Mutated PD1 extracellular domain fragment and CAR and NK cell containing fragment

Abstract

The invention relates to a mutant PD1 extracellular domain fragment and CAR and NK cells containing the fragment. Specifically, the invention provides a polypeptide which is the mutant PD1 extracellular domain fragment and contains an amino acid sequence as shown in SEQ ID NO: 1 or consists of the amino acid sequence as shown in SEQ ID NO: 1. The CAR provided by the invention contains the polypeptide. The invention also provides an NK cell for expressing the CAR. The tumor cell killing capacity of the NK cell expressing the CAR containing the mutant PD1 extracellular domain is obviously higher than that of the NK cell expressing the CAR containing the wild type PD1 extracellular domain.

Description

Mutated PD1 extracellular domain fragment and CAR and NK cells containing the fragment Technical Field

[0001] The present invention relates to a mutant PD1 extracellular domain fragment and a CAR and NK cell containing the fragment. Background Art

[0002] Under normal physiological conditions, activation of PD-1 signaling can suppress excessive inflammatory responses and prevent the development of autoimmune diseases. However, in the tumor environment, PD-1 / PD-L1 signaling is a key pathway for tumor cells to escape immune control. High levels of PD-L1 protein have been detected in the blood and in tumor situ in patients with various cancers, including lung cancer, breast cancer, melanoma, head and neck cancer, and lymphoma. In tumors, PD-L1 interacts with the PD-1 receptor on immune cells, including T and NK cells. The PD-1 receptor acts as a brake signal for these cells. It contains two inhibitory domains within the cell: the ITIM (immunoreceptor tyrosine-based inhibitory motif) and the ITSM (immunoreceptor tyrosine-based switch motif). These domains inhibit TCR signaling and NK activation signals through phosphorylation of the SHP protein. Currently, several antibody therapies targeting PD-1 / PD-L1 have shown some effectiveness in treating tumors such as lymphoma and melanoma, but these remain limited. On the one hand, antibody therapy can only block the interaction between immune cells and tumor cells in the tumor through antibody blocking, but it still has an immunosuppressive effect on the immune cells that have already interacted, which cannot be reversed. On the other hand, the Fc region of the antibody will largely interact with the patient's immune cells through ADCC / ADCP, thereby causing immune system disorders.

[0003] NK-CAR (Chimeric Antigen Receptor NK Cell) technology is a new type of cell therapy that infuses modified NK cells back into the body to directly kill tumors and activate the body's immune system. This technology overcomes the cytotoxicity, drug resistance, and recurrence of traditional treatments and is considered one of the most effective treatments for malignant tumors. NK-CAR technology has achieved remarkable results in the treatment of hematologic malignancies such as acute and chronic lymphocytic leukemia and lymphoma (e.g., B-cell lymphoma and acute and chronic B-cell leukemia).

[0004] NK-CAR cells have strong killing ability, but in the tumor microenvironment, PDL1 and other proteins in the tumor microenvironment will inhibit the activity of cytotoxic NK cells through the immune cell receptor PD1, thereby allowing tumor cells to escape immune surveillance. Therefore, it is hoped that the negative feedback signal of tumor cells (PDL1-PD-1 interaction) can be used to activate immune cells, thereby reducing the inhibitory effect of tumor cells on immune cells and activating NK cells, thereby enhancing the killing effect of NK-CAR on tumor cells.

[0005] Summary of the Invention

[0006] The present invention provides a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0007] The present invention also provides a chimeric antigen receptor, which contains an optional signal peptide, a polypeptide described herein, a hinge region, a transmembrane region, an intracellular co-stimulatory signal domain, and an intracellular signal domain, which are sequentially connected from the N-terminus to the C-terminus.

[0008] In one or more embodiments, the signal peptide is selected from CD8 signal peptide, CD28 signal peptide or CD4 signal peptide; preferably, the signal peptide is CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO: 3, amino acid residues 1 to 20.

[0009] In one or more embodiments, the hinge region is selected from the CD8α hinge region, the IgD hinge region, the IgG1 Fc CH2CH3 hinge region or the IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is the CD8α hinge region; preferably, the amino acid sequence of the CD8α hinge region is as shown in amino acid residues 171 to 225 of SEQ ID NO: 3.

[0010] In one or more embodiments, the transmembrane region is selected from the CD28 transmembrane region, the CD8 transmembrane region, the CD3ζ transmembrane region, the CD134 transmembrane region, the CD137 transmembrane region, the ICOS transmembrane region or the DAP10 transmembrane region; preferably, the transmembrane region is the CD8 transmembrane region; preferably, the amino acid sequence of the CD8 transmembrane region is as shown in amino acid residues 226 to 246 of SEQ ID NO: 3.

[0011] In one or more embodiments, the intracellular costimulatory signaling domain is an intracellular domain of a costimulatory signaling molecule, preferably selected from the intracellular domain of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell costimulatory factor or DNAX activating protein 10; preferably, the intracellular costimulatory signaling domain is the intracellular domain of 4-1BB; preferably, the amino acid sequence of the intracellular domain of 4-1BB is as shown in amino acid residues 247 to 288 of SEQ ID NO: 3.

[0012] In one or more embodiments, the intracellular signaling domain is the CD3ζ intracellular signaling domain or the FcεRIγ intracellular signaling domain; preferably, the intracellular signaling domain is the CD3ζ intracellular signaling domain; preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in amino acid residues 289 to 400 of SEQ ID NO: 3.

[0013] In one or more embodiments, the chimeric antigen receptor contains, from N-terminus to C-terminus, a CD8 signal peptide, the polypeptide of claim 1, a CD8α hinge region, a CD8 transmembrane region, a 4-1BB intracellular domain, and a tyrosine activation motif of CD3ζ.

[0014] In one or more embodiments, the amino acid sequence of the chimeric antigen receptor is amino acid residues 1 to 20 of SEQ ID NO: 3, amino acid residues 171 to 400 of SEQ ID NO: 1, and SEQ ID NO: 3, connected sequentially from N-terminus to C-terminus; or the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 3.

[0015] The present invention provides a nucleic acid molecule encoding the polypeptide chimeric antigen receptor described herein; preferably, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0016] In one or more embodiments, the nucleic acid molecule is the coding sequence of the polypeptide of claim 1, and its nucleotide sequence is shown in the nucleotide sequence at positions 70 to 510 of SEQ ID NO: 2.

[0017] In one or more embodiments, the nucleic acid molecule comprises, from 5' to 3' end, the nucleotide sequence shown in SEQ ID NO: 2, positions 1 to 60, and the nucleotide sequence shown in SEQ ID NO: 2, positions 70 to 1200, or its nucleotide sequence is as shown in SEQ ID NO: 2.

[0018] In one or more embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0019] The present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein.

[0020] In one or more embodiments, the nucleic acid construct is an expression cassette that contains, in addition to the nucleic acid molecule, regulatory sequences.

[0021] In one or more embodiments, the nucleic acid construct is a vector, including expression vectors and integration vectors for integrating the nucleic acid molecule into the genome of a host cell; preferably, the vector is a transposon vector, more preferably, the transposon vector is a eukaryotic expression vector containing a transposable element selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty.

[0022] The present invention also provides a host cell, which contains the nucleic acid construct described herein and / or expresses the polypeptide or CAR described herein; preferably, the host cell is a NK cell.

[0023] The present invention also provides a composition or kit comprising the vector described herein and optionally a transfection agent.

[0024] In one or more embodiments, the kit contains the composition.

[0025] The present invention also provides a pharmaceutical composition comprising the NK cells described herein and a pharmaceutically acceptable carrier or excipient.

[0026] The present invention also provides the use of the polypeptides, chimeric antigen receptors, nucleic acid molecules, nucleic acid constructs, host cells or pharmaceutical compositions described herein in the preparation of drugs for treating or preventing PD1 or PDL1-mediated cancers; preferably, the cancer is selected from gastric cancer, lung cancer (such as non-small cell lung cancer), liver cancer, intrahepatic bile duct cancer, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, bladder cancer, renal cell carcinoma, skin cancer and oral squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1: Sequence of NK-PD1M-CAR targeting PDL1.

[0028] Figure 2: CD56 expression and CAR expression in NK-PD1M-CAR. In the figure, the control ("control") is the same batch of NK cells that were not transferred with the gene encoding the chimeric antigen receptor PD1M-CAR.

[0029] Figure 3: In vitro killing results of NK cells against K562 tumor cell line overexpressing PD-L1.

[0030] Figure 4: Killing results of NK+HAC and NK-PD1M-CAR against A549. Based on the rightmost curve in the figure, from top to bottom, the corresponding curves are: A549; A549+NK+HAC, E / T=0.625; A549+NK, E / T=1.25; A549+NK+HAC, E / T=1.25; A549+NK-PD1M-CAR, E / T=0.625; A549+NK-PD1M-CAR, E / T=1.25.

[0031] Figure 5: Cytotoxicity of NK-PD1WT-CAR and NK-PD1M-CAR against A549. The rightmost curve in the figure, from top to bottom, corresponds to: A549; A549 + NK-GFP + HAC, E / T = 0.625; A549 + NK-PD1WT-CAR, E / T = 0.625; A549 + NK-PD1M-CAR, E / T = 0.625; A549 + NK-GFP + HAC, E / T = 1.25; A549 + NK-PD1WT-CAR, E / T = 1.25; A549 + NK-PD1M-CAR, E / T = 1.25.

[0032] Figure 6: Cytotoxicity of H1299 cells by NK-PD1WT-CAR and NK-PD1M-CAR. The rightmost curve in the figure, from top to bottom, corresponds to: H1299; H1299 + NK-GFP + HAC; H1299 + NK-PD1WT-CAR; H1299 + NK-PD1M-CAR.

[0033] Figure 7: Cytotoxicity of HCC827 cells by NK-PD1WT-CAR and NK-PD1M-CAR. The rightmost curve in the figure, from top to bottom, corresponds to: HCC827; HCC827 + NK-GFP + HAC; HCC827 + NK-PD1WT-CAR; HCC827 + NK-PD1M-CAR.

[0034] Figure 8: Killing effect of NK-PD1M-CAR targeting HCC827.

[0035] Figure 9: PDL1 expression in several cell lines.

[0036] Figure 10: CD56 expression and CAR expression in NK-PD1WT-CAR. In the figure, the control ("control") is the same batch of NK cells that were not transferred with the gene encoding the chimeric antigen receptor PD1WT-CAR.

[0037] Figure 11: Diagram of the in vivo CAR NK action mode.

[0038] Figure 12: In vivo imaging of mice.

[0039] Figure 13: In vivo imaging statistics.

[0040] Figure 14: Tumor growth in mice on day 21 after infusion of different NK cells.

[0041] Figure 15: Mouse survival curve. DETAILED DESCRIPTION

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0043] In the present invention, the term "expression cassette" refers to the complete elements required for expressing a gene, including a promoter, a gene coding sequence, and a Poly A tailing signal sequence.

[0044] The term "coding sequence" refers to the portion of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR). The boundaries of the coding sequence are typically determined by the ribosome binding site (for prokaryotes) immediately upstream of the mRNA 5' end open reading frame and the transcription termination sequence immediately downstream of the mRNA 3' end open reading frame. The coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0045] The term "co-stimulatory molecule" refers to a molecule present on the surface of antigen-presenting cells that can bind to the co-stimulatory molecule receptors on Th cells to generate a co-stimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens but also the reception of co-stimulatory molecule signals. Co-stimulatory signals are transmitted to T cells mainly through the binding of co-stimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells to CD28 molecules on the surface of T cells. B cells can receive co-stimulatory signals through common pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells.

[0046] The term "linker" or hinge refers to a polypeptide segment that connects different proteins or polypeptides. Its purpose is to maintain the spatial conformation of the connected proteins or polypeptides to maintain the function or activity of the proteins or polypeptides. Exemplary linkers include linkers containing G and / or S, and, for example, the Furin 2A peptide.

[0047] The term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0048] The term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0049] The term "disease and / or condition" refers to a physical condition of the subject, which is associated with the diseases and / or conditions described herein.

[0050] The term "subject" or "patient" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, alleviate and / or relieve the disease or condition described in the present invention.

[0051] The term "chimeric antigen receptor" (CAR) is an artificially modified receptor that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens to immune cells (such as T cells), allowing immune cells to recognize tumor antigens or viral antigens and kill tumor cells or virus-infected cells. CARs typically contain, in sequence, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signaling region.

[0052] The present invention utilizes a CAR constructed with a mutant PD1 extracellular segment and expresses the CAR in NK cells. The present invention has been completed by discovering that NK cells expressing a CAR containing a mutant PD1 extracellular segment have a significantly stronger ability to kill tumor cells than NK cells expressing a CAR containing a wild-type PD1 extracellular segment.

[0053] Therefore, the first aspect of the present invention provides a mutant polypeptide, which is a mutant of the extracellular segment of PD1. In some embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1. The polypeptide of the present invention also includes mutations having 1 to 8, preferably 1 to 5, more preferably 1 to 3 amino acid mutations (including insertions, deletions and / or substitutions) compared to SEQ ID NO: 1. The mutation is preferably a conservative substitution well known in the art, for example, replacing one or more (such as 1 to 8, 1 to 5 or 1 to 3) of SEQ ID NO: 1 with amino acid residues having the same or similar properties (such as classification based on side chain groups or classification based on chemical structure). It should be understood that the mutant of SEQ ID NO: 1 retains the biological activity of SEQ ID NO: 1, especially the biological activity of SEQ ID NO: 1 demonstrated herein.

[0054] The second aspect of the present invention provides a chimeric antigen receptor (CAR), which contains, from N-terminus to C-terminus, a signal peptide, a mutant of the PD1 extracellular segment described herein, a hinge region, a transmembrane region, an intracellular co-stimulatory signal domain and an intracellular signal domain connected in sequence.

[0055] A signal peptide is a short peptide chain (5-30 amino acids in length) that directs newly synthesized proteins into the secretory pathway. It often refers to the amino acid sequence at the N-terminus (sometimes not necessarily at the N-terminus) of a newly synthesized polypeptide chain that directs transmembrane transfer (localization) of proteins. It is responsible for directing proteins to subcellular organelles containing different membrane structures in the cell. In some embodiments, the signal peptide used herein can be selected from a CD8 signal peptide, a CD28 signal peptide, or a CD4 signal peptide. Preferably, the signal peptide is a CD8 signal peptide. Preferably, the amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO: 3, amino acid residues 1 to 20.

[0056] The hinge region refers to the region between the CH1 and CH2 functional regions of an immunoglobulin heavy chain. This region is rich in proline, does not form an alpha helix, and is prone to stretching and some degree of distortion. In some embodiments, the hinge region used herein can be selected from the group consisting of a CD8α hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region. Preferably, the hinge region is a CD8α hinge region or an IgG4 Fc CH2CH3 hinge region. More preferably, the amino acid sequence of the CD8α hinge region is as shown in SEQ ID NO: 3, amino acid residues 171 to 225.

[0057] In some embodiments, the transmembrane region used herein may be one of the CD28 transmembrane region, the CD8 transmembrane region, the CD3ζ transmembrane region, the CD134 transmembrane region, the CD137 transmembrane region, the ICOS transmembrane region, and the DAP10 transmembrane region. Preferably, the CD8 transmembrane region is used herein; more preferably, the amino acid sequence of the CD8 transmembrane region is as shown in amino acid residues 226 to 246 of SEQ ID NO: 3.

[0058] In some embodiments, the intracellular costimulatory signaling domain used herein includes the intracellular domain of a costimulatory signaling molecule, including CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell costimulator (ICOS) and the intracellular domain of DNAX activating protein 10. Preferably, the intracellular costimulatory signaling domain is the intracellular domain of 4-1BB. More preferably, the amino acid sequence of the intracellular domain of 4-1BB is as shown in SEQ ID NO: 3 amino acid residues 247 to 288.

[0059] In some embodiments, the intracellular signaling domain used herein is the CD3ζ intracellular signaling domain or the FcεRIγ intracellular signaling domain. Preferably, the CD3ζ intracellular signaling domain is used herein; more preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 3, amino acid residues 289 to 400.

[0060] In some embodiments, the chimeric antigen receptor described herein contains, from N-terminus to C-terminus, a CD8 signal peptide, a mutant of the PD1 extracellular segment described herein, a CD8a hinge region, a CD8 transmembrane region, a 4-1BB intracellular domain, and a tyrosine activation motif of CD3ζ.

[0061] The above-mentioned parts forming the chimeric antigen receptor herein, such as the signal peptide, mutants of the extracellular segment of PD1, hinge region, transmembrane region, intracellular co-stimulatory signal domain and intracellular signal domain, can be directly connected to each other or can be connected through a linker sequence. The linker sequence can be a linker sequence suitable for antibodies well known in the art, such as a linker sequence containing G and S. The length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine in the linker sequence is not particularly limited and is generally 2 to 20, such as 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.

[0062] It should be understood that in gene cloning operations, it is often necessary to design appropriate enzyme cleavage sites (e.g., amino acid residues 21 to 23 of SEQ ID NO: 3), which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, but this does not affect the activity of the target sequence. In addition, in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the protein of the recombinant protein. Therefore, the amino or carboxyl terminus of the CAR herein or between its elements may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

[0063] In some embodiments, the amino acid sequence of the chimeric antigen receptor described herein is amino acid residues 1 to 20 of SEQ ID NO: 3, SEQ ID NO: 1, and amino acid residues 171 to 400 of SEQ ID NO: 3, sequentially connected from the N-terminus to the C-terminus. In some embodiments, the amino acid sequence of the chimeric antigen receptor described herein is as shown in SEQ ID NO: 3.

[0064] The present invention also provides a nucleic acid molecule encoding a mutant polypeptide or chimeric antigen receptor as described in any embodiment herein. The nucleic acid molecule can be a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule. The DNA can be single-stranded or double-stranded.

[0065] The nucleic acid molecules described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein and amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. Long sequences often require two or more PCR amplifications, followed by splicing the fragments amplified from each amplification into the correct order.

[0066] In some embodiments, the nucleic acid molecule is a coding sequence for a polypeptide described herein, and preferably, its nucleotide sequence is as shown in the nucleotide sequence at positions 70 to 510 of SEQ ID NO: 2.

[0067] In some embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, the nucleotide sequence shown in SEQ ID NO: 2, positions 1 to 60, and the nucleotide sequence shown in SEQ ID NO: 2, positions 70 to 1200. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2.

[0068] In some embodiments, the present invention also provides a nucleic acid construct comprising the nucleic acid molecule described in any embodiment of the present invention. In some embodiments, the nucleic acid construct is an expression cassette, which, in addition to comprising the nucleic acid molecule, further comprises a regulatory sequence.

[0069] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is generally operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0070] The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used herein.

[0071] In certain embodiments, the nucleic acid construct is a vector. Specifically, the coding sequence of the CAR herein can be cloned into many types of vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector can be an expression vector, or an integration vector for integrating the nucleic acid molecules described herein into host cells. The expression vector can be provided to the cell in the form of a viral vector. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0072] Typically, a suitable vector comprises an origin of replication, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers that function in at least one organism. For example, in certain embodiments, the present invention utilizes a retroviral vector comprising a replication origin, a 3'LTR, a 5'LTR, a coding sequence for a CAR as described herein, and optionally a selectable marker.

[0073] Suitable promoters include but are not limited to the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence high-level expression that can be operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, it is also possible to consider the use of inducible promoters. The use of inducible promoters provides a molecular switch that can open the expression of the polynucleotide sequence that can be operably connected to the inducible promoter when the deadline is expressed, and close expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0074] Selectable markers include any one or both of a marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population infected by a viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes such as neo. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or a green fluorescent protein gene.

[0075] In certain embodiments, the coding sequence of the chimeric antigen receptor described herein is cloned into a vector (also referred to as an integration vector) for integrating the nucleic acid sequence of interest into the genome of the host cell, particularly a transposon vector. Suitable transposon vectors are eukaryotic expression vectors containing transposable elements selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty. Such transposon vectors contain 5' inverted terminal repeats (5'LTR) of the corresponding transposon and 3' inverted terminal repeats (3'LTR) of the corresponding transposon. The transposase can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system. When using transposases from different transposition systems, the sequences of 5'LTR and 3'LTR in the vector are also changed to sequences compatible with the transposition system, which can be easily determined by those skilled in the art. Between 5'LTR and 3'LTR is the expression cassette of the CAR of the present invention, including the corresponding promoter sequence, the coding sequence of CAR and the polyA tailing signal sequence.

[0076] The present invention also provides a host cell comprising a nucleic acid construct as described herein, and / or expressing a CAR as described herein. In some embodiments, the host cell is a NK cell.

[0077] The nucleic acid constructs of the present invention can be transfected into host cells using techniques well known in the art. Transfection methods are conventional in the art, including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. In certain embodiments, electroporation is used to transfect the vector into the cells of interest.

[0078] The present invention also provides a composition comprising a vector containing the chimeric antigen receptor expression cassette described herein. The composition may also contain suitable reagents, including but not limited to transfection reagents.

[0079] The present invention also provides a kit comprising a vector containing the chimeric antigen receptor expression cassette described herein, or comprising the composition described herein, and the kit may also be equipped with reagents or instruments for transferring the vector into cells.

[0080] The present invention also provides a pharmaceutical composition comprising the NK cells described herein. The pharmaceutical composition may contain a suitable pharmaceutically acceptable carrier or excipient. The pharmaceutical composition comprises a therapeutically or prophylactically effective amount of NK cells. The therapeutically or prophylactically effective amount of NK cells can be determined based on factors such as the patient's condition.

[0081] The present invention also provides mutants of the PD1 extracellular segment described herein, CAR, its coding sequence or complementary sequence, nucleic acid constructs, and host cells for use in the preparation of drugs for treating or preventing cancer. The present invention also provides mutants of the PD1 extracellular segment described herein, CAR, its coding sequence or complementary sequence, nucleic acid constructs, and host cells for treating or preventing cancer. The present invention also provides a method for treating or preventing cancer, comprising administering to a subject in need thereof an effective amount of the NK cells of the present invention or a pharmaceutical composition thereof. The cancer described herein is preferably a PD1 or PDL1-mediated cancer, that is, a cancer that can be treated or prevented by blocking the signaling pathway of PD1 and PDL1, or a disease or condition caused by PD-L1 expression or characterized by PD-L1 expression, including T cell dysfunction diseases, such as cancer and inflammatory diseases. In some embodiments, the cancers described herein include, but are not limited to, gastric cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, intrahepatic bile duct cancer, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, bladder cancer, renal cell carcinoma, skin cancer, and oral squamous cell carcinoma.

[0082] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0083] Example 1: UCB-NK Preparation

[0084] (1) Isolation of CBMCs

[0085] One unit of cord blood was diluted three times with PBS containing 2% EDTA. For every 35 ml of diluted cord blood, 10 mL of Ficoll centrifuge was added vertically from the bottom using a pipette. The cells were centrifuged at 400 g for 35 minutes, with an acceleration of 1 and a deceleration of 0. After centrifugation, the cells were separated and the middle lymphocyte layer was removed. CBMCs were obtained after washing with PBS.

[0086] (2) Removal of CD3+ cells and purification of NK cells

[0087] The above CBMCs were taken and added with PBS containing 1% EDTA to prepare a cell suspension; removal antibodies and magnetic beads were added in sequence according to the kit, incubated for 5 minutes, and the incubated cells were screened using a magnet; after washing with PBS and removing the immunomagnetic beads, CD3-negative lymphocytes were obtained.

[0088] (3) NK purification and maturation

[0089] The CD3-negative lymphocytes obtained by the immunomagnetic bead separation method were taken and the cells were counted and centrifuged on the 5th day of culture to change the medium. The cell concentration was adjusted to 1×10 6 cells / mL, inoculate, and culture; on the 7th day of culture, observe the cell status. If the cell density increases, dilute the cells to a concentration of 0.5×10 6 Cell viability was tested and culture was continued. NK cells were harvested on day 10-14 of the expansion culture.

[0090] Example 2: CAR sequence design and preparation of its expression vector

[0091] The coding genes for UTR, CD8 signal peptide, hPD-1 mutant (PD1M), CD8α hinge region, CD8 transmembrane region, 4-1BB signal region and CD3ζ signal region were prepared respectively. The coding genes for the above-mentioned UTR, CD8 signal peptide, PD-1, CD8α hinge region, CD8 transmembrane region, 4-1BB signal region and CD3ζ signal region were linked together from the 5' end to the 3' end by PCR to obtain the coding gene for the chimeric antigen receptor PD1M-CAR, the schematic diagram of which is shown in Figure 1.

[0092] The amino acid sequence of the hPD-1 mutant is (SEQ ID NO: 1):

[0093] The coding sequence of the chimeric antigen receptor PD1M-CAR is (SEQ ID NO: 2):

[0094] The amino acid sequence of the chimeric antigen receptor PD1M-CAR is:

[0095] The same method was used to obtain a PD1WT-CAR expressing the wild-type PD-1 extracellular segment. The PD1WT-CAR was identical to the PD1M-CAR except that the extracellular segment was the wild-type PD-1 extracellular segment. The amino acid sequence of the wild-type PD-1 extracellular segment is as follows (SEQ ID NO: 4):

[0096] (1) Construction of T7-PD1M-CAR recombinant plasmid

[0097] The PD1M-CAR construct was recombined into a T7 plasmid using PCR and then transformed into competent E. coli DH5α cells. Positive clones were identified by PCR and sequencing. The PCR product, confirmed by gel electrophoresis and sequencing, matched the target fragment size and sequence, indicating successful construction. Large quantities of the plasmid were obtained through in vitro extraction.

[0098] The same method was used to construct the recombinant plasmid of T7-PD1WT-CAR.

[0099] (2) mRNA synthesis

[0100] The PD1M-CAR and PD1WT-CAR sequences were obtained from T7 plasmids by PCR. Modified mRNA was obtained by in vitro capping and tailing, and RNA length and integrity were determined by agarose gel electrophoresis. The purified RNA was dissolved in nuclease-free water / sodium citrate at a concentration of 1-1.5 μg / μl for subsequent RNA electroporation experiments.

[0101] Example 3: NK cell transfection

[0102] The mature NK cells obtained above were centrifuged and the culture medium was removed. The cells were counted using a hemocytometer. Each 20 μl electroporation buffer contained 0.5-1×10 6NK cells were electroporated with the RNA prepared in Example 2 using a LONZA 4D electroporator and then cultured in complete NK medium (CST EXPAND MEDIUM #A5019001) to obtain NK cells expressing the chimeric antigen receptor PD1M-CAR (NK-PD1M-CAR cells). After 8 hours of culture, PD1M-CAR expression was detected by flow cytometry.

[0103] The results are shown in Figure 2. The highest expression of PD1M-CAR was 88.8%. The results in Figure 2 show that the cell activity and CD56 expression of NK-PD1M-CAR were not affected.

[0104] The same method was used to obtain NK cells expressing PD1WT-CAR (NK-PD1WT-CAR cells). The results are shown in Figure 10. The expression of PD1WT-CAR was 87.9%. The results in Figure 10 show that the cell activity and CD56 expression of NK-PD1WT-CAR were no different from those before transfection.

[0105] Example 4: Cytotoxicity of NK cells against PD-L1-overexpressing K562 tumor cell lines in vitro

[0106] In vitro, the NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (negative control group) and target cells (K562 cells expressing PDL1) were co-cultured at 37°C and 5% CO2 at effector-target ratios of 5:1, 2.5:1, 1.25:1 and 0.625:1. After 6 hours of culture, the supernatant was collected after centrifugation at 250g, and the LDH content in the supernatant of each group was detected by an LDH kit to evaluate the killing ability of NK.

[0107] There were two replicates in each group, and the NK cells were derived from three or more sample receptors. The results are shown in Figure 3. As can be seen from Figure 3, the more NK-PD1M-CAR cells are added (i.e., the higher the effect-target ratio), the stronger the killing ability against tumor cells. The above results show that the NK-PD1M-CAR cells targeting PDL1 of the present invention have efficient and specific tumor killing ability, which can prevent tumor cells from escaping immune surveillance.

[0108] Example 5: Cytotoxicity of NK cells against non-small cell lung cancer cell line A549 in vitro

[0109] This example compares the in vitro tumor killing effects of NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (negative control group), NK lymphocytes expressing GFP (negative control group), NK lymphocytes overexpressing PD-1-WT (negative control group), and microbodies using PD-1-M1 alone (negative control). Specific experimental methods include:

[0110] 1. Day 1

[0111] First, the lung cancer cell line A549 was digested by trypsin, counted, and resuspended in NK culture medium. Then, according to the RTCA instrument operation manual of Agilent, 10,000 tumor cells were evenly inoculated into a 96-well electrode plate. The target cells were cultured in the 96-well plate for 10 or 20 hours before the next step.

[0112] 2. Day 2

[0113] The NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (NK, negative control group), NK lymphocytes expressing GFP (NK-GFP, negative control group), and NK lymphocytes overexpressing PD-1-WT (NK-PD1WT-CAR, negative control group) were mixed with target cells at an effector-target ratio of 1.25:1 and 0.625:1 and co-cultured at 37°C and 5% CO2. When the PD-1-M1 microbody (HAC, SEQ ID NO: 5) was added, its addition concentration was 0.25 μM. The death of the target cells was reflected by real-time detection of the electrode plate.

[0114] Experiments using NK-PD1M-CAR cells were divided into the following groups:

[0115] (1)A549;

[0116] (2) A549+NK-GFP, E / T=0.625;

[0117] (3) A549+NK-GFP, E / T=1.25;

[0118] (4) A549+NK-GFP+HAC, E / T=0.625;

[0119] (5)A549+NK-GFP+HAC, E / T=1.25;

[0120] (6)A549+NK-PD1M-CAR, E / T=0.625;

[0121] (7)A549+NK-PD1M-CAR, E / T=1.25.

[0122] Two repetitions per set.

[0123] The experimental groups using NK lymphocytes overexpressing PD-1-WT are as follows:

[0124] (1)A549;

[0125] (2) A549+NK-GFP+HAC, E / T=0.625;

[0126] (3) A549+NK-GFP+HAC, E / T=1.25;

[0127] (4)A549+NK-PD1WT-CAR, E / T=0.625;

[0128] (5)A549+NK-PD1WT-CAR, E / T=1.25;

[0129] (6)A549+NK-PD1M-CAR, E / T=0.625;

[0130] (7)A549+NK-PD1M-CAR, E / T=1.25.

[0131] Two repetitions per set.

[0132] The results are shown in Figures 4 and 5. As can be seen from Figure 4, compared with NK-GFP, NK-PD1M-CAR has a stronger killing effect, and HAC has some enhancement of the killing function of NK-GFP, but it is far lower than the killing ability of NK-PD1M-CAR.

[0133] As shown in Figure 5 , NK-PD1M-CAR has a stronger killing effect than NK-GFP supplemented with HAC, and also has a stronger killing effect than PD-1-WT-CAR.

[0134] Example 6: Cytotoxicity of NK cells against non-small cell lung cancer cell line H1299 in vitro

[0135] This example compares the in vitro tumor killing effects of NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (negative control group), NK lymphocytes expressing GFP (negative control group), and NK lymphocytes overexpressing PD-1-WT (negative control group). The specific experimental methods include:

[0136] 1. Day 1

[0137] First, the lung cancer cell line H1299 was digested with trypsin, counted, and resuspended in NK culture medium. Then, according to the RTCA instrument operation manual of Agilent, 10,000 tumor cells were evenly inoculated into a 96-well electrode plate. The target cells were cultured in the 96-well plate for 20 hours before the next step.

[0138] 2. Day 2

[0139] The NK-PD1M-CAR cells prepared in Example 3, NK lymphocytes expressing GFP (NK-GFP, negative control group), and NK lymphocytes overexpressing PD-1-WT (NK-PD1WT-CAR, negative control group) were mixed with target cells at an effector-target ratio of 1.25:1 and co-cultured at 37 ° C and 5% CO2. When the PD-1-M1 microbody (HAC) was added, the added concentration was 0.25 μM. The death of the target cells was reflected by real-time detection of the electrode plate.

[0140] Experiments using NK-PD1M-CAR cells were divided into the following groups:

[0141] (1)H1299;

[0142] (2) H1299+NK-GFP+HAC, E / T=1.25;

[0143] (3)H1299+NK-PD1WT-CAR, E / T=1.25;

[0144] (4)H1299+NK-PD1M-CAR, E / T=1.25.

[0145] Three repetitions per set.

[0146] As shown in Figure 6 , NK-PD1M-CAR has a stronger killing effect than NK-GFP supplemented with HAC, and also has a stronger killing effect than PD-1-WT-CAR.

[0147] Example 7: Cytotoxic effect of NK cells on non-small cell lung cancer cell line HCC827 in vitro

[0148] This example compares the in vitro tumor killing effects of NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (negative control group), NK lymphocytes expressing GFP (negative control group), and NK lymphocytes overexpressing PD-1-WT (negative control group). The specific experimental methods include:

[0149] 1. Day 1

[0150] First, the lung cancer cell line HCC827 was digested with trypsin, counted, and resuspended in NK culture medium. Then, according to the Agilent RTCA instrument operating manual, 10,000 tumor cells were evenly seeded into a 96-well electrode plate. The target cells were cultured in the 96-well plate for 20 hours before proceeding to the next step. 2. Day 2

[0151] The NK-PD1M-CAR cells prepared in Example 3, NK lymphocytes expressing GFP (NK-GFP, negative control group), and NK lymphocytes overexpressing PD-1-WT (NK-PD1WT-CAR, negative control group) were mixed with target cells at an effector-target ratio of 1.25:1 and co-cultured at 37 ° C and 5% CO2. When the PD-1-M1 microbody (HAC) was added, the added concentration was 0.25 μM. The death of the target cells was reflected by real-time detection of the electrode plate.

[0152] Experiments using NK-PD1M-CAR cells were divided into the following groups:

[0153] (1) HCC827;

[0154] (2) HCC827+NK-GFP+HAC, E / T=1.25;

[0155] (3) HCC827+NK-PD1WT-CAR, E / T=1.25;

[0156] (4) HCC827+NK-PD1M-CAR, E / T=1.25.

[0157] Three repetitions per set.

[0158] As shown in Figure 7 , NK-PD1M-CAR has a stronger killing effect than NK-GFP supplemented with HAC, and also has a stronger killing effect than PD-1-WT-CAR.

[0159] Example 8: Cytotoxic effect of NK cells on the HCC827 tumor cell line with high expression of PDL1 in vitro

[0160] In vitro, the NK-PD1M-CAR cells prepared in Example 3, NK lymphocytes overexpressing PD-1-WT (NK-PD1WT-CAR, negative control group) and target cells (HCC827 cells expressing PDL1) were co-cultured at 37 ° C and 5% CO2 at effector-target ratios of 5:1, 2.5:1, and 1.25:1. After 4 hours of culture, the supernatant was collected after centrifugation at 250g, and the LDH content in the supernatant of each group was detected by an LDH kit to evaluate the killing ability of NK.

[0161] There were two replicates in each group, and the NK cells were derived from three or more sample receptors. The results are shown in Figure 8. As can be seen from Figure 8, the more NK-PD1M-CAR cells are added (i.e., the higher the effect-target ratio), the stronger the killing effect on tumor cells. Compared with NK-PD1WT-CAR, it has a stronger killing effect. The above results show that the NK-PD1M-CAR cells targeting PDL1 of the present invention have efficient and specific tumor killing ability, which can prevent tumor cells from escaping immune surveillance.

[0162] Example 9: PDL1 expression in target cells

[0163] Following conventional cell antibody staining methods, A549, H1299, and HCC827 were digested with trypsin, centrifuged at 300 g for 5 minutes, and resuspended in PBS to obtain a single cell suspension. Count each cell type, and place 1 million cells in a 5-ml round-bottom glass tube. Wash the cells with 2-3 ml of PBS and centrifuge at 300 g for 5 minutes. Discard the supernatant and add 5 μl of isotype-APC (Biolegend #402206) or PDL1-APC (Biolegend #329708), respectively. Stain at 4°C for 30 minutes and analyze by flow cytometry (cytoflex5).

[0164] The detection results are shown in Figure 9. The results show that A549, H1299, and HCC827 all express PDL1, and the expression levels of PDL1 are: HCC827>H1299>A549.

[0165] Example 10: In vivo killing effect of PD-1-M1 CAR NK

[0166] This example uses humanized B-NDG mice inoculated with RKO tumor cells intraperitoneally to verify the killing effect of PD-1-M1 CAR NK in vivo. Tumor cells grow slowly in the peritoneal cavity. 12 days after the RKO cells are inoculated, the tumor cells are clearly tumorous, which is used to simulate the actual tumor formation results of the human body. We used the electroporation method described in Example 3 to obtain NK-PD1M-CAR cells and NK-PD1WT-CAR cells, which were infused 2-3 times a week for 3 courses of treatment, with non-electroporated NK cells as a control. At the same time, cytokines IL2 and IL15 were injected before the NK cells were infused to create a more suitable growth environment for NK. In vivo imaging of small animals was performed on the 7th, 14th and 21st days after the infusion. The experimental method is shown in Figure 11.

[0167] After the first week of treatment, the control group that was only inoculated with tumors showed obvious metastatic lesions. Compared with the NK cell and NK-PD1WT-CAR cell groups, the tumor growth of the NK-PD1M-CAR cell group was significantly inhibited. After the second course of treatment, compared with the control group that was only inoculated with tumors, the reinfusion of NK cells alone could limit the growth of RKO colon cancer to a certain extent, while the NK-PD1WT-CAR cell group and the NK-PD1M-CAR cell group showed a more obvious inhibitory effect on tumor growth (Figures 12 and 13). According to the statistical fluorescence signal values ​​of the mice, at the end of the third course of treatment, in the late stage of tumor growth, the tumor growth of the mice in the control group was very rapid, and the NK-PD1WT-CAR cell group and the NK-PD1M-CAR cell group significantly slowed down the tumor growth rate compared with the reinfusion of NK (Figure 14).

[0168] After three cycles of treatment, the mortality of tumor-bearing mice under various treatment conditions was further observed and recorded. In the late stage of tumor inoculation, some treated mice developed severe ascites. According to ethical regulations for mice, mice that gained 10% or more of their body weight after developing ascites were euthanized. The survival curves are shown below. Compared to the control group inoculated with tumors alone, the NK-PD1WT-CAR cell and NK-PD1M-CAR cell groups significantly prolonged the lifespan of tumor-bearing mice (Figure 15).

Claims

1. A polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:

1.

2. A chimeric antigen receptor, characterized in that From N-terminus to C-terminus, the chimeric antigen receptor contains an optional signal peptide, the polypeptide of claim 1, a hinge region, a transmembrane region, an intracellular co-stimulatory signal domain and an intracellular signal domain, which are sequentially connected.

3. The chimeric antigen receptor according to claim 2, wherein: The signal peptide is selected from CD8 signal peptide, CD28 signal peptide or CD4 signal peptide; preferably, the signal peptide is CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide is as shown in the residues of amino acids 1 to 20 of SEQ ID NO: 3; and / or The hinge region is selected from the group consisting of a CD8α hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region or an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is a CD8α hinge region; preferably, the amino acid sequence of the CD8α hinge region is as shown in amino acid residues 171 to 225 of SEQ ID NO: 3; and / or The transmembrane region is selected from the group consisting of CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region or DAP10 transmembrane region; preferably, the transmembrane region is CD8 transmembrane region; preferably, the amino acid sequence of the CD8 transmembrane region is as shown in amino acid residues 226 to 246 of SEQ ID NO: 3; and / or The intracellular co-stimulatory signal domain is an intracellular domain of a co-stimulatory signal molecule, preferably selected from the intracellular domain of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor or DNAX activation protein 10; preferably, the intracellular co-stimulatory signal domain is the intracellular domain of 4-1BB; preferably, the amino acid sequence of the intracellular domain of 4-1BB is as shown in amino acid residues 247 to 288 of SEQ ID NO: 3; and / or The intracellular signaling domain is a CD3ζ intracellular signaling domain or a FcεRIγ intracellular signaling domain; preferably, the intracellular signaling domain is a CD3ζ intracellular signaling domain; preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in amino acid residues 289 to 400 of SEQ ID NO:

3.

4. The chimeric antigen receptor according to claim 3, characterized in that: The chimeric antigen receptor contains, from N-terminus to C-terminus, a CD8 signal peptide, the polypeptide of claim 1, a CD8α hinge region, a CD8 transmembrane region, a 4-1BB intracellular domain, and a tyrosine activation motif of CD3ζ; Preferably, the amino acid sequence of the chimeric antigen receptor is amino acid residues 1 to 20 of SEQ ID NO: 3, amino acid residues 171 to 400 of SEQ ID NO: 1 and SEQ ID NO: 3 connected sequentially from N-terminus to C-terminus; or the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

3.

5. A nucleic acid molecule encoding the polypeptide of claim 1 or the chimeric antigen receptor of any one of claims 2 to 4; preferably, the nucleic acid molecule is a DNA molecule or an RNA molecule; Preferably, the nucleic acid molecule is the coding sequence of the polypeptide according to claim 1, and its nucleotide sequence is shown in the nucleotide sequence of positions 70 to 510 of SEQ ID NO: 2; Preferably, the nucleic acid molecule contains, from the 5' end to the 3' end, the nucleotide sequence shown in positions 1 to 60 of SEQ ID NO: 2 and the nucleotide sequence shown in positions 70 to 1200 of SEQ ID NO: 2, or its nucleotide sequence is as shown in SEQ ID NO:

2.

6. A nucleic acid construct comprising the nucleic acid molecule according to claim 5; Preferably, the nucleic acid construct is an expression cassette, which contains, in addition to the nucleic acid molecule, a regulatory sequence; or the nucleic acid construct is a vector, including an expression vector and an integration vector for integrating the nucleic acid molecule into the genome of a host cell; preferably, the vector is a transposon vector, more preferably, the transposon vector is a eukaryotic expression vector containing a transposable element selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty.

7. A host cell, comprising the nucleic acid construct of claim 6, and / or expressing the polypeptide of claim 1 or the CAR of any one of claims 2 to 4; preferably, the host cell is a NK cell.

8. A composition or kit comprising the vector according to claim 6 and an optional transfection agent.

9. A pharmaceutical composition comprising the NK cells according to claim 7 and a pharmaceutically acceptable carrier or excipient.

10. Use of the polypeptide of claim 1, the chimeric antigen receptor of claims 2 to 4, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6, the host cell of claim 7 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating or preventing PD1 or PDL1-mediated cancer; preferably, the cancer is selected from gastric cancer, lung cancer (such as non-small cell lung cancer), liver cancer, intrahepatic bile duct cancer, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, bladder cancer, renal cell carcinoma, skin cancer and oral squamous cell carcinoma.

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