A single-domain antibody targeting mesothelin, a chimeric antigen receptor, and their applications

Abstract

The present invention relates to the technical field of immunotherapy. The present invention provides a single-domain antibody targeting mesothelin, a chimeric antigen receptor (CAR) targeting mesothelin constructed using the single-domain antibody, and an engineered immune effector cell comprising the CAR. The present invention also provides the use of the single-domain antibody targeting mesothelin, the chimeric antigen receptor, and the engineered immune effector cell comprising the CAR in the preparation of a medicament for diagnosing, preventing, and / or treating a disease or disorder associated with mesothelin expression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to single-domain antibodies specifically targeting mesothelin, chimeric antigen receptors targeting mesothelin constructed using such single-domain antibodies, engineered immune effector cells comprising such chimeric antigen receptors, and their use in the preparation of drugs for diagnosing, preventing, and / or treating diseases. Background Art

[0002] At present, there are still many obstacles in the clinical treatment of solid tumors. Solid tumors have a dense tissue structure, which makes it more difficult for drugs to enter the tumor tissue and contact with tumor cells. For example, the "tumor microenvironment" (TME) present in tumor tissues greatly limits the efficacy of anti-tumor drugs; there is a lack of antigens with high specificity for solid tumor cells, thus there is a risk of serious adverse reactions caused by drugs attacking normal cells. Among them, the lack of tumor-specific antigens is one of the important reasons for the poor treatment effect of solid tumors. Therefore, to solve the treatment problem of solid tumors, finding an ideal target with high specific expression on tumor cells is a potential solution. By finding a target with high specific expression in tumors, it is hoped to achieve the goal of effectively treating solid tumors while reducing the risk of adverse reactions.

[0003] Mesothelin (MSLN) is a cell surface glycoprotein of about 40 kDa, which is anchored to the cell membrane through glycosylphosphatidylinositol. The mesothelin gene encodes a 69 kDa precursor protein, which is hydrolyzed by furin protease to produce two protein products. Among them, the C-terminal membrane-bound protein of about 40 kDa is the mature mesothelin, and the N-terminal fragment of about 30 kDa, called megakaryocyte potentiating factor (MPF), is shed and released extracellularly. MPF can promote the formation of megakaryocyte clones in vitro, and membrane-anchored MSLN plays an important role in cell adhesion. Currently, in the field of targeted therapy, the mesothelin target usually refers to the fragment anchored to the cell membrane, that is, the protein of the C-terminal fragment of about 40 kDa. Mesothelin is expressed in a narrow range and at a low level in normal tissues. For example, it is expressed at a low level in mesothelial tissues such as the pleura, peritoneum, and pericardium, and is not expressed in other normal tissues. However, in cancerous tissues, the high expression of mesothelin is extremely common. Studies have found that the expression of mesothelin has been observed in various types of solid tumors, such as mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, esophageal cancer, cholangiocarcinoma, gastric cancer, colon cancer, thymic cancer, endometrial cancer, and breast cancer, etc. Based on the characteristics of limited distribution of mesothelin in normal tissues but high expression in certain tumor tissues, it has high specificity as an antigen for various solid tumors, making it a potential target for the treatment of solid tumors.

[0004] Single-domain antibody (SdAb) is an antibody composed only of the variable region amino acids of heavy-chain antibodies, thus different from traditional 4-chain antibodies. Its molecular weight is only 12-15 kDa, so it is also called nanobody (Nb). Camelids and sharks produce antibodies that are naturally lacking in light chains, which are called heavy-chain-only antibodies, or simply heavy-chain antibodies (HCAb). The antigen-binding fragment in each arm of the camelid heavy-chain antibody has a single heavy-chain variable domain, which is called the variable domain of heavy chain of heavy-chain antibody (VHH). Single-domain antibodies possess similar or higher specificity and affinity compared to traditional antibodies. In addition, they also have natural advantages such as good solubility, high stability, strong penetrability, and a wide range of binding epitopes. These advantages have made them not only highly regarded in the field of immunotherapy but also being applied to the treatment of various malignancies, autoimmune diseases, anti-infection, etc. Especially at the present stage, the treatment effect of cell therapy for solid tumors is still not ideal, and the high heterogeneity of tumors and the tumor microenvironment (TME) have largely affected its efficacy. Therefore, single-domain antibodies may become a more advantageous and potential choice for development.

[0005] Chimeric antigen receptor (CAR)-modified T cells, as an immunotherapy strategy, are an emerging and effective means of cancer treatment. Currently, CAR-T cell therapies targeting CD19, CD22, BCMA and other targets have achieved remarkable efficacy in the treatment of hematological malignancies such as B-cell leukemia, B-cell lymphoma, and multiple myeloma. A chimeric antigen receptor is a recombinant polypeptide construct, and its representative structure consists of four parts: an extracellular antigen-binding domain (usually a single-chain antibody with antigen recognition function, scFv), a hinge domain, a transmembrane domain, and an intracellular signal transduction domain. Usually, according to whether co-stimulatory molecules are added and the number of co-stimulatory molecules added in the intracellular signal transduction domain, the classical chimeric antigen receptor structure is divided into the first generation (without co-stimulatory molecules), the second generation (including one co-stimulatory molecule), and the third generation (including two co-stimulatory molecules). So far, the second-generation chimeric antigen receptor structure design has been the most widely used in marketed products and clinical research. The principle of the chimeric antigen receptor is that through genetic engineering modification, T cells express a receptor structure (such as scFv) that can specifically recognize tumor cell surface antigens. After the receptor specifically binds to the tumor cell surface antigen, it activates its downstream immune co-stimulatory molecules and T cells, breaking through the restriction of the Major Histocompatibility Complex (MHC), directly specifically recognizing and killing tumor cells, and achieving the purpose of targeted elimination of malignant tumors. Since the realization of this recognition and killing function requires the use of antibodies with high specificity, good binding activity, and efficient binding epitopes, one of the keys to the success of CAR-T cell therapy lies in screening high-affinity antibodies with good specificity, strong binding force, and effective binding epitopes.

[0006] Therefore, there is still a widespread need to screen for and develop improved mesothelin-targeted single-domain antibodies, mesothelin-targeted chimeric antigen receptors constructed using them, and engineered immune effector cells. In particular, to develop mesothelin-specific single-domain antibodies suitable for more effective and efficient CAR-T cell therapy. Summary of the Invention

[0007] The object of the present invention is to provide a mesothelin-targeting single-domain antibody and its application. The mesothelin-targeting single-domain antibody structure of the present invention is a natural single-chain structure, which has the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability, and does not require additional folding and assembly steps or linker optimization and modification, making it a promising alternative to the relatively large molecular weight scFv single-chain antibody. Moreover, the CAR-T cells constructed using the single-domain antibody have significant tumor cell killing ability.

[0008] According to the first aspect of the present disclosure, there is provided a mesothelin-targeting single-domain antibody. The mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises any one of the amino acid sequences shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49 or 52, wherein CDR2 comprises any one of the amino acid sequences shown in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50 or 53, and wherein CDR3 comprises any one of the amino acid sequences shown in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54.

[0009] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 is any one of the amino acid sequences shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49 or 52, CDR2 is any one of the amino acid sequences shown in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50 or 53, and CDR3 is any one of the amino acid sequences shown in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54.

[0010] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1, CDR2 and CDR3 comprise the amino acid sequences shown in Table 1.

[0011] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1, CDR2 and CDR3 are the amino acid sequences shown in Table 1.

[0012] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:1, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:2, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:3.

[0013] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:4, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:5, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:6.

[0014] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:7, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:8, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:9.

[0015] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:10, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:11, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:12.

[0016] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:13, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:14, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:15.

[0017] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO:16, wherein CDR2 is the amino acid sequence shown in SEQ ID NO:17, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO:18.

[0018] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 19, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 20, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 21.

[0019] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 22, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 23, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 24.

[0020] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 25, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 26, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 27.

[0021] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 28, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 29, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 30.

[0022] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 31, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 32, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 33.

[0023] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 34, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 35, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 36.

[0024] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 37, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 38, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 39.

[0025] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 40, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 41, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 42.

[0026] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 43, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 44, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 45.

[0027] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 46, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 47, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 48.

[0028] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 49, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 50, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 51.

[0029] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 52, wherein CDR2 is the amino acid sequence shown in SEQ ID NO: 53, and wherein CDR3 is the amino acid sequence shown in SEQ ID NO: 54.

[0030] For the technical solution disclosed by the present invention, the specific amino acid sequence information shown in SEQ ID NOs: 1-54 is shown in Table 1.

[0031] Table 1

[0032]

[0033]

[0034] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein the determination of CDR1, CDR2, and CDR3 is based on any one of the IMGT numbering scheme, Kabat numbering scheme, AbM numbering scheme, Chothia numbering scheme, or Contact numbering scheme. The mesothelin-targeting single-domain antibody provided by the present invention comprises CDR1, CDR2, and CDR3 regions; wherein the determination of CDR1, CDR2, and CDR3 is based on the IMGT numbering scheme.

[0035] The single-domain antibody provided by the present invention or its CDR regions cover the CDRs defined by the above numbering scheme or other known numbering schemes. For example, those skilled in the art can understand that the CDR regions determined by any numbering scheme, as long as they are the same as or contain the CDR regions of the present invention, fall within the protection scope of the present invention.

[0036] The mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with any of the amino acid sequences shown in Table 2 or comprising SEQ ID NOs: 55-72.

[0037] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 55.

[0038] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 56.

[0039] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 57.

[0040] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 58.

[0041] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 59.

[0042] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 60.

[0043] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 61.

[0044] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 62.

[0045] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 63.

[0046] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 64.

[0047] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 65.

[0048] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 66.

[0049] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 67.

[0050] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 68.

[0051] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 69.

[0052] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 70.

[0053] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 71.

[0054] In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 72.

[0055] The mesothelin-targeting single-domain antibody provided by the present invention comprises any amino acid sequence shown in Table 2 or SEQ ID NOs: 55-72. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 57. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 59. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 61. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 65. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 66. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 67. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 68. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 69. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 71. In some embodiments, the mesothelin-targeting single-domain antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO: 72.

[0056] The single-domain antibody targeting mesothelin provided by the present invention has any amino acid sequence shown in Table 2 or SEQ ID NOs: 55-72. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 57. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 59. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 61. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 63. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 65. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 66. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 67. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 68. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 69. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 71. In some embodiments, the single-domain antibody targeting mesothelin provided by the present invention has the amino acid sequence shown in SEQ ID NO: 72.

[0057] For the technical solution disclosed by the present invention, the specific amino acid sequence information shown in SEQ ID NO: 55-72 is as shown in Table 2.

[0058] Table 2

[0059]

[0060]

[0061]

[0062] In some embodiments, there is provided a use of a mesothelin-targeting single-domain antibody of the present invention in the preparation of a chimeric antigen receptor (CAR). The single-domain antibody has particular advantages in the use for preparing a chimeric antigen receptor. First, the single-domain antibody exerts the function of binding to an antigen through a single domain. Therefore, the chimeric antigen receptor constructed based on the single-domain antibody has a lower risk of aggregation compared to other types of antibodies such as single-chain antibodies (scFv). Second, the CDR3 region of the single-domain antibody is longer and can access epitopes that cannot be accessed by conventional antibodies. Therefore, the single-domain antibody has a higher affinity as the antigen recognition and binding domain of the chimeric antigen receptor. Moreover, since the sequence homology of the single-domain antibody with the corresponding human region is significantly higher than that of the scFv with its corresponding human sequence, the immunogenicity of the single-domain antibody is lower than that of the scFv, and the risk of generating immunogenicity when used as the antigen recognition domain of the chimeric antigen receptor is lower. In addition, the single-domain antibody also has the advantages of strong tissue permeability, good hydrophilicity, good solubility, high stability, wide binding epitopes, easy modification and optimization, low production cost, and suitability for large-scale industrial production, making it have unique advantages in the use for preparing a chimeric antigen receptor.

[0063] According to the second aspect of the present disclosure, there is provided a chimeric antigen receptor comprising the single-domain antibody of the present invention.

[0064] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an extracellular antigen-binding domain, and the extracellular antigen-binding domain comprises a mesothelin-targeting single-domain antibody as described in the present invention.

[0065] In some embodiments, the chimeric antigen receptor provided by the present invention may further comprise one or more of the following structures: a linker (such as a peptide linker), a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory signal domain, an intracellular signal transduction domain.

[0066] In some embodiments, the chimeric antigen receptor provided by the present invention comprises:

[0067] (a) an extracellular antigen-binding domain,

[0068] (b) Transmembrane domain, and

[0069] (c) Intracellular signaling domain;

[0070] Wherein, the extracellular antigen-binding domain comprises a mesothelin-targeting single-domain antibody as described in the first aspect of the present disclosure.

[0071] In some embodiments, the chimeric antigen receptor provided by the present invention comprises:

[0072] (a) Extracellular antigen-binding domain,

[0073] (b) Transmembrane domain, and

[0074] (c) Intracellular signaling domain;

[0075] Wherein, the extracellular antigen-binding domain comprises a mesothelin-targeting single-domain antibody, and the single-domain antibody comprises CDR1, CDR2 and CDR3 regions, wherein the CDR1, CDR2 and CDR3 regions comprise the CDR1, CDR2 and CDR3 regions of the mesothelin-targeting single-domain antibody as described in the first aspect of the present disclosure.

[0076] In some embodiments, the chimeric antigen receptor provided by the present invention comprises:

[0077] (a) Extracellular antigen-binding domain,

[0078] (b) Transmembrane domain, and

[0079] (c) Intracellular signaling domain;

[0080] Wherein, the extracellular antigen-binding domain comprises a mesothelin-targeting single-domain antibody, and the single-domain antibody comprises CDR1, CDR2 and CDR3 regions, wherein CDR1, CDR2 and CDR3 are the CDR1, CDR2 and CDR3 regions of the mesothelin-targeting single-domain antibody as described in the first aspect of the present disclosure.

[0081] In some embodiments, the chimeric antigen receptor provided by the present invention comprises:

[0082] (a) Extracellular antigen-binding domain,

[0083] (b) Transmembrane domain, and

[0084] (c) Intracellular signaling domain;

[0085] Wherein, the extracellular antigen-binding domain comprises a single-domain antibody targeting mesothelin; wherein, the single-domain antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any of the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72.

[0086] In some embodiments, the chimeric antigen receptor provided by the present invention comprises:

[0087] (a) An extracellular antigen-binding domain,

[0088] (b) A transmembrane domain, and

[0089] (c) An intracellular signaling domain;

[0090] Wherein, the extracellular antigen-binding domain comprises a single-domain antibody targeting mesothelin; wherein, the single-domain antibody comprises any of the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72.

[0091] In some embodiments, the chimeric antigen receptor provided by the present invention comprises:

[0092] (a) An extracellular antigen-binding domain,

[0093] (b) A transmembrane domain, and

[0094] (c) An intracellular signaling domain;

[0095] Wherein, the extracellular antigen-binding domain comprises a single-domain antibody targeting mesothelin; wherein, the single-domain antibody is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with any one of the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72.

[0096] In some embodiments, the chimeric antigen receptor of the present invention comprises:

[0097] (a) an extracellular antigen-binding domain,

[0098] (b) a transmembrane domain, and

[0099] (c) an intracellular signaling domain;

[0100] Wherein, the extracellular antigen-binding domain comprises a single-domain antibody targeting mesothelin; wherein, the single-domain antibody is any one of the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72.

[0101] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the transmembrane domain is derived from CD8α, CD28, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRα, TCRβ, TCRγ, TCRδ, TCRζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1 or any combination thereof.

[0102] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the transmembrane domain is derived from CD8α. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:75. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the transmembrane domain is the amino acid sequence shown in SEQ ID NO:75.

[0103] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signaling domain comprises the major intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the intracellular signaling domain is derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d or any combination thereof.

[0104] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signaling domain is derived from CD3ζ. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:77. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signaling domain is the amino acid sequence shown in SEQ ID NO:77.

[0105] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from MHC class I molecules, BTLA, and Toll ligand receptors. In some embodiments, the co-stimulatory signaling domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (LIGHTR), BTLA, CD8α, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

[0106] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signaling domain is derived from 4-1BB (CD137). In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:76. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the co-stimulatory signaling domain is the amino acid sequence shown in SEQ ID NO:76.

[0107] In some embodiments, the chimeric antigen receptor provided by the present invention further comprises a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0108] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the hinge region is derived from CD8α, CD28, CD137, IgG4, IgG1, or any combination thereof.

[0109] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the hinge region is derived from CD8α. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:74. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the hinge region is the amino acid sequence shown in SEQ ID NO:74.

[0110] In some embodiments, the chimeric antigen receptor provided by the present invention further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide.

[0111] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide is derived from HLA-A, CD8α, CD4, CD33, CD137, GM-CSFRα, IgG1, Igκ, IL-2, or any combination thereof.

[0112] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide is derived from CD8α.

[0113] In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide is derived from HLA-A. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO:73. In some embodiments, the chimeric antigen receptor provided by the present invention, wherein the signal peptide is the amino acid sequence shown in SEQ ID NO:73.

[0114] For the technical solution disclosed by the present invention, wherein the specific amino acid sequence information shown in SEQ ID NO:73-77 is shown in Table 3.

[0115] Table 3

[0116]

[0117]

[0118] In some embodiments, the chimeric antigen receptor polypeptide provided by the present invention sequentially comprises from the N-terminus to the C-terminus: an HLA-A signal peptide or a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge region, a CD8α transmembrane domain, a co-stimulatory signal domain derived from 4-1BB (CD137), and an intracellular signal transduction domain derived from CD3ζ.

[0119] The chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence shown in Table 4 or comprising the amino acid sequences shown in SEQ ID NO:78-95.

[0120] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:78.

[0121] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:79.

[0122] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 80.

[0123] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 81.

[0124] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 82.

[0125] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 83.

[0126] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 84.

[0127] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 85.

[0128] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 86.

[0129] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 87.

[0130] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 88.

[0131] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 89.

[0132] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 90.

[0133] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 91.

[0134] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 92.

[0135] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 93.

[0136] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 94.

[0137] In some embodiments, the chimeric antigen receptor provided by the present invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 95.

[0138] In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequences shown in Table 4 or comprising SEQ ID NOs: 78-95. In some embodiments, the chimeric antigen receptor provided by the present invention comprises as

[0139] the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:80. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:82. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:83. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:84. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:85. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:86. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:87. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:88. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:89. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:90. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:91. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:92. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:93. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:94. In some embodiments, the chimeric antigen receptor provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:95.

[0140] The chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NO: 78-95.

[0141] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 78.

[0142] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 79.

[0143] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 80.

[0144] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 81.

[0145] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 82.

[0146] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 83.

[0147] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 84.

[0148] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 85.

[0149] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 86.

[0150] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 87.

[0151] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 88.

[0152] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 89.

[0153] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 90.

[0154] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 91.

[0155] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 92.

[0156] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 93.

[0157] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 94.

[0158] In some embodiments, the chimeric antigen receptor provided by the present invention is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 95.

[0159] In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in Table 4 or as shown in SEQ ID NOs: 78-95. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 78. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 79. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 80. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 81. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 82. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 83. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 85. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 86. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 87. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 88. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 89. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 90. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 93. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 94. In some embodiments, the chimeric antigen receptor provided by the present invention has the amino acid sequence shown in SEQ ID NO: 95.

[0160] For the technical solution disclosed by the present invention, the specific amino acid sequence information shown in SEQ ID NOs: 78-95 is as shown in Table 4.

[0161] Table 4

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] According to a third aspect of the present disclosure, there is provided an isolated nucleic acid encoding the chimeric antigen receptor of the present invention.

[0168] In some embodiments, the nucleic acid provided by the present invention is in the form of DNA or RNA. In some embodiments, the DNA provided by the present invention includes cDNA, genomic DNA or synthetic DNA. In some embodiments, the DNA provided by the present invention is single-stranded DNA or double-stranded DNA. In some embodiments, the DNA provided by the present invention is coding-strand DNA or non-coding-strand DNA.

[0169] The isolated nucleic acid provided by the present invention comprises the nucleic acid sequences shown in Table 5 or comprising SEQ ID NOs: 96-113. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 96. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 97. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 98. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 99. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 100. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 101. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 102. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 103. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 104. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 105. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 106. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 107. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 108. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 109. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 110. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 111. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 112. In some embodiments, the isolated nucleic acid provided by the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 113.

[0170] For the technical solution disclosed by the present invention, the specific nucleic acid sequence information shown in SEQ ID NOs: 96-113 is shown in Table 5.

[0171] Table 5

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] According to a fourth aspect of the present disclosure, there is provided a vector comprising the isolated nucleic acid of the present invention.

[0184] In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is selected from the group consisting of: DNA, RNA, plasmid, or viral vector. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.

[0185] In some embodiments, the vector is contained in a host cell.

[0186] In some embodiments, the host cell is a killer cell.

[0187] In some embodiments, the killer cell is a T cell or an NK cell.

[0188] In some embodiments, the NK cell is a primary NK cell.

[0189] In some embodiments, the T cell is a peripheral blood T lymphocyte or a cord blood T lymphocyte.

[0190] According to a fifth aspect of the present disclosure, there is provided an engineered immune effector cell comprising the chimeric antigen receptor or the isolated nucleic acid of the present invention.

[0191] In some embodiments, the engineered immune effector cells provided by the present invention can be selected from T cells, B cells, NK cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells (iPSCs), or any combination thereof.

[0192] In some embodiments, the engineered immune effector cells provided by the present invention are T cells. For example, T cells can be selected from CD4+ / CD8+ T cells, CD4+ / CD8- T cells, CD4- / CD8+ T cells, CD4- / CD8- T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, NKT cells, DNT cells (double-negative T cells).

[0193] In some embodiments, the T cells are CD4+ / CD8- T cells, CD4- / CD8+ T cells, CD4+ / CD8+ T cells, CD4- / CD8- T cells, or a combination thereof.

[0194] In some embodiments, T cells produce cytokines such as IL-2, IFNγ, and / or TNFα after expressing a chimeric antigen receptor and binding to target cells (such as mesothelin-expressing tumor cells). In some embodiments, CD8+ T cells lyse antigen-specific target cells after expressing a chimeric antigen receptor and binding to the target cells.

[0195] In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells can be an established cell line, such as NK-92 cells.

[0196] In some embodiments, the immune effector cells can be differentiated from stem cells, such as differentiated from iPSCs.

[0197] According to the sixth aspect of the present disclosure, there is provided a pharmaceutical composition comprising the mesothelin-targeting single-domain antibody, chimeric antigen receptor, engineered immune effector cells of the present invention, and one or more pharmaceutically acceptable excipients and / or carriers.

[0198] Excipients and / or carriers are often also referred to as adjuvants. A pharmaceutically acceptable carrier refers to a carrier acceptable in pharmacy, which is usually not the same substance as the vector used for inserting nucleic acids in genetic engineering. Those skilled in the art can realize that pharmaceutically acceptable excipients and / or carriers are non-toxic or substantially non-toxic to the recipient at the doses and concentrations employed, and are compatible with the active ingredient of the present invention pharmacologically and / or physiologically, for example, without affecting its viability or efficacy. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, release rate, absorption or penetration of the composition. The optimal pharmaceutical composition can be determined according to the expected route of administration, delivery mode and required dose. Examples of pharmaceutically acceptable excipients and / or carriers are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th edition, 1990). Pharmaceutically acceptable excipients and / or carriers refer to any excipient, filler, salt, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, adsorbent, antioxidant, solvent, co-solvent, buffer, chelating agent, surfactant, wetting agent, emulsifying agent, coating agent, isotonic agent, absorption delaying agent, tonicity regulator, diluent, preservative and / or adjuvant. In some embodiments, the excipients and / or carriers that can be used in the present invention include, but are not limited to: water; salt solutions such as sodium chloride, potassium chloride, magnesium chloride; cell culture media; buffers such as neutral buffered salt solutions or sulfate buffered salt solutions, etc.; carbohydrates such as glucose, mannose, sucrose, dextran or mannitol, etc.; glycerol; proteins; polypeptides or amino acids such as glycine, etc.; antioxidants; chelating agents such as EDTA or glutathione, etc.; ethanol; polyols; and one or more of the above excipients and / or carriers. In some embodiments, the excipients and / or carriers that can be used in the present invention include, but are not limited to: one or more of water, salt solution, buffer, glucose, glycerol, ethanol, polyol.

[0199] The pharmaceutical composition of the present invention can be provided in the form of, for example, a sterile preparation. Forms of sterile preparations include, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions, etc. The solvent or medium of the pharmaceutical composition can be one or several of water, ethanol, or polyols. The pharmaceutical composition of the present invention can be made sterile by, for example, filtering through a sterile filtration membrane. When the composition is freeze-dried, this method can be used for sterilization before, during, or after freeze-drying, reconstitution, or dilution. The composition for parenteral administration can be stored in a freeze-dried form or in solution. It can be prepared by conventional methods, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be pierced by a hypodermic needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as orally). The pharmaceutical composition of the present invention can be provided in other forms that are obvious to those skilled in the art, for example, as a sustained-release or controlled-release composition. Such as a sustained-release or controlled-release composition containing the antibody, chimeric antigen receptor, or engineered immune effector cells described in the present invention. Techniques for preparing sustained-release or controlled-release drugs (such as liposomal carriers, bioerodible microparticles, porous beads, or depot injections) are also well known to those skilled in the art. For example, the extended absorption of an injectable composition can be achieved by including absorption-retarding excipients such as monostearic acid or gelatin in the composition. The pharmaceutical composition can be stored in a sterile vial in the form of a solution, suspension, gel, emulsion, viscous composition, solid, crystal, or freeze-dried powder. The pharmaceutical composition can be stored in a ready-to-use form or in a form that is reconstituted and processed before administration. The preparation method of the pharmaceutical composition of the present invention is obvious to those skilled in the art.

[0200] The pharmaceutical composition of the present invention can be used in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The pharmaceutical composition of the present invention can be injected, for example, orally, nasally, intravenously, intraperitoneally, intracerebrally (intraparenchymal), intraventricularly, intramuscularly, intraocularly, intraarterially, via the portal vein, or intralesionally, and can also be administered through a sustained-release system or through an implant device. In some embodiments, the pharmaceutical composition of the present invention is administered parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0201] A therapeutically effective amount of a pharmaceutical composition comprising a mesothelin-targeting single-domain antibody, chimeric antigen receptor or engineered immune effector cell of the present invention will depend, for example, on the degree of treatment and the target. Those skilled in the art will appreciate that appropriate dosage levels for treatment will depend in part on the molecule being delivered, the indication, the route of administration and the patient's circumstances (body weight, body surface or organ size) and / or condition (age and general health). In some embodiments, the clinician may adjust the dosage and change the route of administration to achieve optimal therapeutic effects.

[0202] The frequency of administration will depend on the pharmacokinetic parameters of the pharmaceutical composition of the present invention. Clinicians typically administer the pharmaceutical composition until a dose is reached that achieves the desired effect. Thus, the pharmaceutical composition may be administered as a single dose, or over time in two or more doses (which may or may not contain the same amount of the desired molecule), or by continuous infusion via an implant device or catheter.

[0203] According to a seventh aspect of the present disclosure, the present invention provides the use of a mesothelin-targeting single-domain antibody, chimeric antigen receptor, engineered immune effector cell, or pharmaceutical composition as described above in the preparation of a medicament for the diagnosis, prevention, and / or treatment of a disease or disorder.

[0204] The present invention also provides a method of treating a subject having a disease associated with mesothelin expression, comprising administering to the subject an effective amount of an engineered immune effector cell or pharmaceutical composition according to the present invention. Thus, the present invention provides the use of a mesothelin-targeting single-domain antibody, chimeric antigen receptor, engineered immune effector cell, or pharmaceutical composition as described in the present invention in the preparation of a medicament for the diagnosis, prevention, and / or treatment of a disease or disorder.

[0205] In some embodiments, the disease or disorder comprises a disease or disorder associated with mesothelin expression.

[0206] In some embodiments, the disease or disorder comprises a cancer or tumor associated with mesothelin expression.

[0207] In some embodiments, the disease or disorder comprises a solid tumor associated with mesothelin expression.

[0208] In some embodiments, the disease or disorder comprises mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, cholangiocarcinoma, gastric cancer, colon cancer, rectal cancer, bladder cancer, prostate cancer, cervical cancer, thymic cancer, endometrial cancer, breast cancer, thyroid cancer, or peritoneal cancer.

[0209] In some embodiments, the disease or disorder includes ovarian tumor, lung tumor, non-small cell lung cancer, squamous cell lung cancer, fallopian tube cancer, peritoneal mesothelioma, pleural mesothelioma, epithelial ovarian cancer, non-squamous non-small cell lung cancer, triple-negative breast cancer, colorectal cancer, colorectal tumor, metastatic pancreatic ductal adenocarcinoma, high-grade ovarian serous carcinoma, Glioma, head and neck cancer or Malignant Pleural Effusions (Mpe).

[0210] The single-domain antibodies, nanobodies or heavy-chain antibodies of the present invention can be prepared by conventional methods in the art, such as the well-known phage display technology in the art. Alternatively, the various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with the sequences encoding the various antibodies of the present invention. The transformation can be carried out by any known method, for example, including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedures used depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulating the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus, etc. Mammalian host cell lines that can be used for expression are well known in the art, such as various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma (HepG2) cells, etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantially mesothelin-binding properties.

[0211] The chimeric antigen receptors of the present invention can be prepared by conventional methods in the art. See, for example, Park et al, Trends Biotechnol., 29:550-557, 2011; Grupp et al, N Engl J Med., 368:1509-1518, 2013; Han et al, J. Hematol. Oncol., 6:47, 2013.

[0212] As is well known to those skilled in the art, due to the degeneracy of the genetic code, a large number of nucleic acids can be prepared, all of which encode the chimeric antigen receptor of the present invention. Therefore, in the case where a specific amino acid sequence has been identified, those skilled in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Accordingly, the present invention also relates to polynucleotides that hybridize to the above polynucleotide sequences and have at least 70%, preferably at least 80%, more preferably at least 90% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.

[0213] The nucleic acid full-length sequences or fragments of the single-domain antibodies and chimeric antigen receptors of the present invention can be obtained by conventional techniques. For example, they can generally be obtained by PCR amplification, recombination, or artificial synthesis. Using the method of artificial synthesis to synthesize the relevant sequences is a feasible method, especially for sequences with shorter fragment lengths. Generally, multiple small fragments can be synthesized first and then ligated to obtain longer fragments. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0214] Once the relevant sequences are obtained, they can be obtained in large quantities by recombination. This is usually done by cloning them into a vector, then transferring them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) involved in the present invention include biomolecules in an isolated form. Currently, it is already possible to obtain the DNA sequence encoding the polypeptide of the present invention (or its fragment, or its derivative) entirely by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the polypeptide sequence of the present invention by chemical synthesis.

[0215] The present invention also relates to nucleic acid constructs comprising the above nucleic acid sequences and appropriate promoters or control sequences, such as expression vectors and recombinant vectors. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors usually contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The said sequences (collectively referred to as "flanking sequences" in some embodiments) generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence encoding for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting the nucleic acid encoding the antibody to be expressed, and optional marker elements.

[0216] Nucleic acids can be cloned into vectors using any known molecular cloning method in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters for gene expression in mammalian cells have been disclosed in the prior art, and any promoter known in the art can be used in the present invention. Promoters can be further classified as constitutive promoters or regulatable promoters, such as inducible promoters.

[0217] In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of heterologous genes (also referred to as transgenes) in host cells. Exemplary constitutive promoters of the present invention include, but are not limited to, the cytomegalovirus (CMV) promoter, the human elongation factor-1α (hEF1α) promoter, the ubiquitin C (UbiC) promoter, the phosphoglycerate kinase (PGK) promoter, the simian virus 40 (SV40) early promoter, and the chicken β-actin coupled with the CMV early enhancer (CAGG) promoter. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a large number of studies. For example, Michael C. Milone et al. (Molecular Therapy, 17(8):1453-1464 (2009)) compared the efficiencies of CMV, hEF1α, UbiC, and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF1α promoter not only induced the highest level of transgene expression but also maintained optimal expression in CD4 and CD8 human T cells. In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to the hEF1α promoter.

[0218] In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to an inducible promoter. Inducible promoters belong to the regulatable promoter category. Inducible promoters can be induced by one or more conditions, such as physical conditions, inducers, etc.

[0219] In some embodiments, the expression of endogenous genes in engineered mammalian cells and / or subjects receiving the pharmaceutical composition is induced under the inducing conditions. In some embodiments, the inducing conditions are selected from: inducers, radiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor microenvironment, and the activation state of engineered mammalian cells.

[0220] Viral vector technology is well-known in the art and is described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and other virology and molecular biology manuals.

[0221] In the prior art, many virus-based systems have been developed for gene transfer into mammalian cells. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated in vitro or ex vivo and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells). In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying coding sequences of immunomodulators (e.g., immune checkpoint inhibitors) and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged using protocols known in the art.

[0222] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well-known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase, treated with the CaCl2 method, and the steps used are well-known in the art. Another method is to use MgCl2. In addition, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0223] The obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the nucleic acid molecules of the present invention. Depending on the host cells used, the culture medium used in the culture can be selected from various conventional media. The culture is carried out under conditions suitable for the growth of the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0224] The polypeptide or protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant polypeptide or protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatments, treatment with protein precipitants (salting-out methods), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0225] The engineered immune effector cells of the present invention are prepared by introducing a chimeric antigen receptor into immune effector cells (such as T cells).

[0226] The nucleic acid sequence encoding the chimeric antigen receptor can be introduced into immune effector cells using conventional methods known in the art (such as by transfection, transduction, transformation, etc.).

[0227] Methods for introducing a vector or isolated nucleic acid into immune effector cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical, or biological methods.

[0228] Physical methods for introducing a vector into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for preparing cells containing a vector and / or exogenous nucleic acid are well known to those skilled in the art and are described, for example, in Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. and other virology and molecular biology manuals. In some embodiments, the vector is introduced into immune effector cells by electroporation.

[0229] Biological methods for introducing a vector into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells (such as human cells).

[0230] Chemical methods for introducing a vector into immune effector cells include colloidal dispersion systems, such as including macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vector is liposomes.

[0231] In some embodiments, the vector further comprises a selectable marker gene or a reporter gene to select cells expressing the chimeric antigen receptor from a population of host cells transfected with the lentiviral vector. Appropriate regulatory sequences can flank both the selectable marker and the reporter gene for expression in the host cell. For example, the vector can contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of nucleic acid sequences.

[0232] Reporter genes can be used to identify cells that may have been transfected and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or not expressed by the recipient organism or tissue, and that encodes a polypeptide that expresses some easily detectable property, such as enzyme activity. Expression of the reporter gene is assayed at an appropriate time after introduction of the DNA into the recipient cells. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (see Kumiko Ui-Tei. FEBS Letters, 479:79 - 82 (2000)). Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially. Other methods for confirming the presence of the nucleic acid encoding the chimeric antigen receptor in engineered immune effector cells include: molecular biology test methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; biochemical assay methods, such as detecting the presence or absence of a specific peptide; and immunological methods, such as ELISA.

[0233] In some embodiments, the nucleic acid molecule encoding any of the chimeric antigen receptors described herein can be prepared by conventional methods (such as in vitro transcription) and then introduced into immune effector cells by known methods such as mRNA electroporation (see Peter M Rabinovich. Human Gene Therapy, 17:1027 - 1035 (2006)).

[0234] In some embodiments, transduced or transfected immune effector cells are cultured ex vivo to proliferate after introduction of the vector or the isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days.

[0235] Term Explanation

[0236] Unless otherwise indicated, the meaning of all terms used herein is the same as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0237] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with exactly the same sequence have 100% identity. Those skilled in the art know that some algorithms can be used to determine sequence identity, such as Blast, Blast2. In addition, it can be determined using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN.

[0238] As used herein, the term "derived from" refers to the relationship between two, usually referring to the structural similarity between them. For example, in the case of an intracellular signaling domain derived from CD3ζ, the intracellular signaling domain retains sufficient CD3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. This term does not imply or include a limitation on the specific process for generating the intracellular signaling domain. For example, "derived from" does not mean that in order to provide the intracellular signaling domain, one must start with the CD3ζ sequence and delete unwanted sequences or impose mutations to arrive at the intracellular signaling domain.

[0239] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen (such as a target antigen). An antibody can be a complete immunoglobulin from a natural source or a recombinant source, or an immunoreactive portion of a complete immunoglobulin. Non-limiting examples of antibodies include monoclonal antibodies (including complete antibodies), single-domain antibodies, single-chain antibodies (scFv), heavy-chain antibodies (HCAb), light-chain antibodies (LCAb), monovalent antibodies. "Antibody" also includes antibody fragments or synthetic polypeptides carrying one or more CDR sequences that are capable of exhibiting the desired biological activity, especially antigen-binding fragments, such as Fab, F(ab’)2 and Fv. In some embodiments of the present invention, the terms "immunoglobulin (Ig)" and "antibody" can be used interchangeably.

[0240] As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to at least a part or fragment of a complete antibody or its recombinant variant that contains the antigen-binding site (such as the CDR region) of the complete antibody and thus retains the ability to bind to an antigen. Examples of antibody fragments include, but are not limited to: Fab, Fab’, F(ab)2, scFv, the heavy-chain variable region (VH) of an antibody, linear antibodies, single-domain antibodies, nanobodies, natural ligands of an antigen or functional fragments thereof, etc.

[0241] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0242] As used herein, the term "antigen" or "Ag" refers to a molecule that elicits an immune response. The immune response can involve the production of antibodies or the activation of specific immunologically active cells, or both. One of ordinary skill in the art will appreciate that virtually any macromolecule that includes all proteins or peptides can serve as an antigen. In addition, an antigen can be derived from recombinant or genomic DNA. One of ordinary skill in the art will appreciate that any DNA that contains a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen". An antigen can be produced synthetically, or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or fluids with other biological components.

[0243] As used herein, the term "functional fragment" or "variant" refers to a variant that substantially comprises the amino acid sequence of the parent but contains at least one amino acid modification (e.g., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In some embodiments, the amino acid modification is a conservative modification.

[0244] Heavy chain antibodies are antibodies derived from organisms of the Camelidae family or Chondrichthyes. Compared with 4-chain antibodies, heavy chain antibodies lack the light chain and the heavy chain constant region 1 (CH1), and only contain 2 heavy chains composed of a variable region (VHH) and other constant regions. The variable region is connected to the constant region through a structure similar to the hinge region. Each heavy chain of a Camelidae heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of a Chondrichthyes heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can have the CH2 and CH3 of human IgG Fc.

[0245] Single domain antibodies (sdAbs) can have the same or different origins and can have the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy chain variable domains from only heavy chain antibodies (such as VHHs), binding molecules that are naturally devoid of light chains, single domains (such as VH or VL) derived from conventional 4-chain antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and engineered domains and single domain scaffolds that are not derived from antibodies. Any sdAb known in the art or disclosed by the present invention, including the single domain antibodies disclosed in the present invention, can be used to construct the CARs described herein. SdAbs can be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, sharks, goats, rabbits, and cows. The single domain antibodies of the present invention also include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0246] In the present invention, the terms "single domain antibody", "single domain antibody targeting mesothelin", "heavy chain single domain antibody", "VHH", "nanobody" can be used interchangeably and all refer to single domain antibodies that specifically recognize and bind to mesothelin. Generally, a single domain antibody contains three CDR regions and four FR regions. A single domain antibody is the smallest functional antigen-binding fragment. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region of the heavy chain (CH1), the variable region of the antibody heavy chain is then cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0247] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid sequences within the variable region of an antibody that determine antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable region (such as HCDR1, HCDR2, and HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3).

[0248] The portion of the chimeric antigen receptor comprising an antibody or an antibody fragment thereof can exist in a variety of forms, such as where the antigen-binding domain is expressed as part of a polypeptide chain (including, for example, single-domain antibody fragments (sdAb), single-chain antibodies (scFv), or, for example, human or humanized antibodies), see Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426.

[0249] As used herein, the term "antigen-binding domain" refers to the portion of the chimeric antigen receptor that specifically binds to a target antigen, and the chimeric antigen receptor can direct T cells and / or other immune cells to a selected target using its antigen-binding properties. The antigen-binding domain typically can comprise the variable region of the antibody light chain (VL) and the variable region of the antibody heavy chain (VH), but it need not contain both variable regions simultaneously. For example, it can include Fab fragments, Fab' fragments, single Fv fragments such as scFv, single-domain antibodies, etc. having antigen-binding activity.

[0250] As used herein, the term "transmembrane domain" is used to link the extracellular domain and the intracellular domain of the chimeric antigen receptor. The transmembrane domain can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains include TCRα, TCRβ, TCRγ, TCRδ, TCRζ, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1.

[0251] As used herein, the term "intracellular signaling domain" refers to the functional portion of a protein, i.e., the functional portion of an intracellular signaling domain that is sufficient to transduce effector function signals. The intracellular signaling domain is responsible for primary intracellular signal transduction following antigen binding by the antigen-binding domain, resulting in the activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of an immune cell in which a chimeric antigen receptor is expressed. For example, the effector functions of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines. In one embodiment, the intracellular signaling domain comprised by the chimeric antigen receptor of the invention can be the cytoplasmic sequences of a T cell receptor and co-receptor that act together to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The intracellular signaling domain can comprise a number of immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of intracellular signaling domains include, but are not limited to, FcRγ, FcRβ, TCRζ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d.

[0252] As used herein, the term "hinge region" refers to any oligopeptide or polypeptide used to link a transmembrane domain and an antigen-binding domain. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antigen-binding domain. The hinge region can comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region can be derived in whole or in part from a natural molecule, such as the extracellular region of CD8 or CD28 in whole or in part, or the constant region of an antibody in whole or in part. The hinge region can also be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence.

[0253] The different domains of a chimeric antigen receptor (CAR) can also be fused to each other via peptide linkers. Depending on the structure and / or functional characteristics of the single-domain antibody and / or the various domains, each peptide linker in the chimeric antigen receptor can have the same or different lengths and / or sequences. Those skilled in the art can independently select and optimize each peptide linker. In some embodiments, the peptide linker consists of amino acids linked together by peptide bonds, where the amino acids are selected from the 20 naturally occurring amino acids: glycine, alanine, valine, leucine, isoleucine, serine, cysteine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine, and glutamine. As understood by those skilled in the art, one or more of these amino acids can be glycosylated. In some embodiments, the peptide linker contains flexible residues (such as glycine and serine) such that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0254] The peptide linker can have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids in length. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids in length. In some embodiments, the length of the peptide linker is from about 1 amino acid to about 10 amino acids, from about 1 amino acid to about 20 amino acids, from about 1 amino acid to about 30 amino acids, from about 5 amino acids to about 15 amino acids, from about 10 amino acids to about 25 amino acids, from about 5 amino acids to about 30 amino acids, from about 10 amino acids to about 30 amino acids, from about 30 amino acids to about 50 amino acids, from about 50 amino acids to about 100 amino acids, or from about 1 amino acid to about 100 amino acids.

[0255] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy-chain only antibody can be used as a linker. See, e.g., WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include but are not limited to glycine polymers (G)n, glycine-serine polymers (such as (GS)n, (GSG)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0256] As used herein, the term "costimulatory signal domain" may be an intracellular functional signaling domain derived from a costimulatory molecule, which includes the entire intracellular portion of the costimulatory molecule or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as proliferation, differentiation) of the immune cell. Non-limiting examples of costimulatory domains include, but are not limited to, the intracellular regions of the following proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CDS, ICAM, CD137 (4-1BB), CD276 (B7-H3), CD278 (ICOS), GITR, BAFFR, LIGHT, HVEM (LIGHTR), BTLA, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, ITGB7, NKG2D, NKG2C, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

[0257] As used herein, the term "signal peptide" can direct a nascent protein to the endoplasmic reticulum and subsequently to the cell surface when a chimeric antigen receptor is expressed in a cell (e.g., a T cell). Generally, the core of a signal peptide can contain a long stretch of hydrophobic amino acids that has a tendency to form a single α-helix. A signal peptide directs the translocation and / or secretion of the translated protein across a membrane. At the end of the signal peptide, there is usually an amino acid segment that is recognized and cleaved by signal peptidase. Signal peptidase can cleave during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then degraded by specific proteases. A signal peptide can also be referred to as a targeting signal, transit peptide, localization signal, or signal sequence. For example, a signal sequence can be a co-translational or post-translational signal peptide. Signal peptides commonly used in the art are derived from HLA-A, CD8α, CD4, CD33, CD137, GM-CSFRα, IgG1, Igκ, IL-2, or any combination thereof.

[0258] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, and polymers thereof. The DNA or RNA includes natural, synthetic, or artificial nucleotide analogs or bases. Unless explicitly defined otherwise, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly specified sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more of the three positions of the selected (or all) codons are replaced with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0259] As used herein, the term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template in a biological process for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the corresponding mRNA results in the production of the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is typically provided in the sequence listing, and the non-coding strand that serves as the template for transcribing the gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of one another and encode the same amino acid sequence. Nucleotide sequences encoding a protein or RNA may also include introns.

[0260] As used herein, a "vector" comprising an isolated nucleic acid refers to a nucleic acid vehicle into which a nucleotide encoding a protein can be inserted and which enables expression of the protein. A vector can transform, transduce, or transfect a host cell to cause expression of the genetic elements carried by the vector in the host cell. Non-limiting examples of vectors include plasmid vectors, phage vectors, and viral vectors. A vector can contain one or more elements that control expression. Additionally, a vector can contain an origin of replication.

[0261] As used herein, the term "immune effector cell" refers to an immune cell that can perform immune effector functions (such as cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). For example, immune effector cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and / or NKT cells, or immune cells derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells, etc. When the immune effector cell is a T cell, the T cell can be any T cell, such as T cells cultured in vitro, for example, primary T cells, or T cells from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. T cells can also be concentrated or purified. T cells can be at any stage of development, including but not limited to, CD4+ / CD8+ T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), CD4- / CD8- T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc.

[0262] As used herein, the terms "patient", "subject", "individual", "object" are used interchangeably and include any living organism, preferably an animal, more preferably a mammal (such as a rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

[0263] As used herein, the term "treatment" refers to administering to a subject the treatment method described in the present invention to achieve at least one positive therapeutic effect (such as a reduction in the number of cancer cells, a decrease in tumor volume, a reduction in the rate of cancer cell infiltration into surrounding organs or a reduction in the rate of tumor metastasis or tumor growth). The treatment method for effectively treating a patient can be adjusted according to various factors (such as the disease state, age, weight of the patient and the ability of the therapy to stimulate the anti-cancer response of the subject). BRIEF DESCRIPTION OF THE DRAWINGS

[0264] Figure 1A Shows the expression rate of the CAR molecule targeting mesothelin on the prepared CD4+ CAR-T cells in each group.

[0265] Figure 1B Shows the expression rate of the CAR molecule targeting mesothelin on the prepared CD8+ CAR-T cells in each group.

[0266] Figure 2AShows the cumulative proliferation fold of CD4+ T cells after 11 days of culture of the prepared CAR-T cells.

[0267] Figure 2B Shows the cumulative proliferation fold of CD8+ T cells after 11 days of culture of the prepared CAR-T cells.

[0268] Figure 3 Shows the proportion of T SCM +T CM in CAR-T cells after 11 days of culture of the prepared CAR-T cells.

[0269] Figure 4 Shows the proportion of CAR-T cells expressing Tim3 after 11 days of culture of the prepared CAR-T cells.

[0270] Figure 5 Shows the expression of mesothelin antigen on the surface of various target cells: Nalm6-EGFP-MES cells, Nalm6-EGFP cells, and NCI-H226 cells measured separately.

[0271] Figure 6A Shows the results of flow cytometry for the in vitro killing rate of effector cells in each group against Nalm6-EGFP-MES cells on the 1st day when the effector-to-target ratio (E:T) = 1:1.

[0272] Figure 6B Shows the results of flow cytometry for the in vitro killing rate of effector cells in each group against Nalm6-EGFP-MES cells on the 3rd day when the effector-to-target ratio (E:T) = 1:1.

[0273] Figure 7A Shows the results of flow cytometry for the in vitro killing rate of effector cells in each group against Nalm6-EGFP-MES cells on the 1st day when the effector-to-target ratio (E:T) = 1:3.

[0274] Figure 7B Shows the results of flow cytometry for the in vitro killing rate of effector cells in each group against Nalm6-EGFP-MES cells on the 3rd day when the effector-to-target ratio (E:T) = 1:3.

[0275] Figure 8A Shows the results of flow cytometry for the in vitro killing rate of effector cells in each group against Nalm6-EGFP cells on the 1st day when the effector-to-target ratio (E:T) = 1:1.

[0276] Figure 8B Shows the results of flow cytometry for the in vitro killing rate of effector cells in each group against Nalm6-EGFP cells on the 3rd day when the effector-to-target ratio (E:T) = 1:1.

[0277] Figure 9 Shows the results of the LDH release assay for the in vitro killing rate of effector cells in each group against NCI-H226 cells on the 1st day when the effector-to-target ratio (E:T) = 1:1. Detailed implementation

[0278] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to the conventional methods and conditions in the art or according to the product specifications.

[0279] Example 1. Preparation of a single-domain antibody (VHH) targeting mesothelin

[0280] (1) Animal immunization and immune response test

[0281] Healthy alpacas were selected as the immunization subjects. At the first immunization, complete Freund's adjuvant was mixed with 0.8 mg of Mesothelin antigen Human Mesothelin / MSLN(296-580)Protein,Fc Tag(MALS verified) in a ratio of 1:1 and emulsified, and then injected subcutaneously at multiple points. For subsequent booster immunizations, incomplete Freund's adjuvant was mixed with Mesothelin antigen Human Mesothelin / MSLN(296-580)Protein,Fc Tag(MALS verified) in a ratio of 1:1. The immunization interval was 2 weeks, and a total of 5 immunizations were carried out; Peripheral blood was collected before immunization and after each immunization to isolate serum, and the immune response was monitored by ELISA to confirm the serum titer. After the 5th immunization, the titer reached the level of 100,000, indicating good immune effect. 50 mL of blood was collected, and the lymphocytes were isolated and stored in Trizol for subsequent construction of the antibody phage library.

[0282] (2) Construction of the antibody phage library

[0283] 1) RNA extraction and reverse transcription

[0284] RNA was extracted from the isolated lymphocytes, and then using the Takara reverse transcription kit, the total RNA obtained by extraction was divided into two equal parts and reverse transcribed into cDNA according to the instructions of the reverse transcription kit. The reverse transcription primers were Oligo dT Primer and Random 6-mers respectively.

[0285] 2) PCR amplification

[0286] Specific antibody fragments were amplified from reverse-transcribed cDNA using the PCR method. Using the cDNA as a template, PCR amplification was carried out using Taq DNA Polymerase Hot Start enzyme. All PCR products were subjected to 1% agarose gel electrophoresis, and the bands with a target fragment size of approximately 600 - 700 bp were excised and recovered. These were the products of the first-round PCR amplification and were stored at -20°C.

[0287] The products of the first-round PCR amplification were used as a template for the second-round PCR reaction. After the reaction, 1% agarose gel electrophoresis was performed. Finally, the bands with a single target band and a fragment size of approximately 400 bp were excised and recovered, and a universal DNA purification and recovery kit was used to purify the DNA in the PCR reaction solution to obtain the products of the second-round PCR amplification, namely the VHH target gene fragments.

[0288] 3) Restriction Enzyme Digestion and Ligation

[0289] The VHH target gene fragments obtained by the second-round PCR amplification and the pComb3XSS phagemid vector were digested with the restriction enzymes Spe I and Sac I respectively. After digestion, the VHH target gene fragments were ligated to the pComb3XSS phagemid vector using T4 ligase to construct a recombinant plasmid, obtaining a ligation product.

[0290] 4) Electroporation and Bacterial Library Construction

[0291] Take 100 ng of the above ligation product and electrotransform it into TG1 competent cells. The electroporation parameters are: electroporator BIO-RAD, GENE PUISER XCEll, voltage 1.8 KV, electroporation cuvette 1 mm. Immediately after electroporation, add 1 mL of SOC medium (preheated at 37°C) to the electroporation cuvette, mix well, and shake the bacteria at 37°C and 200 rpm for 1 h for recovery as the recovery product. Continue to take about 20 systems of 100 ng ligation product and perform electroporation reactions using TG1 competent cells according to the above steps. Take 100 μL from the recovery product, dilute it in a 10-fold gradient, and spread it on plates, and culture overnight at 37°C. Spread all the remaining recovery products onto about 20 15-cm culture plates (2×YT medium, containing 100 μg / mL Amp, 2% agarose), and culture them inverted at 37°C overnight. Calculate the number of transformed colonies that can be obtained from all reactions according to the dilution factor and the number of single colonies, which is the library capacity of the bacterial library. At the same time, randomly select several monoclonal colonies from the gradient dilution plates for colony PCR. A single band of about 400 bp in the PCR product is considered a positive clone, and the cloning positive rate of the bacterial library is estimated accordingly. Scrape the colonies on the overnight-cultured plates with 2×YT liquid medium, place them in a 50-mL centrifuge tube, measure their OD600 value, add glycerol with a final concentration of 20%, and store at -80°C.

[0292] 5) Phage library construction and purification

[0293] Inoculate the bacterial library into 100 mL of 2×YT liquid medium (containing 100 μg / mL Amp) to make the initial OD600 value 0.1, and culture at 37 °C and 250 rpm until OD600 reaches 0.5 - 0.55. Add helper phage at a ratio of 1:20 (number of bacteria: number of helper phages), incubate at 37 °C and 250 rpm for 30 min, then add kanamycin with a final concentration of 50 μg / mL, and culture overnight at 30 °C and 250 rpm. Continue to centrifuge the overnight culture and collect the supernatant, then add pre-cooled PEG / NaCl with a volume of 1 / 4 of the supernatant, mix well, incubate on ice for at least 30 min, then centrifuge at 4 °C and 4000 rpm for 20 minutes. After removing the supernatant, add 1 mL of PBS buffer to dissolve the precipitate. Add pre-cooled PEG / NaCl with a volume of 1 / 4 again and incubate on ice for 10 minutes, then centrifuge at 4 °C and 12000 g for 10 minutes. Remove the supernatant and dissolve the precipitate in 1 mL of PBS, and store at -80 °C, which is the purified phage library.

[0294] (3) Phage screening

[0295] 1) First round of screening

[0296] Coat the screening antigen on the immunotubes (50 μg / tube, coating solution is PBS, 2 mL / tube), rotate slowly at 4 °C overnight. At the same time, coat BSA (50 μg in PBS, 2 mL / tube) in parallel as a control. After overnight, discard the supernatant in the immunotubes coated overnight, wash the immunotubes 3 times with PBS buffer at room temperature, rotate for 5 min each time. Then add 2 mL of blocking solution (3% skim milk powder) solution, rotate and block at room temperature for 2 h, then discard the supernatant, and add 2 mL of PBST (1×PBS plus 0.1% Tween20) buffer to wash the immunotubes 3 times at room temperature, rotate for 5 min each time. Discard the washing solution in the immunotubes, add about 10 12 pfu of the prepared phage library as the input phage library for the first round of screening, add PBS buffer to 2 mL, and rotate and incubate at room temperature for 1 h, then discard the supernatant, add 2 mL of PBST (1×PBS plus 0.1% Tween20) buffer to wash the immunotubes 20 times at room temperature, rotate for 5 min each time. Discard the liquid in the immunotubes, add 1 mL of 0.25 mg / mL trypsin solution, rotate and elute at room temperature for 30 min. Then add 10 μL of 10% AEBSF to terminate the elution, transfer the solution in the immunotubes to a new 1.5 mL centrifuge tube, which is the first-round phage eluate.

[0297] 2) Titer detection of the first-round phage eluate

[0298] Take 10 μL of the first-round phage eluate, and dilute it in a 1.5 mL centrifuge tube at a 10-fold gradient for a total of 12 gradients until the concentration reaches 10 -12 . Add 90 μL of TG1 bacterial solution to each dilution centrifuge tube, mix well by shaking, and incubate at 37 °C for 30 min. Take 5 μL from each dilution centrifuge tube and drop it onto a 2×YT solid medium (Amp), let it stand for a few minutes, and then incubate it overnight at 37 °C in an inverted position. Count and calculate the phage titer. Count the number of single colonies at the dilution where single colonies can be clearly distinguished on the plate, and calculate the number of phagemids in each milliliter of the phage solution, that is, the phage library titer, according to the following formula:

[0299] T (pfu / ml) = N × D × 400

[0300] Where T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0301] 3) Amplification of the first-round phage eluate

[0302] Amplify and purify the first-round phage eluate according to the method in "(5) Phage library construction and purification".

[0303] Repeat the above experiment 3 times, and use the phage from the first round as the input phage library for the second-round screening to obtain the second-round phage eluate. Similarly, use the phage from the second round as the input phage library for the third-round screening to obtain the third-round phage eluate.

[0304] (4) Monoclonal ELISA detection

[0305] Take the bacterial solution at an appropriate dilution after the third-round screening, evenly coat it on a solid medium plate containing 100 μg / mL Amp, and incubate it overnight at 37 °C. Randomly pick 192 monoclonal colonies from the overnight-cultured medium plate into a sterile 96-well cell culture plate, add 200 μL of 2×YT medium (containing 100 μg / mL Amp) to each well, and incubate it overnight at 37 °C with static culture. Take 5 μL of the overnight-cultured bacterial solution and transfer it to a new 96-well cell culture plate with 200 μL of 2×YT liquid medium (containing 100 μg / mL Amp) in each well, and incubate it statically at 37 °C for 5 h. Then add the helper phage M13K07 to each well, where the ratio of the number of bacteria to the number of phages is 1:20. After incubating at 37 °C for 30 min, add kanamycin with a final concentration of 50 μg / mL, and incubate it statically overnight at 30 °C. Then centrifuge the 96-well cell culture plate and store it at 4 °C as the centrifuged supernatant for standby.

[0306] Coat an ELISA plate with the screened antigen (1 ng / μL, PBS, 100 μL / well), and simultaneously coat BSA at the same concentration in parallel as a control. Incubate overnight at 4°C. Discard the supernatant, and wash the ELISA plate 3 times with PBS buffer at room temperature for 10 min each time. Add 200 μL of blocking solution (3% BSA in PBST) to each well to block the ELISA plate, and incubate at room temperature for 1 h. Discard the blocking solution, add 200 μL of PBST (1×PBS plus 0.1% Tween 20) buffer to each well, and wash the ELISA plate 3 times at room temperature for 10 min each time. Add 100 μL of blocking solution to each well, and then add 100 μL of the above-prepared centrifuged supernatant. Incubate at room temperature for 2 h. Discard the liquid in the ELISA plate, add 200 μL of PBST (1×PBS plus 0.1% Tween 20) buffer to each well and wash 3 times for 10 min each time. Add M13 Bacteriophage Antibody (HRP), Mouse Mab, diluted in the blocking solution at a ratio of 1:30000, 100 μL / well, and incubate at room temperature for 1 h. Discard the liquid in the ELISA plate, add 200 μL of PBST (1×PBS plus 0.1% Tween 20) buffer to each well and wash 6 times for 5 min each time. Add 100 μL of TMB single-component chromogenic solution to each well, develop color in the dark for 1 - 3 min, add 100 μL of 1 M HCl to each well to terminate the reaction, and read the OD450 value with an ELISA reader. Sequence the positive clone colonies to obtain the gene sequence of the VHH single-domain antibody.

[0307] Example 2. Preparation of Mesothelin-Targeted CAR-T Cells

[0308] (1) Construction of Mesothelin-Targeted CAR

[0309] The CAR nucleotide sequences (SEQ ID NO: 96 - 113) targeting mesothelin were designed and artificially synthesized for each group. The nucleotide sequences of each group included the HLA - A signal peptide (SEQ ID NO: 73), the extracellular antigen - binding domain of Mesothelin - VHH (SEQ ID NO: 55 - 72), the CD8α hinge region (SEQ ID NO: 74), the CD8α transmembrane domain (SEQ ID NO: 75), the 4 - 1BB (CD137) co - stimulatory signal domain (SEQ ID NO: 76), and the CD3ζ intracellular signal transduction domain (SEQ ID NO: 77), which were used to express the complete Mesothelin - CAR polypeptide molecules (SEQ ID NO: 78 - 95) of each experimental group, denoted as groups M1, M5, M8, M9, M14, M16, M17, M18, M23, M28, M29, M31, M34, M41, M44, M46, M49, and M60. The Mesothelin - CAR nucleotide sequences were inserted into the multiple cloning site of the lentiviral expression vector pK1 through homologous recombination to obtain pK1 - Mesothelin - CAR. The successful construction of the lentiviral expression vector sequence was confirmed by electrophoresis and sequencing results.

[0310] In addition, the positive control CAR molecule Meso3 was constructed by the same method. The single - chain antibody sequence against the third region of mesothelin (see the amino acid residues at positions 23 - 272 of SEQ ID NO: 1 in CN109971714A) disclosed in the patent document CN109971714A was used as the antigen - binding domain of the positive control CAR molecule Meso3.

[0311] Amino acid sequence of the positive control CAR molecule Meso3:

[0312] AVMAPRTLLLLLSGALALTQTWAEVQLVESGGGLVQPGGSLRLSCAASGFDLGFYFYACW

[0313] VRQAPGKGLEWVSCIYTAGSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

[0314] CARSTANTRSTYYLNLWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDR

[0315] VTITCQASQRISSYLSWYQQKPGKVPKLLIYGASTLASGVPSRFSGSGSGTDFTLTISSLQPE

[0316] DVATYYCQSYAYFDSNNWHAFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG

[0317] GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEE

[0318] DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

[0319] EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:114)

[0320] Nucleic acid sequence of the positive control CAR molecule Meso3:

[0321] GCTGTGATGGCCCCTAGAACCCTGCTGCTGCTGCTGAGCGGCGCCCTGGCCCTGACACA

[0322] GACCTGGGCCGAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGGGGG

[0323] ATCCCTGAGACTCTCCTGCGCAGCCTCTGGATTCGACCTCGGTTTCTACTTTTACGCCTG

[0324] TTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTCTCATGCATTTATACTGCTG

[0325] GTAGTGGTAGCACGTACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAGAC

[0326] AATTCGAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCG

[0327] TGTATTACTGTGCGAGATCTACTGCTAATACTAGAAGTACTTATTATCTTAACTTGTGGGG

[0328] CCAAGGCACCCTGGTCACCGTCTCCTCAGGCGGAGGCGGATCAGGTGGTGGCGGATCT

[0329] GGAGGTGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGT

[0330] GGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGGATTAGTAGTTACTTATCCT

[0331] GGTATCAGCAGAAACCAGGGAAAGTTCCCAAGCTCCTGATCTATGGTGCATCCACTCTG

[0332] GCATCTGGGGTCCCCTCGCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCAC

[0333] CATCAGCAGCCTGCAGCCTGAAGATGTTGCCACTTACTACTGTCAGAGTTATGCTTATTT

[0334] TGATAGTAATAATTGGCATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGA

[0335] CGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCT

[0336] GCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGA

[0337] CTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCT

[0338] GTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA

[0339] ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGAT

[0340] TTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAG

[0341] ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA

[0342] AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA

[0343] AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT

[0344] GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA

[0345] CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:115)

[0346] (2) Packaging of lentiviral vector

[0347] Resuscitate 293T cells and culture them in DMEM medium containing 10% FBS. After 2 - 3 passages of cell expansion culture, at a density of 4×10 4 cells / cm 2Inoculate at a density into a 2-layer cell factory. Three days after cell inoculation, plasmid transfection is carried out. After adding 40 mL of Opti-MEM into a sterile 50 mL centrifuge tube for plasmid transfection, add viral packaging vectors and viral envelope vectors according to the ratio of pK1-Mesothelin-CAR:pLP1:pLP2:pLP-VSVG = 5:4:3:1, and add 800 μL of PEI transfection reagent. Immediately mix well and incubate at room temperature for 15 min. Then add the plasmid / vector / transfection reagent complex dropwise into the culture flask of 293T cells. After 24 h, collect the viral supernatant into a 50 mL centrifuge tube, centrifuge at 250 g for 5 min. After centrifugation, filter the supernatant with a 0.45 μm filter, and ultracentrifuge the filtered supernatant (25000 g, 4 °C, 3 h) to obtain concentrated Mesothelin-CAR lentivirus. Discard the supernatant after centrifugation, resuspend the lentivirus with pre-cooled PBS at 4 °C, aliquot the resuspended Mesothelin-CAR lentivirus solution, and store it at -80 °C for standby.

[0348] (3) Resuscitation and activation of T cells

[0349] Take out the cryopreserved cord blood and thaw it in a 37 °C water bath. Transfer the cord blood to a 50 mL centrifuge tube, add an appropriate amount of RPMI1640 medium (Shanghai Dart Hill Biotechnology Co., Ltd., Lot: C3010-0500), mix well and take a sample for counting; centrifuge at 500 g for 5 min, discard the supernatant after centrifugation, collect the lower layer of cells, and resuspend them with the medium (RPMI1640 + 10% FBS) to a T lymphocyte density of 1×10 6 cells / mL. Add the activation magnetic beads Enceed TM T cellActivation,human and DNase GMP Grade, add cytokines IL-7 (acrobiosystems, lot: #C24dC1-G002-224MF1-134), IL-15 (acrobiosystems, lot: #S85-216QF1-11H), IL-21 (acrobiosystems, lot: C617C2-G002-233PF1-19P). The addition amount of the activation magnetic beads is: for every 1E6 total cell amount, add 5 μL of magnetic beads, the DNase concentration is 0.05 U / μL, the cytokine IL-7 concentration is 10 ng / mL, the IL-15 concentration is 10 ng / mL, and the IL-21 concentration is 10 ng / mL. Then transfer the cell suspension to a cell culture container and place it in a 37 °C, 5% carbon dioxide incubator for 48 h.

[0350] (4) Sorting and purification of T cells

[0351] After activating T cells for 48 h, use a flow cytometer (model: NovoCyte Penteon UVBYR) to detect and count, and record the numbers of CD4+ and CD8+ T cells. Transfer the cell suspension to a centrifuge tube and centrifuge (300 g, 5 min), discard the supernatant and collect the cells in the lower layer; add MACS Buffer to wash, centrifuge again (300 g, 5 min), collect the cells in the lower layer, and add an appropriate amount of MACS Buffer to resuspend the cells. Calculate the usage amounts of CD4+ magnetic beads (Miltenyi Biotec, lot: 5220100705) and CD8+ magnetic beads (Miltenyi Biotec, lot: 5220809706) according to the cell amount and the magnetic bead instruction manual, and use CD4+ and CD8+ magnetic beads for sorting according to the magnetic bead instruction manual. Obtain the sorted T cells. Use a flow cytometer to detect the numbers of CD4+ and CD8+ T cells in the recovered T cell suspension.

[0352] (5) Preparation of mesothelin-targeted CAR-T cells

[0353] Transduce the constructed lentivirus into T cells, adjust the cell density to about 400 cells / μL for plating, and the volume per well is 400 μL. According to the actual number of T cells per well, add Mesothelin-CAR virus to each group at MOI = 25, and the negative control group without CAR transduction (UnT) does not add virus. After culturing in an incubator at 37 °C and 5% carbon dioxide, detect the proliferation of T cells and the expression rate of Mesothelin-CAR. Regularly observe the cell expansion situation after plating, and supplement fresh culture medium. After culturing the prepared CAR-T cells for 11 days, use Anti-Alpaca IgG, VHH domain (Jackson Immunoresearch, lot: 128-545-230) antibody to detect the expression rate of CAR molecules in each experimental group. The expression rates of Mesothelin-CAR on CD4+ and CD8+ T are as Figure 1A and 1B shown. At the same time, detect and count the proliferation of T cells. The proliferation of T cells is as Figure 2A and 2B shown. Use a flow cytometer to detect the T SCM +T CM phenotype and the proportion of CAR-T cells expressing Tim3. The detection results of the T SCM +T CM phenotype are as Figure 3 shown, and the detection results of CAR-T cells expressing Tim3 are as Figure 4As shown, harvest CAR-T cells for subsequent in vitro killing experiments. The constructed mesothelin-targeted CAR-T cells are M1 (SEQ ID NO:78), M5 (SEQ ID NO:79), M8 (SEQ ID NO:80), M9 (SEQ ID NO:81), M14 (SEQ ID NO:82), M16 (SEQ ID NO:83), M17 (SEQ ID NO:84), M18 (SEQ ID NO:85), M23 (SEQ ID NO:86), M28 (SEQ ID NO:87), M29 (SEQ ID NO:88), M31 (SEQ ID NO:89), M34 (SEQ ID NO:90), M41 (SEQ ID NO:91), M44 (SEQ ID NO:92), M46 (SEQ ID NO:93), M49 (SEQ ID NO:94), M60 (SEQ ID NO:95). The negative control T cell group is UnT (untransduced CAR), and the positive control CAR-T cell group is Meso3 (SEQ ID NO:114).

[0354] Stem cell-like memory T cells (T SCM ) are a subset of T cells with the ability of self-renewal and effector T cell production, and have the characteristic of being able to survive in the body for a long time, playing a role in maintaining long-term anti-tumor ability. Central memory T cells (T CM ) are the later stage of T SCM differentiation. Usually, the in vivo persistence and therapeutic potential of effector T cells are judged by analyzing the proportion of T SCM +T CM . In the present invention, the sum of the percentages of CD45RO-CD62L+ and CD45RO+CD62L+ T cells is analyzed to evaluate the proportion of T SCM +T CM (CD45RO: APC / Cyanine7 anti-human CD45RO Antibody (Biolegend, lot: B378781); CD62L: PE / Cyanine7 anti-human CD62L Antibody (Biolegend, lot: B373155)). The higher the proportion of T SCM +T CM , the stronger the in vivo persistence of CAR-T cells is predicted. Therefore, if a higher proportion of T SCM +T CM can be maintained in CAR-T cells, it will also be beneficial for their use in in vivo treatment.

[0355] The present invention determines the exhaustion status of T cells by analyzing the percentage of T cells expressing Tim3 (Brilliant Violet 421 TM anti-human CD366 (Tim-3) Antibody (Biolegend, lot: B368969)). The higher the proportion of CAR-T cells expressing Tim3, the lower the effector function of CAR-T cells is predicted.

[0356] Example 3. Verification of the tumor cell killing effect of mesothelin-targeted CAR-T cells

[0357] (1) Determination of the expression of mesothelin antigen on the surface of target cells

[0358] Using human lung squamous carcinoma cells NCI-H226 (expressing MES protein) (ATCC: CRL-5826), human B lymphoblastic leukemia cells Nalm6-EGFP (ATCC: CRL-3273, with EGFP green fluorescence label), and Nalm6-EGFP-MES (ATCC: CRL-3273, expressing MES protein, with EGFP green fluorescence label) recombinantly expressing MES protein as target cells, the above cells were all cultured using RPMI1640 medium (containing 10% FBS, where FBS: EXCELL BIO, Lot: 12B067), and the expression of MES protein on the cell surface was detected by anti-hMesothelin (R&D Systems, lot: AANW0521011) antibody staining. The detection results are as Figure 5 shown. The results show that Nalm6 cells are negative for mesothelin antigen expression, and NCI-H226 cells and Nalm6-EGFP-MES cells are positive for mesothelin antigen expression.

[0359] Preparation of Nalm6-EGFP-MES positive cells: Obtain lentivirus expressing mesothelin antigen, and seed 293T cells at 2×10 5 / cm 2Inoculate 293T cells transiently transfected with plasmids at a density in a T-shaped container. After adding 40 mL of Optim-MEM to a sterile 50 mL centrifuge tube for plasmid transfection, add viral packaging vectors and viral envelope vectors according to the ratio of pK1-Mesothelin:pLP1:pLP2:pLP-VSVG = 5:4:3:1, and add 800 μL of PEI transfection reagent. Immediately mix well and incubate at room temperature for 15 min. Then, add the plasmid / vector / transfection reagent complex dropwise to the 293T cells. After 24 h, collect the viral supernatant into a 50 mL centrifuge tube, centrifuge at 250 g for 5 min. After centrifugation, filter the supernatant through a 0.45 μm filter, and ultracentrifuge the filtered supernatant (25,000 g, 4 °C, 3 h) to obtain concentrated mesothelin lentivirus. After centrifugation, discard the supernatant, resuspend the lentivirus with pre-cooled PBS at 4 °C, aliquot the resuspended mesothelin lentivirus solution, and store it at -80 °C for later use. Plate the normally proliferating Nalm6-EGFP cells at a density of 4×10 4 cells / well in a 24-well plate, add the mesothelin lentivirus solution at an MOI of 25, and supplement the volume to a total volume of 500 μL with fresh growth medium; on the 3rd day after virus infection, detect the mesothelin antigen expression of the cells. When the mesothelin expression of the cells is stable for more than 5 passages and the infection efficiency is higher than 90%, perform monoclonal plating in a 96-well plate using the limiting dilution method, adjust the cell density to 0.008 cells / μL, and the plating volume per well is 100 μL. Observe the growth status of the monoclonal at the time points of 8 h, D1, D7, and D14 after plating. After confirming that the Nalm6-EGFP cells are monoclonal, culture the cells for more than 2 months, and then, according to the instructions of the mesothelin fluorescently labeled antibody (Human Mesothelin PE-conjugated Antibody, R&D SYSTEMS, FAB32652P), stain the Nalm6-EGFP-MES monoclonal cells in the dark and use a flow cytometer to detect the mesothelin antigen expression on the cell surface. When the mesothelin antigen expression is determined, the Nalm6-EGFP-MES positive cell line is obtained.

[0360] (2) In vitro killing effect determination of CAR-T cells targeting mesothelin by flow cytometry

[0361] In 48-well plates, add the target cells (900 cells / μL) Nalm6-EGFP-MES and Nalm6-EGFP respectively. According to the effector-to-target ratio (E:T) of 1:1 and 1:3, calculate the amount of effector cells to be added based on the cell amount of CD8+ CAR-T cells, and add the CAR-T cells obtained in Example 2 to the 48-well plates according to the corresponding amount of effector cells. The negative control UnT group without CAR transduction also adds the same amount of UnT cells and target cells according to the corresponding effector-to-target ratio. Complete medium (90% RPMI1640 + 10% FBS + 0.1% 2-mercaptoethanol + 1% L-Glu) is supplemented to 500 μL / well for all groups, and each group is set with 3 replicates. Place the plates in an incubator at 37 °C and 5% CO2 for culture. After 1 day and 3 days of culture, detect the number of target cells in each well by flow cytometry and calculate the killing rate. The results are respectively as Figure 6A , 6B , 7A, 7B, 8A and 8B show.

[0362] The calculation formula is: killing rate % = reduction amount of target cells / number of target cells plated × 100%.

[0363] (3) In vitro killing effect of mesothelin-targeted CAR-T cells Lactate dehydrogenase (LDH) release assay

[0364] Add 6×10 4 target cells NCI-H226 to 96-well plates. According to the effector-to-target ratio (E:T) of 1:1, calculate the amount of effector cells to be added based on the cell amount of CD8+ CAR-T cells, and add the CAR-T cells harvested in Example 2 to the 96-well plates according to the corresponding amount of effector cells. The total volume of each well is 200 μL, so that the final density is 300 target cells / μL and 300 CD8+ CAR-T cells / μL. The negative control UnT group without CAR transduction also adds the same amount of UnT cells and target cells according to the corresponding effector-to-target ratio. At the same time, set up the maximum release well of target cells, the spontaneous release well of target cells, the spontaneous release well of effector cells, the background well (200 μL medium) and the volume correction well (200 μL medium + 20 μL lysis solution), and each group is set with 5 replicates. Incubate the 96-well plates in an incubator at 37 °C and 5% CO2 for 24 h. Add 20 μL of lysis solution to the maximum release well of target cells, then put it back into the incubator at 37 °C for incubation for 1 h, take it out and centrifuge (600 g, 10 min). Take 50 μL of the supernatant from each well to a new 96-well plate, and measure according to the instructions of the LDH cytotoxicity detection kit (CytoTox Non-Radioactive Cytotoxicity Assay G1780). Judge the degree of damage of target cells by detecting the activity of LDH in the cell culture supernatant of each group. The detection results are asFigure 9 as shown

[0365] The calculation formula is as follows:

[0366] Killing rate % = (OD value of experimental group - OD value of spontaneous release hole of effector cells - OD value of spontaneous release hole of target cells) / (OD value of maximum release hole of target cells - OD value of spontaneous release hole of target cells) × 100%;

[0367] OD value of experimental group = measured OD value of experimental group - OD value of background hole;

[0368] OD value of spontaneous release hole of effector cells = measured OD value of spontaneous release hole of effector cells - OD value of background hole;

[0369] OD value of control hole of spontaneous release of target cells = measured OD value of spontaneous release hole of target cells - OD value of background hole;

[0370] OD value of maximum release hole of target cells = measured OD value of control hole of maximum release of target cells - OD value of volume correction hole.

Claims

1. A VHH single-domain antibody targeting mesothelin, characterized in that, The VHH single-domain antibody comprises CDR1, CDR2, and CDR3; wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 10, CDR2 is the amino acid sequence shown in SEQ ID NO: 11, and CDR3 is the amino acid sequence shown in SEQ ID NO:

12.

2. The VHH single domain antibody targeting mesothelin according to claim 1, characterized in that, The VHH single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO:

58.

3. The VHH single-domain antibody targeting mesothelin according to claim 1, characterized in that, The VHH single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:

58.

4. Use of a VHH single-domain antibody targeting mesothelin as described in any one of claims 1-3 for the preparation of a chimeric antigen receptor (CAR).

5. A chimeric antigen receptor, characterized in that, Comprising (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain; wherein the extracellular antigen-binding domain comprises the VHH single-domain antibody targeting mesothelin as described in any one of claims 1-3.

6. The chimeric antigen receptor according to claim 5, wherein wherein the transmembrane domain is derived from CD8α, CD28, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRα, TCRβ, TCRγ, TCRδ, TCRζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

7. The chimeric antigen receptor according to claim 6, wherein wherein the transmembrane domain is derived from CD8α.

8. The chimeric antigen receptor according to claim 5, wherein, wherein the intracellular signaling domain is derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof.

9. The chimeric antigen receptor according to claim 8, wherein, wherein the intracellular signaling domain is derived from CD3ζ.

10. The chimeric antigen receptor according to claim 8, wherein, wherein the intracellular signaling domain further comprises a co-stimulatory signal domain, and the co-stimulatory signal domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (LIGHTR), BTLA, CD8α, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

11. The chimeric antigen receptor according to claim 10, wherein wherein the co-stimulatory signal domain is derived from 4-1BB (CD137).

12. The chimeric antigen receptor according to claim 5, wherein, Further comprises a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

13. The chimeric antigen receptor according to claim 12, wherein Wherein the hinge region is derived from CD8α, CD28, CD137, IgG4, IgG1 or any combination thereof.

14. The chimeric antigen receptor according to any one of claims 5-13, wherein Further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide.

15. The chimeric antigen receptor according to claim 14, wherein Wherein the signal peptide is derived from HLA-A, CD8α, CD4, CD33, CD137, GM-CSFRα, IgG1, Igκ, IL-2 or any combination thereof.

16. A chimeric antigen receptor, characterized in that, It comprises the amino acid sequence shown in SEQ ID NO:

81.

17. An isolated nucleic acid, characterized in that, Comprises a nucleic acid sequence encoding the chimeric antigen receptor according to any one of claims 5-16.

18. The isolated nucleic acid according to claim 17, wherein Comprises the nucleic acid sequence shown in SEQ ID NO:

99.

19. A carrier, characterized in that, The vector comprises the isolated nucleic acid according to claim 17 or 18.

20. An engineered immune effector cell, characterized in that, Comprising a chimeric antigen receptor according to any one of claims 5-16 、 An isolated nucleic acid according to claim 17 or 18, or a vector according to claim 19.

21. The engineered immune effector cell according to claim 20, wherein, The immune effector cells are selected from T cells, B cells, NK cells, NKT cells, DNT cells, macrophages, dendritic cells or any combination thereof.

22. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mesothelin-targeting VHH single-domain antibody according to any one of claims 1-3, the chimeric antigen receptor according to any one of claims 5-16, or the engineered immune effector cell according to any one of claims 20-21, and one or more pharmaceutically acceptable excipients and / or carriers.

23. Use of the mesothelin-targeting VHH single-domain antibody according to any one of claims 1-3, the chimeric antigen receptor according to any one of claims 5-16, the engineered immune effector cell according to any one of claims 20-21, or the pharmaceutical composition according to claim 22 in the preparation of a drug for diagnosing, preventing and / or treating mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, gastric cancer, colon cancer, rectal cancer, cervical cancer, endometrial cancer or triple-negative breast cancer.

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