CN110981973B

CN110981973B - Chimeric receptor targeting human membrane-bound and soluble NKG2D ligand, nucleic acid molecule, immune effector cell and application thereof

Info

Publication number: CN110981973B
Application number: CN201911358647.0A
Authority: CN
Inventors: 郭长江; 陈涵; 张会勇; 牛志远; 支灵通; 朱武凌
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2023-03-31
Anticipated expiration: 2039-12-25
Also published as: CN110981973A

Abstract

The invention discloses a chimeric receptor, a nucleic acid molecule, an immune effector cell and application of a targeted human membrane-bound and soluble NKG2D ligand, and belongs to the technical field of biomedicine or bioengineering. The chimeric receptor targeting the human membrane-bound and soluble NKG2D ligands targets various NKG2D ligands by using the ligand-binding domain of a human natural NKG2D molecule, specifically recognizes tumor-associated surface antigens NKG2DLs through the ligand/receptor, and transmits an activation signal into an immune cell through an intracellular signal transduction molecule, so that the killing activity of the immune cell is activated, and the tumor cell is finally eliminated. Based on the protein complex structure of NKG2D-NKG2DLs and the receptor dimerization activation mechanism, the chimeric receptor uses the monomer type hinge region, so that the chimeric receptor can simultaneously respond to NKG2DLs on the surface of a tumor cell and soluble NKG2DLs shed into a tumor microenvironment, and the immune inhibition effect of the soluble NKG2DLs is converted into the immune activation effect again.

Description

Chimeric receptor targeting human membrane-bound and soluble NKG2D ligand, nucleic acid molecule, immune effector cell and application thereof

Technical Field

The invention relates to a chimeric receptor, a nucleic acid molecule, an immune effector cell and application thereof of a targeted human membrane-bound and soluble NKG2D ligand, belonging to the technical field of biomedicine or bioengineering.

Background

In recent years, tumor immunocyte therapy techniques typified by chimeric antigen receptor-modified T cells (CAR-T) have been attracting attention because of their excellent therapeutic effects on malignant tumors. At present, CD 19-targeted CAR-T therapy has a remarkable effect on hematological malignancies, and opens a new hope gate for human beings to overcome malignancies (Nature Reviews Clinical Oncology,2018, 15. However, there are several drawbacks to the existing CAR-T technology, such as the susceptibility to cytokine storm (CRS), poor treatment of solid tumors, etc. (International Reviews of Immunology,2015,34 (2): 154-187). The treatment of solid tumors is the true primary battlefield for anti-tumor.

Natural Killer (NK) cells have been found to have the potential to replace T cells as ideal vectors for immunotherapy (Molecular Therapy,2017,25 (8): 1769-1781). NK cells are important immune cells and are the first line of defense of the body against tumors. NK cells have many own immunological properties and advantages compared to T cells. As the most commonly used human NK cell line, clinical studies of NK92 cells prove that the cells are toxic to various solid tumor cells, safe to human bodies, free of toxic and side effects and capable of realizing allogeneic therapy, but the curative effect of the cells is very limited (Immunotherapy, 2016,65 (4): 485-492). This suggests that applying the CAR-T design principle to NK cells, using NK92 cells with higher safety and controllability (Immunotherapy, 2017,9 (9): 753-765), is expected to achieve more economical Off-the-Shelf solid tumor therapy.

The primary activating receptor for NK cells, NKG2D, plays a key role in tumor immune surveillance. NKG2D is expressed in NK cells and NKT cells, etc. (International Journal of Molecular Sciences,2018,19 (1): 177), and its activation alone is sufficient to stimulate the activation of NK cells, triggering degranulation and cytokine production. NKG2D is a very potential CAR-NK construction element. The NKG2D receptor is a C-type lectin-like receptor, belongs to the NKG2 family, is encoded by the NK gene complex (NKC) located at 12p12.3-p13.1, and has the ability to allow NK cells to recognize and eliminate infected or tumor cells. The human NKG2D ligands (NKG 2 DLs) are mainly two major classes of MICA/B and human cytomegalovirus protein UL16 binding proteins (ULBPs), and the total number is 8. NKG2DLs are rarely expressed on the surface of normal cells, and the expression of the NKG2DLs mainly occurs in the process of cell infection or malignant transformation, especially the surface of tumor cells usually expresses at least one type of NKG2DL, so the NKG2DLs are called indicators of cell stress states.

However, malignant cells (e.g., tumor cells) have developed multiple pathways to prevent or reduce the expression of NKG2DLs, thereby evading immune surveillance. Research shows that the main mechanism of NKG2DLs positive tumor cell immune escape is that tumor cells often shed NKG2DLs (membrane-bound mNKG2 DLs) on the membrane surface thereof by means of metalloproteinase hydrolysis, and the NKG2DLs (sinkg 2 DLs) are present in the tumor microenvironment in the form of soluble NKG2 DLs. Binding of soluble NKG2DLs to NKG2D promotes receptor internalization, thereby reducing NK cells or CD8 ⁺ Killing by T cells (Frontiers in immunology.2018; 9. Furthermore, the NKG 2D-mediated toxic effects of NK cells are also severely impaired by the persistent activation of NKG2D upon binding of skkg 2 DLs. Therefore, enhancing the recognition effect of NKG2D and NKG2DLs and blocking the immunosuppressive effect caused by sNKG2DL can become a new approach for immunotherapy of solid tumors.

The Chinese patent application with publication number CN 109803983A discloses a specific chimeric antigen receptor combined with human NKG2DL in a targeting way, wherein the amino acid sequence of the specific chimeric antigen receptor is formed by sequentially connecting a leader sequence, a human NKG2DL sequence, a human CD8 transmembrane region sequence, a human CD8 hinge region, a human CD28 intracellular domain sequence, a human 4-1BB intracellular domain sequence and a CD3 zeta intracellular domain sequence in series from an amino terminal to a carboxyl terminal; the Chinese patent application with publication number CN 110028589A discloses a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain and a costimulatory signaling region, and the structure of the chimeric antigen receptor is NKG2D-CD8 alpha hinge-CD8 ^TM -4-1BB-CD3 ζ; the Chinese patent application with publication number CN 110494558A discloses an NKG2D chimeric protein, which is obtained by amplifying an extracellular domain (NKG 2D-ED, uniprot P26718-1, amino acids 83-216) of human NKG2D from a PMBC cDNA library by PCR (polymerase chain reaction), andfusion of NKG2D-ED with CD8 α hinge and transmembrane region (CD 8H-TM, uniprot P01732, amino acids 128-210), CD3 ζ or DAP12 signaling moieties to construct NKG2D chimeric proteins; the chimeric receptors (CARs) involved in the above patent applications all belong to the class I transmembrane proteins, and the key to their function is that the extracellular domain of NKG2D outside the cell membrane can normally adopt its native conformation, which is a prerequisite for NKG2D binding to its ligand. However, natural human NKG2D protein belongs to type II transmembrane protein, and all existing NKG 2D-CARs use the signal peptide and transmembrane domain of the type I transmembrane protein to force them to reverse their functions as type I protein, which undoubtedly affects the formation of the natural conformation of NKG2D ectodomain, and thus affects its specific binding with ligand, thereby causing functional limitation and safety hazard. In addition, conventional Chimeric Antigen Receptors (CARs) use a native CD8 α or IgG-derived hinge region, and the presence of cysteines in the hinge region sequence allows the chimeric receptor to undergo disulfide bond-type polymerization on the cell membrane, thereby forming a dimeric or multimeric structure. Also because of this, traditional CARs can only be activated when they bind to membrane-type ligands on the cell membrane. However, tumor cells often shed NKG2D ligands from their membranes by metalloproteinases, forming soluble ligands that are released into the tumor microenvironment and blood. These soluble ligands can significantly inhibit the activity of traditionally designed NKG 2D-CARs, thereby blocking the immune response to form immune escape, which is very disadvantageous for clinical treatment of tumors, and is also an important reason for poor clinical efficacy of many current CAR therapies.

Disclosure of Invention

The invention aims to provide a chimeric receptor targeting human membrane-bound and soluble NKG2D ligand, which is designed based on a receptor dimerization activation mechanism and a structural model of NKG2D and ligand binding trimer for the first time, and the chimeric receptor which can be activated by the soluble NKG2D ligand is constructed by using a special monomer type hinge region, so that the immunosuppressive action of the soluble NKG2D ligand is reversed, and the tumor immune escape is hopefully overcome, thereby greatly improving the clinical application curative effect.

The invention also provides a nucleic acid molecule encoding the chimeric receptor targeting the human membrane-bound and soluble NKG2D ligand.

The invention also provides a viral vector comprising the nucleic acid molecule described above.

The invention also provides an immune effector cell modified by the chimeric receptor gene.

The invention also provides a preparation method of the immune effector cell modified by the chimeric receptor gene.

The invention also provides an application of the chimeric receptor of the targeted human membrane-bound and soluble NKG2D ligand or the immune effector cell genetically modified by the chimeric receptor in preparing anti-tumor immunotherapy drugs.

The invention also provides an anti-tumor immunotherapy medicament.

In order to realize the purpose, the technical scheme adopted by the invention is as follows:

a chimeric receptor targeting human membrane-bound and soluble NKG2D ligands comprises a signal peptide region, an NKG2D ligand-binding domain based on a human NKG2D molecule, a hinge region, a transmembrane region and an intracellular signal domain which are connected in series in sequence; the NKG2D ligand binding domain comprises SEQ ID NO:1, or an engineered amino acid sequence having 85% -99% identity thereto; the hinge region is a monomeric hinge region structure and is selected from IgG4 molecules or CD8 alpha molecules.

The ligand molecules that bind to the NKG2D ligand binding domains described above are associated with malignancies, including but not limited to any NKG2D ligand such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and combinations thereof.

The chimeric receptor targeting human membrane-bound and soluble NKG2D ligands (NKG 2D-CAR or chNKG 2D) of the present invention targets a variety of NKG2D ligands (NKG 2 DLs) using the ligand-binding domain of a human natural NKG2D molecule, specifically recognizes tumor-associated surface antigens NKG2DLs (e.g., MICA, MICB) through the "ligand/receptor", and transmits an activation signal into immune cells via intracellular signaling molecules, thereby activating the killing activity of the immune cells, and finally eliminating tumor cells. Based on the protein complex structure of NKG2D-NKG2DLs and the receptor dimerization activation mechanism, the invention creatively uses the monomer hinge region to enable the chimeric receptor to simultaneously respond to NKG2DLs (mNKG 2 DLs) on the surface of tumor cells and soluble NKG2DLs (sNKG 2 DLs) shed into a tumor microenvironment, thereby converting the immunosuppression effect of the sNKG2DLs into the immunoactivation effect.

In the chimeric receptor targeting the human membrane-bound and soluble NKG2D ligand, a signal peptide region (or extracellular signal peptide structure) has a signal peptide sequence related to protein secretion, and can be selected from I-type transmembrane protein signal peptides such as CD8 alpha, PD1, DAP10, DNAM-1, CD137 (or 4-1 BB) and the like; further preferred is a CD 8. Alpha. Signal peptide. The CD8 a signal peptide comprises SEQ NO:2, and the sequence of the nucleic acid molecule for coding the sequence is shown as SEQ ID NO: shown at 9.

In the chimeric receptors targeting human membrane-bound and soluble NKG2D ligands, the hinge region is preferably a monomeric IgG4 hinge region (i.e., an IgG4 molecule). The IgG4 molecule comprises SEQ NO:3, and the sequence of the nucleic acid molecule for coding the sequence is shown as SEQ ID NO: shown at 10.

In the chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, the transmembrane region (or transmembrane domain) may be selected from the transmembrane regions of type I transmembrane proteins such as TCR α, TCR β, TCR γ, CD3 ζ, CD4, CD16, CD8 α, CD28, PD1, DAP10, DNAM1, CD137 (or 4-1 BB); more preferably, the CD28 transmembrane domain. The CD28 transmembrane region comprises SEQ NO:4, and the sequence of the nucleic acid molecule for coding the sequence is shown as SEQ ID NO: shown at 11.

In the chimeric receptor targeting human membrane-bound and soluble NKG2D ligands, the intracellular signaling domain comprises an intracellular signaling domain and/or an intracellular costimulatory signaling domain, and the intracellular signaling domain and intracellular costimulatory signaling domain can be selected from the group consisting of CD3 ζ, CD2, CD7, CD27, CD28, CD30, CD40, CD83, CD137 (or referred to as 4-1 BB), 2B4, OX40, DAP10, DNAM1, LIGHT, NKG2C, B7-H3, DAP10, and like intracellular domains of activating receptors, or any combination thereof; further, the intracellular signaling domain is preferably CD3 ζ, and the intracellular costimulatory signaling domain is preferably CD28 and CD137. The CD3 ζ comprises SEQ NO:5, and the sequence of the nucleic acid molecule of the sequence is shown as SEQ ID NO: shown at 12. The CD28 comprises SEQ NO:6, and the sequence of the nucleic acid molecule for coding the sequence is shown as SEQ ID NO: shown at 13. The CD137 comprises SEQ NO:7, and the sequence of the nucleic acid molecule of the sequence is shown as SEQ ID NO: as shown at 14.

In the chimeric receptor targeting the human membrane-bound and soluble NKG2D ligand, a Flag tag sequence is also connected between a signal peptide region and an NKG2D ligand binding domain, and comprises SEQ NO:17, and the sequence of the nucleic acid molecule for coding the sequence is shown as SEQ ID NO:18, respectively.

Further, the chimeric receptor targeting human membrane-bound and soluble NKG2D ligands has the sequence as shown in SEQ NO:15, or a pharmaceutically acceptable salt thereof.

A nucleic acid molecule encoding the nucleotide sequence of the chimeric receptor targeting the human membrane-bound and soluble NKG2D ligand.

Specifically, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 16.

A viral vector comprising the above-described nucleotide sequence encoding a chimeric receptor targeting a human membrane-bound and soluble NKG2D ligand.

A chimeric receptor genetically engineered immune effector cell (i.e., a genetically engineered immune effector cell) comprising a nucleotide sequence or viral vector encoding a chimeric receptor that targets human membrane-bound and soluble NKG2D ligands.

The immune effector cells modified by the chimeric receptor gene can express the chimeric receptor of a targeted human membrane-bound and soluble NKG2D ligand, and have the amino acid sequence shown in SEQ NO:15, or a pharmaceutically acceptable salt thereof. The human membrane-bound and soluble NKG2D ligand-targeted chimeric receptor-modified NK cell (NKG 2D-CAR-NK 92) disclosed by the invention can be specifically bound with the NKG2D ligand on the surface of a tumor cell and in a tumor microenvironment (as shown in figure 10), so that a specific killing effect is generated on the tumor cell, and further, the killing effect is not inhibited by sNKG2 DLs.

The invention is based on a transmembrane protein three-level structure high-precision modeling method, and through the change of an NKG2D ectodomain sequence selection range and the optimization of a hinge region sequence, the chimeric receptor has an extracellular conformation which is highly consistent with that of a natural NKG2D protein, the specific binding capacity of the chimeric receptor and each ligand is retained to the maximum extent, and the chimeric receptor can simultaneously respond to a membrane-bound NKG2D ligand on the surface of a cell membrane and a fallen soluble NKG2D ligand. The invention is also based on a heterotrimeric structure of NKG2D combined with a ligand thereof (namely two NKG2D molecules are combined with one NKG2D ligand), a monomeric CAR molecule is constructed by using a ligand binding domain outside the NKG2D molecule, and the limit that the traditional CAR molecule can only respond to a membrane-bound ligand and is inhibited by a soluble ligand is broken through a ligand-mediated CAR molecule dimerization activation mode. In addition, the invention uses a transmembrane protein structure modeling rational design technology (Molecular Immunology,2019,114, 108-113), successfully solves the problem of conformational change existing when the II-type transmembrane protein NKG2D constructs CAR molecules, and constructs a novel chimeric receptor which can realize the clinical treatment application of solid tumors and can target human membrane-bound and soluble NKG2D ligands.

A method for preparing an immune effector cell genetically modified by a chimeric receptor comprises the following steps: and transferring the virus vector into an immune effector cell, and screening to obtain the chimeric receptor gene modified immune effector cell.

The immune effector cell can be selected from T lymphocyte, NK cell, hematopoietic stem cell, pluripotent stem cell or embryonic stem cell culture differentiated immune cell; further preferably, NK cells.

The human membrane-bound and soluble NKG2D ligand-targeted chimeric receptor-modified NK cell (NKG 2D-CAR-NK 92) can be specifically bound with the NKG2D ligand on the surface of a tumor cell and in a tumor microenvironment, so that a specific killing effect is generated on the tumor cell, and the killing effect is not inhibited by sNKG2 DLs.

An application of chimeric receptor of targeted human membrane-bound and soluble NKG2D ligand or immune effector cell modified by chimeric receptor gene in preparing antineoplastic immunotherapy medicine.

In particular to application of chimeric receptors targeting human membrane-bound and soluble NKG2D ligands or immune effector cells modified by chimeric receptor genes in preparation of drugs for treating solid tumors.

An anti-tumor immunotherapy medicine takes immune effector cells modified by chimeric receptor genes as a drug effect component.

The anti-tumor immunotherapy medicament has extremely high clinical application value in treating NKG2D ligand positive solid tumors, can provide a broader target spot for tumor immunotherapy, and simultaneously reverses the immunosuppressive action of soluble immunosuppressive molecules in a tumor microenvironment.

The invention has the beneficial effects that:

the invention designs a chimeric receptor targeting human membrane-bound and soluble NKG2D ligands by utilizing the interaction principle of an NK cell activation receptor NKG2D and a ligand thereof and a receptor dimerization activation mechanism. Unlike the classical architecture of traditional chimeric antigen receptors, this is a completely new, natural receptor-based chimeric receptor that simultaneously targets soluble NKG2D ligands in response to membrane-bound and shed on the cell membrane surface. The chimeric receptors of the invention that target human membrane-bound and soluble NKG2D ligands comprise a ligand-binding domain based on the natural human NKG2D molecule, a haplotype hinge region, a transmembrane region, and an intracellular signaling region, among others; immune cells Modified by the Chimeric Receptor, namely NKG2D-CAR Chimeric Receptor Modified NK92 cells (Chimeric NKG2D Receptor Modified NK92 cells, chNKG2D-NK 92). The chimeric receptor targeting the human membrane-bound and soluble NKG2D ligand and the immune effector cell thereof have better NKG2D ligand target specificity and broad-spectrum tumor killing effect than the traditional chimeric antigen receptor, the use of the natural human NKG2D molecule can realize the simultaneous specific targeting response to 8 ligands thereof, and the soluble NKG2D ligand dropped in the tumor microenvironment can activate the chimeric receptor without inhibition. The chimeric receptor also has better safety, is different from a chimeric antigen receptor which takes a single-chain antibody as a construction element, and cannot generate heterologous immunogenicity when the ligand of the natural human NKG2D molecule is targeted for immunotherapy by using the natural human NKG2D molecule, so the chimeric receptor and the immune effector cell thereof have high clinical application value in treating NKG2D ligand positive solid tumors.

An important mechanism for recognition and response of soluble ligands by receptors on cell membranes is ligand-induced receptor dimerization activation, such as protein tyrosine kinase type receptors. The key to this activation mechanism is that one ligand (or ligand complex) can bind to two or more receptors simultaneously, thereby inducing a receptor dimerization activation effect. According to the invention, through protein structure information analysis, the combination of NKG2D and the ligand thereof just meets the condition that one NKG2D ligand is combined with two NKG2D molecules. Therefore, the invention provides that a haplotype CAR molecule is constructed by using the extracellular ligand-binding domain of the NKG2D molecule, and the ligand-mediated CAR molecule dimerization activation mode breaks through the limitation that the traditional CAR molecule can only respond to a membrane-bound ligand and is inhibited by a soluble ligand, so that the problem of immune escape caused by the fallen soluble ligand molecule in the clinical application of tumor immunotherapy is well solved, and a better clinical application curative effect is obtained.

The innovation points of the invention are mainly embodied in the following three aspects: 1) Optimizing the sequence and the extracellular conformation of the chimeric receptor using a transmembrane protein structure modeling technique; 2) Designing a chimeric receptor with dimerization activation function by combining an NKG 2D-ligand structure; 3) The construction of chimeric receptors which can be activated simultaneously by membrane-type ligands and soluble ligands to block tumor immune escape.

In conclusion, the novel chimeric receptor constructed by the invention has obvious structural difference and functional advantage compared with the traditional design: based on a receptor dimerization activation mechanism and an NKG2D-NKG2DL trimer structure model, a transmembrane protein structure modeling technology is combined, and a monomer type hinge region is used for the first time to successfully construct a chimeric receptor capable of simultaneously responding to two types of ligands, namely a membrane type ligand and a soluble ligand. The chimeric receptor can effectively avoid the defect that the traditional chimeric receptor is easily inhibited by soluble molecules (the defect is often the key causing failure of clinical application), thereby having the advantage of robustness of anti-tumor functions which the chimeric receptor does not have, having great potential for greatly improving the clinical treatment effect and being very beneficial to the development, clinical popularization and popularization of novel tumor immune cell therapies in China.

Drawings

FIG. 1 is a schematic structural view and a vector of a chimeric receptor targeting human membrane-bound and soluble NKG2D ligand (NKG 2D-CAR) constructed in example 1 of the present invention;

FIG. 2 is a schematic diagram of the construction of the tertiary junction of a chimeric receptor (NKG 2D-CAR) protein targeting human membrane-bound and soluble NKG2D ligands constructed in example 1 of the present invention;

FIG. 3 is a microscopic image of NK92 cells after transfection of lentivirus in example 4 of the present invention;

FIG. 4 is a graph showing the results of flow cytometry detecting the expression of NKG2D molecules on the surface of untransfected NK92 cells (a) and NKG 2D-CAR-transfected NK92 cells (b) in example 4 of the present invention;

FIG. 5 is a graph showing the results of flow cytometry for detecting the expression of MICA/B molecules, which are the main ligands of NKG2D, on the surfaces of cervical cancer Hela cells (a), gastric cancer SGC-7901 cells (B), osteosarcoma U2OS cells (c) and lung cancer H1299 cells (D) in test example 1 of the present invention;

FIG. 6 is a graph showing the in vitro cytotoxicity of untransfected NK92 cells and NKG 2D-CAR-transfected NK92 cells on four tumor cells (Hela, SGC7901, U2OS, H1299) in experimental example 1 of the present invention by lactate dehydrogenase method;

FIG. 7 is a graph showing the in vitro cytotoxicity change of the NK92 cells transfected by the non-transfected and NKG2D-CAR cells on SGC7901 cells when the NK92 cells are blocked by the lactate dehydrogenase method in the test example 1 of the present invention at different concentrations of NKG2D antibody;

FIG. 8 shows the amount of IFN-. Gamma.released by ELISA after incubation of MICA protein at different concentrations in Experimental example 2 with untransfected NK92 cells and NKG2D-CAR-NK92 cells for 24 h;

FIG. 9 shows the effect of MICA protein treatment on NKG2D-CAR-NK92 cytotoxicity in vitro in test example 2;

FIG. 10 is a schematic diagram of the binding of chimeric receptor genetically engineered immune effector cells to tumor cell surface or shed NKG2D ligands in accordance with the present invention.

Detailed Description

The following examples are intended to illustrate the invention in further detail, but are not to be construed as limiting the invention in any way.

The chimeric receptor targeting human membrane-bound and soluble NKG2D ligands (NKG 2D-CAR) of the present invention comprises a signal peptide region, an NKG2D ligand binding domain based on a human NKG2D molecule, a hinge region, a transmembrane region, and an intracellular signaling domain, connected in series in sequence.

The term "NKG 2D ligand binding domain based on human NKG2D molecules" as used herein refers to an NKG2D ligand specific binding domain derived from the amino acid sequence of a natural human NKG2D protein.

The amino acid sequence of natural human NKG2D molecule contains 216 amino acids (UniProt: P26718), is II type transmembrane protein, and removes the intracellular domain and transmembrane domain (1-72) at the N terminal to obtain the amino acid sequence (73-216) of the ligand binding domain thereof, such as SEQ ID NO:1 is shown. The domain has the ability to specifically bind NKG2D ligands (e.g., MICA, MICB).

The term "identity" of amino acid sequences as used herein refers to the degree of similarity between amino acid sequences as determined by a sequence alignment software such as BLAST.

Typically, an engineered amino acid sequence may be obtained by substitution, deletion and/or addition of one or more amino acid residues to a known amino acid sequence. For example, by conventional protein engineering means (e.g., amino acid conservative substitutions, etc.), the amino acid sequence of SEQ ID NO:1, the NKG2D ligand binding domain shown in SEQ ID NO:1, and having substantially the same ability to bind an NKG2D ligand.

The chimeric receptors of the invention targeting human membrane-bound and soluble NKG2D ligands contain an extracellular signal peptide structure (i.e., a signal peptide region). The extracellular signal peptide structure can be obtained from natural protein, and can be selected from molecules such as CD8 alpha, PD1, DAP10, DNAM-1, CD137 (or called 4-1 BB), and the like, preferably CD8 alpha, as shown in SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

The chimeric receptor targeting human membrane-bound and soluble NKG2D ligands of the invention comprises a haplotype hinge region structure based on IgG4 molecules (shown as SEQ ID NO: 3) or CD8 alpha molecules, and is a flexible amino acid sequence for connecting the NKG2D ligand-binding domain and the transmembrane domain. The hinge structure not only can make the NKG2D-CAR of the invention obtain a conformation close to a natural NKG2D molecule, but also can ensure that the NKG2D-CAR molecule exists on a cell membrane in a monomer form.

The term "transmembrane domain" (TM) is used herein interchangeably with "transmembrane domain" (TM) and refers to a protein domain that is thermodynamically stable anchored within the cell membrane. The transmembrane region can be obtained from a native protein and can be selected from molecules such as TCR α, TCR β, TCR γ, CD3 ζ, CD4, CD16, CD8 α, CD28, PD1, DAP10, DNAM1, CD137 (or referred to as 4-1 BB), and preferably CD28, as shown in SEQ ID NO: 4.

The term "intracellular signaling domain" or "intracellular signaling region" as used herein refers to a region of a protein structure that is capable of signaling a cellular effector function and directing the cell to perform a specific function. The intracellular signaling domain may include a signaling domain and/or a costimulatory signaling domain. The intracellular signaling domain is derived from a native protein and is selected from molecules such as CD3 ζ, CD2, CD7, CD27, CD28, CD30, CD40, CD83, CD137 (or called 4-1 BB), 2B4, OX40, DAP10, DNAM1, LIGHT, NKG2C, B7-H3, DAP10, etc., the intracellular signaling domain is preferably CD3 ζ, and the intracellular costimulatory signaling domain is preferably CD28 and CD137, as shown in SEQ ID NO: 5. 6 and 7.

Example 1

Rational design and construction of chimeric receptors targeting human membrane-bound and soluble NKG2D ligands in this example:

the NKG2D-CAR fusion gene fragment was designed in the following order of the encoding genes: extracellular signal peptide, flag tag, NKG2D ligand binding domain based on NKG2D molecule, hinge region, transmembrane region, intracellular costimulatory signal domain and intracellular signaling domain. FIG. 1 is a schematic structural diagram and a schematic vector diagram of a chimeric receptor targeting human membrane-bound and soluble NKG2D ligand (NKG 2D-CAR) constructed in example 1 of the present invention. FIG. 1a is a schematic structural diagram of the NKG2D-CAR of the present invention, wherein the signal peptide part is preferably CD8 a molecule, the transmembrane domain part is preferably CD28 molecule, and the signaling domains are exemplified by CD28, CD137 and CD3 zeta signaling molecules. FIG. 1b is a schematic diagram of the structure of NKG2D-CAR expression vector constructed in the present invention.

Since the NKG2D molecule is a type II transmembrane protein and the other elements are type I transmembrane proteins, which have diametrically opposite transmembrane orientations, it is desirable to optimally design the NKG2D-CAR molecule such that its ectodomain and transmembrane domain have a three-dimensional conformation similar to that of native human NKG2D ectodomain and transmembrane domain by transmembrane protein structure modeling analysis.

The ectodomain and transmembrane domains of NKG2D-CAR were modeled three-dimensionally using the transmembrane protein structure prediction tool PredMP (https:// predmp.com) and the protein structure comprehensive prediction tool I-TASSER (https:// zhanglab.ccmb.med.umi/I-TASSER), wherein PredMP was used for prediction of transmembrane domain structure and I-TASSER was used for reconstruction and optimization of NKG2D ectodomain structure. The results show that NKG 2D-CARs linked using a monomeric IgG4 hinge structure have a near-native NKG2D conformation (as shown in figure 2). FIG. 2 is a schematic diagram of the construction of the tertiary junction of a chimeric receptor (NKG 2D-CAR) protein targeting human membrane-bound and soluble NKG2D ligands constructed in example 1 of the present invention. FIG. 2a is the tertiary structure of the transmembrane domain and extracellular domain of a native human NKG2D protein molecule; FIG. 2b is a three-level structural diagram of the ligand binding domain, igG4 hinge region and CD28 transmembrane domain of human NKG2D protein molecule. The modeling method is the combination of a transmembrane protein structure modeling tool PredMP and a protein structure comprehensive modeling tool I-TASSER.

Through the above structural modeling rational design, the NKG2D-CAR fusion gene fragment of embodiment 1 of the present invention is constructed as follows: CD8 alpha extracellular signal peptide (1-63 bp), flag tag sequence (64-87 bp), NKG2D ligand binding domain based on human NKG2D molecule (88-492 bp), haplotype IgG4 hinge region (493-528 bp), CD28 transmembrane region (529-609 bp), intracellular costimulatory signal domain (CD 28:610-732bp, CD137:733-858 bp) and intracellular signaling domain (CD 3 ζ:859-1197 bp).

Example 2

In this example, synthesis of nucleic acid molecules encoding chimeric receptors targeting human membrane-bound and soluble NKG2D ligands and construction of viral vectors (i.e., synthesis of NKG2D-CAR fusion genes and construction of expression vectors):

the nucleotide sequence (SEQ ID NO: 16) encoding the chimeric receptor (NKG 2D-CAR) molecule targeting the human membrane-bound and soluble NKG2D ligands obtained in example 1 was first synthesized by a whole-gene synthesis method, and a gene synthesis technology was provided by Nanjing King-Shirui Biotech Co., ltd.

The synthetic fusion gene fragment was cloned into pCDH-CMV-MCS-P2A-copGFP-T2A-Puro lentiviral vector as shown in FIG. 1. The lentiviral vector and the gene fragment (see table 1) were digested with restriction enzymes Xba I and EcoR I, respectively, to obtain the linearized lentiviral vector after digestion and the digested NKG2D-CAR gene fragment, and incubated with T4DNA ligase system at 16 ℃ for 3h (see table 2). Then transforming escherichia coli Stbl3 competent cells, coating a culture medium plate containing ampicillin, selecting a plurality of clone colonies for plasmid extraction, and carrying out enzyme digestion identification and sequencing comparison to construct a successful vector named as pCDH-NKG2D-CAR.

TABLE 1 digestion reaction System of lentiviral vector and NKG2D-CAR fusion Gene

TABLE 2 ligation reaction System of Lentiviral vectors with NKG2D-CAR fusion genes

Note: n is a radical of _{Vector fragment} :N _{Target gene} Mass of 1000bp DNA of 0.03pmol = 0.3pmol and 1mol was about 0.66. Mu.g, and the volume of the linearized vector and the gene fragment added in the ligation reaction was determined according to the above relationship.

Example 3

Packaging, concentration and titer determination of viral vectors (i.e., NKG2D-CAR lentiviral expression vectors) in this example:

(I) packaging of NKG2D-CAR lentiviral expression vectors

Taking 8X 10 ⁶ HEK293T cells (purchased from ATCC under the accession number CRL-1573) were seeded at 175cm ² The cell culture flask of (4) was cultured overnight at 37 ℃ and, when the cell density reached 75% to 85%, 25mL of a fresh penicillin-and streptomycin-free DMEM medium (Hyclone, cat # SH 30022.01) containing 6% FBS (BI, cat # 04-001-1 ACS) was replaced by 1 hour in advance, and the flask was further placed in an incubator for culture, and virus packaging was carried out after 1 hour.

Packaging with calcium phosphate kit (Biyuntian, with a product number of C0508), and packaging: the recombinant NKG2D-CAR expression plasmid (pCDH-NKG 2D-CAR) extracted in example 2, was mixed with helper plasmids pSPAX2, pmd2.G in a ratio of 4 ₂ And (3) in the solution (provided by the kit), beating and mixing uniformly, adding the mixed solution into BBS (barium-based phosphate buffer solution) dropwise, beating and mixing uniformly, incubating at room temperature for 15-30min, clarifying the liquid until no white precipitate is generated, adding the mixed solution into HEK293T cells, mixing uniformly, putting the mixture into an incubator to continue culturing for 8-16h, removing supernatant, washing once by PBS (phosphate buffered saline), replacing 35mL of fresh DMEM (dimethyl Ether) culture medium containing 10 FBS, and collecting the virus after 48h, 72h and 96h respectively. The collected lentivirus supernatant was centrifuged at 3500g for 10 min to remove cell debris pellet.

Concentration of (di) NKG2D-CAR lentiviral expression vectors

Adding 5 XPEG into the harvested lentivirus supernatant, standing at 4 ℃ for 24h,3500g, centrifuging at 4 ℃ to collect virus particle precipitates, and taking precooled PBS for resuspension. Concentrating with a bottle of 175cm ² The virus supernatant harvested from the culture flask is concentrated to 200-300 mu L. And (3) subpackaging the concentrated lentivirus stock solution into freezing storage tubes, and storing at-80 ℃ to avoid repeated freezing and thawing.

(III) determination of the titer of NKG2D-CAR Lentiviral expression vectors

The titer of the lentivirus concentrate was determined using the qPCR lentivirus titer kit (Aiben Meng, cat. LV 900), which gave a lentivirus titer of 3X 10 in this example ⁹ Left and right.

The recombinant NKG2D-CAR lentiviral expression vector obtained by the above method was named pCDH-NKG2D-CAR, while the original lentiviral expression vector without the fusion gene was named pCDH.

Example 4

The chimeric receptor gene modified immune effector cell (NKG 2D-CAR modified NK92 cell) and the preparation method thereof in the embodiment comprise the following steps:

lentiviral transfection of NK92 cells

Taking 3-5X 10 ⁵ NK92 cells (ATCC, cat # CRL-2407) were seeded in a 24-well plate, pCDH-NKG2D-CAR lentivirus concentrate (MOI = 20-80) was added thereto, polybrene (Saint Renzinobios, cat # 40804ES 76) was added thereto at a final concentration of 8. Mu.g/mL, and the mixture was mixed, incubated in an incubator at 37 ℃ for 12 to 15 hours, centrifuged, and the virus supernatant was removed and the medium was replaced with fresh one. After 72h, the expression efficiency of NKG2D-CAR was determined by fluorescence quantitative real-time qPCR method using untransfected NK92 cells and NK92 cells transfected by unloaded pCDH as controls. After the verification, resistance screening is carried out by puromycin with the concentration of 800ng/ml, and the NK92 cell line which stably expresses the NKG2D-CAR is obtained after 1-2 weeks. NKG2D-CAR-NK92 cells were observed under a fluorescence microscope, and the morphology of the cells under a 20X-fold microscope is shown in FIG. 3. FIG. 3a is a white light channel view and FIG. 3b is a green fluorescence channel view. In this figure, the lower right short line represents 100. Mu.m. Detection of green fluorescence indicates successful expression of the NKG2D-CAR fusion gene.

(II) flow cytometry detection of expression of NKG2D-CAR protein molecules in transfected cells

1X 10 times of each of the NKG2D-CAR-NK92 cells and the untransfected NK92 cells which were transfected with lentivirus successfully ⁶ Each of the cells was centrifuged at 300g for 5min, washed 2 times with 1% FBS-containing PBS (hereinafter referred to as wash buffer), added to 100. Mu.L of the system with APC-labeled mouse anti-human NKG2D monoclonal antibody (Thermo Fisher, cat. No. 1997140), incubated on ice in the dark for 30min, and washed 2 to 3 times with wash buffer. Finally, 400. Mu.L of PBS was added to resuspend the cells, and the cells were analyzed by flow cytometry (Guava easy cytology 8), and the results are shown in FIG. 4. FIG. 4 is a graph showing the results of flow cytometry detecting the expression of NKG2D molecules on the surface of untransfected NK92 cells (a) and NKG 2D-CAR-transfected NK92 cells (b).

As can be seen from the figure, the expression rate of NK92 cells is 1.42%, the expression rate of NKG2D-CAR-NK92 cells is 96.2%, and it can be seen that NKG2D-CAR molecules are successfully expressed in the NK92 cells transfected by the molecules and are localized on the cell membrane.

Example 5

In the embodiment, the immune effector cells modified by the chimeric receptor gene are applied to preparing anti-tumor immunotherapy medicaments and the anti-tumor immunotherapy medicaments take the immune effector cells modified by the chimeric receptor gene as medicinal components and are used for tumor immunotherapy.

Test example 1

Function of NKG2D-CAR-NK92 cell in vitro antitumor activity:

(I) detection of tumor cell surface NKG2D Primary ligand MICA/B expression

Respectively taking cervical cancer Hela cells, gastric cancer SGC-7901 cells, osteosarcoma U2OS cells and lung cancer H1299 cells, wherein the cell number is 1 × 10 ⁶ The results of the flow assay using a mouse anti-human MICA/B antibody (BioLegend, cat. No. 320906) labeled with PE are shown in FIG. 5. FIG. 5 is a graph showing the results of flow cytometry on the expression of MICA/B molecules as the main ligands of NKG2D on the surface of cervical cancer Hela cells (a), gastric cancer SGC-7901 cells (B), osteosarcoma U2OS cells (c) and lung cancer H1299 cells (D).

As can be seen from the figure, the expression rates of MICA/B on the surfaces of four solid tumor cells are respectively 53.3%, 63.5%, 73.4% and 97.9%, and the expression rates all show high-level MICA/B expression, and can be used as target cells to detect the anti-tumor activity of NKG2D-CAR-NK92 cells.

(II) in vitro toxic Effect of NKG2D-CAR-NK92 cells on four tumor cells

Respectively taking Hela cells, SGC-7901 cells, U2OS cells and H1299 cells in logarithmic growth phase, inoculating the cells in a 96-well plate, and ensuring that each well is 1 multiplied by 10 ⁴ Each cell, setting 3 duplicate wells per cell, placing in 5% CO ₂ And cultured overnight in an incubator at 37 ℃. Using CytoTox96 ^R Non-Radioactive cytoxicity Assay (Promega, cat # G1781). NKG2D-CAR-NK92 cells were placed 5% co ₂ Incubated for 6h in 37 ℃ incubator with untransfected NK92 cells are controls. At the same time, 3 sets of controls, i.e., target cell control wells, sample maximal enzyme activity control wells (target cell wells not treated with effector cells for subsequent lysis), blank cell control wells, were set, with a total volume of 100 μ L per well.

45min before the predetermined detection time point, the 96-well plate was removed, and lysine Solution (10X) was added to the "sample maximum enzyme activity control well" in an amount of 10% of the original culture Solution volume. Adding lysine Solution, repeatedly beating and mixing, and culturing in incubator for 45min. After the co-incubation time was reached, centrifugation was carried out at 300g for 5 minutes. The supernatant of each well (50. Mu.L) was collected and transferred to a new 96-well plate (designated as plate (1)). In the corresponding wells, the following measurements were performed. The sample determination procedure was as follows: add 50. Mu.L of CytoTox96 to each well of plate (1) ^R (kit supply), incubating the plate (1) in the dark for 30min at room temperature, adding 50 μ L of Stop Solution, measuring absorbance at 490nm by using a microplate reader, subtracting the absorbance of a background blank control well from the measured absorbance of each well, and calculating the cytotoxicity of each experimental group according to the following formula:

cytotoxicity (%) = (experimental group-effector cell control well-target cell control well)/(sample maximum enzyme activity control well-target cell control well) × 100.

The results show that: the cytotoxicity of NKG2D-CAR-NK92 was significantly higher on 4 MICA/B positive tumor cells than on untransfected NK92 cells at different effective target ratios (wherein the effective target ratio was 5.

As can be seen from the figure, NKG2D-CAR transfected NK92 cells all showed higher killing activity than transfected NK92 cells for the four MICA/B positive tumor cells, and the cytotoxic effect (i.e. lethality) was close to 100%. This shows that the NKG2D-CAR-NK92 cells constructed by the invention have strong killing ability on a plurality of tumor cells positive to NKG2D ligands.

(III) NKG2D antibody inhibits NKG2D-CAR-NK92 cell toxicity in vitro

In the example of gastric cancer SGC-7901, the in vitro toxicity test procedure was followed, and the test was divided into 3 groups, and the ligand binding domain of NKG2D-CAR was blocked by adding NKG2D antibody (10575-MM 02, manufactured by Kazu Katsuki), 2.5. Mu.g/mL, and 10. Mu.g/mL, respectively, and the tumor cell killing activity was examined at different antibody concentrations, as shown in FIG. 7, where ". Star" represents P < 0.001.

FIG. 7 is a lactate dehydrogenase assay to examine the in vitro cytotoxic changes of untransfected NK92 cells versus NKG 2D-CAR-transfected NK92 cells on SGC7901 cells when blocked with NKG2D antibody at different concentrations. The result shows that the NKG2D antibody blocking obviously reduces the tumor killing activity of NKG2D-CAR transfected NK92 cells, and the higher the antibody concentration is, the stronger the inhibition effect is. This indicates that the tumor killing activity of NKG2D-CAR-NK92 cells is derived from the NKG2D-CAR constructed by the present invention.

Test example 2

Effect of soluble MICA protein on NKG2D-CAR-NK92 cells:

(one) IFN-gamma release amount of NKG2D-CAR-NK92 cells under MICA protein treatment of different concentrations

Taking NKG2D-CAR-NK92 cells in logarithmic growth phase and untransfected NK92 cells, inoculating the cells in a 96-well plate, and ensuring that each well is 1 multiplied by 10 ⁵ Each cell treated with 0, 0.5, 2.5, 5, 25, 50, 250, 500ng/mL MICA protein, each cell group was set to 3 replicate wells, placed 5% CO ₂ And an incubator at 37 ℃ for 24 hours. Cell supernatants were collected and assayed for IFN-. Gamma.secretion by ELISA kits (Ebotaike, cat. RK 00015) and the results are shown in FIG. 8, where ". X" indicates P < 0.001.

FIG. 8 shows that the release amount of IFN-gamma is measured by ELISA after different concentrations of MICA protein are respectively incubated with untransfected NK92 cells and NKG2D-CAR-NK92 cells for 24h. The ordinate represents IFN-. Gamma.release concentration and the abscissa represents concentration of MICA protein incubated therewith.

As can be seen from the figure, the release amount of IFN-gamma of NKG2D-CAR-NK92 cells is obviously higher than that of NK92 cells when the MICA protein is treated at the same concentration. For the NKG2D-CAR-NK92 cell group, the IFN-gamma release amount showed a tendency of increasing and then decreasing with increasing MICA protein concentration, wherein the IFN-gamma release amount reached the highest peak (about 350 pg/mL) at 50ng/mL MICA protein treatment, while no significant correlation was shown between the IFN-gamma release amount of the NK92 cell group and the MICA protein concentration. It can thus be seen that NKG2D-CAR-NK92 cells have the ability to respond to soluble MICA protein and exhibit dose-dependent effects.

(II) Effect of different treatment modalities of MICA protein on the in vitro cytotoxicity of NKG2D-CAR-NK92 cells

Taking SGC-7901 gastric cancer cells in logarithmic growth phase, inoculating the cells in a 96-well plate, and ensuring that each well is 1 multiplied by 10 ⁴ Each cell was plated with 3 duplicate wells and cultured overnight in an incubator containing 5% CO2 at 37 ℃. Set up 5 cases of effector cells, 0ng/ml MICA protein treated group, 5ng/ml MICA protein treated group, 50ng/ml MICA protein treated group, 5ng/ml MICA protein treated group 24h ahead of time and 50ng/ml MICA protein treated group 24h ahead of time, effective target ratio was 5, 1, 10 respectively, with untransfected NK92 cells as control. The detection method was the same as in test example 1 (one). The results are shown in fig. 9, where "×" indicates P < 0.001.

FIG. 9 is a graph showing the effect of MICA protein treatment on NKG2D-CAR-NK92 cytotoxicity in vitro. The killing effect of the parental NK92 cells and NKG2D-CAR-NK92 cells on SGC-7901 gastric cancer cells is detected by a luciferase method under different MICA protein concentrations and different treatment modes when the effective target ratio is 5. Where the abscissa is MICA concentration and treatment pattern, "(24)" indicates that killing is detected after cells 24 are pretreated with MICA protein, and the others indicate that MICA protein incubation is performed during the killing process.

It can be seen from the figure that the in vitro toxic effect of NKG2D-CAR-NK92 cells on SGC-7901 gastric cancer cells was not significantly changed under different MICA protein treatments, i.e. the killing activity of NKG2D-CAR-NK92 cells on MICA/B high expressing tumor cells was not inhibited by soluble MICA protein. Namely, the cytotoxicity of NKG2D-CAR-NK92 under different MICA protein treatment modes is obviously higher than that of NK92 cells, and under the action of higher-concentration MICA protein, the killing effect is still close to 100%, and the cytotoxicity is extremely obvious. Therefore, the killing activity of NKG2D-CAR-NK92 cells against MICA/B high expressing tumor cells was not inhibited by soluble MICA protein.

The above examples, test examples and results demonstrate that the immune effector cells engineered with the chimeric receptor gene targeting human membrane-bound and soluble NKG2D ligands of the present invention can not only target and efficiently kill a variety of MICA/B positive solid tumor cells, but also can be activated by soluble MICA protein to avoid its inhibitory effects.

Another advantage of the present invention is: compared with the traditional CAR-T/NK technology which targets a single tumor-associated antigen, the NKG2D-CAR constructed based on the natural human NKG2D molecule can target and kill tumor cells by specifically recognizing eight NKG2D ligands, and the NKG2D-CAR provided by the invention has broad-spectrum tumor resistance because most solid tumors at least express one NKG2D ligand.

Yet another advantage of the present invention is that: the NKG2D-CAR-NK92 cells prepared based on the safe and in-vitro amplifiable NK92 cells can break through the limitation of the traditional autologous CAR-T/NK cell therapy, and potentially realize allogeneic adoptive therapy, so that the NKG2D-CAR-NK cell therapy becomes a real off-the-shelf (off-the-shelf) cell immunotherapy, the treatment period is greatly shortened, the treatment cost is reduced, and the clinical popularization and the popularization of the tumor immunocytotherapy are finally realized.

The above-mentioned experimental methods which are not described in detail are all conventional techniques in the art, and can be realized by literature or technical means before the filing date.

Claims

1. A chimeric receptor targeting human membrane-bound and soluble NKG2D ligands characterized by: the chimeric receptor comprises a signal peptide region, an NKG2D ligand binding domain based on a human NKG2D molecule, a hinge region, a transmembrane region and an intracellular signal domain which are sequentially connected in series; the NKG2D ligand binding domain is SEQ ID NO: 1; the hinge region is a monomer type hinge region structure; the haplotype hinge region is a haplotype IgG4 hinge region, and the haplotype IgG4 hinge region is SEQ ID NO: 3; the transmembrane region is a CD28 transmembrane region; the intracellular signaling domain comprises an intracellular signaling domain and an intracellular costimulatory signaling domain; the intracellular signaling domain is CD3 ζ; the costimulatory signaling domains are CD28 and CD137.

2. The chimeric receptor targeting a human membrane-bound and soluble NKG2D ligand according to claim 1, characterized in that: the chimeric receptor targeting the human membrane-bound and soluble NKG2D ligand is as shown in SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.

3. A nucleic acid molecule, characterized in that: the nucleic acid molecule is a nucleotide sequence encoding the chimeric receptor targeting the human membrane-bound and soluble NKG2D ligand of any one of claims 1-2.

4. The nucleic acid molecule of claim 3, wherein: the nucleic acid molecule is shown as SEQ ID NO: 16.

5. A viral vector, characterized by: the viral vector comprising the nucleotide sequence of claim 3 or 4 encoding a chimeric receptor targeting human membrane-bound and soluble NKG2D ligands.

6. A chimeric receptor genetically engineered immune effector cell, characterized by: the immune effector cell comprises the nucleic acid molecule of claim 3 or 4, or the viral vector of claim 5.

7. A method of preparing an immune effector cell genetically modified with a chimeric receptor according to claim 6, wherein the method comprises the steps of: the method comprises the following steps: transferring the virus vector of claim 5 into immune effector cells, and screening to obtain the immune effector cells with chimeric receptor gene modification.

8. Use of a chimeric receptor targeting a human membrane-bound and soluble NKG2D ligand according to any one of claims 1-2 or an immune effector cell genetically modified from a chimeric receptor according to claim 6 for the preparation of a medicament for anti-tumor immunotherapy.

9. An anti-tumor immunotherapy medicament, which is characterized in that: the antitumor immunotherapeutic agent comprising the chimeric receptor gene-modified immune effector cell according to claim 6 as a pharmaceutically effective ingredient.