Mutant PD1 extracellular domain fragments and CAR and NK cells containing said fragments

Abstract

The present invention relates to a fragment of a mutant PD1 extracellular domain, and a CAR and NK cells comprising the fragment. Specifically, the present invention provides a polypeptide that is a fragment of the mutant PD1 extracellular domain, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:1. A CAR of the present invention comprises the polypeptide. The present invention also provides NK cells expressing the CAR. In the present invention, the tumor cell-killing ability of NK cells expressing a CAR comprising a mutant PD1 extracellular domain is clearly stronger than that of NK cells expressing a CAR comprising a wild-type PD1 extracellular domain.

Description

[Technical Field]

[0001] The present invention relates to a fragment of a mutant PD1 extracellular domain, and a CAR and an NK cell comprising said fragment. [Background technology]

[0002] Under normal physiological conditions, activation of PD-1 signaling suppresses excessive inflammatory responses and prevents the development of autoimmune diseases. However, in the tumor environment, PD-1 / PD-L1 signaling is an important pathway for tumor cell immune evasion. High levels of PD-L1 protein are detected in the blood and at primary tumor sites of patients with various tumors, including lung cancer, breast cancer, melanoma, head and neck cancer, and lymphoma. PD-L1 interacts with the PD-1 receptor on immune cells such as T cells and NK cells in tumor patients. The PD-1 receptor acts as a brake on immune cells such as T cells and NK cells. It contains two inhibitory domains, immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM), which suppress TCR signaling and NK activation signaling via phosphorylation of the SHP protein. Currently, several PD-1 / PD-L1-related antibody therapies are available and have shown some efficacy in tumors such as lymphoma and melanoma, but challenges remain. On the other hand, antibody therapy relies solely on the blocking mechanism of antibodies, and can only inhibit the interaction between immune cells in tumors to a certain extent, but cannot reverse the immunosuppressive effects on immune cells that are already interacting. On the other hand, the Fc region of antibodies is likely to interact with immune cells in the patient's body through ADCC / ADCP, which can disrupt the immune system.

[0003] NK-CAR (chimeric antigen receptor NK cell) technology is a new cell therapy in which modified NK cells are reinjected into the human body to directly kill tumors and activate the immune system. It is considered one of the most effective treatments for malignant tumors because it overcomes the cytotoxicity, drug resistance, and recurrence problems of conventional treatments. Currently, NK-CAR technology has demonstrated remarkable efficacy in hematological malignancies such as acute and chronic lymphocytic leukemia and lymphoma (B-cell lymphoma, acute and chronic B-lymphocytic leukemia, etc.).

[0004] Although NK-CAR cells have relatively strong killing ability, in the tumor microenvironment, PDL1 and other molecules in the tumor microenvironment suppress the activity of lethal NK cells via the immune cell receptor PD1, allowing tumor cells to escape immune surveillance. Therefore, by utilizing the negative feedback signal of tumor cells (PDL1-PD-1 interaction) to activate immune cells, it is hoped that the suppressive effect of tumor cells on immune cells can be reduced, and NK-activated cells can be activated, enhancing the killing effect of NK-CAR on tumor cells. Summary of the Invention

[0005] The present invention provides a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:1.

[0006] The present invention further provides a chimeric antigen receptor comprising, sequentially linked from the N-terminus to the C-terminus, an optional signal peptide, a polypeptide described herein, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.

[0007] In one or more embodiments, the signal peptide is 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 set forth in amino acid residues 1 to 20 of SEQ ID NO:3.

[0008] In one or more embodiments, the hinge region is selected from 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 set forth in amino acid residues 171 to 225 of SEQ ID NO:3.

[0009] In one or more embodiments, the transmembrane domain is selected from the CD28 transmembrane domain, the CD8 transmembrane domain, the CD3ζ transmembrane domain, the CD134 transmembrane domain, the CD137 transmembrane domain, the ICOS transmembrane domain, or the DAP10 transmembrane domain. Preferably, the transmembrane domain is the CD8 transmembrane domain. Preferably, the amino acid sequence of the CD8 transmembrane domain is as set forth in amino acid residues 226 to 246 of SEQ ID NO:3.

[0010] In one or more embodiments, the intracellular costimulatory signal domain is the intracellular domain of a costimulatory signal molecule, preferably selected from the intracellular domains 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 signal domain is the intracellular domain of 4-1BB. Preferably, the amino acid sequence of the intracellular domain of 4-1BB is as set forth in amino acid residues 247 to 288 of SEQ ID NO:3.

[0011] 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 set forth in amino acid residues 289 to 400 of SEQ ID NO:3.

[0012] In one or more embodiments, the chimeric antigen receptor comprises, sequentially 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-based activation motif of CD3ζ.

[0013] In one or more embodiments, the amino acid sequence of the chimeric antigen receptor is, sequentially linked from the N-terminus to the C-terminus, 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. Alternatively, the amino acid sequence of the chimeric antigen receptor is as set forth in SEQ ID NO:3.

[0014] The present invention provides nucleic acid molecules encoding the polypeptides or chimeric antigen receptors described herein. Preferably, the nucleic acid molecules are DNA or RNA molecules.

[0015] In one or more embodiments, the nucleic acid molecule is a coding sequence for the polypeptide of claim 1, the nucleotide sequence of which is as set forth in the sequence of nucleotides 70 to 510 of SEQ ID NO:2.

[0016] In one or more embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, the nucleotide sequence set forth at positions 1 to 60 of SEQ ID NO:2 and the nucleotide sequence set forth at positions 70 to 1200 of SEQ ID NO:2, or the nucleotide sequence is as set forth in SEQ ID NO:2.

[0017] In one or more embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:2.

[0018] The present invention further provides nucleic acid constructs comprising the nucleic acid molecules described herein.

[0019] In one or more embodiments, the nucleic acid construct is an expression cassette and includes regulatory sequences in addition to the nucleic acid molecule.

[0020] In one or more embodiments, 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-based vector, and more preferably, the transposon-based vector is a eukaryotic expression vector containing a transposon element selected from piggybac, sleeping beauty, frog prince, Tn5, or Ty.

[0021] The present invention further provides a host cell comprising a nucleic acid construct described herein and / or expressing a polypeptide or CAR described herein. Preferably, the host cell is a NK cell.

[0022] The present invention further provides compositions or kits comprising the vectors described herein and optional transfection reagents.

[0023] In one or more embodiments, the kit comprises the composition.

[0024] The present invention further provides pharmaceutical compositions comprising the NK cells described herein and a pharmaceutically acceptable carrier or excipient.

[0025] The present invention further provides use of a polypeptide, chimeric antigen receptor, nucleic acid molecule, said nucleic acid construct, host cell, or pharmaceutical composition described herein in the manufacture of a medicament for treating or preventing a PD1- or PDL1-mediated cancer. Preferably, the cancer is selected from gastric cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, intrahepatic cholangiocarcinoma, 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. [Brief explanation of the drawings]

[0026] [Figure 1] The sequence of NK-PD1M-CAR targeting PDL1 is shown. [Figure 2] This figure shows the CD56 expression and CAR expression in NK-PD1M-CAR. In the figure, the control ("control") is an NK cell from the same batch that was not transfected with the gene encoding the chimeric antigen receptor PD1M-CAR. [Figure 3] Figure 1 shows the in vitro killing of NK cells against the PD-L1-overexpressing K562 tumor cell line. [Figure 4] The killing results of NK+HAC and NK-PD1M-CAR against A549 cells are shown. The curves on the far right correspond, from top to bottom, to 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, and A549+NK-PD1M-CAR, E / T=1.25. [Figure 5] The killing results of NK-PD1WT-CAR and NK-PD1M-CAR against A549 cells are shown. The curves on the far right correspond, from top to bottom, 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, and A549+NK-PD1M-CAR, E / T=1.25. [Figure 6] The graph shows the killing results of NK-PD1WT-CAR and NK-PD1M-CAR against H1299. The curves on the far right of the graph correspond, from top to bottom, to H1299, H1299+NK-GFP+HAC, H1299+NK-PD1WT-CAR, and H1299+NK-PD1M-CAR. [Figure 7]The graph shows the killing results of NK-PD1WT-CAR and NK-PD1M-CAR against HCC827. The curves on the far right in the graph correspond, from top to bottom, to HCC827, HCC827+NK-GFP+HAC, HCC827+NK-PD1WT-CAR, and HCC827+NK-PD1M-CAR. [Figure 8] Shows the killing effect of NK-PD1M-CAR targeting HCC827. [Figure 9] 1 shows the PDL1 expression status in several cell lines. [Figure 10] This figure shows the CD56 expression and CAR expression in NK-PD1WT-CAR. In the figure, the control ("control") is an NK cell from the same batch that was not transfected with the gene encoding the chimeric antigen receptor PD1WT-CAR. [Figure 11] A schematic diagram of CAR NK action in vivo is shown. [Figure 12] 1 shows an imaging diagram of a living mouse. [Figure 13] Bioimaging statistics are shown. [Figure 14] Figure 1 shows tumor growth in mice 21 days after reinfusion with different NK cells. [Figure 15] Mouse survival curves are shown. DETAILED DESCRIPTION OF THE INVENTION

[0027] 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 (for example, in the Examples) can be combined with each other to form preferred embodiments.

[0028] In the present invention, the term "expression cassette" refers to all the elements necessary to express one gene, including a promoter, a coding sequence of the gene, and a PolyA tailing signal sequence.

[0029] The term "coding sequence" refers to that portion of a 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 (in prokaryotes) upstream of and adjacent to the open reading frame at the 5' end of the mRNA and the transcription termination sequence downstream of and adjacent to the open reading frame at the 3' end of the mRNA. Coding sequences include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0030] The term "costimulatory molecule" refers to a molecule present on the surface of antigen-presenting cells that binds to a costimulatory molecule receptor on Th cells to generate a costimulatory signal. Lymphocyte proliferation requires not only antigen binding but also signals from costimulatory molecules. Costimulatory signals are transmitted to T cells by the binding of the costimulatory molecules CD80 and CD86, which are primarily expressed on the surface of antigen-presenting cells, to CD28 molecules on the T cell surface. B cells can receive costimulatory signals via common pathogen components such as LPS, complement components, or CD40L on the surface of activated, antigen-specific Th cells.

[0031] The term "linker" or hinge refers to a polypeptide segment that connects different proteins or polypeptides so that the linked proteins or polypeptides maintain their respective spatial conformations and maintain the function or activity of the proteins or polypeptides. Exemplary linkers include linkers containing G and / or S, and Furin 2A peptides.

[0032] 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, and is well known in the art (see, e.g., 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 (e.g., Tween-80). Ionic strength enhancers include, but are not limited to, sodium chloride.

[0033] The term "effective amount" refers to a dose that is capable of treating, preventing, alleviating, and / or ameliorating a disease or condition according to the present invention in a subject.

[0034] The term "disease and / or condition" refers to a physical condition of said subject that is associated with a disease and / or condition according to the present invention.

[0035] The term "subject" or "patient" refers to a patient or other animal, particularly a mammal (e.g., a human, dog, monkey, cow, horse, etc.), who receives a pharmaceutical composition of the present invention to treat, prevent, alleviate, and / or ameliorate a disease or condition of the present invention.

[0036] The term "chimeric antigen receptor" (CAR) refers to an artificially engineered receptor that immobilizes a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen on an immune cell (e.g., a T cell), allowing the immune cell to recognize tumor or viral antigens and kill tumor cells or virus-infected cells. CARs typically sequentially comprise an optional signal peptide, a tumor cell membrane antigen-binding polypeptide, a hinge region, a transmembrane region, and an intracellular signal region.

[0037] The present invention constructs a CAR using a mutant PD1 extracellular domain and expresses the CAR in NK cells. The present invention discovered that the tumor cell-killing ability of NK cells expressing a CAR containing a mutant PD1 extracellular domain is significantly stronger than that of NK cells expressing a CAR containing a wild-type PD1 extracellular domain, thereby completing the present invention.

[0038] Thus, a first aspect of the present invention provides mutant polypeptides that are variants of the PD1 extracellular domain. In some embodiments, the polypeptides comprise or consist of the amino acid sequence set forth in SEQ ID NO:1. The polypeptides of the present invention also include variants having 1 to 8, preferably 1 to 5, and more preferably 1 to 3 amino acid mutations (including insertions, deletions, and / or substitutions) compared to SEQ ID NO:1. The mutations are preferably conservative substitutions well known in the art. For example, one or more (e.g., 1 to 8, 1 to 5, or 1 to 3) amino acid residues in SEQ ID NO:1 are substituted with amino acid residues having the same or similar properties (e.g., based on classification by side chain group or chemical structure). It should be understood that the variants of SEQ ID NO:1 retain the biological activity of SEQ ID NO:1, particularly the biological activity of SEQ ID NO:1 set forth herein.

[0039] A second aspect of the present invention provides a chimeric antigen receptor (CAR) comprising, sequentially linked from the N-terminus to the C-terminus, a signal peptide, a mutant of the PD1 extracellular domain described herein, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.

[0040] A signal peptide is a short peptide chain (5-30 amino acids in length) that directs newly synthesized proteins into the secretory pathway. It usually refers to the N-terminal amino acid sequence of a newly synthesized polypeptide chain (although it may not be present at the N-terminus) that directs the transmembrane transport (localization) of proteins and plays a role in directing proteins to suborganelles with different membrane structures within the cell. In some embodiments, the signal peptide used herein can be selected from the CD8 signal peptide, the CD28 signal peptide, or the CD4 signal peptide. Preferably, the signal peptide is the CD8 signal peptide. Preferably, the amino acid sequence of the CD8 signal peptide is as set forth in amino acid residues 1-20 of SEQ ID NO:3.

[0041] Hinge region refers to the region between the CH1 and CH2 functional regions of an immunoglobulin heavy chain; this region is proline-rich, does not form an α-helix, and is prone to extension and some degree of twisting. In some embodiments, the hinge region used herein may be selected from the CD8α hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region, and 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 set forth in amino acid residues 171 to 225 of SEQ ID NO:3.

[0042] In some embodiments, the transmembrane domain used herein may be any one of the CD28 transmembrane domain, the CD8 transmembrane domain, the CD3ζ transmembrane domain, the CD134 transmembrane domain, the CD137 transmembrane domain, the ICOS transmembrane domain, and the DAP10 transmembrane domain. Preferably, the CD8 transmembrane domain is used herein. More preferably, the amino acid sequence of the CD8 transmembrane domain is as set forth in amino acid residues 226 to 246 of SEQ ID NO:3.

[0043] In some embodiments, the intracellular costimulatory signal domain used herein comprises the intracellular domain of a costimulatory signal molecule, including the intracellular domains of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T-cell costimulator (ICOS), and DNAX-activating protein 10. Preferably, the intracellular costimulatory signal domain is the intracellular domain of 4-1BB. More preferably, the amino acid sequence of the intracellular domain of 4-1BB is as set forth in amino acid residues 247 to 288 of SEQ ID NO:3.

[0044] 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 set forth in amino acid residues 289 to 400 of SEQ ID NO:3.

[0045] In some embodiments, the chimeric antigen receptors described herein comprise, sequentially from N- to C-terminus, a CD8 signal peptide, a variant of the PD1 extracellular domain described herein, a CD8α hinge region, a CD8 transmembrane region, a 4-1BB intracellular domain, and a tyrosine-based activation motif of CD3ζ.

[0046] The above-mentioned components forming the chimeric antigen receptor of the present invention, such as the signal peptide, PD1 extracellular domain mutant, hinge region, transmembrane region, intracellular costimulatory signal domain, and intracellular signal domain, may be linked directly to each other or via a linker sequence. The linker sequence may be a linker sequence used in antibodies well known in the art, such as a linker sequence containing G and S. The linker may be 3 to 25 amino acid residues in length, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited and is typically 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker can include other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q).

[0047] Gene cloning procedures often require the design of appropriate restriction enzyme cleavage sites (e.g., amino acid residues 21-23 of SEQ ID NO:3), which inevitably introduces one or more extraneous residues at the end of the expressed amino acid sequence. However, it should be understood that this does not affect the activity of the target sequence. Furthermore, it is often necessary to add amino acids to the N-terminus, C-terminus, or other appropriate region of a recombinant protein to construct a fusion protein, promote recombinant protein expression, obtain a recombinant protein that is automatically secreted outside of host cells, or facilitate recombinant protein purification. Therefore, one or more polypeptide fragments may be included as protein tags at the amino or carboxyl terminus of the CAR herein, or between each element thereof. Any suitable tag may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-Tag II, AU1, EE, T7, 4A6, ε, B, gE, or Ty1. These tags can be used for protein purification.

[0048] In some embodiments, the amino acid sequence of a chimeric antigen receptor described herein is, sequentially from N- to C-terminus, amino acid residues 1-20 of SEQ ID NO:3, SEQ ID NO:1, and amino acid residues 171-400 of SEQ ID NO:3. In some embodiments, the amino acid sequence of a chimeric antigen receptor described herein is as set forth in SEQ ID NO:3.

[0049] The present invention further provides a nucleic acid molecule encoding a mutant polypeptide or chimeric antigen receptor described in any of the embodiments herein. The nucleic acid molecule may be a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule. The DNA may be single-stranded or double-stranded.

[0050] 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 relevant sequences can be obtained by amplifying a commercially available cDNA library or a cDNA library prepared by a common method known to those skilled in the art as a template. For long sequences, PCR amplification usually needs to be performed twice or multiple times, and then the amplified fragments must be linked in the correct order.

[0051] In some embodiments, the nucleic acid molecule is a coding sequence for a polypeptide described herein, preferably the nucleotide sequence of which is as set forth in nucleotides 70 to 510 of SEQ ID NO:2.

[0052] In some embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, the nucleotide sequence set forth at positions 1 to 60 of SEQ ID NO:2 and the nucleotide sequence set forth at positions 70 to 1200 of SEQ ID NO:2. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO:2.

[0053] In some embodiments, the present invention further provides a nucleic acid construct comprising a nucleic acid molecule described in any of the embodiments herein, hi some embodiments, the nucleic acid construct is an expression cassette and comprises, in addition to the nucleic acid molecule, a regulatory sequence.

[0054] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence from which the protein is to be expressed. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0055] The regulatory sequence may be a suitable transcription terminator sequence, i.e., a sequence recognized by a 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 functions in the selected host cell can be used herein.

[0056] In some 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 may be an expression vector or an integration vector for integrating the nucleic acid molecule described herein into a host cell. The expression vector may be provided to a 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.

[0057] Typically, a suitable vector will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers. For example, in some embodiments, the invention uses retroviral vectors that contain an origin of replication, a 3' LTR, a 5' LTR, a coding sequence for a CAR described herein, and an optional selectable marker.

[0058] Suitable promoters include, but are not limited to, the immediate-early cytomegalovirus (CMV) promoter sequence. Such promoter sequences are strong constitutive promoter sequences capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor-1α (EF-1α). However, other constitutive promoter sequences can also be used. These include, but are not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the use of inducible promoters is contemplated. The use of inducible promoters provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when expression is desired and turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0059] Selectable markers include either a marker gene or a reporter gene, or both, for identifying and selecting expressing cells from a population of cells infected with the viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes such as neo. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein.

[0060] In some embodiments, the coding sequence of a chimeric antigen receptor described herein is cloned into a vector (also called an integrating vector), particularly a transposon vector, for integrating a nucleic acid sequence of interest into the genome of a host cell. Suitable transposon vectors are eukaryotic expression vectors containing transposon elements selected from piggybac, sleeping beauty, frog prince, Tn5, or Ty. This type of transposon vector contains the 5' inverted repeat (5' LTR) and the 3' inverted repeat (3' LTR) of the corresponding transposon. The transposase may be derived from the piggybac, sleeping beauty, frog prince, Tn5, or Ty transposon system. When a transposase from a different transposon system is used, the sequences of the 5' LTR and 3' LTR in the vector are also appropriately changed to sequences compatible with the transposon system, which can be easily determined by one of ordinary skill in the art. Between the 5'LTR and 3'LTR is an expression cassette for the CAR of the present invention, which contains the corresponding promoter sequence, the coding sequence for the CAR, and a polyA tailing signal sequence.

[0061] The present invention further provides a host cell comprising a nucleic acid construct described herein and / or expressing a CAR described herein. In some embodiments, the host cell is a NK cell.

[0062] The nucleic acid constructs of the present invention can be transfected into host cells using techniques well known in the art. Transfection methods are common in the art and include, but are not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, electroporation, etc. In some embodiments, the vector is transfected into the cells of interest using electroporation.

[0063] The present invention further provides compositions comprising vectors containing the chimeric antigen receptor expression cassettes described herein, which may also include suitable reagents, including but not limited to transfection reagents.

[0064] The present invention further provides kits comprising a vector containing a chimeric antigen receptor expression cassette described herein or comprising a composition described herein, and may further comprise reagents or instruments for introducing the vector into cells.

[0065] The present invention further provides pharmaceutical compositions comprising the NK cells described herein. The pharmaceutical compositions may include a suitable pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions include 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.

[0066] The present invention further provides use of the PD1 extracellular domain mutants, CARs, their coding sequences or complementary sequences, nucleic acid constructs, and host cells described herein in the manufacture of a medicament for treating or preventing cancer. The present invention further provides the PD1 extracellular domain mutants, CARs, their coding sequences or complementary sequences, nucleic acid constructs, and host cells described herein for the treatment or prevention of cancer. The present invention also provides a method for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an NK cell or pharmaceutical composition described herein. The cancer described herein is preferably a PD1- or PDL1-mediated cancer, i.e., a cancer that can be treated or prevented by blocking the PD1 and PDL1 signaling pathway, or a disease or condition caused by or characterized by PD-L1 expression, including T cell dysfunction diseases such as cancer and inflammatory diseases. In some embodiments, cancers according to the present invention include, but are not limited to, gastric cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, intrahepatic cholangiocarcinoma, 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.

[0067] The present invention will be further described below with reference to specific examples. It should be noted that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In the following examples, experimental methods for which specific conditions are not specified generally follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. [Example]

[0068] Example 1: Preparation of UCB-NK (1) Isolation of CBMC One unit of cord blood was diluted three times with 2% EDTA-containing PBS. 10 mL of Ficoll centrifugal solution was added vertically to each 35 mL of diluted cord blood using a pipette. The mixture was centrifuged at 400 g for 35 minutes, with an acceleration rate of 1 and a deceleration rate of 0. After centrifugation, the cells were separated into layers, and the middle lymphocyte layer was collected. After washing with PBS, CBMCs were obtained.

[0069] (2) Purification of NK cells by depletion of CD3+ cells The CBMCs were collected and a cell suspension was prepared by adding 1% EDTA in PBS. Following the kit instructions, the depletion antibody and magnetic beads were added sequentially, incubated for 5 minutes, and the incubated cells were sorted using a magnet. After washing with PBS and removing the immunomagnetic beads, CD3-negative lymphocytes were obtained.

[0070] (3) Purification and maturation of NK cells CD3-negative lymphocytes obtained by the above immunomagnetic bead separation method were collected, and the cell number was counted on the fifth day of culture. The medium was replaced by centrifugation, and the cell concentration was adjusted to 1 × 10 6 The cell concentration was adjusted to 0.5 × 10 cells / mL and then inoculated and cultured. The state of the cells was observed on the 7th day of culture, and if the cell density increased, the cell concentration was reduced to 0.5 × 10 cells / mL. 6 The cells were diluted to 10-14 days after expansion and culture, and then the NK cells were collected.

[0071] Example 2: Design of the CAR sequence and preparation of its expression vector The genes encoding the UTR, CD8 signal peptide, hPD-1 mutant (PD1M), CD8α hinge region, CD8 transmembrane region, 4-1BB signal region, and CD3ζ signal region were prepared. The genes encoding the UTR, CD8 signal peptide, PD-1, CD8α hinge region, CD8 transmembrane region, 4-1BB signal region, and CD3ζ signal region were sequentially linked from the 5' to 3' end by PCR to obtain the gene encoding the chimeric antigen receptor PD1M-CAR. A schematic diagram of the PD1M-CAR is shown in Figure 1.

[0072] The amino acid sequence of the hPD-1 mutant is (SEQ ID NO:1): FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV.

[0073] The coding sequence for this chimeric antigen receptor PD1M-CAR is (SEQ ID NO:2):

[0074] The amino acid sequence of this chimeric antigen receptor PD1M-CAR is: ALPVTALLLPLALLLHAARPMGSFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSNTSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*(SEQ ID NO:3).

[0075] Using the same method, we obtained PD1WT-CAR, which expresses the wild-type PD-1 extracellular domain, which is identical to PD1M-CAR except that its extracellular domain is the wild-type PD-1 extracellular domain.

[0076] The amino acid sequence of the wild-type PD-1 extracellular domain is (SEQ ID NO:4): FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV.

[0077] (1) Construction of T7-PD1M-CAR recombinant plasmid PD1M-CAR was recombined into a T7 plasmid by PCR and then transformed into E. coli DH5α competent cells. Positive clones were identified by PCR and sequenced. Gel electrophoresis and sequencing of the PCR products confirmed that the fragment size and sequence matched the desired sequence, indicating successful construction. Large quantities of the plasmid were obtained by in vitro extraction. The recombinant plasmid of T7-PD1WT-CAR was constructed in a similar manner.

[0078] (2) mRNA synthesis The PD1M-CAR and PD1WT-CAR sequences were isolated from T7 plasmids by PCR, followed by in vitro capping and tailing to obtain modified mRNA. The length and integrity of the RNA were detected by agarose gel electrophoresis. The purified RNA was dissolved in nuclease-free water / sodium citrate solvent at a concentration of 1–1.5 μg / µL and used in subsequent RNA electroporation experiments.

[0079] Example 3: Transfection of NK cells After centrifugation of the mature NK cells obtained above, the medium was removed, and the cell number was counted using a hemocytometer. 0.5-1 x 10 cells were cultured per 20 ul of electroporation buffer. 6 NK cells were 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.

[0080] The results are shown in Figure 2. PD1M-CAR expression was highest at 88.8%. The results in Figure 2 showed that NK-PD1M-CAR cell activity and CD56 expression were not affected.

[0081] Using the same method, NK cells expressing PD1WT-CAR (NK-PD1WT-CAR cells) were obtained. The results are shown in Figure 10. PD1WT-CAR expression was 87.9%. The results in Figure 10 showed that the cell activity and CD56 expression of NK-PD1WT-CAR were no different from those before transfection.

[0082] Example 4: In vitro killing effect of NK cells against PD-L1-overexpressing K562 tumor cell line 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 to target cell ratios of 5:1, 2.5:1, 1.25:1, and 0.625:1. After 6 hours of culture, the supernatants were collected by centrifugation at 250 g, and the LDH content of the supernatants in each group was detected using an LDH kit to evaluate the NK killing ability.

[0083] Each group was repeated twice, and NK cells were derived from three or more sample recipients. The results are shown in Figure 3. Figure 3 shows that the greater the amount of NK-PD1M-CAR cells added (i.e., the higher the effector-to-target cell ratio), the stronger the tumor cell killing ability. The above results demonstrated that the NK-PD1M-CAR cells targeting PDL1 of the present invention have highly efficient and specific tumor killing ability and can prevent tumor cells from escaping immune surveillance.

[0084] Example 5: In vitro killing effect of NK cells against non-small cell lung cancer cell line A549 In this example, the in vitro tumor-killing effects of the NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (negative control group), GFP-expressing NK lymphocytes (negative control group), PD-1-WT-overexpressing NK lymphocytes (negative control group), and a microbody using PD-1-M1 alone (negative control) were compared. The specific experimental method was as follows.

[0085] 1. Day 1 First, lung cancer cell line A549 was trypsinized, counted, and resuspended in NK medium. Following the Agilent RTCA instrument operating manual, 10,000 tumor cells were uniformly seeded onto a 96-well electrode plate. Target cells were cultured in the 96-well plate for 10 or 20 hours before proceeding.

[0086] 2. Day 2 NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (NK, negative control), GFP-expressing NK lymphocytes (NK-GFP, negative control), and PD-1-WT-overexpressing NK lymphocytes (NK-PD1WT-CAR, negative control) were mixed with target cells at effector-to-target cell ratios of 1.25:1 and 0.625:1, and co-cultured at 37°C under 5% CO2. When PD-1-M1 microbody (HAC, SEQ ID NO:5) was added, its concentration was 0.25 μM. The death status of target cells was reflected by real-time detection on the electrode plate.

[0087] The experimental grouping using NK-PD1M-CAR cells was as follows: (1) A549; (2) A549+NK-GFP, E / T=0.625; (3) A549+NK-GFP, E / T=1.25; (4) A549+NK-GFP+HAC, E / T=0.625; (5) A549+NK-GFP+HAC, E / T=1.25; (6) A549+NK-PD1M-CAR, E / T=0.625; (7) A549+NK-PD1M-CAR, E / T=1.25. Each group was repeated twice.

[0088] The experimental grouping using NK lymphocytes overexpressing PD-1-WT was as follows: (1) A549; (2) A549+NK-GFP+HAC, E / T=0.625; (3) A549+NK-GFP+HAC, E / T=1.25; (4) A549+NK-PD1WT-CAR, E / T=0.625; (5) A549+NK-PD1WT-CAR, E / T=1.25; (6) A549+NK-PD1M-CAR, E / T=0.625; (7) A549+NK-PD1M-CAR, E / T=1.25. Each group was repeated twice.

[0089] The results are shown in Figures 4 and 5. Figure 4 shows that NK-PD1M-CAR has a stronger killing effect than NK-GFP, and HAC slightly enhances the killing function of NK-GFP, but the killing ability is much lower than that of NK-PD1M-CAR.

[0090] Figure 5 shows that NK-PD1M-CAR has a stronger killing effect than NK-GFP with HAC, and at the same time, it also has a stronger killing effect than PD-1-WT-CAR.

[0091] Example 6: In vitro killing effect of NK cells against non-small cell lung cancer cell line H1299 In this example, the in vitro tumor-killing effects of the NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (negative control group), GFP-expressing NK lymphocytes (negative control group), and PD-1-WT-overexpressing NK lymphocytes (negative control group) were compared. The specific experimental method was as follows.

[0092] 1. Day 1 First, lung cancer cell line H1299 was trypsinized, counted, and resuspended in NK medium. Following the Agilent RTCA instrument operating manual, 10,000 tumor cells were uniformly seeded onto a 96-well electrode plate. Target cells were cultured in the 96-well plate for 20 hours before proceeding.

[0093] 2. Day 2 NK-PD1M-CAR cells prepared in Example 3, GFP-expressing NK lymphocytes (NK-GFP, negative control), and PD-1-WT-overexpressing NK lymphocytes (NK-PD1WT-CAR, negative control) were mixed with target cells at an effector-to-target cell ratio of 1.25:1 and co-cultured at 37°C under 5% CO2. When PD-1-M1 microbody (HAC) was added, its concentration was 0.25 μM. The death status of target cells was reflected by real-time detection on the electrode plate.

[0094] The experimental grouping using NK-PD1M-CAR cells was as follows: (1) H1299; (2) H1299+NK-GFP+HAC, E / T=1.25; (3) H1299+NK-PD1WT-CAR, E / T=1.25; (4) H1299+NK-PD1M-CAR, E / T=1.25. Each group was repeated three times.

[0095] Figure 6 shows that NK-PD1M-CAR has a stronger killing effect than NK-GFP with HAC, and at the same time, it also has a stronger killing effect than PD-1-WT-CAR.

[0096] Example 7: In vitro killing effect of NK cells against non-small cell lung cancer cell line HCC827 In this example, the in vitro tumor-killing effects of the NK-PD1M-CAR cells prepared in Example 3, untreated NK cells (negative control group), GFP-expressing NK lymphocytes (negative control group), and PD-1-WT-overexpressing NK lymphocytes (negative control group) were compared. The specific experimental method was as follows.

[0097] 1. Day 1 First, lung cancer cell line HCC827 was trypsinized, counted, and resuspended in NK medium. Following the Agilent RTCA instrument operating manual, 10,000 tumor cells were uniformly seeded onto a 96-well electrode plate. Target cells were cultured in the 96-well plate for 20 hours before proceeding.

[0098] 2. Day 2 NK-PD1M-CAR cells prepared in Example 3, GFP-expressing NK lymphocytes (NK-GFP, negative control), and PD-1-WT-overexpressing NK lymphocytes (NK-PD1WT-CAR, negative control) were mixed with target cells at an effector-to-target cell ratio of 1.25:1 and co-cultured at 37°C under 5% CO2. When PD-1-M1 microbody (HAC) was added, its concentration was 0.25 μM. The death status of target cells was reflected by real-time detection on the electrode plate.

[0099] The experimental grouping using NK-PD1M-CAR cells was as follows: (1) HCC827; (2) HCC827+NK-GFP+HAC, E / T=1.25; (3) HCC827+NK-PD1WT-CAR, E / T=1.25; (4) HCC827+NK-PD1M-CAR, E / T=1.25. Each group was repeated three times.

[0100] Figure 7 shows that NK-PD1M-CAR has a stronger killing effect than NK-GFP with added HAC, and at the same time, it also has a stronger killing effect than PD-1-WT-CAR.

[0101] Example 8: In vitro killing effect of NK cells against PDL1-highly expressing HCC827 tumor cell line 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 effector to target cell ratios of 5:1, 2.5:1, and 1.25:1 at 37°C under 5% CO2. After 4 hours of culture, the supernatants were collected by centrifugation at 250g, and the LDH content of the supernatants in each group was detected using an LDH kit to evaluate the NK killing ability.

[0102] Each group was repeated twice, and NK cells were derived from eight or more sample recipients. The results are shown in Figure 3. Figure 8 shows that the greater the amount of NK-PD1M-CAR cells added (i.e., the higher the effector-to-target cell ratio), the stronger the tumor cell killing effect. Compared to NK-PD1WT-CAR, the cells had a stronger killing effect. The above results demonstrate that the NK-PD1M-CAR cells targeting PDL1 of the present invention have highly efficient and specific tumor killing ability and can prevent tumor cells from escaping immune surveillance.

[0103] Example 9: PDL1 expression status of target cells Following standard cell antibody staining procedures, A549, H1299, and HCC827 cells were digested with trypsin, centrifuged at 300 g for 5 min, and resuspended in PBS to obtain a single-cell suspension. The cell numbers of each cell type were counted, and 1 million cells were placed in a 5 ml round-bottom glass tube. 2–3 ml of PBS was added to wash the cells, and the cells were centrifuged at 300 g for 5 min. The supernatant was discarded. Five microliters of isotype-APC (Biolegend #402206) or PDL1-APC (Biolegend #329708) were added, and the cells were stained for 30 min at 4°C before detection using a flow cytometer (Cytoflex 5).

[0104] The detection results are shown in Figure 9. The results showed that A549, H1299, and HCC827 all expressed PDL1, with the PDL1 expression levels being HCC827>H1299>A549.

[0105] Example 10: In vivo killing efficacy of PD-1-M1 CAR NK In this example, we tested the in vivo killing effect of PD-1-M1 CAR NK using humanized B-NDG mice inoculated intraperitoneally with RKO tumor cells. The tumor cells grew slowly within the peritoneal cavity, and obvious tumor formation was observed 12 days after RKO cell inoculation, which was used to simulate actual tumor formation in humans. NK-PD1M-CAR cells and NK-PD1WT-CAR cells were obtained using the electroporation method described in Example 3. Non-electroporated NK cells served as controls and were reinjected 2–3 times per week for three cycles. At the same time, the cytokines IL2 and IL15 were injected prior to NK cell reinfusion to provide a more suitable growth environment for NK cells. Small animal bioimaging was performed on days 7, 14, and 21 after reinfusion. For experimental methods, see Figure 11.

[0106] After the first week of treatment, obvious metastatic lesions were observed in the tumor-only control group, whereas tumor growth was significantly suppressed in the NK-PD1M-CAR cell group compared with the NK cell reinjection group and the NK-PD1WT-CAR cell group. After the second course of treatment, NK cell injection alone was able to moderately suppress the growth of RKO colon cancer compared with the tumor-only control group, while the NK-PD1WT-CAR cell and NK-PD1M-CAR cell groups exhibited significant tumor growth suppression (Figures 12 and 13). Fluorescence signal analysis of the mice revealed that, after the third course of treatment, tumor growth in the control group was very rapid, while the NK-PD1WT-CAR and NK-PD1M-CAR cell groups significantly slowed tumor growth compared with the NK cell reinjection group (Figure 14).

[0107] After the three courses of treatment, the mortality of tumor-bearing mice under each treatment condition was continuously observed and statistically analyzed. In the later stages of tumor inoculation, severe ascites was observed in some treated mice. In accordance with ethical guidelines, mice with a 10% weight gain due to ascites had to be euthanized. The survival curves for these mice were as follows: Compared to the control group inoculated with tumor alone, the NK-PD1WT-CAR cell group and the NK-PD1M-CAR cell group significantly extended the lifespan of tumor-bearing mice (Figure 15).

Claims

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

1.

2. A chimeric antigen receptor comprising, in order from the N-terminus to the C-terminus, an optional signal peptide, the polypeptide of claim 1, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.

3. the signal peptide is 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 set forth in amino acid residues 1 to 20 of SEQ ID NO:3; and / or the hinge region is selected from 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 set forth in amino acid residues 171 to 225 of SEQ ID NO:3; and / or the transmembrane domain is selected from the CD28 transmembrane domain, the CD8 transmembrane domain, the CD3ζ transmembrane domain, the CD134 transmembrane domain, the CD137 transmembrane domain, the ICOS transmembrane domain, or the DAP10 transmembrane domain, preferably the transmembrane domain is the CD8 transmembrane domain, preferably the amino acid sequence of the CD8 transmembrane domain is as set forth in amino acid residues 226 to 246 of SEQ ID NO:3; and / or the intracellular costimulatory signal domain is an intracellular domain of a costimulatory signal molecule, preferably selected from the intracellular domains 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 domain of 4-1BB, preferably the amino acid sequence of the intracellular domain of 4-1BB is as set forth in amino acid residues 247 to 288 of SEQ ID NO: 3; and / or The chimeric antigen receptor according to claim 2, wherein the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain, preferably the intracellular signaling domain is a CD3ζ intracellular signaling domain, and preferably the amino acid sequence of the CD3ζ intracellular signaling domain is as set forth in amino acid residues 289 to 400 of SEQ ID NO:

3.

4. the polypeptide of claim 1, a CD8α hinge region, a CD8 transmembrane region, a 4-1BB intracellular domain, and a tyrosine-based activation motif of CD3ζ, The chimeric antigen receptor according to claim 3, wherein the amino acid sequence of the chimeric antigen receptor is preferably the 1st to 20th amino acid residues of SEQ ID NO: 3, SEQ ID NO: 1, and the 171st to 400th amino acid residues of SEQ ID NO: 3 linked sequentially from the N-terminus to the C-terminus, or the amino acid sequence of the chimeric antigen receptor is as set forth 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, wherein the nucleic acid molecule is preferably a DNA molecule or an RNA molecule; Preferably, the nucleic acid molecule is a coding sequence for the polypeptide of claim 1, the nucleotide sequence of which is as set forth in the sequence of nucleotides 70 to 510 of SEQ ID NO: 2, Preferably, the nucleic acid molecule comprises, in order from the 5' end to the 3' end, the nucleotide sequence set forth at positions 1 to 60 of SEQ ID NO:2 and the nucleotide sequence set forth at positions 70 to 1200 of SEQ ID NO:2, or the nucleotide sequence is as set forth in SEQ ID NO:

2.

6. A nucleic acid construct comprising the nucleic acid molecule of claim 5, Preferably, the nucleic acid construct is an expression cassette and comprises a regulatory sequence in addition to the nucleic acid molecule, or the nucleic acid construct is a vector and comprises 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 comprising a transposon element selected from piggybac, sleeping beauty, frog prince, Tn5, or Ty.

7. 10. 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, wherein the host cell is preferably a NK cell.

8. A composition or kit comprising the vector of claim 6 and optional transfection reagents.

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

10. 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 manufacture of a medicament for treating or preventing a PD1- or PDL1-mediated cancer, wherein the cancer is preferably selected from gastric cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, intrahepatic cholangiocarcinoma, 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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