CN112500497A

CN112500497A - CLTX-NKG2D bispecific chimeric antigen receptor cell and preparation method and application thereof

Info

Publication number: CN112500497A
Application number: CN202011471739.2A
Authority: CN
Inventors: 代红久; 徐慧; 李晓宇; 朱靓婧
Original assignee: Nanjing Kaidi Medical Technology Co ltd
Current assignee: Nanjing Kaidi Medical Technology Co ltd
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2021-03-16
Anticipated expiration: 2040-12-14
Also published as: CN112500497B

Abstract

The invention belongs to the field of chimeric antigen receptor cells, and particularly relates to a coding amino acid sequence of a bispecific chimeric antigen receptor targeting human CLTX-R and NKG2DL, an immune response cell modified by the coding amino acid sequence, and a preparation method and application of the immune response cell. The invention relates to a bispecific chimeric antigen receptor of a protein or a functional variant thereof for bispecific targeting human NKG2DL and a chlorotoxin receptor (CLTX-R), a modified immune response cell thereof, a preparation method and application thereof; the obtained novel functional immune response cell can effectively target and attack various tumors and prepare a preparation for treating malignant tumors. The immune response cell modified by the bispecific chimeric antigen receptor targeting CLTX-R and NKG2DL can enhance the combination with tumor cells and obviously improve the anti-tumor activity.

Description

CLTX-NKG2D bispecific chimeric antigen receptor cell and preparation method and application thereof

Technical Field

The invention belongs to the field of chimeric antigen receptor cells, and relates to an amino acid coding sequence of a bispecific chimeric antigen receptor targeting human CLTX-R and NKG2DL, an immune response cell modified by the amino acid coding sequence, a preparation method of the amino acid coding sequence and an application of the amino acid coding sequence in medicine preparation.

Background

With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer treatment.

Cancer immunotherapy mainly comprises adoptive cell therapy, immunomodulators, tumor vaccines, immune binding site blocking therapy and the like. Among them, in the field of cell therapy, CAR-T therapy has undoubtedly become a star in the dispute of research institutions and pharmaceutical companies as "pursuit".

The principle of the immunotherapy represented by CAR-T (Chimeric Antigen Receptor T-Cell) is mainly that Chimeric Antigen Receptor modification is carried out on T cells extracted from patients by genetic engineering means to form CAR-T cells, and the T cells can specifically recognize tumor surface associated antigens (tumor Cell markers) so as to kill tumors in a targeted manner.

Recent studies have shown that the expression of the NKG2DL protein is an indicator of "stress" in cells, and is rarely or only transiently expressed in healthy tissues, while it is usually expressed at higher levels on the surface of various tumor cells from different sources. The receptor of the NKG2DL protein is NKG2D, and research shows that the NKG2D-NKG2DL system plays an important role in the anti-tumor immunity of the organism, and NKG2D transmits an activation signal through recognizing NKG2DL generated on the surface of a tumor cell and activates the immune system, thereby playing a killing role on the tumor cell. In addition, the research finds that serum of patients with autoimmune diseases contains soluble NKG2DL, and the NKG2D-NKG2DL system plays a certain role in treating autoimmune diseases, resisting inflammation, resisting aging and the like (see Legroup L et al, Frontiers in Immunology, (2019)). Therefore, the expression of NKG2DL is used as a specific change on tumor cells when an organism generates tumors, provides a more accurate target point for the immunotherapy of the tumors, and provides a hint for the development of related new therapies and medicaments.

Chlorotoxin (CLTX) is reported to bind to a variety of membrane proteins, CLTX-R being a receptor for CLTX including MMP-2, CLCN3, ANXA2, etc., specifically membrane associated matrix metalloproteinase 2(MMP2), chloride channel CLCN3, and phospholipoprotein annexin a2(ANXA2) (see Wang et al, sci. trans. med., 2020). CLTX has been shown to bind widely and specifically to gliomas and other neuroectodermal tumors, while exhibiting very low cross-reactivity with non-malignant cells in the brain and other sites. Some embryo-associated tumors, including melanoma, have also been shown to express MMP-2 and bind CLTX. CLTX itself is not cytotoxic to tumor and normal tissues and can be safely used to specifically target tumors. There are studies showing that CLTX-CAR T cells treated in situ xenograft tumor model mice can regress tumors, and that CLTX-CAR T cells exhibit no observable off-target effector activity either on normal cells or after adoptive transfer to mice.

In conclusion, we constructed novel highly specific killing immunoresponsive cells based on the activating target NKG2D of the NKG2D-NKG2DL system and modified using CLTX and its mutant peptide as bispecific chimeric antigen receptor targeting tumor regions for tumor treatment.

Disclosure of Invention

In view of the above problems and/or other problems of the related art, it is an object of the present invention to overcome the problems of poor specificity and low killing efficiency of effector cells in the intra-tumor environment encountered in the existing tumor clinical technologies, and to provide a bispecific targeting binding polypeptide domain targeting human CLTX-R and NKG2DL or a functional variant thereof, a bispecific chimeric antigen receptor targeting human CLTX-R and NKG2DL or a functional variant thereof, a coding nucleotide sequence and an expression vector thereof, an engineered bispecific chimeric antigen receptor modified immunoresponsive cell targeting CLTX-R and NKG2DL, and uses thereof. The immune response cell modified by the engineered bispecific chimeric antigen receptor targeting the CLTX-R and NKG2DL bodies can improve the specific killing efficiency of tumor cells, avoid the problem of safe treatment toxicity caused by off-target, and enhance the combination with the tumor cells, thereby providing a new means for tumor treatment with application prospect.

Technical scheme

A chimeric antigen receptor modified immune cell specifically targeting human CLTX-R and NKG2DL, characterized in that the immune cell contains a chimeric antigen receptor, and the amino acid sequence of the chimeric antigen receptor is as follows:

the amino acid sequence of a leader sequence, the amino acid sequence of an extracellular recognition domain targeted to bind human CLTX-R and NKG2DL, the amino acid sequence of a hinge region, the amino acid sequence of a transmembrane domain and the amino acid sequence of an intracellular signal domain which are sequentially connected from an amino terminal to a carboxyl terminal;

or the amino acid sequence of a leader sequence, the amino acid sequence of an extracellular recognition domain targeted to bind human NKG2DL and CLTX-R, the amino acid sequence of a hinge region, the amino acid sequence of a transmembrane domain, the amino acid sequence of an intracellular signal domain, which are sequentially connected from an amino terminal to a carboxyl terminal;

the extracellular recognition structural domain amino acid sequence of the targeted combined human CLTX-R is as follows: an amino acid sequence shown as SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4 which is combined with CLTX; or a variant which is produced by amino acid modification and has 80-99% homology with the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.

The amino acid sequence of the extracellular recognition domain targeted to bind human NKG2DL is as follows: the amino acid sequence of human NKG2D shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10 and targeted to combine with human NKG2DL protein; or a variant which is produced by amino acid modification and has 80-99% homology with the amino acid sequence shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

Wherein the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;

the immune cell is characterized in that:

the nucleic acid molecule for coding the chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL comprises a nucleotide sequence for coding the guide sequence, a nucleotide sequence for coding the human NKG2D protein receptor targeted to bind human NKG2DL, a nucleotide sequence for coding an extracellular recognition domain targeted to bind human CLTX-R, a nucleotide sequence for coding a hinge region, a nucleotide sequence for coding the transmembrane domain and a nucleotide sequence for coding the intracellular signal domain which are connected in series from 5 'to 3';

or a nucleic acid molecule encoding the chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL, comprising a nucleotide sequence encoding the leader sequence, a nucleotide sequence encoding the extracellular recognition domain targeted to bind human CLTX-R, a nucleotide sequence encoding the human NKG2D protein receptor targeted to bind human NKG2DL, a nucleotide sequence encoding the hinge region, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, which are connected in series from 5 'to 3';

a recombinant vector or expression plasmid specifically targeting a chimeric antigen receptor of human CLTX-R and NKG2DL, characterized in that it comprises said nucleic acid molecule.

The recombinant vector or expression plasmid, characterized in that the recombinant vector or expression plasmid contains a promoter, wherein the promoter comprises the EF1 α long promoter, or the EFs short promoter.

A recombinant virus comprising the nucleotide sequence of said recombinant vector and a viral particle; the virus includes lentivirus, adenovirus, adeno-associated virus or retrovirus.

The immune cell is applied to preparing medicaments for resisting brain cancer, lung cancer, breast cancer, stomach cancer, liver cancer, prostatic cancer, lymph cancer, leukemia, intestinal cancer, cervical cancer, ovarian cancer, bladder cancer, esophagus cancer, kidney cancer or pancreatic cancer.

Detailed description of the invention

In a first aspect, the present application provides a chimeric antigen receptor that specifically targets human CLTX-R and NKG2 DL:

the CLTX amino acid sequence of the targeted binding human CLTX-R is as follows: an amino acid sequence of chlorotoxin that binds CLTX-R as shown in SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4; or a variant which is produced by amino acid modification and has 80-99% homology with the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.

The human NKG2D protein receptor that targets binding to human NKG2DL or a functional variant (analog) thereof comprising a sequence selected from the group consisting of: an amino acid sequence shown as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10, or a functional variant resulting from one or more amino acid modifications; wherein the functional variant modified by amino acid is polypeptide with 80-99% homology with amino acid sequence shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

The inventor continuously performs amino acid sequence design and sequence permutation, combination and screening through creative work, performs random screening test and targeting function verification (for example, tests of constructing a virus vector, further infecting T cells, obtaining modified T cells, detecting in vitro activity of the obtained modified T cells and the like) on sequences of hundreds of CAR molecules, then performs sequence adjustment according to comparison of a plurality of random combination results, and finally screens out sequences with best effect, thereby obtaining the amino acid sequence of the human NKG2D protein receptor which is efficiently targeted and combined with human NKG2DL and functional variants thereof.

The inventor continuously designs amino acid sequences, arranges, combines and screens the sequences by creative labor, analyzes the biological characteristics by software, and selects the high-titer CLTX amino acid sequence with good stability and high binding force and functional variants thereof.

In certain non-limiting embodiments, a chimeric antigen receptor that specifically targets human CLTX-R and NKG2DL may further comprise a linking structure represented by the amino acid sequence (GGGGS) n, where 3. ltoreq. n.ltoreq.8.

In a second aspect, the present application provides a chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL, comprising the amino acid sequence of a leader sequence, sequentially linked from amino terminus to carboxy terminus, the CLTX targeted to bind to human CLTX-R and the human NKG2D protein receptor amino acid sequence targeted to bind to human NKG2DL, the amino acid sequence of the hinge region, the amino acid sequence of the transmembrane domain, the amino acid sequence of the intracellular signaling domain. The amino acid sequence targeted to bind to the extracellular recognition domain of human NKG2DL comprises the human NKG2D protein receptor targeted to bind to human NKG2DL or a functional variant thereof as described in the first aspect of the present application.

The extracellular recognition domain (also referred to as the extracellular domain or simply consisting of the recognition element it contains) comprises a recognition element that specifically binds to a molecule present on the cell surface of a target cell.

In some non-limiting examples, the leader sequence is covalently linked to the 5' end of the extracellular antigen-binding domain.

In some embodiments, the chimeric antigen receptor that specifically targets human CLTX-R and NKG2DL comprises a hinge region.

In some embodiments, the transmembrane domain comprises a transmembrane region.

In some embodiments, the amino acid sequence of the human CD8 polypeptide of the hinge region is selected from the group consisting of the polypeptide set forth in SEQ ID No.11 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence set forth in SEQ ID No. 11.

In some embodiments, the amino acid sequence of human CD8 of the transmembrane region is selected from the group consisting of the polypeptide of SEQ ID No.12 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence of SEQ ID No. 12.

In some embodiments, the amino acid sequence of human CD28 of the transmembrane region is selected from the group consisting of the polypeptide of SEQ ID No.13 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence of SEQ ID No. 13.

In some embodiments, the human 4-1BB intracellular domain is selected from: a polypeptide having an amino acid sequence as shown in SEQ ID No. 14; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 14.

In some embodiments, the human CD28 intracellular domain is selected from the group consisting of: a polypeptide having an amino acid sequence as shown in SEQ ID No. 15; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 15.

In some embodiments, the human OX40 intracellular domain is selected from the group consisting of: a polypeptide having an amino acid sequence as shown in SEQ ID No. 16; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 16.

In some embodiments, the CD3 ζ intracellular domain is selected from: a polypeptide having an amino acid sequence as shown in SEQ ID No. 17; or a functional variant with amino acid modifications. Wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 17.

In some non-limiting embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;

in some non-limiting embodiments, the chimeric antigen receptor that specifically targets human CLTX-R and NKG2DL is recombinantly expressed or expressed from a vector.

In certain non-limiting embodiments, the intracellular domain of the chimeric antigen receptor of the present application that specifically targets human CLTX-R and NKG2DL further comprises at least one costimulatory signaling region comprising at least one costimulatory ligand molecule that provides optimal lymphocyte activation.

In certain non-limiting embodiments, the chimeric antigen receptor that specifically targets human CLTX-R and NKG2DL may further comprise a spacer (spacer) that links the antigen binding domain to the transmembrane domain. The spacer may be sufficiently flexible to allow the antigen binding domain to be oriented in different directions to facilitate antigen recognition. The spacer may be a hinge region from IgG1, or part of the CH2CH3 region and CD3 of an immunoglobulin.

In certain non-limiting embodiments, the intracellular domain of a chimeric antigen receptor that specifically targets human CLTX-R and NKG2DL may comprise a human CD3 ζ polypeptide that can activate or stimulate cells (e.g., T cells of lymphoid lineage).

In certain non-limiting embodiments, the intracellular domain of a Chimeric Antigen Receptor (CAR) that specifically targets human CLTX-R and NKG2DL further comprises at least one costimulatory signaling region comprising at least one costimulatory molecule that provides optimal lymphocyte activation. As used herein, "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to an antigen. The at least one co-stimulatory signaling region may comprise a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide (not based on a protein associated with an immune response), or a combination thereof.

In some embodiments, the costimulatory signaling region of the intracellular domain of the CAR comprises two costimulatory molecules: CD28 and 4-1BB, 4-1BB and OX40 or CD28 and OX 40.

In a third aspect, the present application provides a nucleic acid molecule encoding the chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL according to the second aspect, the nucleic acid molecule comprising a nucleotide sequence encoding a leader sequence, a nucleotide sequence encoding human NKG2D, a nucleotide sequence encoding a transmembrane domain, and a nucleotide sequence encoding an intracellular signaling domain, which are connected in series from 5 'to 3'.

In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a hinge region. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;

polynucleotides encoding extracellular recognition domains that target binding to human NKG2DL can be modified by codon optimization. Codon optimization can alter naturally occurring and recombinant gene sequences to achieve the highest possible level of productivity in any given expression system.

In a fourth aspect, the present application provides a recombinant vector or expression plasmid comprising the chimeric antigen receptor of the second aspect of the present application or the nucleic acid of the third aspect of the present application.

Genetic modification of immune-responsive cells (e.g., T cells, CTL cells, NK cells) can be achieved by transducing substantially homologous cellular compositions with recombinant DNA or RNA constructs. In one embodiment, the vector is a retroviral vector (e.g., a gammaretrovirus or lentivirus) that can introduce a DNA or RNA construct into the genome of a host cell. For example, polynucleotides that specifically target chimeric antigen receptors of human CLTX-R and NKG2DL can be cloned into retroviral vectors and expression can be driven from their endogenous promoters, retroviral long terminal repeats, or from alternative internal promoters.

Non-viral vectors or RNA may also be used. Random chromosomal integration or targeted integration can be used (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and/or regularly clustered interspaced short palindromic repeats (CRISPRs) or transgene expression (e.g., using natural or chemically modified RNAs)).

In some embodiments, the vector is selected from the group consisting of a gamma-retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector.

In an exemplary embodiment, the vector is a gamma-retroviral vector.

In a fifth aspect, the present application provides a recombinant virus capable of expressing a chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL according to the second aspect of the invention and capable of infecting immunoresponsive cells.

In some embodiments, the immunoresponsive cell is a cytotoxic T lymphocyte, an NK cell, an NKT cell, a helper T cell, or a macrophage.

In an exemplary embodiment, the immunoresponsive cell is a cytotoxic T lymphocyte.

In some embodiments, the virus is a lentivirus, adenovirus, adeno-associated virus, retrovirus, or the like.

In an exemplary embodiment, the virus is a lentivirus.

In an exemplary embodiment, the virus is a retrovirus.

In a sixth aspect, the present application provides an isolated modified immunoresponsive cell comprising a chimeric antigen receptor according to the second aspect of the present application transformed with a recombinant vector or expression plasmid according to the third aspect of the present application.

For initial genetic modification of cells to provide the chimeric antigen receptor-modified immunoresponsive cells specifically targeting human CLTX-R and NKG2DL, transduction is typically performed using retroviral vectors, although any other suitable viral vector or non-viral delivery system may be used. Retroviral gene transfer (transduction) has also proven effective for subsequent genetic modification of cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands. Combinations of retroviral vectors and suitable assembly lines are also suitable, wherein the capsid proteins are functional for infecting human cells.

In some embodiments, the immunoresponsive cell further comprises at least one exogenous co-stimulatory ligand.

Possible transduction methods also include direct co-culture of the cells with the producer cells. The transduced viral vectors can be used to express a co-stimulatory ligand (e.g., 4-1BBL) in immune-responsive cells. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression.

In some embodiments, preferably, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof, or more preferably, the co-stimulatory ligand is 4-1 BBL.

In some embodiments, the immunoresponsive cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a Cytotoxic T Lymphocyte (CTL), a regulatory T cell, a macrophage, a human embryonic stem cell, and a pluripotent stem cell that can differentiate into lymphoid cells, preferably a T cell and a Natural Killer (NK) cell, more preferably a T cell.

Multiple T cell subsets isolated from patients can be transduced with vectors for CAR expression.

In an exemplary embodiment, wherein the modified immunoresponsive cell is a CAR-T cell.

Genetically modified central memory T cells can be prepared using the chimeric antigen receptors specifically targeting human CLTX-R and NKG2DL and then cryopreserved.

Seventh aspect the present application provides a method of making an isolated chimeric antigen receptor modified immunoresponsive cell of the sixth aspect of the application, comprising the steps of:

firstly, the nucleic acid molecule of the third aspect is connected into an expression vector by a molecular cloning mode to obtain the expression vector of the chimeric antigen receptor specifically targeting human CLTX-R and NKG2 DL;

then, transfecting 293T cells with the obtained chimeric antigen receptor expression vector specifically targeting human CLTX-R and NKG2DL to obtain virus solution;

finally, the virus liquid is used for infecting immune response cells, and the immune response cells expressing the chimeric antigen receptor modification specifically targeting human CLTX-R and NKG2DL are obtained from the infected cells.

In some non-limiting embodiments, the modified immunoresponsive cells of the invention can be cells of lymphoid lineage. The cells of the lymphoid lineage are selected from B, T and Natural Killer (NK) cells, and provide functions such as antibody production, regulation of cellular immune system, detection of foreign substances in blood, detection of foreign cells in a host, and the like. Non-limiting examples of cells of lymphoid lineage include T cells, Natural Killer (NK) cells, Cytotoxic T Lymphocytes (CTLs), regulatory T cells, macrophages, embryonic stem cells, and pluripotent stem cells (e.g., pluripotent stem cells that can differentiate into lymphoid cells).

In some embodiments, the immunoresponsive cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a Cytotoxic T Lymphocyte (CTL), a regulatory T cell, a macrophage, a human embryonic stem cell, and a pluripotent stem cell that can differentiate into lymphoid cells, preferably a T cell or a Natural Killer (NK) cell.

In some exemplary embodiments, the T cells are lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system.

In some non-limiting embodiments, T cells include, but are not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells (e.g., TEM cells and TEMRA cells), regulatory T cells (also referred to as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, and γ δ T cells.

In some embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, and combinations thereof. In one embodiment, the co-stimulatory ligand is 4-1 BBL.

In a preferred embodiment, the isolated modified immunoresponsive cell is a T cell.

In a preferred embodiment, the isolated modified immunoresponsive cell is a Natural Killer (NK) cell.

In some non-limiting embodiments, the isolated modified immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

In an eighth aspect, the present application provides a pharmaceutical composition comprising an effective amount of the isolated modified immunoresponsive cell of the sixth aspect of the invention and a pharmaceutically acceptable excipient.

The application discloses a pharmaceutical composition comprising an isolated modified immunoresponsive cell expressing said chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL and a pharmaceutically acceptable carrier.

Administration of the pharmaceutical composition may be autologous or non-autologous. For example, immunoresponsive cells expressing the chimeric antigen receptors specifically targeting human CLTX-R and NKG2DL and compositions comprising the same may be obtained from one subject and administered to the same subject or to different compatible subjects. Peripheral blood-derived T cells of the presently disclosed subject matter or progeny thereof (e.g., in vivo, ex vivo, or derived in vitro) can be administered by including catheter administration, intravenous injection, or parenteral administration. When a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising the described immunoresponsive cells specifically targeting the chimeric antigen receptor of human CLTX-R and NKG2 DL) is administered, it is typically formulated in a unit dose injectable form (solution, suspension, emulsion).

The compositions of the present application may be formulations. The immunoresponsive cells expressing the Chimeric Antigen Receptors (CARs) specifically targeting human CLTX-R and NKG2DL disclosed herein and compositions comprising the same may be conveniently provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition can comprise a carrier, which can be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

Various additives may be added that enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers.

According to the present application, any carrier, diluent or additive used must be compatible with the immunoresponsive cells expressing the Chimeric Antigen Receptors (CARs) specifically targeting human CLTX-R and NKG2DL of the presently disclosed subject matter.

If desired, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. The selection of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a time-release form or a liquid-fill form).

In a ninth aspect, the present application provides a kit for the treatment or prevention of a disease comprising an immunoresponsive cell of the sixth aspect of the invention or a nucleic acid of the third aspect of the invention.

In a tenth aspect, the present application provides the use of CLTX amino acids targeted to bind human CLTX-R and human NKG2D protein receptor or functional variant thereof targeted to bind human NKG2DL, the chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL of the second aspect, the recombinant vector or expression plasmid of the fourth aspect, the recombinant virus of the sixth aspect, the isolated modified immunoresponsive cell of the seventh aspect, and the kit of the ninth aspect in a product for the treatment, or prevention of a disease, disorder, or health disorder.

In some embodiments, the disease treated or prevented includes anti-tumor, anti-aging, autoimmune, anti-bacterial, and the like.

Principle of action

The chimeric antigen receptor modified immune response cell specifically targeting human CLTX-R and NKG2DL disclosed by the invention has a killing effect on tumor cells by identifying tumor cell surface antigens CLTX-R and NKG2DL to transmit an activation signal and activate an immune system (as shown in figure 1); the immune response cell modified by the bispecific chimeric antigen receptor targeting CLTX-R and NKG2DL can improve the specific killing efficiency on tumor cells, avoid the problem of safe treatment toxicity caused by off-target, and enhance the combination with the tumor cells, thereby providing a new means for tumor treatment with application prospect.

Advantageous effects

The invention utilizes the chimeric antigen receptor modified T cell technology to prepare the chimeric antigen receptor modified engineering immune cell specifically targeting human CLTX-R and NKG2DL, the preparation method has simple steps, the obtained novel engineering immune cell can specifically identify the tumor cell, can more effectively target and attack the tumor cell, has high killing rate to the tumor, and can be used for preparing anti-tumor products, in particular for preparing the tumor which treats NKG2DL positive and is combined with CLTX; the specifically targeted human CLTX-R and NKG2DL chimeric antigen receptor modified engineering immune cell enhances the combination with tumor cells, thereby obviously improving the killing efficiency of the engineering immune cells to the tumor cells. The invention can be hopefully used for preparing antitumor products, in particular for preparing medicaments for resisting brain cancer, lung cancer, breast cancer, stomach cancer, liver cancer, prostatic cancer, lymph cancer, leukemia, intestinal cancer, cervical cancer, ovarian cancer, bladder cancer, esophagus cancer, kidney cancer or pancreatic cancer, and has good industrial application prospect.

Drawings

FIG. 1 shows schematic diagrams of the working modes of two CAR from KD-618 engineered cells of the invention, FIG. 1A is CLTX-NKG 2D-CAR; FIG. 1B is NKG 2D-CLTX-CAR.

FIG. 2 is a schematic diagram showing the connection sequence of the parts of the chimeric antigen receptor in example 1, in which KD-025 is shown in FIG. 2A and KD-618 is shown in FIG. 2B.

FIG. 3 shows the secondary structure diagram of the protein receptor human NKG2D protein targeting human NKG2DL in the chimeric antigen receptor of the present invention, and FIGS. 3A-F show the extracellular region of the amino acid sequence of human NKG2D, respectively, and the characteristics of the inventors are analyzed by software, that is, based on the crystal structure (PDB number: 4S0U) of the complex formed by NKG2D and its ligand NKG2DL, the amino acids of the key sites affecting affinity are first obtained by alanine scanning, and then the saturation mutation of single-point mutation is performed, and according to the result of the saturation mutation, the secondary structure diagram of 6 sequences with good stability and high ligand binding force is selected by performing the calculation of multiple-point mutation.

FIG. 4 shows the secondary structure diagram of CLTX protein targeting and binding human CLTX-R in the chimeric antigen receptor of the invention, and FIGS. 4A-C are the amino acid sequences of CLTX, and the secondary structure diagram of 3 sequences with good stability and high ligand binding force selected by mutation calculation is analyzed by software for the biological characteristics of interaction between the CLTX and its ligand.

FIG. 5 shows the results of flow cytometry for T cell purity in example 3.

FIG. 6 shows the results of the KD-618CAR-T cell in vitro expression assay in example 4.

FIG. 7 shows the results of expression assays of KD-618 virus transfected 293T cells in example 5. Wherein, FIG. 7A shows the expression of NKG2D, and FIG. 7B shows the result of the experiment for detecting the binding of KD-618 and MMP 2-His.

Fig. 8 is the results of the in vitro killing experiment of KD-618CAR-T cells in example 6, where fig. 8A is the killing of KD-618CAR-T cells on U251 cells, fig. 8B is the killing of KD-618CAR-T cells on U87 cells, fig. 8C is the killing of KD-618CAR-T cells on a549 cells, and fig. 8D is the killing of KD-618CAR-T cells on MCF-7 cells.

FIG. 9 shows the results of the in vitro cytokine IFN- γ release assay for KD-618CAR-T cells in example 7, wherein FIG. 9A shows U251 cells and FIG. 9A shows U87 cells.

FIG. 10 is the safety experiment of KD-618CAR-T in example 8 in mice, wherein FIG. 10A is the picture of HE staining of mouse visceral organs, and FIG. 10B is the survival rate of mice.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "functional variant" is a modification of the parent structure, and refers to a variant of the structure that has the same or similar biological function and properties as the parent, e.g., the same targeted binding function as the parent. By way of non-limiting example, a "functional variant" may be obtained by making one or more conservative substitutions in the parent. Functional variants in this application are modifications of the resulting structure binding to the human NKG2DL target on the basis of the receptor for human NKG2DL (human NKG2D amino acid sequence) and modifications of the resulting structure binding to the human CLTX-R target on the basis of the CLTX amino acid sequence.

The term "analog" refers to a structurally related polypeptide that has the function of the reference polypeptide molecule. In the application, the polypeptide refers to a polypeptide which is related to the amino acid sequence structure of human NKG2D and has a polyamino acid structure combined with human NKG2DL in a targeted way; and a polyamino acid structure that is structurally related to the CLTX amino acid sequence and has targeted binding to human CLTX-R.

The term "amino acid modification" refers to a conservative amino acid modification that does not significantly affect or alter the binding characteristics of a CAR (e.g., extracellular recognition domain) of the present disclosure comprising an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.

The term "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid within the same group.

The term "homology": refers to a high proportion of amino acids or nucleotides that are matched by comparison of a target amino sequence or target nucleotide sequence to a reference sequence. Homology herein can be determined using standard software such as BLAST or FASTA.

The term "Chimeric Antigen Receptor (CAR)": the chimeric antigen receptor includes a leader peptide portion, an extracellular target identification domain, a transmembrane domain, and an intracellular domain.

CARs can both bind antigen and transduce functions of T cell activation that are independent of MHC restriction. Thus, CARs are "universal" immune antigen receptors that can treat a population of patients with antigen-positive tumors regardless of their HLA genotype. Adoptive immunotherapy using T lymphocytes expressing tumor-specific CARs can be a powerful therapeutic strategy for treating cancer.

The term "recognition" refers to selective binding to a target. The term "specifically binds" or "specifically binds to" or "specifically targets" as used herein refers to a polypeptide or fragment thereof that recognizes and binds to a biological molecule of interest (e.g., a polypeptide), but which does not substantially recognize other molecules in a sample, e.g., other molecules in a biological sample that naturally includes a polypeptide of the invention.

The term "specific binding" refers to the association between two molecules (e.g., a ligand and a receptor) characterized by the ability of one molecule (ligand) to bind to another specific molecule (receptor), even in the presence of many other different molecules, i.e., the ability to show preferential binding of one molecule to another in a heterogeneous mixture of molecules. Specific binding of the ligand to the receptor was also demonstrated as follows: in the presence of excess unlabeled ligand, the detectably labeled ligand has reduced binding to the receptor (i.e., a binding competition assay).

The term "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to an antigen.

The term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with appropriate control elements and which can transfer gene sequences into a cell. Thus, the term includes cloning and expression vectors, as well as viral vectors and plasmid vectors.

The term "expression vector" refers to a recombinant nucleic acid sequence, i.e., a recombinant DNA molecule, which contains the desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. The nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers and terminators, as well as polyadenylation signals.

The term "immunoresponsive cell" as used herein refers to a cell that plays a role in an immune response, or a progenitor cell thereof, or a progeny cell thereof.

The term "isolated cell" refers to an immune cell that is separated from the molecules and/or cellular components that naturally accompany the cell.

The term "modulate" as used herein refers to a change, either positively or negatively.

The term "exogenous" as used herein refers to a nucleic acid molecule or polypeptide that is not endogenously present in the cell or is not present at a level sufficient to achieve the functional effect achieved upon overexpression. Thus, the term "exogenous" is intended to include any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous, heterologous, and overexpressed nucleic acid molecules and polypeptides.

The term "exogenous nucleic acid molecule or polypeptide" as used herein refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or in a sample obtained from a cell. The nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

The present invention will be further described with reference to the following examples, but the present invention is not limited to these specific embodiments. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of expression plasmids of chimeric antigen receptors specifically targeting human CLTX-R and NKG2DL

The overall design is as follows:

1. determination of the amino acid sequence of chimeric antigen receptors specifically targeting CLTX-R and NKG2DL

First, the full-length amino acid sequence of human NKG2D (NP-031386.2) was searched from the Genbank database of the national library of medicine (NCBI), and the full-length amino acid sequence of CLTX protein was searched from the Uniprot website (Uniprot: P45639).

Secondly, a chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL was constructed, i.e. the amino acid sequence of the chimeric antigen receptor molecule was determined:

from amino terminal to carboxyl terminal, the amino acid sequence of leader peptide (shown as SEQ ID No. 1), the amino acid sequence of CLTX (shown as SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4), the amino acid sequence of human NKG2D (shown as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10), the amino acid sequence of human CD8 hinge region (shown as SEQ ID No. 11), the amino acid sequence of the human CD8 transmembrane region (shown as SEQ ID No. 12) or the amino acid sequence of the human CD28 transmembrane region (shown as SEQ ID No. 13), the amino acid sequence of the human 4-1BB intracellular domain (shown as SEQ ID No. 14) or the amino acid sequence of the human CD28 intracellular domain (shown as SEQ ID No. 15) or the amino acid sequence of the human OX40 intracellular domain (shown as SEQ ID No. 16) or the combination of the two, and the amino acid sequence of the human CD3 zeta domain (shown as SEQ ID No. 17) are connected in series in sequence (shown as FIG. 2);

A-F shown in FIG. 3 are secondary structure diagrams of 6 sequences which are selected by the inventor through calculation of multi-point mutation and have good stability and high ligand binding force according to the result of saturation mutation, wherein the characteristics of the amino acids are analyzed by using software according to the extracellular region of the amino acid sequence of human NKG2D, namely based on the crystal structure (PDB number: 4S0U) of a compound consisting of NKG2D and ligand NKG2DL, the amino acids of key sites influencing affinity are firstly obtained through alanine scanning, then the saturation mutation of single-point mutation is carried out, and the calculation of the multi-point mutation is carried out according to the result of the saturation mutation;

FIG. 4 is a sequence with good stability and high binding force selected by the inventor through mutation calculation according to the amino acid sequence of CLTX and by analyzing the biological characteristics of the interaction between the CLTX and the ligand thereof by using software; fig. 4A-C are secondary structure diagrams of 3 CLTX sequences with high binding force with CLTX-R.

The codon optimized sequences include: nucleotide sequence encoding a leader sequence, a nucleotide sequence encoding CLTX, a nucleotide sequence encoding a human NKG2D sequence, a nucleotide sequence encoding a human CD8 hinge region, a nucleotide sequence encoding a human CD8 or CD28 transmembrane region, a nucleotide sequence encoding a human 4-1BB or CD28 or OX40 intracellular domain, a nucleotide sequence encoding a CD3 zeta domain, figure 2 shows a schematic representation of the order of the linkage of the parts of the chimeric antigen receptor.

Table 1 shows the binding rate of CLTX and MMP-2, which is determined by the following method, when different CLTX amino acid sequences targeting human CLTX-R and the amino acid sequence of NKG2D (SEQ ID No.5, refer to the best NKG2D sequence in the patent CN 111978412A) are combined, and corresponding virus-infected 293T cells are obtained according to the vector constructed by the following method; the results show that: when the CLTX amino acid sequence (shown as SEQ ID No.2) is combined, the CLTX and MMP-2 combination rate is optimal, and the combination can be used as a subsequent activity verification experiment.

TABLE 1 shows the binding rates of CLTX to MMP-2 in different sequence combinations

NKG2D sequence	CLTX sequence	Binding rate of CLTX to MMP-2
			SEQ ID No.5	SEQ ID No.2	67.3％
SEQ ID No.5	SEQ ID No.3	56.7％
			SEQ ID No.5	SEQ ID No.4	61.8％

2. Construction of chimeric antigen receptor molecular plasmids expressing specific targets to human CLTX-R and NKG2DL

The invention selects a CAR sequence in the step 1, which is named as KD-618 (the amino acid sequence is shown as SEQ ID No.18, and the nucleotide is shown as SEQ ID No. 19) to complete the pharmacodynamic validation test of the subsequent embodiments, and the specific sequence is as follows:

the polypeptide is formed by sequentially connecting an amino acid sequence of a leader peptide (shown as SEQ ID No. 1), an amino acid sequence of CLTX (shown as SEQ ID No.2), an amino acid sequence of human NKG2D (shown as SEQ ID No. 5), an amino acid sequence of a human CD8 hinge region (shown as SEQ ID No. 11), an amino acid sequence of a human CD8 transmembrane region (shown as SEQ ID No. 12), an amino acid sequence of a human 4-1BB intracellular domain (shown as SEQ ID No. 14) and an amino acid sequence of a human CD3 zeta domain (shown as SEQ ID No. 17) in series;

the nucleotide sequence (Nanjing-one biosynthesis) of the chimeric antigen receptor molecule specifically targeting human CLTX-R and NKG2DL is shown in SEQ ID No.19, and is connected to a lentivirus vector lentiGuide-Puro (Addgene, USA) in a molecular cloning manner to construct a full-length CAR sequence expression frame with a single coding frame, and the full-length CAR sequence expression frame is expressed by utilizing an EF1 alpha promoter or an EFS promoter.

The specific operation steps are as follows:

the nucleotide sequence of the chimeric antigen receptor molecule specifically targeting human CLTX-R and NKG2DL is synthesized through whole gene synthesis, the CAR molecule sequence is artificially synthesized through PCR amplification, the CAR molecule sequence is recovered through an Axygen gel recovery kit (Hangzhou Zhang Heng), and homologous recombination and connection are carried out on the CAR molecule sequence and a vector lentiGuide-Puro (Addgene, USA) digested by restriction enzymes SmaI and MluI to form a KD-618 expression vector.

The specific recombination and ligation reaction system and conditions are as follows:

recombination and connection system:

PCR product 5. mu.l recovered from the gel, SmaI and MluI enzyme digestion lentiGuide-Puro plasmid (Addgene, USA) 3. mu.l recovered from the gel; 4X 1402 quick cloning Kit (Nanjing Kinuomei) 5. mu.l; 7 mu l of deionized water; the volume of the ligation reaction system is 20 μ l;

recombinant ligation conditions: and (3) placing the reaction system in a water bath at 50 ℃, reacting for 15min, and then placing on ice for 1 min.

10ul of the recombinant ligation product was transformed with competent Stbl3, using the following procedure.

Mu.l of the ligation product was added to 50. mu.l of competent cells (Stbl3, purchased from Invitrogen, USA) and ice-cooled for 30min at 42 ℃ for 45s for 2min, and then 500. mu.l of non-resistant LB liquid medium was added and shake-cultured at 37 ℃ and 200rpm for 40min, spread on ampicillin-resistant LB solid plates, and left overnight in a 37 ℃ incubator. After single colonies appeared, 5 colonies with proper size were picked, plasmids were extracted and sent to a commercial sequencer for sequencing, and the sequencing results were compared with the fitted nucleotides (i.e., the nucleotides of the chimeric antigen receptors specifically targeting human CLTX-R and NKG2 DL) to confirm that the sequences were completely correct, thus confirming that the plasmids (KD-618 expression plasmids) specifically targeting the chimeric antigen receptors of human CLTX-R and NKG2DL were obtained.

Extraction and purification of chimeric antigen receptor expression plasmids specifically targeting human CLTX-R and NKG2 DL.

The Stbl3 strain containing the KD-618 expression Plasmid was cultured in LB medium in large quantities and subjected to high-purity endotoxin-free extraction using Qiagen Plasmid Midi Kit (Qiagen, Germany) for infection. (see Qiagen plasmid extraction kit instructions for the specific detection procedures).

Example 2: preparation of viral solution of lentivirus vector

The recombinant plasmid (KD-618 expression plasmid) specifically targeting the chimeric antigen receptor of human CLTX-R and NKG2DL obtained in example 1 and the packaging vectors psPAX2 and VSVG were ligated in the same manner as described in 10: 8: 5, using Lipofectamine^TM6000 transfection reagent (purchased from Biyuntian, product model is C0526) co-transfects 293T cells (the specific transfection operation process is shown in the transfection instruction), a complete culture medium (purchased from hyclone, product model is SH30243.01) is replaced after 6 hours of transfection, cell supernatants rich in lentiviral particles are collected respectively after 48 hours and 72 hours of culture, and virus supernatants are concentrated by ultracentrifugation to obtain a virus solution (hereinafter abbreviated as KD-618 virus solution) of the lentiviral vector carrying the chimeric antigen receptor specifically targeting human CLTX-R and NKG2 DL.

Example 3: isolated culture of T cells

Fresh peripheral blood of a healthy donor is taken,separating fresh peripheral blood mononuclear cells by density gradient centrifugation; then, paramagnetic beads (purchased from Invitrogen, USA, and the product information is shown in the specification) coupled with anti-CD3 antibody and anti-CD 28 antibody are used

Human T-Activator CD3/CD28) to enrich CD3+ T cells, specifically, peripheral blood mononuclear cells are diluted to a concentration of (10-30). times.10⁶The individual cells/ml were mixed with Magnetic beads at a ratio of 3:1, incubated at room temperature for 2-3 hours, and CD3+ T cells were enriched using a Magnetic particle collector (MPC, available from Invitrogen, USA). The enriched CD3+ T cells were finally resuspended in culture medium (purchased from Life Technologies, USA, under the product information OpTsizer^TMT-Cell Expansion SFM), adjusted to a Cell concentration of 1X 10⁶One/ml, finally 5% CO at 37 ℃₂The culture was carried out in an incubator for 2 days, and the results of the measurement are shown in FIG. 5.

Example 4: preparation of chimeric antigen receptor T cells (KD-618 CAR-T) specifically targeting human CLTX-R and NKG2DL

First, the CD3+ T cells obtained in example 3 were seeded in a 24-well plate at a seeding concentration of 1 to 10X 10⁵Cell/ml at 37 ℃ with 5% CO₂The culture is carried out in the environment for about 24 hours (the culture time depends on the specific practice, and generally, the cell confluence rate is ensured to be between 50 and 70 percent when the virus liquid is infected). Then, the virus solutions of KD-618 collected in example 2 were taken, and added to a cell culture flask together according to an MOI of 10 to 40, and the flask was sealed, placed in a flat angle centrifuge, centrifuged at a low speed (500g to 1000g/min) for 1 hour, and then placed in an incubator to be cultured at 37 ℃. Chimeric antigen receptor T cells (KD-618 CAR-T) specifically targeting human CLTX-R and NKG2DL are obtained 48 hours after infection, and a next functional experiment can be carried out, as shown in figure 1, the working diagram of the engineering cells is shown, namely the engineering cells transmit activation signals and activate an immune system by recognizing CLTX-R and NKG2DL generated on the surface of tumor cells, so that the tumor cells are killed;

detection of CAR protein expression using flow cytometry analysis:

the cells were centrifuged, washed twice with PBS and resuspended in FACS fluid (PBS with 0.1% sodium azide and 0.4% BSA); Anti-CD314 Antibody (APC-Anti-human CD314(NKG2D), biolegend, 320808) was added to the cell suspension, incubated at 4 ℃ for 1h, and Isotype control group (APC Mouse IgG1, kappa Isotype Ctrl Antibody, biolegend, 400120) was set; after washing the cells twice, 200. mu.L of FACS solution was added to resuspend the cells; BD facscan II was used to obtain stained cells and FlowJo was used to analyze the results. As shown in figure 6, the control group was T cells infected with empty viral fluid, and expression of the CAR molecule was barely detectable; the experimental group is T cells infected with KD-618 virus liquid, and the expression rate of NKG2D is 61.8%;

example 5: KD-618 AND MMP2 BINDING DETECTION

The specific operation steps are as follows:

first, 293T cells (ATCC, USA) were inoculated into a 24-well plate at a concentration of 1-10X 10⁵Cell/ml at 37 ℃ with 5% CO₂The culture is carried out in the environment for about 24 hours (the culture time depends on the specific practice, and generally, the cell confluence rate is ensured to be between 50 and 70 percent when the virus liquid is infected). Then, the KD-618 virus solution collected in example 2 was collected and 293T cells were infected at an MOI of 10 to 40.

Culturing for 48h, collecting cells, and centrifuging at 4 deg.C for 5min at 300 g; washed twice with PBS and resuspended in FACS fluid (PBS with 0.1% sodium azide and 0.4% BSA); Anti-CD314 antibodies (APC-Anti-human CD314(NKG2D), biolegend, 320808) were added to the cell suspension, incubated at 4 ℃ for 1h, and Isotype controls (APC Mouse IgG1, kappa Isotype Ctrl Antibody, biolegend, 400120) were set. Adding MMP2 primary antibody (Recombinant Human MMP-2, C377) into the cell suspension, incubating for 4h at 4 ℃, and setting a control group; after washing the cells twice, 200. mu.L of FACS solution was added to resuspend the cells; adding a secondary Antibody (Human IgG Fc PE-conjugated Antibody, 130-120-787) into the cell suspension, and incubating for 1h at 4 ℃; after washing the cells twice, 200. mu.L of FACS solution was added to resuspend the cells; BD facscan II was used to obtain stained cells and FlowJo was used to analyze the results. As shown in FIG. 7A, the expression rate of NKG2D of 293T infected by KD-618 virus is 97.8%; as shown in fig. 7B, the control group was 293T cells not infected with viral fluid, and hardly bound to MMP 2; the binding rate of 293T cells infected with KD-618 virus liquid and MMP2 in the experimental group is 67.3%.

Example 6: KD-618CAR-T cell in vitro killing experiment

Setting an experimental group and 3 control groups corresponding to each target cell line, wherein the experimental group is added with the cell suspension of the CAR-T cells specifically targeting human CLTX-R and NKG2DL obtained in example 4; the blank control group was supplemented with T cells not infected with virus (i.e., CD3+ T cells obtained in example 3); the KD-019 control group is added with CAR-T cells targeting CD19 (the preparation method is referred to CN 109803983B); the KD-025 control group was supplemented with CAR-T cells targeting human NKG2DL (see CN109803983B for preparation method).

First, KD-019 CAR-T, KD-025CAR-T, KD-618CAR-T cells were prepared by infection as described in example 4, cultured for 72 hours after infection and then subjected to killing inoculation, each target cell line was subjected to CSFE fluorescent staining, and then the target cells were counted under a fluorescent microscope to adjust the cell density to about 2X 10⁶cells/ml, 20. mu.l/well, target cells were seeded in 96-well culture plates; adding T, KD-019 CAR-T, KD-025CAR-T, KD-618CAR-T cells into effector cells according to an effective target ratio of 0.25:1, 1:1 and 4: 1; then placing the cells in an incubator at 37 ℃ for culturing for 24 h; finally, the killing of the target cell line by the KD-618CAR-T cells was evaluated using 7-AAD/CFSE cytotoxicity test kit (purchased from Biovision, Inc., cat # K315-100) according to the instructions for the kit. As shown in FIG. 8, FIGS. 8A and 8B are the killing results of U251 and U87 cells, respectively, FIGS. 8C and 8D are the killing results of A549 and MCF-7 cells, respectively, and KD-025CAR-T and KD-618CAR-T have a certain killing effect on U251; KD-618CAR-T has higher killing effect on U251, U87, A549 and MCF-7 cells than KD-025CAR-T, which shows that CLTX expressed by KD-618CAR-T increases the combination with tumor cells and improves the killing effect.

Example 7: KD-618CAR-T cell in vitro cytokine release experiment

Experiment set up one experimental group and 2 control groups, wherein, the experimental group adds the cell suspension of the CAR-T cell specifically targeting human CLTX-R and NKG2DL obtained in example 4; the blank control group was supplemented with T cells not infected with virus (i.e., CD3+ T cells obtained in example 3); the KD-019 control group was supplemented with unrelated CAR-T cells targeting CD 19.

First, KD-019 CAR-T, KD-618CAR-T cells were prepared by infection as described in example 4, and after infection and further culture for 72 hours, killer inoculation was carried out with the target cell line adjusted to a cell density of about 1.5X 10⁶cells/ml, 20. mu.l/well, target cells were seeded in 96-well culture plates; according to the effective target ratio of 5:1 adding effector cells T, KD-019 CAR-T, KD-618CAR-T cells; then placing the cells in an incubator at 37 ℃ for culturing for 24 h; finally, the release of killer cytokines from KD-618CAR-T cells on the above-mentioned target cell line was evaluated using a Human IFN-. gamma.ELISA Kit II Kit (purchased from BD Co., Ltd., cat # 550612) according to the instructions for the Kit. As shown in fig. 9, after KD-618CAR-T cells were co-cultured with tumor cell U251, and KD-618CAR-T cells were co-cultured with tumor cell U87, INF- γ was significantly increased compared to the control group, indicating that KD-618CAR-T had a very good anti-tumor effect.

Example 8: safety experiments of KD-618CAR-T in mice

First, KD-618CAR-T cells were prepared by infection as in example 4, and the effect of KD-618CAR-T cells on the major organs and survival cycle of mice is shown in fig. 10. As can be seen from fig. 10, the KD-618CAR-T cells did not cause inflammation, edema and necrosis of major organs such as heart, liver, lung and kidney of mice (fig. 10A), and did not have any negative effect on the life cycle of mice (fig. 10B).

In a word, the virus vector specifically targeting the chimeric antigen receptor of human CLTX-R and NKG2DL and the engineering immune cell modified by the virus vector can be applied to treating various tumors, including brain cancer, lung cancer, breast cancer, gastric cancer, liver cancer, prostate cancer, lymph cancer, leukemia, intestinal cancer, cervical cancer, ovarian cancer, bladder cancer, esophageal cancer, renal cancer or pancreatic cancer.

Sequence listing

<110> Nanjing Kaidi medical technology Co., Ltd

<120> CLTX-NKG2D bispecific chimeric antigen receptor cell and preparation method and application thereof

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<213> leader sequence (2 Ambystoma latex x Ambystoma jeffersonanium)

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 2

<211> 36

<212> PRT

<213> CLTX sequence 1(2 Ambystoma laterale x Ambystoma jeffersonoanum)

<400> 2

Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys

1 5 10 15

Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln

20 25 30

Cys Leu Cys Arg

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<210> 3

<211> 36

<212> PRT

<213> CLTX sequence 2(2 Ambystoma laterale x Ambystoma jeffersonanum)

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Met Cys Met Pro Cys Phe Thr Thr Asp His Lys Met Ala Lys Lys Cys

1 5 10 15

Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Gln

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Cys Leu Cys Arg

35

<210> 4

<211> 36

<212> PRT

<213> CLTX sequence 3(2 Ambystoma laterale x Ambystoma jeffersonanum)

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Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys

1 5 10 15

Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Tyr Gly Pro Arg

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Cys Leu Cys Arg

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<211> 136

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<213> NKG2D sequence 1(2 Ambystoma laterale x Ambystoma jeffersonia)

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Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

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Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

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Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

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Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

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Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

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<210> 6

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<213> NKG2D sequence 2(2 Ambystoma laterale x Ambystoma jeffersonia)

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Ser Leu Phe Asn Lys Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

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Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

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Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

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<210> 7

<211> 136

<212> PRT

<213> NKG2D sequence 3(2 Ambystoma laterale x Ambystoma jeffersonia)

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Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

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Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

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Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Lys Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Lys Arg Thr Val

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<210> 8

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<212> PRT

<213> NKG2D sequence 4(2 Ambystoma laterale x Ambystoma jeffersonia)

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Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

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Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Lys

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 9

<211> 136

<212> PRT

<213> NKG2D sequence 5(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 9

Ser Leu Phe Asn Lys Glu Val Lys Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 10

<211> 136

<212> PRT

<213> NKG2D sequence 6(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 10

Ser Leu Phe Asn Lys Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Lys Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 11

<211> 45

<212> PRT

<213> CD8 hinge region (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 11

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 12

<211> 24

<212> PRT

<213> CD8 transmembrane region (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 12

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 13

<211> 27

<212> PRT

<213> CD28 transmembrane region (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 13

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 14

<211> 42

<212> PRT

<213> 4-1BB intracellular Domain (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 14

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 15

<211> 41

<212> PRT

<213> CD28 Domain (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 15

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 16

<211> 36

<212> PRT

<213> OX40 Domain (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 16

Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly

1 5 10 15

Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr

20 25 30

Leu Ala Lys Ile

35

<210> 17

<211> 112

<212> PRT

<213> CD3 zeta Domain (2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 17

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 18

<211> 436

<212> PRT

<213> KD-618CAR molecule (2 Ambystoma laterale x Ambystoma jeffersonoanum)

<400> 18

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Met Cys Met Pro Cys Phe Thr Thr Asp His Gln

20 25 30

Met Ala Arg Lys Cys Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys

35 40 45

Cys Tyr Gly Pro Gln Cys Leu Cys Arg Gly Gly Gly Gly Ser Gly Gly

50 55 60

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Leu Phe

65 70 75 80

Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys

85 90 95

Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp

100 105 110

Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn

115 120 125

Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys

130 135 140

Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn

145 150 155 160

Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu

165 170 175

Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser

180 185 190

Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys

195 200 205

Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

210 215 220

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

225 230 235 240

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

245 250 255

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

260 265 270

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

275 280 285

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

290 295 300

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

305 310 315 320

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

325 330 335

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

340 345 350

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

355 360 365

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

370 375 380

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

385 390 395 400

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

405 410 415

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

420 425 430

Leu Pro Pro Arg

435

<210> 19

<211> 1308

<212> DNA

<213> KD-618CAR molecule nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonanium)

<400> 19

atggctctcc cggtaacggc gcttctgttg cctttggcac tcctgcttca cgctgccagg 60

cctatgtgta tgccgtgctt caccactgat caccagatgg cgagaaagtg tgacgattgc 120

tgcggcggca aaggcagagg caaatgctac ggccctcagt gcctttgtcg cggaggtggg 180

ggttcaggag gaggaggtag tgggggaggt ggcagcgggg gtggcggaag cagtcttttt 240

aaccaagaag tccaaatacc gctcaccgag tcttactgtg gcccatgtcc aaaaaactgg 300

atctgctaca aaaataactg ttatcagttc tttgatgaga gtaagaactg gtacgaatct 360

caggcttctt gcatgagcca aaatgcttca ttgctcaaag tttatagtaa ggaggaccag 420

gacctcctca aattggtaaa atcatatcac tggatgggct tggtgcatat acctacaaat 480

gggagttggc agtgggagga cggtagcatc ctgagtccaa acctgcttac cataattgag 540

atgcaaaaag gcgactgtgc tctgtacgcc agtagtttta aggggtatat agaaaactgc 600

agtacgccaa atacctatat ctgtatgcag cggacggtaa cgactacgcc tgctcctcgc 660

cccccgacac cggctcccac catcgcgtct caacctcttt ccttgcggcc cgaagcgtgt 720

cgaccagcag ccggaggtgc cgtacatacg agggggttgg acttcgcgtg cgatatttac 780

atttgggcac cgctcgccgg cacttgcggc gtactcttgc ttagtctcgt cattactctc 840

tactgcaaga gaggaaggaa aaaactgctt tatattttta aacagccctt catgcggccc 900

gtacagacta ctcaggaaga ggatggatgt tcttgcaggt tcccggagga ggaagaaggc 960

ggatgcgagc tcagggtcaa attttcacga tccgcggatg ctcctgcata tcaacaggga 1020

cagaatcagc tctacaatga gttgaatctg gggcggcgag aggaatacga cgtgctggat 1080

aagaggagag gacgggaccc tgaaatggga ggcaagccaa ggcgaaaaaa cccccaggag 1140

ggcctgtata atgagcttca gaaggataaa atggcggaag cttacagtga aataggtatg 1200

aaaggagaac gccgaagggg taaagggcac gacggtctgt atcaaggctt gtctaccgca 1260

acaaaagaca cttatgacgc actgcacatg caagctctgc ccccgcga 1308

Claims

1. A chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL, characterized by:

the chimeric antigen receptor comprises:

amino acid sequence of extracellular recognition domain targeting human CLTX-R protein: the amino acid sequence of CLTX targeted to be combined with CLTX-R protein shown as SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4; or a variant modified by an amino acid having 80 to 99% homology with the amino acid sequence;

amino acid sequence of extracellular recognition domain targeting human NKG2DL protein: the amino acid sequence of human NKG2D shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10 and targeted to combine with human NKG2DL protein; or a variant modified by an amino acid having 80 to 99% homology with the above amino acid sequence.

2. The chimeric antigen receptor according to claim 1, wherein: the amino acid sequence of the chimeric antigen receptor is as follows:

the amino acid sequence of a guide sequence, the amino acid sequence of an extracellular recognition domain targeted to be combined with human CLTX-R protein, the amino acid sequence of an extracellular recognition domain targeted to be combined with human NKG2DL protein, the amino acid sequence of a hinge region, the amino acid sequence of a transmembrane domain and the amino acid sequence of an intracellular signal domain which are sequentially connected from an amino terminal to a carboxyl terminal; or the amino acid sequence of a guide sequence, the extracellular recognition domain amino acid sequence targeted to be combined with human NKG2DL protein, the extracellular recognition domain amino acid sequence targeted to be combined with human CLTX-R, the hinge region amino acid sequence, the transmembrane domain amino acid sequence and the intracellular signal domain amino acid sequence which are sequentially connected from an amino terminal to a carboxyl terminal;

the transmembrane domain comprises a CD8 transmembrane domain, or a CD28 transmembrane domain, or a 4-1BB transmembrane domain, or an OX40 transmembrane domain, or an ICOS transmembrane domain;

the immunoreceptor tyrosine activation motif comprises an intracellular signaling domain of the CD3 zeta chain or an fcsri gamma intracellular signaling structure;

the costimulatory signaling domain comprises a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, or an ICOS intracellular signaling domain;

the costimulatory signaling domain comprises a CD28 and 4-1BB intracellular signaling domain, or a 4-1BB and OX40 intracellular signaling domain, or a CD28 and OX40 intracellular signaling domain.

3. Nucleic acid molecule encoding the chimeric antigen receptor specifically targeting human NKG2DL and CLTX-R according to claim 2, characterized in that:

comprises a nucleotide sequence which is connected in series from 5 'to 3' and is used for coding the guide sequence, a nucleotide sequence which is used for coding a targeting CLTX-R, a nucleotide sequence which is used for coding a targeting human NKG2DL, a nucleotide sequence which is used for coding a hinge region, a nucleotide sequence which is used for coding the transmembrane domain and a nucleotide sequence which is used for coding the intracellular signal domain;

or comprises a nucleotide sequence coding the guide sequence, a nucleotide sequence coding the targeting human NKG2DL, a nucleotide sequence coding the targeting CLTX-R, a nucleotide sequence coding the hinge region, a nucleotide sequence coding the transmembrane domain and a nucleotide sequence coding the intracellular signal domain which are connected in series from 5 'to 3'.

4. A recombinant vector specifically targeting a chimeric antigen receptor of human CLTX-R and NKG2DL, characterized in that it comprises a nucleic acid molecule as claimed in claim 3.

5. A recombinant virus comprising the recombinant vector of claim 4 and a viral particle; the virus includes lentivirus, adenovirus, adeno-associated virus or retrovirus.

6. A functionalized immune-responsive cell obtained by infecting an immune effector cell with the recombinant virus of claim 5; the immune effector cells include cytotoxic T lymphocytes, NK cells, NKT cells, helper T cells, or macrophages.

7. A biological product characterized by comprising the amino acid sequence of claim 2; or comprising the nucleic acid molecule of claim 3; or a recombinant vector according to claim 4; or a recombinant virus according to claim 5; or comprising an immune cell according to claim 6.

8. Use of a biological product according to claim 7 in the manufacture of a medicament for the treatment of cancer, autoimmune diseases or viral bacterial infections.

9. The use of claim 8, wherein the cancer is brain, lung, breast, stomach, liver, prostate, lymph, leukemia, bowel, cervix, ovary, bladder, esophagus, kidney, or pancreas.