e.g., tumors), oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or infiltration stage. Cancer cells can form solid tumors or excess tumor cells in the blood (e.g., hematological cancers). Alternatively or additionally, it can include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or infiltration stage.

[0034] In the present invention, the term "pharmaceutically acceptable" or "pharmacologically compatible" generally refers to a material that does not have biological or other undesirable effects. For example, the material can be incorporated into a pharmaceutical composition administered to a subject without causing any significant undesirable biological effects or interacting in a deleterious manner with any other component of the composition in which it is contained. Pharmaceutically acceptable adjuvants or excipients preferably have met the required toxicological and manufacturing testing standards and / or are included in the inactive ingredient guidelines established by the Food and Drug Administration.

[0035] In the present invention, the term "immune cell" generally refers to cells that participate in an immune response, such as cells that promote immune effector responses. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. For example, the immune cells of the present invention may include cells derived from artificial pluripotent stem (iPS) cells, PBMC cells, and / or tumor infiltrating lymphocytes. For example, the immune cells of the present invention may be obtained by differentiation of iPS cells. The term also includes engineered immune cells, such as immune cells that are genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0036] In the present invention, the term "PBMC" or "human peripheral blood mononuclear cell" generally refers to cells in peripheral blood that have a single nucleus. For example, any blood cell with a round nucleus (i.e., lymphocyte, monocyte, or macrophage). These blood cells are key components of the immune system to fight infection and adapt to invaders. The lymphocyte population is composed of CD4 + and CD8 + T cells, B cells, NKT cells and natural killer cells, CD14 + Monocytes and basophils / neutrophils / eosinophils / dendritic cells. Usually, FICOLL TM(a hydrophilic polysaccharide that separates blood), these cells are separated from whole blood, where monocytes and lymphocytes form the buffy coat below the plasma layer. For example, "PBMC" refers to a cell population that contains at least T cells, and optionally NK cells, NKT cells, and antigen-presenting cells.

[0037] In the present invention, the term "killing ability" refers to the killing of cells by contacting the cells with an effective amount of active substances. For example, the TCR of the present invention can enhance the killing ability of cells. For example, cells expressing the TCR of the present invention can show enhanced killing ability. For example, the combination of cells expressing the TCR of the present invention and other immune cells, antibodies, immunoconjugates (e.g., conjugates of binding molecules targeting immune checkpoints with other active molecules), and / or bispecific / multispecific molecules can show enhanced killing ability. The method may include killing cells that express positive antigens, optionally in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms.

[0038] In the present invention, the terms "about" and "approximately" generally refer to a statistically significant range of values. Such a range can be within an order of magnitude of a given value or range, can be included within 50%, can be included within 20%, can be included within 10%, can be included within 5%. The permissible variations contained in the terms "about" or "approximately" can depend on the specific system under study and can be easily understood by those of ordinary skill in the art. The terms "above", "below", "at most" and "at least" can include this number.

[0039] Detailed Description of the Invention

[0040] In one aspect, the present invention provides a T cell receptor (TCR) or an antigen-binding fragment thereof, wherein the TCR comprises a beta chain variable region (Vβ), the Vβ comprising a CDR3, the CDR3 of the Vβ comprising the sequence shown in any one of SEQ ID NOs: 14-26, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0041] For example, the TCR comprises a beta chain variable region (Vβ), wherein the Vβ comprises CDR1 and CDR2, wherein the CDR1 of the Vβ comprises the sequence of SEQ ID NO: 28, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. For example, the TCR comprises a beta chain variable region (Vβ), wherein the Vβ comprises CDR1 and CDR2, wherein the CDR2 of the Vβ comprises the sequence of SEQ ID NO: 27, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0042] For example, the TCR comprises a beta chain variable region (Vβ), wherein the Vβ comprises the sequence shown in any one of SEQ ID NOs: 1-13, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0043] For example, the TCR comprises a beta chain constant region comprising the sequence shown in SEQ ID NO: 29 or 30, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0044] For example, the TCR comprises an α chain variable region (Vα), the Vα comprises a CDR3, the CDR3 of the Vα comprises the sequence shown in SEQ ID NO: 31, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0045] For example, the TCR comprises an α chain variable region (Vα), the Vα comprising CDR1 and CDR2, the CDR1 of the Vα comprising the sequence of SEQ ID NO: 33, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. For example, the TCR comprises an α chain variable region (Vα), the Vα comprising CDR1 and CDR2, the CDR2 of the Vα comprising the sequence of SEQ ID NO: 32, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0046] For example, the TCR comprises an α chain variable region (Vα), wherein Vα comprises the sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0047] For example, the TCR comprises an α chain constant region comprising the sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0048] For example, the TCR comprises an α chain, the full length of which comprises the sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 80% to 99%, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0049] For example, the sequences mentioned herein include sequences that are at least about 95% homologous to the sequence. For example, the sequences mentioned herein include sequences that are at least 80% to 99% homologous to the sequence, such as at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% of the sequence, at least about 97% of the sequence, at least about 98% of the sequence, or at least about 99% of the sequence. Preferably, the sequences mentioned herein also include sequences that have one or more amino acid insertions, deletions, or substitutions, particularly conservative amino acid substitutions.

[0050] For example, the TCR comprises an α chain variable domain and a β chain variable domain (SEQ ID NO number) of a TCR molecule selected from the following table:

[0051] In certain embodiments, the present invention provides a TCR of the present invention comprising one, two, or all three of the CDRs of Vα or Vβ provided herein, for example, wherein the CDRs can be defined according to the Kabat or IMGT numbering system and division.

[0052] In one aspect, the present invention provides the use of the TCR described herein in preparing a fusion protein. For example, the TCR described herein can be operably linked to another molecule. In one aspect, the present invention provides a fusion protein comprising the TCR described herein or an antigen-binding fragment thereof.

[0053] The present invention also provides TCR-derived antibodies or derived CARs. The disclosure also provides antibodies or their antigen-binding fragments or derived CARs containing any one or more CDRs as described herein. In some embodiments, the derived antibody or antigen-binding fragment or derived CAR contains variable heavy and light chains, and the variable heavy and light chains are included in the CDR1, CDR2 and / or CDR3 contained in the α chain and the CDR1, CDR2 and / or CDR3 contained in the β chain. In some embodiments, the antibody or antigen-binding fragment comprises one or more CDRs with at least about 80% to 99% identity to the CDR sequence of the present invention, or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0054] In one aspect, the present invention provides a nucleic acid molecule encoding the TCR or antigen-binding fragment thereof of the present invention and / or the fusion protein of the present invention.

[0055] In another aspect, the present invention provides a vector, which may comprise the nucleic acid of the present invention.

[0056] In another aspect, the present invention provides a composition comprising the TCR or antigen-binding fragment thereof described herein, the fusion protein described herein, the nucleic acid molecule described herein, and / or the vector described herein, and optionally a pharmaceutically acceptable adjuvant, such as, for example, an excipient, a disintegrant, a binder, a lubricant, a diluent, a buffer, an isotonicity agent, a preservative, a wetting agent, an emulsifier, a dispersant, a stabilizer, and a cosolvent.

[0057] On the other hand, the present invention provides a cell, which may contain the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the vector described in the present invention and / or the composition described in the present invention.

[0058] For example, the cell may comprise an immune cell. For example, the cell may comprise NK cells, NKT cells and / or T cells. For example, the cell may comprise cells derived from artificial pluripotent stem (iPS) cells, totipotent stem cells, pluripotent stem cells, PBMC cells and / or tumor infiltrating lymphocytes. For example, the cell of the present invention may be obtained by differentiation of iPS cells. For example, the cell of the present invention may be an HSC (hematopoietic stem cell). For example, the cell of the present invention may be obtained by differentiation of HSC cells. In some embodiments, the T cell is a type 1 T helper cell and a type 2 T helper cell. In some embodiments, the T cell expressing this receptor is an αβ-T cell. In an alternative embodiment, the T cell expressing this receptor is a γδ-T cell.

[0059] For example, compared to the expression level, functional activity and / or anti-tumor activity of the same TCR in similar cells (but wherein the expression of endogenous TCR has been reduced or eliminated), when the cell expresses the TCR of the present invention, the engineered TCR is expressed at a similar or improved level on the cell surface, exhibiting similar or higher functional activity (e.g., cell killing activity) and / or exhibiting similar or higher anti-tumor activity. For example, when expressed in a cell, the expression level of the engineered TCR as described herein on the cell surface is at least about 80% to 120% of the cell expressing endogenous TCR, such as at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120%. For example, cells expressing the TCR of the present invention or its antigen-binding fragment can exhibit NKT cell-related cellular activity.

[0060] In some embodiments, a TCR or antigen binding fragment thereof or a TCR derived binding molecule as described herein can increase the immune response, activity or number of cells by at least 10% to 20 fold, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold or 20-fold. In some embodiments, a TCR or antigen binding fragment thereof or a TCR derived binding molecule can increase serum concentrations of IFN-γ in the presence of an antigen of interest. In some embodiments, activation can induce an increase in serum concentration of IFN-γ by at least 10% to 1000-fold, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold or 1000-fold. In some embodiments, activation can induce an increase in specific killing of target cells by at least 10% to 5-fold, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold or 5-fold. In some embodiments, specific killing of target cells is determined by the killing efficacy of target cells using the methods described herein.

[0061] In one aspect, the present invention provides a method for preparing the TCR of the present invention, comprising culturing the cells of the present invention under conditions such that the TCR is expressed.

[0062] In one aspect, the present invention provides a kit comprising the TCR or antigen-binding fragment thereof described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the vector described in the present invention, the composition described in the present invention and / or the cell described in the present invention.

[0063] In one aspect, the present invention provides a method for culturing cells, which may comprise administering a TCR or antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule, a vector, a composition, a cell, and / or a kit. For example, the method for culturing cells of the present invention may involve transducing a vector expressing a TCR or fusion protein of the present invention into the cell.

[0064] In one aspect, the present invention provides use of the TCR or antigen-binding fragment thereof, the fusion protein, the nucleic acid molecule, the vector, the composition, the cell, and / or the kit of the present invention in the preparation of a medicament for preventing and / or treating a disease and / or symptom. For example, the disease comprises a tumor.

[0065] In one aspect, the present invention provides the TCR or antigen-binding fragment thereof, the fusion protein, the nucleic acid molecule, the vector, the composition, the cell, and / or the kit of the present invention, which can be used to prevent, alleviate, and / or treat a disease. For example, the disease comprises a tumor.

[0066] In one aspect, the present invention provides a method for preventing, alleviating, and / or treating a disease, comprising providing the TCR or antigen-binding fragment thereof, the fusion protein, the nucleic acid molecule, the vector, the composition, the cell, and / or the kit of the present invention. For example, the disease comprises a tumor.

[0067] According to the present invention, the tumor is selected from the group consisting of lymphoma, melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.

[0068] Without intending to be bound by any theory, the following examples are merely intended to illustrate the proteins, methods and uses of the present invention and are not intended to limit the scope of the present invention.

[0069] Example

[0070] Example 1 Cell flow sorting and single cell sequencing

[0071] PBMCs from donor 1 were cultured in X-VIVO 15 medium supplemented with 500 IU IL-2, 10% FBS, and 100 ng / ml α-GalCer. After 14 days of culture, TCRVα24-positive NKT cells were isolated using a flow cytometer (using the Biolegend 360004 antibody). Figure 1 shows the results of the sorting of CD45-positive and TCRVα24-positive cell populations.

[0072] The 10X Genomic Single-Cell Sequencing Sample Preparation Kit was used to label and amplify TCR sequences in the sorted cells, and the corresponding transcriptome was extracted and reverse transcribed. The resulting TCR sequences and cDNA library were subsequently sequenced, and the resulting TCR sequences were ranked by abundance as shown in Table 1.

[0073] Table 1

[0074] Figure 2 shows the results of single-cell transcriptome sequencing of cells corresponding to each group of TCRs. After analysis and screening, the results showed that the cell populations corresponding to TCR-001, 002, 003, 004, and 005 of the TCR of the present invention can collectively express four NKT-related marker genes (NCAM1 (CD56), KLRK1 (NKG2D), CXCR6, and ZBTB16). Therefore, the TCR of the present invention can have a high correlation with NKT cells. Other TCR sequences, such as TCR-006 or TCR-007, may have no correlation with NKT cells.

[0075] Among them, the cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 1 is TCR-001,

[0076] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 2 is TCR-002,

[0077] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 3 is TCR-003,

[0078] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 4 is TCR-004,

[0079] The cell population corresponding to the TCRB chain V+D+J region shown in SEQ ID NO: 5 is TCR-005.

[0080] Similarly, PBMCs from donor 2 were stimulated and expanded with 100 ng / ml α-GalCer. They were then stained with an α-GalCer-loaded CD1d tetramer that recognizes the NKT TCR (MBL TS-HCG-1), and the tetramer-positive cell population was isolated by flow cytometry. Figure 3 shows the results of the tetramer-positive cell population isolation.

[0081] The sequencing steps were the same as in Example 1. The 10X Genomics single-cell sequencing sample preparation kit was used to perform TCR sequence tagging and corresponding cDNA library construction. The obtained TCR sequences were sorted by abundance as shown in Table 2.

[0082] Table 2

[0083] Figure 4 shows the results of single-cell transcriptome sequencing of cells corresponding to each group of TCRs. After analysis and screening, the results showed that the cell populations corresponding to TCRs of the present invention, TCR-008 to 015, can collectively express four NKT-related marker genes (NCAM1 (CD56), KLRK1 (NKG2D), CXCR6, and ZBTB16). Therefore, the TCRs of the present invention can be highly correlated with NKT cells.

[0084] Among them, the cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 6 is TCR-008,

[0085] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 7 is TCR-009,

[0086] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 8 is TCR-010,

[0087] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 9 is TCR-011,

[0088] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 10 is TCR-012,

[0089] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 11 is TCR-013,

[0090] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 12 is TCR-014;

[0091] The cell population corresponding to the V+D+J region of the TCRB chain shown in SEQ ID NO: 13 is TCR-015.

[0092] Example 2 TCR expression detection

[0093] Plasmids expressing the iNKT TCR of the present invention and a previously published iNKT TCR (control iTCR, whose α chain is shown in SEQ ID NO: 38 and whose β chain is shown in SEQ ID NO: 37) were constructed and packaged using lentivirus. Jurkat cells in which the endogenous TCR had been knocked out were transduced. Expression was monitored using a CD1d tetramer loaded with α-GalCer ( Figure 5 ). The results demonstrated that the iNKT TCR of the present invention could be expressed in Jurkat cells and specifically bound by the CD1d tetramer loaded with α-GalCer.

[0094] HSC cells were transduced with a lentivirus expressing the iNKT TCR of the present invention, and expression was monitored during differentiation. The results showed that the TCR of the present invention can be normally expressed on the cell membrane at the terminal stage of differentiation and promotes the differentiation of HSC cells into T cells.

[0095] Example 3 TCR-expressing cell function experiment

[0096] Detection of TCR signal strength in Jurkat cells using luciferase activity downstream of TCR

[0097] Jurkat cell lines carrying the NFAT-Luc reporter were purchased from BPS Bioscience, and the TCR α and β chains were knocked out. Control iTCR and the iNKT TCR of the present invention were respectively introduced using lentivirus. After expression was confirmed using α-GalCer-CD1d tetramer, they were mixed with CD1d-expressing C1R cells (C1R-CD1d cell line) at a 1:1 ratio and cultured overnight in culture medium with or without 100 ng / ml α-GalCer. The next day, the cells were detected using a One-Lite Luciferase assay system (Vazyme) kit on a BioTek Synergy HTX microplate reader (Table 3). The results showed that many of the iNKT TCRs of the present invention exhibited responses to α-GalCer-CD1d stimulation that were similar to or even better than those of the control iTCR (reflected by the ratio of +α-GalCer / -α-GalCer, where +α-GalCer indicates GalCer stimulation and -α-GalCer indicates no GalCer stimulation). This experiment was performed twice, and the average of the two +α-GalCer / -α-GalCer ratios was taken as the average response multiple.

[0098] Table 3

[0099] Among them, the average corresponding multiples are expressed as A, B, and C:

[0100] A: greater than 1 times, less than or equal to 3 times;

[0101] B: greater than 3 times, less than or equal to 5 times;

[0102] C: more than 5 times,

[0103] Cell expansion

[0104] HSC-derived iNKT cells expressing the iNKT TCR of the present invention were harvested. Figures 6A-6B show that during the differentiation process, the T cell phenotypes such as CD3 and CD4 / CD8 of the cells expressing the iTCR of the present invention were significantly better than those of the group without iTCR (no iTCR).

[0105] At the same time, the expansion times at different stages of differentiation in the iTCR group were significantly better than those in the group without iTCR (Table 4);

[0106] Table 4

[0107] During the PBMC feeder cell expansion stage, the effect of PBMC feeder cells in promoting the expansion of iNKT cells derived from HSCs expressing iNKT TCR was significantly better than that of the group without iTCR (Table 5).

[0108] Table 5

[0109] Cell killing

[0110] iNKT cells differentiated from HSCs and expanded through trophoblast stimulation were harvested and co-cultured with the target C1R-CD1d-mcherry cell line at an effector-to-target ratio of 1:4. 100 ng / ml α-GalCer was added to the culture medium. After co-culture, residual target cells were detected using an Incucyte assay. Target cells were added every 48 hours for nine rounds of in vitro killing. The results showed that the iTCR of the present invention could activate HSC-differentiated cells and effectively kill target cells (Figure 7).

[0111] Cell killing

[0112] T cells derived from peripheral blood (PBMC) were harvested to express the iTCR of the present invention, as well as a previously disclosed iTCR (control iTCR, whose α chain is shown in SEQ ID NO: 38 and whose β chain is shown in SEQ ID NO: 37). They were co-cultured with the target cell C1R-CD1d-mcherry cell line at an effector-target ratio of E:T = 1:4. 100 ng / ml α-GalCer was added to the culture medium. After 96 hours, the residual target cells were detected, and the killing rate of the effector cells against the target cells was calculated ( Figure 8 ). The results showed that cells expressing the iTCR of the present invention can effectively kill target cells, and the killing effect is better than that of known iTCRs.

[0113] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments of the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A T cell receptor (TCR) or an antigen-binding fragment thereof, wherein the TCR comprises a β chain variable region (Vβ), the Vβ comprises a CDR3, and the CDR3 of the Vβ comprises a sequence shown in any one of SEQ ID NOs: 14-26.

2. The TCR or antigen-binding fragment thereof of claim 1, wherein the TCR comprises a β chain variable region (Vβ), the Vβ comprises CDR1 and CDR2, the CDR1 of the Vβ comprises the sequence shown in SEQ ID NO: 28, and the CDR2 of the Vβ comprises the sequence shown in SEQ ID NO:

27.

3. The TCR or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the TCR comprises a β chain variable region (Vβ), and the Vβ comprises the sequence shown in any one of SEQ ID NOs: 1 to 13. 4 . The TCR or antigen-binding fragment thereof according to any one of claims 1 to 3 , wherein the TCR comprises a β chain constant region, and the β chain constant region comprises the sequence shown in SEQ ID NO: 29 or 30.

5. The TCR or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the TCR comprises an α chain variable region (Vα), the Vα comprises a CDR3, and the CDR3 of the Vα comprises the sequence shown in SEQ ID NO:

31.

6. The TCR or antigen-binding fragment thereof of any one of claims 1-5, wherein the TCR comprises an α chain variable region (Vα), the Vα comprises CDR1 and CDR2, the CDR1 of the Vα comprises the sequence shown in SEQ ID NO: 33, and the CDR2 of the Vα comprises the sequence shown in SEQ ID NO:

32.

7. The TCR or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the TCR comprises an α chain variable region (Vα), and the Vα comprises the sequence shown in SEQ ID NO:

34.

8. The TCR or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the TCR comprises an α chain constant region, and the α chain constant region comprises the sequence shown in SEQ ID NO:

35.

9. A fusion protein, wherein the fusion protein comprises the TCR or antigen-binding fragment thereof according to any one of claims 1 to 8. 10 . A nucleic acid molecule, wherein the nucleic acid molecule encodes the TCR or antigen-binding fragment thereof according to any one of claims 1 to 8 and / or the fusion protein according to claim 9 . A vector, wherein the vector comprises the nucleic acid molecule of claim 10.

12. A composition comprising the TCR or antigen-binding fragment thereof according to any one of claims 1 to 8, the fusion protein according to claim 9, the nucleic acid molecule according to claim 10, and / or the vector according to claim 11, and optionally a pharmaceutically acceptable adjuvant.

13. A cell, wherein the cell comprises the TCR or antigen-binding fragment thereof according to any one of claims 1 to 8, the fusion protein according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11 and / or the composition according to claim 12.

14. The cell of claim 13, wherein the cell comprises an immune cell.

15. The cell according to any one of claims 13-14, wherein the cell comprises a NK cell, a NKT cell and / or a T cell.

16. The cell according to any one of claims 13-15, wherein the cell comprises an artificial pluripotent stem cell (iPSC)-derived cell, a PBMC cell and / or a tumor infiltrating lymphocyte.

17. A kit, wherein the kit comprises the TCR or antigen-binding fragment thereof according to any one of claims 1-8, the fusion protein according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the composition according to claim 12 and / or the cell according to any one of claims 13-16.

18. A method for influencing cell growth, comprising administering the TCR or antigen-binding fragment thereof according to any one of claims 1 to 8, the fusion protein according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the composition according to claim 12, the cell according to any one of claims 13 to 16 and / or the kit according to claim 17.

19. Use of the TCR or antigen-binding fragment thereof according to any one of claims 1 to 8, the fusion protein according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the composition according to claim 12, the cell according to any one of claims 13 to 16 and / or the kit according to claim 17 in the preparation of a drug, wherein the drug is used to prevent and / or treat a disease and / or symptom.

20. A drug for preventing and / or treating diseases and / or symptoms, comprising the TCR or antigen-binding fragment thereof according to any one of claims 1 to 8, the fusion protein according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the composition according to claim 12, the cell according to any one of claims 13 to 16 and / or the kit according to claim 17 as active ingredients.

21. A method for preventing and / or treating a disease and / or symptom, comprising administering to a subject in need thereof the TCR or antigen-binding fragment thereof of any one of claims 1-8, the fusion protein of claim 9, the nucleic acid molecule of claim 10, the vector of claim 11, the composition of claim 12, the cell of any one of claims 13-16 and / or the kit of claim 17.

22. The TCR or antigen-binding fragment thereof according to any one of claims 1 to 8, the fusion protein according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the composition according to claim 12, the cell according to any one of claims 13 to 16 and / or the kit according to claim 17, for use in preventing and / or treating diseases and / or symptoms.

23. The use according to claim 19, the medicament according to claim 20, the method according to claim 21, and / or the TCR or its antigen-binding fragment, fusion protein, nucleic acid molecule, vector, composition, cell and / or kit for use according to claim 22, wherein the disease and / or symptom comprises a tumor.

24. The use according to claim 19, the medicament according to claim 20, the method according to claim 21, and / or the TCR or its antigen-binding fragment, fusion protein, nucleic acid molecule, vector, composition, cell and / or kit for use according to claim 22, wherein the disease and / or symptom comprises a hematological tumor and / or a solid tumor.

25. The use according to claim 19, the medicament according to claim 20, the method according to claim 21, and / or the TCR or its antigen-binding fragment, fusion protein, nucleic acid molecule, vector, composition, cell and / or kit for use according to claim 22, wherein the disease and / or symptom comprises one or more selected from the following groups: lymphoma, melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer and kidney cancer.

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