Anti-PD-1 antibody, car-t cell, preparation method therefor and use thereof

Abstract

Provided are an anti-PD-1 antibody, a CAR-T cell, a preparation method therefor and the use thereof. The anti-PD-1 antibody contains LCDR-1-3 of SEQ ID NOs: 1-3, respectively, and HCDR-1-3 of SEQ ID NOs: 4-6, respectively. Further provided is a new element for efficiently blocking PD-1 by means of CAR-T cell expression. The element is a cell membrane-anchored single-chain antibody targeting PD-1. A chimeric antigen receptor and the cell membrane-anchored anti-PD-1 scFv are linked via a 2A self-cleaving protein. The provided membrane-anchored anti-PD-1 scFv expressed by the CAR-T cell can almost completely block PD-1 expression on the T cell membrane surface, thereby blocking the PD-1 / PD-L1 binding signaling pathway, enhancing the ability of the CAR-T cell to antagonize T cell depletion caused by tumors, and achieving the purpose of enhancing the anti-tumor effect of the CAR-T cell.

Description

Anti-PD-1 antibodies, CAR-T cells, their preparation methods and applications

[0001] This application claims priority to Chinese patent application No. 2024115970108, filed on November 8, 2024, entitled "Anti-PD-1 antibody, CAR-T cells, preparation method thereof and application". Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to anti-PD-1 antibodies, CAR-T cells, their preparation methods and applications. Background Technology

[0003] T-cell adoptive immunotherapy is a promising approach for cancer treatment. This immunotherapy utilizes genetically modified isolated human T cells to enhance their specificity for specific tumor-associated antigens. Genetic modification can involve the expression of chimeric antigen receptors or exogenous T-cell receptors to specifically transplant antigens onto T cells. Compared to exogenous T-cell receptors, the specificity of chimeric antigen receptors derives from the variable domains of monoclonal antibodies. Therefore, T cells expressing chimeric antigen receptors (CAR-T cells) induce tumor immune reactivity in a major histocompatibility complex-free manner. T-cell adoptive immunotherapy has been used clinically for many cancers, including B-cell malignancies, multiple myeloma, neuroblastoma, glioblastoma, advanced glioma, ovarian cancer, mesothelioma, melanoma, prostate cancer, and pancreatic cancer.

[0004] Despite the potential usefulness of CAR-T cells as a cancer treatment, adoptive immunotherapy using CAR-T cells in the treatment of solid tumors still faces significant challenges. A key issue in CAR-T cell therapy for solid tumors is the presence of tumor heterogeneity and the tumor immunosuppressive microenvironment. The tumor microenvironment, composed of immunosuppressive signals mediated by immune checkpoints, plays a crucial role in promoting tumor immune escape. After CAR-T cells are activated by antigens, their proliferation and cytokine secretion functions are inhibited by binding to relevant ligands. Particularly on tumor-infiltrating CAR-T cells, the expression of these inhibitory molecules is further upregulated, thus significantly limiting the anti-tumor immune effect of CAR-T cells at the solid tumor site. PD-1 is one of the representative inhibitory factors expressed on the surface of T cells, and its binding with PD-L1 on tumor cells mediates T cell exhaustion. In light of this, several drugs targeting the PD-1 / PD-L1 binding signaling pathway have been marketed, such as PD-1 and PD-L1 monoclonal antibody drugs, and CAR-T cells that can secrete PD-1 or PD-L1 monoclonal antibodies. However, existing drugs that block the PD-1 / PD-L1 signaling pathway either have short half-lives requiring multiple injections (antibody drugs) or have uncontrollable secretion levels, posing significant safety risks (antibody-secreting CAR-T). Therefore, it is necessary to develop a novel drug that can block the PD-1 / PD-L1 signaling pathway to solve the above problems. Summary of the Invention

[0005] Currently, CAR-T therapy faces numerous challenges in the treatment of solid tumors, primarily due to tumor heterogeneity and the tumor immunosuppressive microenvironment. When CAR-T cells enter a tumor, ligands expressed on the tumor surface bind to inhibitory factors expressed on the T cell surface, leading to T cell exhaustion. Therefore, addressing the poor therapeutic effect of CAR-T cells in solid tumors requires resolving the tumor-induced T cell exhaustion problem. PD-1 is a recognized T cell inhibitor, and monoclonal antibody anti-tumor drugs targeting PD-1 are already on the market. Furthermore, CAR-T cells secreting PD-1 antibodies have been reported to be used in clinical trials. However, existing drugs targeting the PD-1 / PD-L1 signaling pathway, including those in clinical trials, either suffer from short half-lives and large molecular weights (e.g., antibody drugs) or uncontrollable secretion levels (e.g., antibody-secreting CAR-T cells).

[0006] One object of the present invention is to provide an anti-PD-1 antibody that is particularly suitable for use with chimeric antibody receptors, which can completely block the expression of PD-1 on the cell surface.

[0007] Another objective of this invention is to achieve synchronous expression of PD-1 with CAR through a novel cell membrane-anchored anti-PD-1 scFv. This anchored anti-PD-1 scFv is non-secretory and can be continuously expressed synchronously with CAR on the surface of T cells. Data from the embodiments show that this anchored anti-PD-1 scFv almost completely blocks the expression of PD-1 on the cell surface, solving the problems existing in the prior art.

[0008] Another objective of this invention is to provide a method for preparing CAR-T cells that simultaneously express novel PD-1 blocking elements and their applications.

[0009] In the first aspect, this article provides an anti-PD-1 antibody or its antigen-binding fragment, comprising light chain complementarity-determining regions (LCDR) 1-3 and heavy chain complementarity-determining regions (HCDR) 1-3, wherein the amino acid sequence of LCDR-1 is SASSVSYMH (SEQ ID NO:1), the amino acid sequence of LCDR-2 is GTSNLAS (SEQ ID NO:2), the amino acid sequence of LCDR-3 is QQWSSYPLT (SEQ ID NO:3), the amino acid sequence of HCDR-1 is DHIIN (SEQ ID NO:4), the amino acid sequence of HCDR-2 is RIYPVSGETNYNQKFKG (SEQ ID NO:5), and the amino acid sequence of HCDR-3 is WDGYYAMDY (SEQ ID NO:6).

[0010] In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the light chain complementarity-determining regions LCDR1-3 of SEQ ID NO:7, and the heavy chain variable region comprises the heavy chain complementarity-determining regions HCDR1-3 of SEQ ID NO:8. In one embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7. In one embodiment, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8. In one embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.

[0011] In one embodiment, the antigen-binding fragment is one or more of Fab', Fab, F(ab')2, Fd fragment, dAb fragment, camel antibody, nanobody, and single-chain Fv.

[0012] In a second aspect, this document provides fusions or protein constructs comprising a first portion or a first and a second portion, wherein the first portion comprises, from the N-terminus to the C-terminus, an optional first signal peptide, an anti-PD-1 antibody or an antigen-binding fragment thereof as described herein, and a first transmembrane domain; and, when present, the second portion comprises, optionally, a second signal peptide and a chimeric antigen receptor; the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, an antigen-binding domain, a second transmembrane domain, a co-stimulatory domain, and a signal transduction domain; the first portion is located at the C-terminus or N-terminus of the second portion. In one embodiment, the second portion is located at the N-terminus of the first portion. In one embodiment, the anti-PD-1 antibody or an antigen-binding fragment thereof is in the form of scFv. In one embodiment, the anti-PD-1 antibody or an antigen-binding fragment thereof is directly linked to the first transmembrane domain or linked via a linker. In one embodiment, the first transmembrane domain is a CD8α transmembrane domain. In one embodiment, the first and second portions are linked by a self-cleaving protein. In one embodiment, the self-cleaving protein is a 2A cleaving protein. In one embodiment, the 2A cleaving protein is selected from P2A, T2A, and F2A. In one embodiment, the first signal peptide and the second signal peptide may be the same or different. In one embodiment, the first signal peptide and the second signal peptide are each independently a CD8 precursor.

[0013] In one embodiment, the fusion comprises the structure of CD8 signal peptide-anti-MSLN scFv-CD8α transmembrane domain-CD28 costimulatory domain-4-1BB costimulatory domain-CD3ζ signal transduction domain-P2A cleavage protein-CD8 signal peptide-anti-PD-1scFv-CD8α transmembrane domain.

[0014] In one embodiment, the antigen-binding domain binds to a cell surface cancer antigen, said cell surface cancer antigen being one or more of the following: Mesothelin, GD2, CD171, CD19, CD20, BCMA, GPC3, TERT, PTEN, PD-1, PD-L1, NKG2D ligand, CD44v6, FR, CD138, PSMA, NY-ESO, EGFR, CEA, HER2, CD22, CD30, CD123, CD5, CD7, CD33, CEA, EGFR, BRAF, HER-2, MUC1, PSCA, GPC3, or VEGF. In one embodiment, the co-stimulatory domain is CD28, 4-1BB, or a combination of CD28 and 4-1BB. In one embodiment, the signal transduction domain is CD3ζ. In one embodiment, the second transmembrane domain is a CD8α transmembrane domain. In one embodiment, CD28 comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9. In one embodiment, the 4-1BB comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:10. In one embodiment, the signal transduction domain comprises a combination of CD28 and 4-1BB. In one embodiment, the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:11. In one embodiment, the chimeric antigen receptor comprises the HCDR-1 sequence DYAMH (SEQ ID NO:18), the HCDR-2 sequence VISTYNGNINYNQKFKG (SEQ ID NO:19), the HCDR-3 sequence GGYDGTGFDY (SEQ ID NO:20); the LCDR-1 sequence SASSSISYMH (SEQ ID NO:21), the LCDR-2 sequence DTSKLAS (SEQ ID NO:22), and the LCDR-3 sequence QQWSSPPT (SEQ ID NO:23). In one embodiment, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:12.

[0015] In a third aspect, this document provides compositions or conjugates comprising the anti-PD-1 antibody or its antigen-binding fragment described herein. In one embodiment, the composition is a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In one embodiment, the conjugate further comprises a cytotoxic agent.

[0016] In a fourth aspect, this document provides a first nucleic acid encoding the anti-PD-1 antibody or its antigen-binding fragment described herein. In one embodiment, the first nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:13 or a degenerate sequence thereof, for example, a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:13.

[0017] In a fifth aspect, this document provides a second nucleic acid encoding the fusion described herein. In one embodiment, the coding sequence of the first portion comprises the nucleotide sequence shown in SEQ ID NO:14 or a degenerate sequence thereof, for example, a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:14. In one embodiment, the coding sequence of the second portion comprises the nucleotide sequence shown in SEQ ID NO:15 or a degenerate sequence thereof, for example, a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:15. The second nucleic acid may comprise the nucleotide sequence shown in SEQ ID NO:24 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:24.

[0018] In a sixth aspect, this document provides an expression cassette comprising either the first nucleic acid or the second nucleic acid described herein and further comprising a control element operatively linked to said nucleic acid. In one embodiment, the control element is one or more of a promoter, an enhancer, and a terminator. In one embodiment, the promoter is selected from one or more of the CMV and EF1a promoters.

[0019] In a seventh aspect, this document provides a vector comprising the expression cassette described herein. In one embodiment, the vector is a viral vector. In another embodiment, the vector is a lentiviral vector or an adeno-associated virus vector.

[0020] In the eighth aspect, this document provides a virus comprising the expression cassette or vector described herein. In one embodiment, the virus is a lentivirus or an adeno-associated virus.

[0021] In a ninth aspect, this document provides the use of the anti-PD-1 antibody described herein or its antigen-binding fragment, or the composition or conjugate described herein, in the preparation of a medicament for treating a disease. Alternatively, this document provides a method of treating a disease in a subject in need, comprising administering to a subject the anti-PD-1 antibody described herein or its antigen-binding fragment, or the composition or conjugate described herein. Alternatively, this document also provides the use of the anti-PD-1 antibody described herein or its antigen-binding fragment, or the composition or conjugate described herein, for treating a disease in a subject in need. In one embodiment, the disease is cancer, particularly a disease involving the PD-1 / PD-L1 signaling pathway. In one embodiment, the cancer is one or more of melanoma, non-small cell lung cancer, nasopharyngeal carcinoma, glioblastoma, colon adenocarcinoma, hepatocellular carcinoma, urothelial carcinoma, multiple myeloma, ovarian cancer, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, lymphoma such as Hodgkin's lymphoma, and leukemia.

[0022] In a tenth aspect, this document provides a cell comprising the first nucleic acid described herein, the second nucleic acid described herein, the expression cassette described herein, the vector described herein, or the virus described herein. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell comprises the first and second portions described herein, the first and second portions being anchored to the cell surface via first and second transmembrane domains, respectively. In one embodiment, the immune cell is one or more of T cells, macrophages, and NK cells. In one embodiment, the cell is one or more of cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T (NKT) cells.

[0023] In the eleventh aspect, this document provides a method for preparing the cells or immune cells described herein, comprising introducing the first nucleic acid, second nucleic acid, expression cassette, vector, or virus described herein into the cells to block the expression of PD-1 on the surface of T cells, thereby blocking the PD-1 / PD-L1 binding signaling pathway. In one embodiment, the method includes one or more of the following steps: 1) viral vector construction; 2) lentiviral packaging using 293T cells; 3) PBMC isolation and T cell sorting; 4) T cell activation; 5) lentiviral transfection of activated T cells; 6) culture of infected CAR-T cells and flow cytometry detection of CAR positivity; 7) cellularity assay of CAR-T cells; and 8) detection of tumor suppression effect of CAR-T cells in a tumor model (preferably a mouse model).

[0024] In a twelfth aspect, this document provides the use of the immune cells described herein in the preparation of medicaments for the treatment and / or prevention of cancer. Alternatively, this document also provides methods for the treatment and / or prevention of cancer, comprising administering CAR-T cells or pharmaceutical compositions containing CAR-T cells to a subject. Alternatively, this document also provides CAR-T cells or pharmaceutical compositions containing CAR-T cells for use in the treatment and / or prevention of cancer. In one embodiment, the cancer is selected from one or more of liver cancer, bladder cancer, various types of leukemia, multiple myeloma, malignant lymphoma, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, head and neck cancer, multiple squamous cell tumors, esophageal cancer, colorectal cancer, melanoma, osteosarcoma, rectal cancer, anal cancer, bile duct cancer, uterine cancer, ovarian cancer, gastric cancer, prostate cancer, meningioma, pancreatic cancer, breast cancer, and medulloblastoma.

[0025] The beneficial effects of this invention include:

[0026] 1. A novel anti-PD-1 antibody or its antigen-binding fragment is provided, which can bind to PD-1 with high binding affinity and specificity; it has a similar binding affinity to nivolumab anti-PD-1 scFv.

[0027] 2. An anchored anti-PD-1 scFv is provided, which can be used to completely block the expression of PD-1 on the cell surface and solve the problem of T cell exhaustion caused by tumors.

[0028] 3. A novel fusion protein or construct and its expression cassette are provided for the simultaneous expression of chimeric antigen receptor and anti-PD-1scFv.

[0029] 4. It provides a cell membrane-anchored anti-PD-1 scFv polynucleotide molecule that can almost completely block the expression of PD-1 on the surface of T cells, thereby blocking the PD-1 / PD-L1 signaling pathway.

[0030] 5. Expression cassettes, vectors, and viruses containing the polynucleotide molecules described herein are provided to facilitate the development of universal CAR-T cells capable of simultaneously expressing cell membrane-anchored anti-PD-1scFv. Attached Figure Description

[0031] Figure 1 is a schematic diagram of the novel CAR structure for simultaneous expression of highly efficient PD-1 blocking and a conventional CAR. The only difference between the novel CAR for simultaneous expression of highly efficient PD-1 blocking and the conventional CAR in Figure 1 is that the conventional CAR does not contain the 2A cleavage protein and the membrane-anchored PD-1 single-chain antibody.

[0032] Figure 2 is an agarose gel electrophoresis image of the double-enzyme digestion product of the recombinant plasmid for synchronously expressing the novel PD-1 blocking CAR element of the present invention. M: 1kb DNA marker; 1: Recombinant plasmid for synchronously expressing the novel PD-1 blocking CAR element (undigested); 2: Recombinant plasmid digestion product; 3: pLenti-MCS-EF1-GFP lentiviral expression plasmid; 4: Target fragment for synchronously expressing the novel PD-1 blocking CAR element.

[0033] Figure 3 shows the flow cytometry results. Figure 3 shows that MSLN CAR-T cells synchronously expressing cell membrane-anchored anti-PD-1 scFv accounted for 72.64% of the total T cells, while ordinary MSLN CAR-T cells accounted for 75.81% of the total T cells.

[0034] Figure 4 shows the flow cytometry results for PD-1 expression levels. Compared to PD-1 expression in uninfected T cells and in ordinary MSLN CAR-T cells, PD-1 expression in MSLN CAR-T cells simultaneously expressing membrane-anchored anti-PD-1 scFv was almost completely blocked.

[0035] Figure 5 shows a flow cytometry analysis of MSLN expression in tumor cells. Figure 5 illustrates MSLN-target-positive tumor cells (ovarian tumor cells SKOV3 and intestinal tumor cells HCT116) and MSLN-non-target-expressing tumor cells (ovarian tumor cells ES-2).

[0036] Figure 6 shows the killing efficiency curves detected by a Real-Time Intelligent Cell Monitoring Instrument (RIMI) (brand: Six Broad Beans, model: CM100-α). Figure 6 shows the killing efficiency of CAR-T cells against MSLN-target-positive SKOV3 cells and HCT116 cells (bottom) and non-MSLN-target-negative ES2 cells (top). MSLN CAR-T cells that simultaneously express cell membrane-anchored anti-PD-1scFv have a higher killing efficiency against tumor cells than ordinary MSLN CAR-T cells.

[0037] Figure 7 shows the bar chart of ELISA detection of IFNγ and TNFα factors 24 h after ES2 cells, SKOV3 cells, or HCT116 cells were killed. The CAR-T cells of this invention, when co-cultured with SKOV3 cells, secreted a large amount of IFNγ factor. Furthermore, the MSLN CAR-T cells simultaneously expressing cell membrane-anchored anti-PD-1scFv showed higher efficiency in secreting IFNγ and TNFα factors than ordinary MSLN CAR-T cells. Detailed Implementation

[0038] The following definitions are provided to enable those skilled in the art to understand the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0039] As used in this article, "programmed cell death molecule 1 (PD-1)," also known as CD279, is a receptor molecule expressed on the surface of activated T cells, natural killer T cells, B cells, and macrophages. PD-1 negatively regulates T cell receptor-mediated signaling pathways. "Programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1, and it downregulates T cell activation and cytokine secretion after binding to PD-1.

[0040] As used herein, the term "antibody" refers to a naturally occurring or partially or wholly synthetic (e.g., recombinant) immunoglobulin. Antibodies include any protein having a binding domain homologous to or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies encompass a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, synthetic antibodies, recombinant antibodies, human antibodies, non-human antibodies (e.g., murine antibodies), humanized antibodies, chimeric antibodies, and intracellular antibodies. In this document, an antibody may be an anti-PD1 antibody. Preferably, the anti-PD-1 antibody comprises 3 LCDRs and 3 HCDRs, wherein the amino acid sequence of LCDR-1 is SASSVSYMH (SEQ ID NO:1), the amino acid sequence of LCDR-2 is GTSNLAS (SEQ ID NO:2), the amino acid sequence of LCDR-3 is QQWSSYPLT (SEQ ID NO:3), the amino acid sequence of HCDR-1 is DHIIN (SEQ ID NO:4), the amino acid sequence of HCDR-2 is RIYPVSGETNYNQKFKG (SEQ ID NO:5), and the amino acid sequence of HCDR-3 is WDGYYAMDY (SEQ ID NO:6). Preferably, the light chain variable region of the anti-PD-1 antibody comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0041] As used herein, the terms "complementarity-determining region" and "CDR" can be used interchangeably and refer to one of several CDRs within each variable region that together form the antigen-binding site of the antibody. Each variable region domain contains three CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable region domain contains three CDRs, designated VCDR1, VCDR2, and VCDR3; the heavy chain variable region domain contains three CDRs, designated HCDR1, HCDR2, and HCDR3. The three CDRs in the variable region are not sequential along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within the loop of the parallel chain connecting the β-sheet of the variable region. As described herein, those skilled in the art know and can identify CDRs based on Kabat or Chothia numbering (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, AIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917). In this paper, the CDR sequence of the antibody can be determined using Kabat numbering.

[0042] As used herein, the percentage sequence identity between two sequences (amino acid or nucleotide sequences) is a function of the number of common positions shared by the sequences relative to the length of the sequences being compared (i.e., % identity = number of common positions / total number of positions compared x 100). This calculation takes into account the number of any gaps and the length of each gap, which are introduced to maximize the degree of sequence identity between the two sequences. Sequence comparisons and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art (see, for example, US 8008449, etc.). When referring to sequence identity, any point value and any range between 80% and 99% can be used, including at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.

[0043] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule of a non-intact antibody that binds to an antigen and / or epitope, including portions of the intact antibody. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-chain antibody molecules (e.g., scFv), etc. In this document, the antigen-binding fragment may be in the form of scFv.

[0044] As used herein, the term "scFv fragment" refers to an antibody fragment comprising a light chain variable region (VL) and a heavy chain variable region (VH) covalently linked in any order via a polypeptide linker. In one embodiment, the scFv may comprise the three LCDRs and three HCDRs described herein. Preferably, the single-chain Fv comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16.

[0045] As used herein, a fusion refers to a protein formed from two or more protein moieties with different functions. In one embodiment, the fusion may comprise a first part and a second part, the first part comprising an anti-PD-1 scFv and a transmembrane domain, and the second part comprising a chimeric antigen receptor. The first part may be located at the N-terminus of the fusion, and the second part may be located at the C-terminus of the fusion. Alternatively, the first part may be located at the C-terminus of the fusion, and the second part may be located at the N-terminus of the fusion. In one embodiment, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a scFv targeting a cancer antigen, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain. The first part and the second part may each comprise a signal peptide (e.g., a signal peptide located at the N-terminus). In one embodiment, the structure of the fusion comprises a CD8 signal peptide-anti-MSLN scFv-CD8α transmembrane domain-CD28 co-stimulatory domain-4-1BB co-stimulatory domain-CD3ζ signal transduction domain-P2A cleavage protein-CD8 signal peptide-anti-PD-1 scFv-CD8α transmembrane domain. In one embodiment, the structure of the fusion comprises a CD8 signal peptide-anti-PD-1scFv-CD8α transmembrane domain.

[0046] As used herein, the term “optional” means that the portion used in conjunction with the term may or may not be present. For example, where the fusion may include an optional signal peptide-anti-PD-1 antigen-binding domain-transmembrane domain structure, the fusion may include either an anti-PD-1 antigen-binding domain-transmembrane domain structure or both.

[0047] As used herein, a "chimeric antigen receptor" or "CAR" refers to a modified receptor that confers or transfers antigen specificity to immune effector cells (e.g., human T cells). A chimeric antigen receptor comprises at least an extracellular ligand-binding domain or portion, a transmembrane domain, and an intracellular domain comprising one or more signal transduction domains and / or co-stimulatory domains. The extracellular ligand-binding domain or portion may be an antibody or antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, or linear antibodies. The extracellular ligand-binding domain or portion may be in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, providing specificity for a particular epitope or antigen. The scFv may be attached via a linker sequence. The scFv may be murine, humanized, or fully human. Intracellular stimulatory domains may include one or more cytoplasmic signaling domains that activate the transmission of signals to T cells upon antigen binding. Intracellular stimulatory domains may also include one or more intracellular co-stimulatory domains that transmit proliferation and / or cell survival signals upon ligand binding. Intracellular co-stimulatory domains may be those known in the art, including but not limited to 4-1BB (CD137), CD27, CD28, CD8, OX40, CD30, CD40, etc. Chimeric antigen receptors also include additional structural elements, including transmembrane domains attached to extracellular ligand-binding domains via hinge or spacer sequences, such as subunits of T cell receptors, such as the CD8α domain. The hinge region refers to any polynucleotide or polypeptide that serves to connect the transmembrane domain to the extracellular ligand-binding domain. The hinge region may be derived entirely or partially from naturally occurring molecules, such as all or part of the extracellular region derived from CD8, CD4, or CD28.

[0048] As used herein, the term "conjugate" refers to a complex formed by linking two or more different molecules together by chemical or biological methods. In one embodiment, the conjugate comprises an anti-PD-1 antibody as the targeting moiety. In one embodiment, the conjugate comprises a cytotoxic agent as the effector moiety to kill cancer cells.

[0049] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu), chemotherapeutic agents (e.g., methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), melphalan, mitomycin C, etc.); growth inhibitors; enzymes and their fragments, such as lysozymes; antibiotics; and toxins, such as small molecule toxins.

[0050] As used herein, the term "polynucleotide molecule" or "nucleic acid" refers to a chain-like compound composed of nucleotides. Nucleotide monomers can be nucleotides or deoxynucleotides. Polynucleotide molecules can encompass both DNA and RNA molecules. The nucleic acids described herein can be those encoding anti-PD-1scFv or those encoding fusions. When referring to "first" and "second" nucleic acids, "first" and "second" have no specific meaning and are used only to distinguish the nucleic acids being referred to.

[0051] As used herein, the term "expression cassette" is a single-stranded or double-stranded polynucleotide used to express a protein (e.g., a fusion). Such expression cassettes can be used alone or in conjunction with a vector. Figure 1 shows an exemplary expression cassette in this document. This expression cassette may contain coding sequences for a promoter and a chimeric antigen receptor from the 5' to 3' ends, and optionally may also contain coding sequences for anti-PD-1scFv.

[0052] As used herein, a “vector” or “recombinant DNA vector” can be a construct comprising a replication system and sequence capable of transcribing and translating polypeptide-coding sequences in a given host cell. If a vector is used, the choice of vector depends on the method to be used for transforming the host cell, as is well known to those skilled in the art. Vectors can include, but are not limited to, viral vectors, such as recombinant lentiviral vectors or AAV vectors. Those skilled in the art are well aware that a vector must contain genetic elements necessary for successful transformation, selection, and propagation.

[0053] As used herein, “human T cell” or “T cell” refers to a T cell isolated from a donor (especially a human donor). T cells and cells derived therefrom include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions.

[0054] As used herein, the term "effective function" refers to a specific function of a cell. For example, the effector function of T cells can be cytolytic activity or helper activity, including the secretion of cytokines.

[0055] As used in this article, diseases involving the PD-1 / PD-L1 signaling pathway can be one or more of the following: melanoma, non-small cell lung cancer, nasopharyngeal carcinoma, glioblastoma, colon adenocarcinoma, hepatocellular carcinoma, urothelial carcinoma, multiple myeloma, ovarian cancer, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, lymphomas such as Hodgkin's lymphoma, and leukemia.

[0056] As used herein, “subject” includes any human or non-human animal. The term “non-human animal” includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, birds such as chickens, and reptiles. In a preferred embodiment, the subject is a human.

[0057] Anti-PD-1 antibody or its antigen-binding fragment

[0058] This document provides an anti-PD-1 antibody or its antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the light chain complementarity-determining regions LCDR 1-3 in SEQ ID NO:7, and the heavy chain variable region comprises the heavy chain complementarity-determining regions HCDR 1-3 in SEQ ID NO:8. For example, an anti-PD-1 antibody or its antigen-binding fragment comprises LCDR 1-3 and HCDR 1-3, wherein: the amino acid sequence of LCDR-1 is SASSVSYMH (SEQ ID NO:1), the amino acid sequence of LCDR-2 is GTSNLAS (SEQ ID NO:2), the amino acid sequence of LCDR-3 is QQWSSYPLT (SEQ ID NO:3), the amino acid sequence of HCDR-1 is DHIIN (SEQ ID NO:4), the amino acid sequence of HCDR-2 is RIYPVSGETNYNQKFKG (SEQ ID NO:5), and the amino acid sequence of HCDR-3 is WDGYYAMDY (SEQ ID NO:6).

[0059] In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0060] Light chain variable region SEQ ID NO:7:

[0061] Heavy chain variable region SEQ ID NO:8:

[0062] In one embodiment, the antibody is a single-chain Fv containing the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:16.

[0063] Single-stranded Fv SEQ ID NO:16:

[0064] Fusion

[0065] The fusion compound may contain an optional signal peptide-anti-PD-1 antigen-binding domain-transmembrane domain structure. The signal peptide may be a CD8 signal peptide. The anti-PD-1 antigen-binding domain is the anti-PD-1 antibody or its antigen-binding fragment described herein, preferably an anti-PD-1 scFv. The transmembrane domain may be a CD8α transmembrane domain.

[0066] The fusion compound may comprise an optional first signal peptide, an antigen-binding domain targeting the cancer antigen, a first transmembrane domain, a co-stimulatory domain, a signal transduction domain, a self-cleaving protein, an optional second signal peptide, an anti-PD-1 antigen-binding domain, and a second transmembrane domain. The first and second signal peptides may each be independently a CD8 signal peptide. The anti-PD-1 antigen-binding domain is the anti-PD-1 antibody or its antigen-binding fragment described herein, preferably an anti-PD-1 scFv. The first and second transmembrane domains may be CD8α transmembrane domains. The antigen-binding domain targeting the cancer antigen may be an anti-MSLN scFv. The self-cleaving protein is a 2A cleaving protein. Preferably, the 2A cleaving protein is selected from P2A, T2A, and F2A cleaving proteins. The co-stimulatory domain is one or a combination of a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, preferably a combination of the CD28 co-stimulatory domain and the 4-1BB co-stimulatory domain.

[0067] In one embodiment, the CD28 co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9. In one embodiment, the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:10. In one embodiment, the CD8 signal peptide comprises the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:17. In one embodiment, the P2A cleavage protein comprises the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0068] CD28 co-stimulatory domain SEQ ID NO:9:

[0069] 4-1BB co-stimulatory domain SEQ ID NO:10:

[0070] Combination of CD28 costimulatory domain and 4-1BB costimulatory domain:

[0071] CD8α transmembrane domain SEQ ID NO:11:

[0072] CD8 signal peptide SEQ ID NO:17:

[0073] Anti-MSLN scFv SEQ ID NO:12:

[0074] (SEQ ID NO:12), in which Kabat analysis revealed that the HCDR-1 sequence is DYAMH (SEQ ID NO:18), the HCDR-2 sequence is VISTYNGNINYNQKFKG (SEQ ID NO:19), and the HCDR-3 sequence is GGYDGTGFDY (SEQ ID NO:20); the LCDR-1 sequence is SASSSISYMH (SEQ ID NO:21), the LCDR-2 sequence is DTSKLAS (SEQ ID NO:22), and the LCDR-3 sequence is QQWSSPPT (SEQ ID NO:23).

[0075] The following provides the structure and amino acid sequence of the chimeric antigen receptor for efficient PD-1 blocking of a novel full-length synchronous expression element used in the embodiments. Those skilled in the art will understand that this structure and amino acid sequence are merely exemplary and do not limit the scope of protection of this invention.

[0076] A chimeric antigen receptor structure for efficient full-length synchronous expression of novel PD-1 blocking elements: CD8 signal peptide-anti-MSLN scFv-CD8α transmembrane domain-CD28 co-stimulatory domain-4-1BB co-stimulatory domain-CD3ζ signal transduction domain-P2A cleavage protein-CD8 signal peptide-anti-PD-1 scFv-CD8α transmembrane domain.

[0077] The amino acid sequence of the chimeric antigen receptor that efficiently blocks novel PD-1 elements through full-length synchronous expression is: MALPVTALLLPLALLLHAARP (CD8 signal peptide, first signal peptide) MAQVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHARSLEWIGVISTYNGNINYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGGYDGTGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTPPKRWIYDTSKLASGVPARFSGSGSGTSYSLTLSSMEAEDVATYYCQQWSSPPTFGVGTKLELKR (anti-MSLN single-chain antibody, i.e., anti-MSLN scFv)

[0078] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY(CD8α transmembrane domain, first transmembrane domain)

[0079] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (CD28 co-stimulatory domain)

[0080] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(4-1BB co-stimulatory domain)

[0081] RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(CD3ζ signal conduction domain)

[0082] GSGATNFSLLKQAGDVEENPGP (P2A cleavage protein)

[0083] MALPVTALLLPLALLLHAARP (CD8 signal peptide, second signal peptide, same as the first signal peptide)

[0084] MCPGQTEQSGAELASPGASVTLSCKASGYTFTDHIINWVKKRPGQGLEWIGRIYPVSGETNYNQKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARWDGYYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIE LTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGGGTKLEIKRYPYDVPDYAGS (anti-PD-1 single chain antibody, namely anti-PD-1scFv)

[0085] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACFWVLVVVGGVLACYSLLVTVAFIIFWV(CD8α transmembrane domain, second transmembrane domain, same as the first transmembrane domain) (SEQ ID NO:28, with each domain in full length shown in parentheses).

[0086] Nucleic acid or polynucleotide

[0087] The nucleic acid or polynucleotide molecules described herein can encode anti-PD-1 antibodies or their antigen-binding fragments. The nucleic acid or polynucleotide molecules described herein can encode cell membrane-anchored anti-PD-1 scFv (i.e., optional signal peptide-anti-PD-1 scFv-transmembrane domain). The nucleic acid or polynucleotide molecules described herein can encode the fusions described herein, such as CD8 signal peptide-anti-MSLN scFv-CD8α transmembrane domain-CD28 co-stimulatory domain-4-1BB co-stimulatory domain-CD3ζ signal transduction domain-P2A cleavage protein-CD8 signal peptide-anti-PD-1 scFv-CD8α transmembrane domain.

[0088] In one embodiment, the nucleic acid encoding the anti-PD-1 antibody comprises the nucleotide sequence shown in SEQ ID NO:13 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:13.

[0089] SEQ ID NO:13:

[0090] In one embodiment, the nucleic acid encoding the anti-PD-1 antibody comprises the nucleotide sequence shown in SEQ ID NO:14 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:14.

[0091] ATGTGCCCAGGTCAAACTGAGCAGTCAGGAGCTGAGCTGGCGAGTCCTGGGGCATCAGTGACACTGTCCTGCAAGGCTTCTGGCTACACATTTACTGACCATATTATAAATTGGGTTAAAAAGAGGCCTGGACAGGGCCTTGAATGGATTGGAAGGATTTATCCAGTAAGTGGTGAAACTAACTACAATCAAAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATGGGATGGTTACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAGTTACCCACTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGTACCCATACGATGTTCCAGATTACGCTGGATCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG(SEQ ID NO:14;(Anti-PD-1 antibody + CD8α transmembrane domain);

[0092] In one embodiment, the nucleic acid encoding the anti-PD-1 antibody comprises the nucleotide sequence shown in SEQ ID NO:15 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:15.

[0093]

[0094] The following provides the nucleotide sequence (SEQ ID NO:24) of the chimeric antigen receptor for efficient blocking of novel PD-1 elements using full-length simultaneous expression, as used in the embodiments:

[0095] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (CD8 signal peptide, first signal peptide) - ATGGCCCAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGCGCCCCGGCGTGAGCGTGAAGATCAGCTGCAAGGGCAGCGGCTACACCTTCACCGACTACGCCATGCACTGGGTGAAGCAGAGCCACGCCCGCAGCCTGGAGTGGATCGGCGTGATCAGCACCTACAACGGCAACATCAACTACAACCAGAAGTTCAAGGGCAAGGCCACCATGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGGCCCGCCTGACCAGCGAGGACAGCGCCATCTACTACTGCGCCCGCGGCGGCTACGACGGCACCGGCTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCATCAGCTACATGCACTGGTACCAGCAGAAGCCCGGCACCCCCCCCAAGCGCTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCCTGAGCAGCATGGAGGCCGAGGACGTGGCCACCTACTACTGCCAGCAGTGGAGCAGCCCCCCCACCTTCGGCGTGGGCACCAAGCTGGAGCTGAAGCGC (Anti-MSLN single-chain antibody,That is, anti-MSLN scFv)-ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTAC (CD8α transmembrane domain,The first transmembrane domain)-AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (CD28 co-stimulatory domain)-AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (4-1BB co-stimulatory domain)-AGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (CD3ζ signaling domain)-GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT (P2A cleavage protein)-ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG (CD8 signal peptide, second signal peptide,-ATGTGCCCAGGTCAAACTGAGCAGTCAGGAGCTGAGCTGGCGAGTCCTGGGGCATCAGTGACACTGTCCTGCAAGGCTTCTGGCTACACATTTACTGACCATATTATAAATTGGGTTAAAAAGAGGCCTGGACAGGGCCTTGAATGGATTGGAAGGATTTATCCAGTAAGTGGTGAAACTAACTACAATCAAAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATGGGATGGTTACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGAGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAACCCTGGATTTATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTGGAGTAGTTACCCACTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGTACCCATACGATGTTCCAGATTACGCTGGATCC(anti-PD-1 single-chain antibody,This is the anti-PD-1scFv)-ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (CD8α transmembrane domain, second transmembrane domain, identical to the first transmembrane domain) (SEQ ID NO:24).

[0096] Expression Box

[0097] An expression cassette may contain a nucleic acid or polynucleotide molecule and a promoter that directs the nucleic acid or polynucleotide molecule. The nucleic acid or polynucleotide molecule and the promoter that directs the nucleic acid or polynucleotide molecule are in an operably linked manner, such that the promoter can effectively direct the expression of the nucleic acid or polynucleotide molecule. The type of promoter can be conventionally determined by those skilled in the art based on the directed nucleic acid or polynucleotide molecule. The nucleic acid or polynucleotide molecule may encode a cell membrane-anchored anti-PD-1 scFv or a chimeric antigen receptor. In this case, the promoter may be selected from CMV and EF1a promoters. The expression cassette may contain, from the 5' end to the 3' end, a promoter, a polynucleotide molecule encoding a chimeric antigen receptor, a P2A cleavage protein, and a cell membrane-anchored anti-PD-1 scFv. The expression cassette may also contain other regulatory elements, such as terminators.

[0098] Chimeric antigen receptor

[0099] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T Immunotherapy) is a novel cellular immunotherapy technology that has rapidly developed in recent years. Based on the theory of immune system recognition and activation, it uses genetic engineering to artificially overexpress single-chain antibody variable region gene fragments on the surface of T cells that recognize specific tumor surface antigens. This allows T cells to recognize specific antigens and kill target cells expressing those antigens. The core theoretical basis of CAR-T immunotherapy is the recognition and activation of T lymphocytes. It primarily involves different scFvs recognizing different specific antigens on tumor cells, and then transmitting signals through the hinge and transmembrane domain of the CD8 molecule to the CD28 or 4-1BB and TCR co-stimulatory activation region within the T lymphocyte membrane. This activates the body's own T lymphocytes, allowing them to specifically attack and kill the recognized tumor cells. Furthermore, because CAR-T cells use an antibody-based antigen recognition model, they are not subject to MHC restrictions.

[0100] CAR-T immunotherapy has achieved unprecedented success in the treatment of hematologic malignancies. Since the first CAR-T drug for hematologic malignancies was approved for marketing in 2017, five CAR-T drugs for hematologic malignancies have been approved by the FDA, and one has been introduced and marketed in China.

[0101] The chimeric antigen receptor described herein comprises at least an extracellular ligand-binding domain or portion, a transmembrane domain, and an intracellular domain comprising one or more signal transduction domains and / or co-stimulatory domains. The extracellular ligand-binding domain or portion may be an antibody or an antibody fragment. In this paper, the antibody fragment may be an scFv antibody fragment targeting MSLN. However, it should be understood that the extracellular ligand-binding domain or portion may be a binding moiety targeting any suitable antigen. The chimeric antigen receptor may target surface antigens selected from the group consisting of: Mesothelin, GD2, CD171, CD19, CD20, BCMA, GPC3, TERT, PTEN, PD-1, PD-L1, NKG2D ligand, CD44v6, FR, CD138, PSMA, NY-ESO, EGFR, CEA, HER2, CD22, CD30, CD123, CD5, CD7, CD33, CEA, EGFR, BRAF, HER-2, MUC1, PSCA, GPC3, or VEGF. The chimeric antigen receptor described in this paper may also include a CD8 precursor as a signal peptide. The chimeric antigen receptor described in this paper may also include CD8α as a transmembrane domain. The chimeric antigen receptor described in this paper may also include CD28 and / or 4-1BB co-stimulatory domains. The chimeric antigen receptor described in this paper may also include a CD3ζ signaling domain.

[0102] carrier

[0103] Constructs or expression cassettes can be delivered using known transfection and / or transduction vectors, including but not limited to lentiviral vectors, adeno-associated viruses, etc. Lentiviral vectors are a preferred vector type, capable of delivering large amounts of viral nucleic acid into host cells. Lentivirals are characterized by their unique ability to infect / transduce non-dividing cells, and after transduction, lentiviruses integrate their nucleic acid into the host cell's chromosome, but they themselves are not replicable. Lentivirals have three major genes encoding packaging proteins: gag, pol, and vsv-g, as well as the regulatory gene rev.

[0104] Lentiviral vector systems or lentivirus particles

[0105] Lentiviral virions (particles) are expressed by a vector system encoding essential viral proteins to produce non-replicating lentiviral virions (viral particles). At least one vector exists containing a nucleic acid sequence encoding a lentiviral pol protein essential for reverse transcription and integration, operatively linked to a promoter. For example, the pol protein is expressed by multiple vectors. Vectors containing a nucleic acid sequence encoding a lentiviral gag protein, essential for forming a viral capsid operatively linked to a promoter, may also exist. This gag nucleic acid sequence may be located on a vector different from at least some of the pol nucleic acid sequences. The gag nucleic acid may be located on a vector separate from all the pol nucleic acid sequences encoding the pol protein.

[0106] The Gag-pol, rev, and vsv-g vectors contain nucleotides of the lentiviral genome that package lentiviral RNA, called the lentiviral packaging sequence. As described above, lentiviral vector systems typically include at least two or three helper plasmids containing at least one of the gag, pol, or rev genes. Each of the gag, pol, and rev genes can be provided on a separate plasmid, or one or more genes can be provided together on the same plasmid. The gag, pol, and rev genes are provided on the same or separate plasmids, while vsv-g is provided on a single plasmid.

[0107] T cells

[0108] The CAR-T cells of this invention possess the property of self-blocking PD-1 expression on the cell surface, thereby blocking the PD-1 / PD-L1 signaling pathway and enhancing the anti-tumor function of CAR-T cells. The T cells of this invention introduce a polynucleotide molecule (cell membrane-anchored anti-PD-1 scFv) that blocks PD-1 expression and have the ability to completely block PD-1 expression on the surface of CAR-T cells. Compared with control T cells without the polynucleotide molecule and ordinary CAR-T cells, PD-1 expression in MSLN CAR-T cells of this invention, which simultaneously express membrane-anchored anti-PD-1 scFv, can be completely blocked. The T cells of this invention are particularly suitable for adoptive immunotherapy.

[0109] The T cells of the present invention can be prepared by the following method, which includes introducing the polynucleotide molecules, expression cassettes, vectors, or viral particles described herein into T cells to block the expression of PD-1 on the surface of CAR-T cells. Specifically, the method includes one or more of the following steps: 1) viral vector construction; 2) lentiviral packaging using 293T cells; 3) PBMC isolation and T cell sorting; 4) T cell activation; 5) lentiviral transfection of activated T cells; 6) CAR-T cell culture after infection, and flow cytometry detection of CAR positivity and CD3 positivity; 7) CAR-T cell killing assay.

[0110] Methods and uses

[0111] The T cells of this invention can be used to treat various cancers. Those skilled in the art can readily determine the type of cancer for CAR-T cell therapy based on the chimeric antigen receptor expressed by the T cells. Cancers include, but are not limited to, liver cancer, bladder cancer, various types of leukemia, multiple myeloma, malignant lymphoma, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, head and neck cancer, multiple squamous cell tumors, esophageal cancer, colorectal cancer, melanoma, osteosarcoma, rectal cancer, anal cancer, bile duct cancer, uterine cancer, ovarian cancer, gastric cancer, prostate cancer, meningioma, pancreatic cancer, breast cancer, and medulloblastoma. The treatment methods of this invention may include administering CAR-T cells to cancer patients. This invention also provides the use of CAR-T cells in the preparation of drugs or kits for treating cancers, such as one or more of the cancers listed above.

[0112] Example

[0113] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The content mentioned in the embodiments is not intended to limit the present invention.

[0114] Example 1: Screening and Binding Activity Assay of Anti-PD-1scFv

[0115] 1. Screening process for anti-PD-1scFv:

[0116] Recombinant human PD-1 protein (brand: Nearshore Protein, catalog number: CM81) was used as an immunogen to immunize mice. Specifically, a PD-1 protein emulsion was prepared by dissolving 0.1 mg of protein in 350 μL of PBS buffer and adding the solution to a 1 mL syringe. An equal volume of Freund's adjuvant was then added to another 1 mL syringe. Air was expelled, and the two syringes were connected using a Luer connector. The syringes were injected back and forth for 15-30 minutes until complete emulsification. A drop of the emulsion was gently dropped onto a surface of water; if it remained intact for more than 10 minutes, emulsification was considered successful. Three 6-8 week old female BALB / c mice were marked and immunized using a subcutaneous multi-site injection method. The mice were initially immunized with 30 μg of PD-1 protein (200 μL emulsion) per mouse. Fourteen days later, a booster immunization was performed using the same dose, consisting of a 1:1 (v / v) emulsion of PD-1 protein in PBS solution and Freund's incomplete adjuvant. A second booster immunization was performed 14 days later, for a total of three immunizations, resulting in the production of anti-human PD-1 antibodies in the mice.

[0117] Mouse spleen cells were collected, and an antibody cDNA library was generated through RNA isolation, PCR amplification, and cloning into a phage display vector. Specifically, RNA was extracted from spleen cells using Trizol (Thermo Fisher Scientific, catalog number: 15596026), and the RNA was reverse transcribed using a reverse transcription kit (Takara, catalog number: [missing information]) to obtain cRNA sequences. The cDNA was then subjected to PCR using library construction primers (primer synthesis company: Sangon Biotech) to obtain the library fragment. The fragment was then double-digested with sfiI (NEB, catalog number: R0123V) and NotI-HF (NEB, catalog number: R3189V), and the pCANTAB5E backbone was ligated and integrated into TG1 competent cells (Lucigen, catalog number: 60502-2) via electroporation.

[0118] Then, the library underwent multiple rounds of panning. Specifically: the library was infected with phages, and the phage solution was harvested. The day before use, the phages were panned with the recombinant human PD-1 protein-coated ELISA plate, with a coating volume of 100 μL per well. Three pannings were performed, with coating concentrations of 50 μg / mL, 20 μg / mL, and 10 μg / mL of recombinant human PD-1 protein in PBS solution. Phages that were positive for PD-1 antigen protein binding were obtained and sent for sequencing (sequencing company: Sangon Biotech) to obtain the nucleotide and amino acid sequences of anti-PD-1scFv.

[0119] Simultaneously, we used the VH and VL sequences of commercially available nivolumab (see US Patent No. 8,008,449) to obtain the nucleotide and amino acid sequences of nivolumab against PD-1 scFv as a positive control. Nivolumab is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell function (US Patent No. 8,008,449).

[0120] The anti-MSLN scFv nucleotide and amino acid sequences of this patent were used as negative controls. Plasmids expressing the anti-PD-1 scFv sequence, the nivolumab anti-PD-1 scFv sequence, and the anti-MSLN scFv sequence were obtained through gene synthesis (Sangon Biotech), respectively (all containing a mouse Fc tag, i.e., an mFc tag, directly linked to the C-terminus of the scFv). Protein production was outsourced to a protein manufacturing company (Nearshore Proteins) to obtain the screened mFc-anti-PD-1 scFv, mFc-nivolumab anti-PD-1 scFv, and mFc-anti-MSLN scFv for subsequent validation experiments.

[0121] The mFc amino acid sequence (P99-K324) used in this patent is an intrinsic sequence, derived from protein number AAK53870.1. The mFc amino acid sequence (P99-K324) used in this patent is as follows:

[0122] The corresponding nucleotide sequences are as follows:

[0123] 2. ELISA method to test the binding affinity of mFc-anti-PD-1 scFv, mFc-nivolumab-anti-PD-1 scFv, and mFc-anti-MSLN scFv to human PD-1.

[0124] 2.1 Coating antigen reagent:

[0125] Using PBS, dilute human PD-1 recombinant protein (brand: Nearshore Protein, catalog number: CM81) to the working concentration for ELISA (1 μg / mL). Add 100 μL of the human PD-1 recombinant protein working solution to a 96-well ELISA plate and incubate overnight at 4°C.

[0126] 2.2 Sealing and Washing:

[0127] Pour out the working solution of human PD-1 recombinant protein from the microplate, wash 5 times with PBST washing buffer, then add 100 μL of 1x blocking buffer for blocking, and incubate at 37°C for one hour.

[0128] The blocking solution was prepared as follows: First, 1x buffer (Coolaber, catalog number: PM5090-50x2L) was prepared using ddH2O; then, 1x buffer (i.e., 1x blocking solution) containing 1% BSA was prepared using BSA (Yisheng Biotechnology, catalog number: 36101ES60).

[0129] 2.3 Preparation and incubation of working solutions for mFc-anti-PD-1 scFv, mFc-nivolumab-anti-PD-1 scFv, and mFc-anti-MSLN scFv (all with mFc tags):

[0130] Wash the microplate five times. Dilute the selected mFc-anti-PD-1 scFv, mFc-nivolumab-anti-PD-1 scFv, and mFc-anti-MSLN scFv to different working concentrations using PBS (see Table 1). Then add the working solutions of mFc-anti-PD-1 scFv, mFc-nivolumab-anti-PD-1 scFv, and mFc-anti-MSLN scFv to the microplate at a volume of 100 μL / well and incubate at 37°C for one hour.

[0131] 2.4 Incubation with secondary antibody (goat anti-mouse IgG1 Fc antibody, HRP-labeled, specifically recognizing the mFc tag):

[0132] Pour out the working solutions of mFc-anti-PD-1 scFv, mFc-nivolumab anti-PD-1 scFv, and mFc-anti-MSLN scFv (all with mFc tags), wash the microplate 5 times, dilute the secondary antibody (brand: SIGMA, catalog number: A0168) to the working concentration (1:50000) with PBS, then add the secondary antibody working solution to the microplate at a volume of 100 μL / well and incubate at 37°C for one hour.

[0133] 2.5 Color Development and Detection:

[0134] Discard the secondary antibody working solution, wash the microplate five times, and use a TMB colorimetric kit (brand: Sangon, catalog number: C520026-0500) to add 100 μL of TMB solution to each well. Incubate at room temperature in the dark for 3 minutes, then add 50 μL of stop solution to each well. Read the data from the microplate at 450 nm using a microplate reader and perform calculations and analysis.

[0135] 3. Results

[0136] 3.1 Sequencing Results

[0137] Sequencing by Sangon Biotech revealed that the anti-PD-1scFv variant possesses a light chain variable region (SEQ ID NO:7) and a heavy chain variable region (SEQ ID NO:8).

[0138] SEQ ID NO:7:

[0139] SEQ ID NO:8:

[0140] Kabat analysis revealed the following amino acid sequence in the light chain variable region of the anti-PD-1scFv enzyme: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGGGTKLEIKRYPYDVPDYAGS (SEQ ID NO:7), where CDR-1 is SASSVSYMH (SEQ ID NO:1), CDR-2 is GTSNLAS (SEQ ID NO:2), and CDR-3 is QQWSSYPLT (SEQ ID NO:3); the heavy chain variable region amino acid sequence is: MCPGQTEQSGAELASPGASVTLSCKASGYTFTDHIINWVKKRPGQGLEWIGRIYPVSGETNYNQKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARWDGYYAMDYWGQGTTVTVSS (SEQ ID NO:7). NO:8), of which CDR-1 sequence is DHIIN (SEQ ID NO:4), CDR-2 sequence is RIYPVSGETNYNQKFKG (SEQ ID NO:5), and CDR-3 sequence is WDGYYAMDY (SEQ ID NO:6).

[0141] 3.2 Combining activity results

[0142] Table 1 shows the binding affinity data of the screened anti-PD-1 scFv, indicating that the screened anti-PD-1 scFv has good binding affinity to recombinant human PD-1 protein. Analysis of the comparison results with the nivolumab anti-PD-1 scFv positive control shows that the screened anti-PD-1 scFv has comparable binding affinity to the nivolumab anti-PD-1 scFv positive control.

[0143] The data in Table 1 show that the screened anti-PD-1 scFv and nivolumab anti-PD-1 scFv positive control both exhibited binding affinity to recombinant human PD-1 protein, while the negative control and PBS did not show binding affinity to recombinant human PD-1 protein. The anti-PD-1 scFv prepared in this study has comparable binding affinity to nivolumab anti-PD-1 scFv.

[0144] Example 2: Preparation of CAR-T cells

[0145] This embodiment describes the preparation of MSLN CAR-T cells synchronously expressing membrane-anchored anti-PD-1scFv: A polynucleotide molecule (SEQ ID NO: 24) containing a chimeric antigen receptor, P2A cleavage protein, and cell membrane-anchored anti-PD-1scFv was prepared. Gene synthesis and sequencing were performed by Shanghai Sangon Biotech Co., Ltd. The synthesized insert sequence was inserted into the pLenti-MCS-EF1-GFP plasmid (purchased from ALSTEM, catalog number: LV010). This plasmid includes: an EF1a promoter; the insertion site of the foreign gene; WPRE elements; an SV40 polyA sequence; regulatory elements such as promoters and enhancers; and key elements such as lentiviral packaging elements. This plasmid is a lentiviral expression vector, which can serve as the most effective vector for expressing the target gene in almost all mammalian cells, including non-dividing and dividing cells. It can accommodate a large foreign gene fragment, has high transfection efficiency, and can achieve satisfactory transfection results in T cells.

[0146] Figure 1 shows a schematic diagram of the CAR structure for simultaneous expression of cell membrane-anchored anti-PD-1 scFv and a conventional CAR. The conventional CAR does not contain cell membrane-anchored anti-PD-1 scFv or the P2A cleavage protein; the amino acid sequences of the other parts are identical to those of the CAR for simultaneous expression of cell membrane-anchored anti-PD-1 scFv. The promoter nucleotide sequence in Figure 1 is as follows:

[0147] The synthesis and sequencing of expression cassettes (hereinafter referred to as insert sequences) for synchronously expressing cell membrane-anchored anti-PD-1 scFv CARs and conventional CARs were performed by Shanghai Sangon Biotech Co., Ltd. A schematic diagram of the insert sequences is shown in Figure 1. Two insert sequences were constructed: one containing a promoter and a polynucleotide encoding a novel CAR element for synchronously expressing highly efficient PD-1 blocking; the other containing a promoter and a polynucleotide encoding a CAR, used as a control (conventional CAR).

[0148] The synthesized insertion sequence was inserted into the pLenti-MCS-EF1-GFP plasmid (purchased from ALSTEM, catalog number: LV010), transformed into competent DH5α cells, and plated on agar plates containing ampicillin. Multiple clones were picked from the agar plates and inoculated into 5 mL of liquid LB medium (containing ampicillin), and cultured in a constant temperature shaker for 12-16 h at 37°C and 250 rpm.

[0149] Plasmids were extracted according to the instructions of the plasmid miniprep kit (catalog number: DP103-03) purchased from Tiangen Biotech Co., Ltd. Each cloned plasmid (pLenti-MCS-EF1-GFP plasmid containing a polynucleotide sequence of a novel CAR element that synchronously and efficiently blocks PD-1 expression or a conventional CAR) was sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing to verify the accuracy of the inserted sequences. Based on the sequencing data provided by Shanghai Sangon Biotech Co., Ltd., the bacterial cultures of clones with correct sequences were selected for mass inoculation and shake-flask culture.

[0150] The expression vector plasmid was extracted using the MN endotoxin-free plasmid large-scale extraction kit, and its concentration and purity were measured using a spectrophotometer. Subsequently, the extracted expression vector plasmid was double-digested with AgeI-HF (NEB, catalog number: R3552S) and HpaI (NEB, catalog number: R3575S) and verified by agarose gel electrophoresis (Figure 2). Finally, the expression vector plasmid was sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing to verify the accuracy of the inserted sequence. Figure 2 is an agarose gel electrophoresis image of the double-digested products of the recombinant plasmid for simultaneously expressing and efficiently blocking the novel PD-1 element CAR of this invention. M: 1kb DNA marker; 1: Recombinant plasmid for simultaneously expressing and efficiently blocking the novel PD-1 element CAR (undigested); 2: Recombinant plasmid digestion product; 3: pLenti-MCS-EF1-GFP lentiviral expression plasmid; 4: Target fragment for simultaneously expressing and efficiently blocking the novel PD-1 element CAR.

[0151] Three packaging plasmids, pPACKH1-GAG, pPACKH1-REV, and pVSV-G (purchased from SBI, catalog number LV550A-1), were extracted using the MN Endotoxin-Free Plasmid Large-Scale Extraction Kit. Concentration and purity were measured using a spectrophotometer. Lentiviral packaging was performed using a four-plasmid packaging system according to the kit instructions. The four plasmids were a lentiviral expression plasmid containing a CAR structure that simultaneously expresses a novel element that efficiently blocks PD-1, and the lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV, and pVSV-G (purchased from SBI, catalog number LV550A-1). 293T cells were used.

[0152] The specific implementation steps are as follows:

[0153] (1) Plating within 24 hours before transfection: Generally, 293T cells with a passage number of no more than 3 times are selected. The cell density is adjusted according to the cell growth density and status. 293T cells with a growth density of 80% are then plated.

[0154] (2) Once the growth density reaches 60-90% and the cells are in good condition, virus packaging can be carried out;

[0155] (3) Use lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV and pVSV-G, as well as the lentiviral plasmids constructed in this paper, and perform lentiviral plasmid preparation according to the plasmid instructions.

[0156] (4) The transfection reagent used is lipofectamine 2000 (stored at 4℃), and the amount added is 2μL / μg plasmid;

[0157] (5) Mix the plasmid mixture in (3) and the transfection reagent mixture in (4) into one tube, let it stand at room temperature for 20 min, then add it to the cells in the medium and continue culturing;

[0158] (6) Collect the culture supernatant after 48h and 72h respectively, and filter it through a 0.45μm filter membrane;

[0159] (7) The collected viral fluid was concentrated using the PEG8000 concentration method, and the viral titer was determined by infecting 293T cells and by subsequent flow cytometry detection of the CAR positivity of infected 293T cells. The virus was stored at -80℃ for later use.

[0160] Example 3: PBMC isolation and T cell sorting, T cell activation, lentiviral infection and culture

[0161] 1. PBMC separation

[0162] (1) Take 6 mL of human peripheral blood (blood for research purposes);

[0163] (2) Dilution: Add an equal volume of PBS at room temperature and gently mix by pipetting.

[0164] (3) Sample addition: Take a 50mL centrifuge tube and add 6mL of Ficoll (lymphocyte separation solution) into the centrifuge tube. The volume ratio of Ficoll to the blood before dilution is 1:1. Tilt the centrifuge tube at 45° and slowly add the diluted blood to the Ficoll tube about 1cm above the Ficoll surface.

[0165] (4) Centrifugation: 18-20℃, 2000rpm, 30min. After centrifugation, the tube will separate into four layers from the bottom to the surface of the liquid: red blood cells and granulocytes, layered liquid, mononuclear cells, and plasma.

[0166] (5) Recovery: Insert the pipette directly into the cloud layer (or first remove the upper layer of plasma), gently aspirate the cloud layer, and place it into a new centrifuge tube;

[0167] (6) Washing: Add PBS at least 3 times the volume of PBMCs (peripheral blood mononuclear cells), 18-20℃, 2000rpm, 10min, twice;

[0168] (7) Cell counting: Discard the supernatant, add 1 mL of lymphocyte culture medium, mix well by pipetting, and prepare a PBMC cell suspension. Count using a hemocytometer: Mix one drop of PBMC suspension with one drop of 2% trypan blue staining solution and add to a hemocytometer. Count the total number of cells within 4 large squares under a microscope. Cell count / mL = Total number of cells in 4 large squares / 4 × 10⁻⁶ 4 x 2 (dilution factor).

[0169] 2. T cell sorting, activation, lentiviral infection and cell sorting

[0170] (1) Day -1: PBMCs were centrifuged and resuspended, then incubated with EasySep. TM The Human T Cell Isolation Kit (STEMCELL) is used to sort T cells according to the kit's instructions.

[0171] (2) 24-well plate coating: Take a Corning 24-well plate, taking 2 wells as an example, add 175 μL of CD3 monoclonal antibody (concentration: 5 μg / ml, purchased from Tongli Haiyuan, catalog number: GMP-TL101) and 175 μL of CD28 monoclonal antibody (concentration: 5 μg / ml, purchased from Tongli Haiyuan, catalog number: GMP-TL102) to each well. After adding, gently vortex to mix, seal the well with sealing film, and place in a refrigerator at 4°C overnight. The function of these two monoclonal antibodies is to activate T cells.

[0172] (3) Day 0: Cleaning the coated plate: Take out the 24-well plate coated on day -1, discard the supernatant, wash twice with PBS, and then add PBS for later use;

[0173] (4) T cell plating: Count the sorted T cells and adjust the final concentration to 5 x 10⁻⁶. 6 Add 400 μL of cell suspension to each well, i.e., add 2 x 10 cells / mL. 6 One cell;

[0174] (5) Viral infection: Infection was carried out with MOI=5. 1 mL of virus culture medium suspension was prepared, added to a 24-well plate, centrifuged at 1000g for 30 min, and the centrifuge temperature was adjusted to 32℃.

[0175] (6) Days 1-2: Observe cell status;

[0176] (7) Day 3: Transfer all cells from the 24-well plate to a 75cm culture medium containing 20mL of culture medium. 2 Observe the cell state in the culture flask;

[0177] (8) Day 9: Observe cell status and cell number. Detect MSLN CAR positivity rate by flow cytometry (as detailed in Example 4) (Figure 3). Subsequent experiments such as cell killing detection or cell cryopreservation will be conducted.

[0178] Example 4: Identification and Detection of MSLN CAR-T Cells

[0179] 1. Detection of CAR positive expression rate

[0180] (1) The obtained negative control (NC) group T cells (not infected with the virus) and sample group cells (infected with the virus prepared in Example 2, as prepared in Example 3) (i.e. MSLN CAR-T cells that simultaneously express cell membrane anchored anti-PD-1scFv and ordinary MSLN CAR-T cells) were gently washed twice with PBS + 2% BSA at 1500 rpm for 6 min, and the waste liquid was discarded.

[0181] (2) Add 200 μL of PBS to the NC tube and resuspend; add 100 μL of PBS to the sample tube, resuspend, and then add 100 μL of Biotinylated Human Mesothelin (brand: ACRO, catalog number: MSN-H82E9) working solution (3 μg / mL) and mix well;

[0182] (3) Incubate at room temperature for 1 hour, 1500 rpm for 3 minutes, then discard the waste liquid;

[0183] (4) Add 200 μL PBS, mix gently and resuspend, 1500 rpm, 3 min, and discard the waste liquid;

[0184] (5) Add 200 μL of PBS to the NC tube and resuspend; add 200 μL of PBS to the sample tube, resuspend, and then add 5 μL of APC-conjugated streptavidin (brand: Biolegend, catalog number: 405207) working solution and mix well;

[0185] (6) After incubating at room temperature in the dark for 1 hour, centrifuge at 1500 rpm for 3 min, discard the waste liquid, wash gently 3 times with PBS + 2% BSA, centrifuge at 1500 rpm for 3 min, and discard the waste liquid.

[0186] (7) Add 100 μL PBS, mix gently, resuspend, and then test on the instrument (brand: ACEA, model: NovoCyte D3000).

[0187] As shown in Figure 3, MSLN CAR-T cells that synchronously express cell membrane-anchored anti-PD-1 scFv accounted for 72.64% of the total number of T cells, while ordinary MSLN CAR-T cells accounted for 75.81% of the total number of T cells.

[0188] 2. Detection of PD-1 positive expression rate

[0189] (1) The obtained negative control (NC) group T cells (not infected with the virus) and sample group cells (infected with the virus prepared in Example 2, as prepared in Example 3) (i.e. MSLN CAR-T cells and ordinary MSLN CAR-T cells that simultaneously express cell membrane anchored anti-PD-1scFv) were gently washed twice with PBS + 2% BSA at 1500 rpm for 6 min, and the waste liquid was discarded.

[0190] (2) Add 200 μL of PBS to the NC tube and resuspend; add 195 μL of PBS to the sample tube, resuspend, and then add 5 μL of APC anti-human CD279(PD-1) Antibody (brand: Biolegend, catalog number: 367406) and mix well;

[0191] (3) Incubate at room temperature for 0.5 h, 1500 rpm for 3 min, then discard the waste liquid;

[0192] (4) Add 200 μL PBS, mix gently and resuspend, 1500 rpm, 3 min, and discard the waste liquid;

[0193] (5) Add 100 μL PBS, mix gently and resuspend, then run on the instrument (brand: ACEA, model: NovoCyte D3000) for detection.

[0194] As shown in Figure 4, compared with the PD-1 expression of uninfected T cells and ordinary MSLN CAR-T cells, the PD-1 expression of MSLN CAR-T cells that simultaneously express cell membrane anchored anti-PD-1 scFv was almost completely blocked.

[0195] Example 5: Detection of MSLN positive expression rate and real-time cell killing detection using Intelligent Cell Monitoring Instrument (RIMI)

[0196] 1. Detection of MSLN positive expression rate

[0197] (1) The obtained ovarian cancer cells SKOV3, intestinal tumor cells HCT116, and ovarian cancer cells ES-2 were gently washed twice with PBS + 2% BSA at 1500 rpm for 6 min, and the waste liquid was discarded.

[0198] (2) Add 200 μL of PBS to the NC tube and resuspend; add 195 μL of PBS to the sample tube, resuspend, and then add 5 μL of Biotin anti-human Mesothelin Antibody (brand: Biolegend, catalog number: 530203) and mix well;

[0199] (3) Incubate at room temperature for 0.5 h, 1500 rpm for 3 min, then discard the waste liquid;

[0200] (4) Add 200 μL PBS, mix gently and resuspend, 1500 rpm, 3 min, and discard the waste liquid;

[0201] (5) Add 200 μL of PBS to the NC tube and resuspend; add 200 μL of PBS to the sample tube, resuspend, and then add 5 μL of APC-conjugated streptavidin (brand: Biolegend, catalog number: 405207) working solution and mix well;

[0202] (6) After incubating at room temperature in the dark for 1 hour, centrifuge at 1500 rpm for 3 minutes, discard the waste liquid, wash gently 3 times with PBS + 2% BSA, centrifuge at 1500 rpm for 3 minutes, and discard the waste liquid.

[0203] (7) Add 100 μL PBS, mix gently, resuspend, and then test on the instrument (brand: ACEA, model: NovoCyte D3000).

[0204] 2. Intelligent Cell Real-Time Monitoring Instrument (RIMI) for Real-Time Cell Killing Detection

[0205] (1) Taking human ovarian cancer cells SKOV3 or intestinal tumor cells HCT116 as examples (SKOV3 and HCT116 are MSLN target-positive cells), MSLN target-negative cells ES-2 were used as controls (see Figure 5). After digestion, tumor cell suspensions were prepared, mixed by pipetting, and then cell counting was performed;

[0206] (2) Dilute the tumor cell suspension to 2 x 10 5 Cells / mL concentration, keep on ice for later use;

[0207] (3) Turn on program 1 of the intelligent cell real-time monitoring instrument (brand: Six Broad Beans, model: CM100-α), take out the RIMI detection plate, and add 100μL of tumor cell suspension / well;

[0208] (4) Place the RIMI detection plate into the intelligent cell real-time monitor and observe whether program 1 is running normally. After confirming that it is running normally, proceed to the next step.

[0209] (5) After 24 hours, once the tumor cells have adhered to the culture plate, pause program 1, remove the detection plate, and add 100 μL of effector cell suspension to the corresponding well. Remove effector cells (including uninfected T cells, MSLN CAR-T cells that synchronously express cell membrane-anchored anti-PD-1scFv, and ordinary MSLN CAR-T cells, as prepared above) from the culture flask, centrifuge, wash, count, and prepare effector cells according to an effector-to-target ratio of 0.5:1.

[0210] (6) After adding the effector cell suspension, place the detection plate into the intelligent cell real-time monitor and continue to run program 1 for daily observation.

[0211] Figure 5 shows a flow cytometry analysis of MSLN expression in tumor cells. Figure 5 illustrates MSLN-target-positive tumor cells (ovarian tumor cells SKOV3 and intestinal tumor cells HCT116) and MSLN-non-target-expressing tumor cells (ovarian tumor cells ES-2).

[0212] Figure 6 shows the killing efficiency of MSLN CAR-T cells synchronously expressing cell membrane-anchored anti-PD-1 scFv and ordinary MSLN CAR-T cells against ES-2, SKOV3, and HCT116 tumor cells as detected by RIMI. In Figure 6, 0.5:1 indicates that the ratio of effector cells to target cells is 0.5:1; "culture medium" indicates that no effector cells were added, only tumor target cells; uninfected T cells indicate that the effector cells are uninfected T cells; ordinary MSLN CAR-T cells indicate that the effector cells are ordinary MSLN CAR-T cells; MSLN CAR-T cells synchronously expressing novel PD-1 blocking elements indicate that the effector cells are T cells infected with CAR-structured viruses synchronously expressing cell membrane-anchored anti-PD-1 scFv (i.e., MSLN CAR-T cells synchronously expressing cell membrane-anchored anti-PD-1 scFv).

[0213] As shown in Figure 6, MSLN CAR-T cells simultaneously expressing cell membrane-anchored anti-PD-1 scFv exhibit a significant killing effect on MSLN-targeted positive tumor cells. The killing effect of MSLN CAR-T cells simultaneously expressing cell membrane-anchored anti-PD-1 scFv on MSLN-targeted positive tumor cells is significantly superior to that of ordinary MSLN CAR-T cells.

[0214] Example 6: ELISA detection of cytokine IFNγ and TNFα secretion

[0215] (1) Dilute 1x coating buffer (Coolaber, catalog number: PM5090-50x2L) with ddH2O to prepare 250x coating antibodies for IFNγ and TNFα detection, respectively. These are the capture antibodies for IFNγ and TNFα, and were purchased from Sino. The IFNγ detection kit contains IFNγ capture antibody, IFNγ detection antibody, and IFNγ standard, purchased from Sino, catalog number: SEKA11725. The TNFα detection kit contains TNFα capture antibody, TNFα detection antibody, and TNFα standard, purchased from Sino, catalog number: SEKA10602. Add 8μL of 250x coating antibody to 2mL of coating buffer;

[0216] (2) Add 100 μL / well of the coating solution prepared in step 1) to the Corning 9018 ELISA high affinity 96-well plate, seal and place in a 4°C refrigerator overnight;

[0217] (3) The coated 96-well plate was washed three times with PBST (0.05% Tween 20);

[0218] (4) Prepare 1x blocking buffer with ddH2O: First, prepare 1x buffer solution (Coolaber, catalog number: PM5090-50x2L) with ddH2O; then prepare 1x buffer solution (i.e. 1x blocking buffer) containing 1% BSA with BSA (Yisheng Biotechnology, catalog number: 36101ES60), add 200μL / well, and block at room temperature for 1h;

[0219] (5) Prepare the IFNγ and TNFα standards by adding 1x blocking buffer according to the requirements of the aforementioned test kit, and perform 7 serial dilutions. Dilute the samples 5-fold. The samples here are the supernatants of CAR-T cells co-cultured with HCT116, SKOV3, or ES-2 for 24 hours in the above RIMI assay.

[0220] (6) Wash the blocked plate with PBST 5 times, add the standard and diluted sample solution, and incubate at room temperature for 2 hours or at 4°C overnight;

[0221] (7) Wash with PBST 4 times;

[0222] (8) Dilute 250x of IFNγ and TNFα detection antibodies (which are reagents in the aforementioned detection kit) with 1x blocking buffer, add 100 μL / well, and incubate at room temperature for 1 h;

[0223] (9) Wash 4 times with PBST, dilute 250xHRP with 1x blocking buffer, add 100μL / well, and incubate at room temperature for 30min;

[0224] (10) Wash 5 times with PBST, add 100 μL of the colorimetric solution from the EL-TMB colorimetric kit (brand: Sangon, catalog number: C520026) to each well, and incubate at room temperature for 15 min;

[0225] (11) Stop the color development by adding 50 μL / well of the stop solution from the EL-TMB colorimetric kit;

[0226] (12) OD value was detected by microplate reader at 450nm.

[0227] Figure 7 shows the bar chart of ELISA detection of IFNγ and TNFα factors 24 h after ES2 cells, SKOV3 cells, or HCT116 cells were killed. The CAR-T cells of this invention (MSLN CAR-T cells simultaneously expressing cell membrane-anchored anti-PD-1scFv and ordinary MSLN CAR-T cells) secreted a large amount of IFNγ factor after co-culturing with SKOV3 cells. The MSLN CAR-T cells simultaneously expressing cell membrane-anchored anti-PD-1scFv secreted IFNγ and TNFα factors more efficiently than ordinary MSLN CAR-T cells. In Figure 7, * (P < 0.05), ** (P < 0.01), and *** (P < 0.001) all indicate statistically significant differences.

[0228] In summary, this invention provides a method for preparing CAR-T cells that simultaneously express a novel, highly efficient PD-1-blocking element and its application. This CAR-T cell simultaneously expresses a cell membrane-anchored anti-PD-1 scFv. This element is anchored on the surface of T cells, requires no secretion, and is continuously expressed, avoiding the problems of uncontrollable secretion and antibody half-life. This CAR-T cell continuously blocks the expression of PD-1 on the surface of T cells, thus blocking the PD-1 / PD-L1 signaling pathway that causes T cell exhaustion. The PD-1 / PD-L1 signaling pathway causing T cell exhaustion is widely recognized in the field. Flow cytometry results in this paper show that PD-1 expression on the surface of T cells is essentially completely blocked. Since the PD-1 / PD-L1 signaling pathway involves the binding of PD-L1 expressed by tumor cells to PD-1 on the surface of T cells, the blocking of PD-1 expression on the T cell surface can be considered as the blocking of the PD-1 / PD-L1 signaling pathway. This enhances the anti-tumor function of CAR-T cells.

[0229] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the examples described above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. An anti-PD-1 antibody or its antigen-binding fragment, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the light chain complementarity-determining region LCDR 1-3 in SEQ ID NO:7, and the heavy chain variable region comprises the heavy chain complementarity-determining region HCDR 1-3 in SEQ ID NO:

8. Preferably, the amino acid sequence of LCDR-1 is SASSVSYMH (SEQ ID NO:1), the amino acid sequence of LCDR-2 is GTSNLAS (SEQ ID NO:2), the amino acid sequence of LCDR-3 is QQWSSYPLT (SEQ ID NO:3), the amino acid sequence of HCDR-1 is DHIIN (SEQ ID NO:4), the amino acid sequence of HCDR-2 is RIYPVSGETNYNQKFKG (SEQ ID NO:5), and the amino acid sequence of HCDR-3 is WDGYYAMDY (SEQ ID NO:6).

2. The anti-PD-1 antibody or its antigen-binding fragment according to claim 1, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8; Optionally, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

8.

3. The anti-PD-1 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antigen-binding fragment is one or more of Fab', Fab, F(ab')2, Fd fragment, dAb fragment, camel antibody, nanobody and single-chain Fv; preferably, the single-chain Fv comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

4. A fusion comprising a first part or a first part and a second part, wherein: The first portion comprises, from the N-terminus to the C-terminus, an optional first signal peptide, an anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims 1-3, and a first transmembrane domain; The second part includes an optional second signal peptide and a chimeric antigen receptor; the chimeric antigen receptor includes an antigen-binding domain, a second transmembrane domain, a co-stimulatory domain, and a signal transduction domain from the N-terminus to the C-terminus. When a second part exists, the first part is located at the C-end or N-end of the second part; preferably, the second part is located at the N-end of the first part. Preferably, the anti-PD-1 antibody or its antigen-binding fragment is in scFv form. Preferably, the anti-PD-1 antibody or its antigen-binding fragment is directly linked to the first transmembrane domain or linked through a linker; Preferably, the first transmembrane domain is a CD8α transmembrane domain; Preferably, the first part and the second part are connected by a self-cleaving protein; Preferably, the self-cleaving protein is a 2A cleaving protein; Preferably, the 2A cleavage protein is selected from P2A, T2A, and F2A; Preferably, the first signal peptide and the second signal peptide are the same or different, and optionally each is an independent CD8 precursor; Preferably, the antigen-binding domain binds to a cell surface cancer antigen, wherein the cell surface cancer antigen is one or more of the following: Mesothelin, GD2, CD171, CD19, CD20, BCMA, GPC3, TERT, PTEN, PD-1, PD-L1, NKG2D ligand, CD44v6, FR, CD138, PSMA, NY-ESO, EGFR, CEA, HER2, CD22, CD30, CD123, CD5, CD7, CD33, CEA, EGFR, BRAF, HER-2, MUC1, PSCA, GPC3, or VEGF; Preferably, the co-stimulatory domain is a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or a combination of a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain; Preferably, the signal conduction structure domain is a CD3ζ signal conduction structure domain; Preferably, the second transmembrane domain is a CD8α transmembrane domain; Preferably, the CD28 co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:9; The 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:10; Preferably, the co-stimulatory domain comprises a combination of CD28 and 4-1BB; Preferably, the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:11; Preferably, the antigen-binding domain of the chimeric antigen receptor comprises the HCDR-1 sequence DYAMH (SEQ ID NO:18), the HCDR-2 sequence VISTYNGNINYNQKFKG (SEQ ID NO:19), and the HCDR-3 sequence GGYDGTGFDY (SEQ ID NO:20); the LCDR-1 sequence SASSSISYMH (SEQ ID NO:21), the LCDR-2 sequence DTSKLAS (SEQ ID NO:22), and the LCDR-3 sequence QQWSSPPT (SEQ ID NO:23); preferably, the antigen-binding domain of the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:12; Preferably, the fusion comprises the structure of CD8 signal peptide-anti-MSLN scFv-CD8α transmembrane domain-CD28 co-stimulatory domain-4-1BB co-stimulatory domain-CD3ζ signal transduction domain-P2A cleavage protein-CD8 signal peptide-anti-PD-1scFv-CD8α transmembrane domain.

5. A composition or conjugate comprising an anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims 1-3; Preferably, the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Preferably, the conjugate also contains a cytotoxic agent.

6. A first nucleic acid encoding an anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims 1-3, preferably, the first nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:13 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:

13.

7. A second nucleic acid, encoding the fusion compound according to claim 4; Preferably, the coding sequence of the first part comprises the nucleotide sequence shown in SEQ ID NO:14 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:14; Preferably, the coding sequence of the second part comprises the nucleotide sequence shown in SEQ ID NO:15 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:15; Preferably, the second nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:24 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:

24.

8. An expression cassette comprising a first nucleic acid according to claim 6 or a second nucleic acid according to claim 7 and further comprising a control element operatively connected to said nucleic acid, preferably, the control element being one or more of a promoter, an enhancer, and a terminator; preferably, said promoter is selected from one or more of CMV and EF1a promoters.

9. A carrier comprising the expression cassette according to claim 8; Preferably, the vector is a viral vector; Preferably, the vector is a lentiviral vector or an adeno-associated virus vector.

10. A virus comprising the expression cassette of claim 8 or the vector of claim 9; preferably, wherein the virus is a lentivirus or an adeno-associated virus.

11. Use of an anti-PD-1 antibody or its antigen-binding fragment according to any one of claims 1-3, or a composition or conjugate according to claim 5, in the preparation of a medicament for treating a disease, preferably cancer, preferably a disease involving the PD-1 / PD-L1 signaling pathway; preferably, the cancer is one or more of melanoma, non-small cell lung cancer, nasopharyngeal carcinoma, glioblastoma, colon adenocarcinoma, hepatocellular carcinoma, urothelial carcinoma, multiple myeloma, ovarian cancer, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, lymphoma such as Hodgkin's lymphoma, and leukemia.

12. A cell comprising the first nucleic acid according to claim 6, the second nucleic acid according to claim 7, the expression cassette according to claim 8, the vector according to claim 9, or the virus according to claim 10, preferably, the cell is an immune cell, preferably one or more of T cells, macrophages, and NK cells; preferably, the T cell is one or more of cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T cells.

13. An immune cell comprising the first portion or the first and second portions as described in claim 4, wherein the first portion is anchored to the cell surface via a first transmembrane domain, or the first and second portions are anchored to the cell surface via first and second transmembrane domains, respectively, preferably one or more of T cells, macrophages, and NK cells; preferably, the T cells are one or more of cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T cells.

14. A method for preparing cells according to claim 12 or immune cells according to claim 13, comprising introducing a first nucleic acid according to claim 6, a second nucleic acid according to claim 7, an expression cassette according to claim 8, a vector according to claim 9, or a virus according to claim 10 into the cells, thereby blocking the expression of PD-1 on the surface of T cells, and thus blocking the PD-1 / PD-L1 binding signaling pathway.

15. The method according to claim 14, comprising one or more of the following steps: 1) viral vector construction; 2) lentivirus packaging using 293T cells; 3) PBMC isolation and T cell sorting; 4) T cell activation; 5) Lentiviral transfection of activated T cells; 6) CAR-T cell culture after infection and detection of CAR positivity; 7) CAR-T cell killing assay; and 8) Detection of tumor suppression effect of CAR-T cells in tumor models.

16. The use of the immune cells of claim 13 in the preparation of a medicament for treating and / or preventing cancer, preferably wherein the cancer is selected from one or more of liver cancer, bladder cancer, various types of leukemia, multiple myeloma, malignant lymphoma, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, head and neck cancer, multiple squamous cell tumors, esophageal cancer, colorectal cancer, melanoma, osteosarcoma, rectal cancer, anal cancer, bile duct cancer, uterine cancer, ovarian cancer, gastric cancer, prostate cancer, meningioma, pancreatic cancer, breast cancer, and medulloblastoma.

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