CN111978412B - Armed targeting TGF-beta specific chimeric antigen receptor cell and preparation method and application thereof - Google Patents

Abstract

The invention relates to an encoding amino acid sequence of a chimeric antigen receptor which is armed with a TGF-beta-targeting specificity and targets human NKG2DL, an immune response cell modified by the same, and a preparation method and application of the immune response cell. The invention relates to a method for modifying immune response cells by arming a recombinant vector of a TGF-beta-targeting specificity-targeting chimeric antigen receptor of human NKG2 DL; the obtained novel functional immune response cell can effectively target and attack various tumors, and the prepared preparation for treating malignant tumors. The engineered immune cell modified by the chimeric antigen receptor of the armed targeting TGF-beta specifically targeting human NKG2DL inhibits immunosuppression in a tumor microenvironment and prevents immune escape of tumor cells, and can obviously improve the killing efficiency of the tumor cells.

Description

Armed targeting TGF-beta specific chimeric antigen receptor cell and preparation method and application thereof

Technical Field

The invention belongs to the technical field of immunotherapy biomedicine, and relates to an amino acid coding sequence of a chimeric antigen receptor of a armed targeting TGF-beta specificity targeting human 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".

CAR-T (Chimeric Antigen Receptor T-Cell) immunotherapy mainly adopts the principle that the genetic modification of Chimeric Antigen Receptor is carried out by taking patient self T cells, and the CAR-T cells can specifically recognize tumor-associated antigens (tumor Cell markers), so that the T cells are guided to target tumors. Compared with conventional immune cells, the targeting, killing activity and durability of the CAR-T cells are higher, and the CAR-T cells can overcome local immunosuppression microenvironment of tumors and break immune tolerance states of hosts. This therapy has significant efficacy in the treatment of acute leukemia and non-hodgkin's lymphoma, and is considered to be one of the most promising modes of tumor treatment.

However, 90% of cancers are solid tumors, and a greater variety of solid tumors and a greater number of tumor surface-specific target antigens remain to be further identified. CAR-T immunotherapy for the treatment of solid tumors faces many challenges, including that CAR-T cells have very high specificity requirements for expression on tumor cells, otherwise they are prone to cause sustained T cell activation to kill normal cells, or release of large amounts of cytokines causing serious side effects. Among them, the tumor cells in the tumor microenvironment secrete a large amount of immunosuppressive factors TGF-beta, and the infiltrating immune cells are domesticated to inhibit the anti-tumor effect of the tumor cells, so that the Chimeric Antigen Receptor (CAR) T cell therapy aiming at the solid tumor cannot achieve the optimal curative effect.

Therefore, the key to the application of CAR-T technology is to identify at least one suitable tumor-targeting antigen that is highly expressed on the surface of tumor cells and is not or is poorly expressed on the surface of normal cells. Currently, most CAR developments involve only a single target, which may lead to selective survival and proliferation of antigen-negative tumors due to tumor heterogeneity. Therefore, it is crucial to design novel CAR-T cells that can recognize and target multiple sites on solid tumors rather than a single site.

In recent years, 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 often 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.

Recent studies have found that TGF-beta RII ligand TGF-beta plays a very important role in both tumorigenesis and tumor development. TGF-beta has three subtypes, TGF-beta 1, TGF-beta 2 and TGF-beta 3, and all three subtypes can bind to the same receptor on the cell surface. TGF-. beta.RII has a high affinity for TGF-. beta. 1/3 and a low affinity for TGF-. beta.2. TGF-. beta.s inhibit tumor growth in the early stages of tumors and convert to a tumor-promoting factor in the later stages. In advanced cancer, most tumor cells secrete TGF-beta to form an immunosuppressive microenvironment, and once TGF-beta levels are increased, immature T cells can be blocked from differentiating into Th1 cells, transformation of the T cells into a Treg subpopulation is promoted, and antigen presentation function of dendritic cells and ATP-coupled respiration of T cells are inhibited, thereby causing immune escape of tumor cells (see Sarah Dimeloe et al, Science Signaling, (2019)). With the development of tumor, TGF-beta signal in tumor Epithelial cells promotes Epithelial Mesenchymal Transition (EMT) by inducing the expression of transcription factors such as Snail1/2, ZEB1/2 and HMGA2, and the process converts Epithelial cells with polarity into Mesenchymal cells with activity, thereby acquiring the capacity of invasion and migration, and the conversion process is very important in the generation, the deterioration and the metastasis of tumor. Tumor metastasis is a very important cause of death in cancer patients, with up to 90% of patients with solid tumors dying from tumor metastasis. Therefore, TGF-beta can be used as an effective drug target for treating solid tumors.

In conclusion, the engineering immune response cell modified by the specific chimeric antigen receptor with double targets of NKG2DL and TGF-beta has a great application prospect. The engineering cells transmit activation signals and activate an immune system by recognizing NKG2DL generated on the surface of the tumor cells, so that the tumor cells are killed; meanwhile, various TGF-beta subtypes such as TGF-beta 1, TGF-beta 2 and TGF-beta 3 cytokines in the tumor microenvironment are identified, immunosuppression in the tumor microenvironment is inhibited, and immune escape of tumor cells is prevented, so that the killing efficiency of the tumor cells can be obviously improved. Meanwhile, the engineered cell also has certain functions of treating autoimmune diseases, resisting inflammation, resisting aging and the like, and finally achieves good clinical treatment effect.

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 tumor environment encountered in the existing tumor clinical technologies, and to provide an immunoresponsive cell modified by a chimeric antigen receptor specifically targeting human NKG2DL, a gene and a recombinant expression vector thereof, an engineered specifically targeting TGF- β, and a chimeric antigen receptor specifically targeting TGF- β 1, TGF- β 2, TGF- β 3 cytokines and human NKG2DL in the tumor microenvironment, and an application thereof. The immune response cell modified by the TGF-beta in the targeted tumor microenvironment and the chimeric antigen receptor specifically targeted to the human NKG2DL can inhibit immunosuppression in the tumor microenvironment and prevent immune escape of tumor cells, and can obviously improve the killing efficiency of the tumor cells, thereby providing a new means for tumor treatment with application prospect. Meanwhile, the engineered cell also has certain functions of treating autoimmune diseases, resisting inflammation, resisting aging and the like.

Technical scheme

An immune cell armed with a chimeric antigen receptor modification targeting TGF- β specifically to human NKG2DL, wherein said immune cell comprises a chimeric antigen receptor having the amino acid sequence:

an amino acid sequence of a leader sequence, an extracellular recognition domain amino acid sequence targeted to bind human NKG2DL, a hinge region amino acid sequence, a transmembrane domain amino acid sequence, an intracellular signaling domain amino acid sequence, and an amino acid sequence armed with a targeting TGF-beta module, sequentially linked from amino terminus to carboxy terminus;

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

wherein the amino acid sequence of the extracellular recognition domain targeted to bind human NKG2DL is: the amino acid sequence of human NKG2D shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7 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.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7.

The amino acid sequence of the armed targeting TGF-beta is as follows: a TGF-beta targeting amino acid sequence shown as SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20 or SEQ ID No. 21; or variants which are produced by amino acid modification and have 80-99% homology with the amino acid sequences shown in SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20 or SEQ ID No. 21. The immune cell is characterized in that:

the nucleic acid molecule for encoding the chimeric antigen receptor which is armed with the TGF-beta targeting specificity and targets human NKG2DL comprises a nucleotide sequence which is connected in series from 5 'to 3' and encodes the guide sequence, a nucleotide sequence which encodes the human NKG2D protein receptor which is targeted to bind human NKG2DL, a nucleotide sequence which encodes a hinge region, a nucleotide sequence which encodes the transmembrane domain, a nucleotide sequence which encodes the intracellular signal domain and a nucleotide sequence which encodes the armed TGF-beta targeting;

a recombinant vector or expression plasmid armed with a chimeric antigen receptor targeting TGF- β specifically to human NKG2DL, comprising 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 medicines for resisting liver cancer, glioma, acute leukemia, pancreatic cancer, gastric cancer and lung cancer.

Chimeric antigen receptor-modified immune cells that are specifically targeted to human NKG2DL that are armed with targeting TGF- β,

detailed description of the invention

In a first aspect, the present application provides a chimeric antigen receptor specifically targeting human NKG2DL armed with TGF- β, said chimeric antigen receptor targeting a human NKG2D protein receptor that binds 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.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7, 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.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7.

The amino acid sequence of the armed targeting TGF-beta is as follows: a TGF-beta targeting amino acid sequence shown as SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20 or SEQ ID No. 21; or variants which are produced by amino acid modification and have 80-99% homology with the amino acid sequences shown in SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20 or SEQ ID No. 21. 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 amino acid sequence which has good stability and high ligand binding force and is combined with human TGF-beta RII ligand in a high-valence targeting way and functional variants thereof.

In a second aspect, the present application provides a chimeric antigen receptor specifically targeting human NKG2DL armed with TGF- β comprising an amino acid sequence of a leader sequence joined sequentially from amino terminus to carboxy terminus, an amino acid sequence of the human NKG2D protein receptor targeted to bind human NKG2DL, an amino acid sequence of the hinge region, an amino acid sequence of the transmembrane domain, an amino acid sequence of the intracellular signaling domain, an amino acid sequence of the TGF- β armed with targeting. 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 by the recognition element it contains) comprises a recognition element that specifically binds to a molecule present on the cell surface of the 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 specifically targeting human NKG2DL armed with TGF- β 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 shown in SEQ ID No.8 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 shown in SEQ ID No. 8.

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.9 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. 9.

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.10 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. 10.

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. 11; 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. 11.

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. 12; 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. 12.

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. 13; 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. 13.

In some embodiments, the CD3 δ intracellular domain is selected from the group consisting of: a polypeptide having an amino acid sequence as shown in SEQ ID No. 14; 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. 14.

In some embodiments, the amino acid sequence that arms the TGF- β module comprises: the nucleotide sequence of Furin-2A as shown in SEQ ID No.15, wherein the amino acid sequence targeted to bind to human TGF-beta comprises a protein receptor of human TGF-beta or a functional variant thereof according to the first aspect of the application.

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, chimeric antigen receptors specifically targeting human NKG2DL armed with TGF- β targeting are recombinantly expressed or expressed from vectors.

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

In some non-limiting embodiments, the intracellular domain of a chimeric antigen receptor specifically targeting human NKG2DL armed with TGF- β comprises a costimulatory signaling region comprising two costimulatory molecules: CD28 and 4-1BB, 4-1BB and OX40 or CD28 and OX 40.

In a non-limiting embodiment, the chimeric antigen receptor intracellular domain specifically targeting human NKG2DL armed with TGF- β of the present application comprises a 4-1BB polypeptide.

In certain non-limiting embodiments, chimeric antigen receptors that are specifically targeted to human NKG2DL armed with TGF- β 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 specifically targeting human NKG2DL armed with TGF- β may comprise a human CD3 δ polypeptide that can activate or stimulate cells (e.g., cells of the lymphoid lineage, e.g., T cells).

In certain non-limiting embodiments, the intracellular domain of a Chimeric Antigen Receptor (CAR) specifically targeting human NKG2DL armed with TGF- β also 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 non-limiting embodiment, the 4-1BB polypeptide has the amino acid sequence of the contiguous portion of SEQ ID NO. 11.

In a third aspect, the present application provides a nucleic acid molecule encoding the TGF- β armed targeting chimeric antigen receptor specifically targeting human NKG2DL of the second aspect, said nucleic acid molecule comprising a nucleotide sequence encoding a leader sequence, a nucleotide sequence encoding human NKG2D, a nucleotide sequence encoding a transmembrane domain, a nucleotide sequence encoding an intracellular signaling domain, and a nucleotide sequence armed with TGF- β, connected in series, in that order 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, a polynucleotide armed with a chimeric antigen receptor specifically targeting human NKG2DL targeting TGF- β can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, a retroviral long terminal repeat or from an alternative internal promoter.

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 NKG2DL armed with TGF- β 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 armed with TGF- β specifically targeting human 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. Transduction viral vectors can be used to express co-stimulatory ligands (e.g., 4-1BBL and IL-12) 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 receptor specifically targeting human NKG2DL armed with TGF- β 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 which is equipped with the TGF-beta targeted chimeric antigen receptor specifically targeting human NKG2 DL;

then, transfecting 293T cells with the obtained chimeric antigen receptor expression vector which is armed with the TGF-beta-targeting specificity and targeted to human NKG2DL to obtain virus solution;

finally, the virus liquid is used for infecting immune response cells, and the immune response cells which express the chimeric antigen receptor modified by the armed targeting TGF-beta and specifically targeting human 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 present application discloses pharmaceutical compositions comprising isolated modified immunoresponsive cells expressing said TGF- β armed chimeric antigen receptor specifically targeting human NKG2DL and a pharmaceutically acceptable carrier.

Administration of the pharmaceutical composition may be autologous or non-autologous. For example, immunoresponsive cells expressing such chimeric antigen receptors specifically targeting human NKG2DL armed with TGF- β and compositions comprising the same may be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived T cells of the presently disclosed subject matter or progeny thereof (e.g., derived in vivo, ex vivo, or in vitro) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administered, the pharmaceutical compositions of the presently disclosed subject matter (e.g., pharmaceutical compositions comprising such TGF- β -armed immunoresponsive cells specifically targeting the chimeric antigen receptor of human NKG2 DL) are typically formulated in unit-dose injectable forms (solutions, suspensions, emulsions).

The compositions of the present application may be formulations. The immunoresponsive cells expressing the Chimeric Antigen Receptor (CAR) armed with a TGF-beta targeting specificity to human NKG2DL disclosed herein and compositions comprising the same may be conveniently provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, 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.

Any carrier, diluent or additive used according to the present application must be compatible with the immunoresponsive cells expressing the TGF- β -armed Chimeric Antigen Receptor (CAR) specifically targeting human 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 a human NKG2D protein receptor targeted to bind human NKG2DL or a functional variant thereof according to the first aspect of the invention, a chimeric antigen receptor specifically targeting human NKG2DL armed with TGF- β according to the second aspect, a recombinant vector or expression plasmid according to the fourth aspect, a recombinant virus according to the sixth aspect, an isolated modified immunoresponsive cell according to the seventh aspect, or a kit according to 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 engineering cell transmits an activation signal and activates an immune system by identifying NKG2DL generated on the surface of a tumor cell, so that the tumor cell is killed; meanwhile, a targeting TGF-beta structure is armed to an immune response cell, namely, a plurality of TGF-beta RII ligands such as TGF-beta 1, TGF-beta 2 and TGF-beta 3 cytokines in a tumor microenvironment are identified through a secretion mode, the formation of an immunosuppressive microenvironment is inhibited, the immune escape of the tumor cell is prevented, and therefore the killing efficiency of the tumor cell is obviously improved.

Advantageous effects

The invention utilizes the chimeric antigen receptor modified T cell technology to prepare the engineering immunocyte modified by the chimeric antigen receptor of the armed targeting TGF-beta specificity targeting human NKG2DL, the preparation method has simple steps, the obtained novel engineering immunocyte 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 and treating NKG2DL positive tumor; especially glioma, the tumor blood vessel is few, distribute multiple TGF-beta R II ligand in the tumor microenvironment, the ones that armed targeting TGF-beta of the invention target the engineering immunocyte that the idiocrasy antigen receptor modifies inhibit the formation of the immunosuppressive microenvironment and prevent the immune escape of the tumor cell, thus obviously raise the killing efficiency of the tumor cell. The invention can be hopefully used for preparing anti-tumor products, in particular for preparing medicines for treating pancreatic cancer, gastric cancer, glioma and liver cancer, and has good industrial application prospect.

Drawings

FIG. 1 shows a schematic diagram of the working mode of KD-275 engineered cells of the present invention.

FIG. 2 is a schematic representation of the sequence of the linkage of the parts of the chimeric antigen receptor armed in example 1, A being KD-025 and B being KD-275.

FIG. 3 shows the secondary structure diagram of the protein receptor human NKG2D protein targeting binding human NKG2DL in the chimeric antigen receptor armed according to the present invention, wherein A-F are respectively the extracellular region of the amino acid sequence of human NKG2D, and the characteristics of the protein receptor human NKG2D protein are analyzed by software, i.e. based on the crystal structure (PDB number: 4S0U) of the compound formed by NKG2D and its ligand NKG2DL, the amino acids of the key sites influencing affinity are firstly obtained by alanine scanning, then the saturation mutation of single-point mutation is carried out, 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 calculation of multi-point mutation.

FIG. 4 shows the secondary structure diagram of the protein receptor human TGF-beta RII protein which is targeted to bind human TGF-beta RII ligand in the armed chimeric antigen receptor of the present invention, A-F are the secondary structure diagram of 6 sequences which are selected by the inventor through mutation calculation according to the amino acid sequence of human TGF-beta RII and the biological characteristics of the interaction between the human TGF-beta RII and the ligand thereof by using software.

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

FIG. 6 is the results of flow assays of the expression of chimeric antigen receptors specifically targeting human NKG2DL armed with TGF- β targeting in example 4.

FIG. 7 shows the results of in vitro TGF- β RII assay of KD-275CAR-T cells in example 5, where A is a TGF- β RII working standard curve, and B-D are MOI 10, 20, and 40, respectively, and the TGF- β RII content in the culture supernatant was determined.

FIG. 8 shows the results of the KD-275CAR-T cell killing experiment in example 6, wherein A is NUGC4 target cell killing and B is Bxpc-3 target cell killing.

FIG. 9 shows the results of the in vitro cytokine release assay for KD-275CAR-T cells in example 7.

FIG. 10 is the in vivo safety assay for KD-275CAR-T mice in example 8, wherein A is the image of HE staining of mice and B 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 structures that bind to the human NKG2DL target that result from modification of the receptor for human NKG2DL (human NKG2D amino acid sequence).

The term "analog" refers to a structurally related polypeptide that has the function of the reference polypeptide molecule. In the present application, the polypeptide refers to a polyamino acid structure which is related to the amino acid sequence structure of the ligand human NKG2D and has the targeted binding of human NKG2 DL.

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 in this context 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 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 armed with chimeric antigen receptors targeting specifically human NKG2DL targeting TGF-. beta.

The overall design is as follows:

1. determination of amino acid sequences of chimeric antigen receptors specifically targeting human NKG2DL armed with TGF-beta targeting

First, the full-length amino acid sequence of human NKG2D (NP-031386.2) and the full-length amino acid sequence of human TGF-. beta.RII protein (NP-003233.4) were searched from the Genbank database of the American national library of medicine (NCBI).

Secondly, a chimeric antigen receptor which is specifically targeted to human NKG2DL and is armed with TGF-beta targeted is constructed, namely the amino acid sequence of the armed chimeric antigen receptor molecule is determined:

from the amino terminal to the carboxyl terminal, the amino acid sequence of a leader peptide (shown as SEQ ID No. 1), the amino acid sequence of human NKG2D (shown as SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7), the amino acid sequence of a human CD8 hinge region (shown as SEQ ID No. 8), the amino acid sequence of a human CD8 transmembrane region (shown as SEQ ID No. 9) or the amino acid sequence of a human CD28 transmembrane region (shown as SEQ ID No. 10), the amino acid sequence of a human 4-1BB intracellular domain (shown as SEQ ID No. 11) or the amino acid sequence of a human CD28 intracellular domain (shown as SEQ ID No. 12) or the amino acid sequence of a human OX40 intracellular domain (shown as SEQ ID No. 13) or a combination of the two, the amino acid sequence of a human CD3 delta domain (shown as SEQ ID No. 14), the amino acid sequence of Furin Furin-2A (shown as SEQ ID No. 15) or a combination thereof, the amino acid sequence of a human CD 358583 delta domain (shown as SEQ ID No. 14), The amino acid sequence (shown as SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20 or SEQ ID No. 21) of the targeted human TGF-beta is connected in series in sequence;

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;

A-F shown in FIG. 4 are secondary structure diagrams of 6 sequences which are selected by the inventor according to the amino acid sequence of human TGF-beta RII and analyzed with software for the biological characteristics of the interaction with the ligand, and are good in stability and high in ligand binding force by mutation calculation.

The codon optimized sequences include: nucleotide sequence encoding a leader sequence, nucleotide sequence encoding a human NKG2D sequence, nucleotide sequence encoding a human CD8 hinge region, nucleotide sequence encoding a human CD8 or CD28 transmembrane region, nucleotide sequence encoding a human 4-1BB or CD28 or OX40 intracellular domain, nucleotide sequence encoding a CD3 delta domain, nucleotide sequence encoding Furin-2A, nucleotide sequence encoding a targeted human TGF-beta, figure 2 shows a schematic representation of the order of the ligation of the various parts of the armed chimeric antigen receptor.

Table 1 shows that when different NKG2D amino acid sequences targeting human NKG2DL and nucleotide sequences targeting human TGF-beta are combined, the vector constructed according to the following method, the obtained virus and the obtained corresponding CAR-T cell are detected according to the following method, and the expression of CAR and the content of TGF-beta RII in culture supernatant are detected according to the following method; the results show that: when the amino acid sequence of human NKG2D (shown as SEQ ID No. 2) and the amino acid sequence of targeting human TGF-beta (shown as SEQ ID No. 16) are combined, the CAR expression and the TGF-beta RII content are optimal, and the combined gene can be used as a subsequent activity verification experiment.

TABLE 1 content of different sequence combinations CAR expression and TGF-. beta.RII

2. Construction of plasmids expressing chimeric antigen receptor molecules specifically targeting human NKG2DL armed with TGF- β targeting (KD-275 Lentiviral vectors)

The invention selects a CAR sequence which is best expressed in the step 1 and is named as KD-275 (the amino acid sequence is shown as SEQ ID No.22, and the nucleotide is shown as SEQ ID No. 23) to complete the efficacy verification test of the subsequent embodiment, and the specific sequence is as follows:

from the amino terminal to the carboxyl terminal, 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 human NKG2D (shown as SEQ ID No. 2), an amino acid sequence of a transmembrane region of human CD8 (shown as SEQ ID No. 9), an amino acid sequence of a human 4-1BB intracellular domain (shown as SEQ ID No. 11), an amino acid sequence of a human CD3 delta domain (shown as SEQ ID No. 14), an amino acid sequence of Furin-2A (shown as SEQ ID No. 15) and an amino acid sequence of a targeted human TGF-beta (shown as SEQ ID No. 16) in series.

The nucleotide sequence (Nanjing-one biosynthesis) of the chimeric antigen receptor molecule of the specific targeting human NKG2DL of the armed targeting TGF-beta is synthesized in a whole gene and shown in SEQ ID No.23, and is connected to a lentivirus vector lentiGuide-Puro (Addgene, USA) in a molecular cloning mode to construct a full-length CAR sequence expression frame with a single coding frame, and the expression is carried out by utilizing an EF1 alpha promoter or an EFS promoter.

The specific operation steps are as follows:

the complete gene is synthesized into a nucleotide sequence of a chimeric antigen receptor molecule of a armed TGF-beta targeting specificity human NKG2DL, a CAR molecule sequence is artificially synthesized by PCR amplification, recovered by an Axygen gel recovery kit (Hangzhou Zhang Heng), and subjected to homologous recombination and connection with a vector lentiGuide-Puro (Addgene, USA) digested by restriction enzymes SmaI and MluI to form a KD-275 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 1402quick 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., nucleotides of the chimeric antigen receptor of the armed targeting TGF- β specifically targeting human NKG2 DL) to confirm that the sequences were completely correct, confirming that a plasmid expressing the chimeric antigen receptor of the armed targeting TGF- β specifically targeting human NKG2DL (KD-275 expression plasmid) was obtained.

Extraction and purification of chimeric antigen receptor expression plasmids targeting specifically human NKG2DL targeted to armed targeting TGF- β.

The Stbl3 strain containing the KD-275 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) to prepare 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 expressing a chimeric antigen receptor specifically targeting human NKG2DL armed with TGF- β (KD-275 expression plasmid) and the packaging vectors psPAX2 and VSVG obtained in example 1 were packaged according to 10: 8: 5, using Lipofectamine^TM6000 transfection reagent (purchased from ThermoFisher corporation, product model 11668019) co-transfects 293T cells (see ThermoFisher transfection instruction for a specific transfection operation process), the medium is replaced by complete medium (purchased from Life Technologies, product model 11995-.

Example 3: isolated culture of T cells

Taking fresh peripheral blood of a healthy donor, and centrifugally separating the fresh peripheral blood mononuclear cells by density gradient; 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 solubility 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 specifically targeting human NKG2DL (KD-275CAR-T) armed with TGF-beta targeting

First, the CD3+ T cells obtained in example 3 were seeded in a 24-well plate at a concentration of 1X 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 solution of KD-275 collected in example 2 was 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 ℃. Expression of chimeric antigen receptor T cells specifically targeting human NKG2DL armed with targeting TGF-beta 48 hours post infection

Then, the virus solution of KD-275 collected in example 2 was 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 ℃. The chimeric antigen receptor T cell (KD-275CAR-T) which expresses the armed targeting TGF-beta and specifically targets human NKG2DL is obtained 48 hours after infection, and a next functional experiment can be carried out, as shown in figure 1, the working schematic diagram of the engineering cell is shown, namely the engineering cell transmits an activation signal and activates an immune system by recognizing NKG2DL generated on the surface of a tumor cell, so that the tumor cell is killed; and meanwhile, various TGF-beta RII ligands such as TGF-beta 1, TGF-beta 2 and TGF-beta 3 cytokines in the tumor microenvironment are recognized, the formation of an immunosuppressive microenvironment is inhibited, and the immune escape of tumor cells is prevented, so that the killing efficiency of the tumor cells is obviously improved.

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-275 virus liquid, and the expression rate of CAR molecules is 67.0 percent respectively.

Example 5: ELISA detection of TGF-beta RII secretion in culture supernatant of KD-275CAR-T cells

The specific operation steps are as follows:

first, KD-275CAR-T cells were prepared by infection as in example 4, and 24 hours after infection, centrifuged and counted with trypan blue, and the cells were each 2.5X 10⁴ 5X 10 per hole⁴1X 10 per hole⁵2X 10 per hole⁵Inoculating the cells into a 96-well plate, correspondingly making auxiliary wells, and making uninfected T cells as a control; the 96-well plate was placed at 37 ℃ in 5% CO₂Culturing in an incubator.

Culturing for 48h, collecting culture supernatant, and centrifuging at 4 deg.C for 5min at 300 g; TGF-beta RII detection is carried out by using a Human TGF-beta 1DuoSet ELISA Kit (purchased from R & D company, product model number DY240) and a DuoSet ELISA Ancillary Reagent Kit 1 (purchased from R & D company, product model number DY007) (the specific detection operation process is shown in the specification of the R & D detection Kit). The detection result is shown in fig. 7, wherein when A is a TGF-beta RII working standard curve and B-D are MOI 10, 20 and 40 respectively, the content of TGF-beta RII in the culture supernatant is detected.

Example 6: KD-275CAR-T cell in vitro killing experiment

An experimental group and 3 control groups are correspondingly arranged for each target cell line, wherein the experimental group is added with the cell suspension of the CAR-T cells which are specifically targeted to human NKG2DL and are provided with the human TGF-beta targets obtained in the 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 109803983A); the KD-025 control group was supplemented with CAR-T cells targeting human NKG2DL (see CN109803983A for preparation method).

First, according to the examplesPreparing KD-019, KD-025 and KD-275CAR-T cells by infection by the method 4, continuously culturing for 72 hours after infection, performing killing inoculation, performing CSFE fluorescent staining on each target cell line, counting the target cells under a fluorescent microscope, and adjusting the cell density to about 2 × 10⁶cells/mL, 20. mu.l/well, target cells were seeded into 96-well plates (tumor cells were resuspended in 2 groups on average, T cell medium for group A, T cell medium +250pg/mL TGF-. beta.1 for group B (purchased from R)&Company D, product model DY 240); according to the effective target ratio of 5: 1 adding T, KD-019 effector cells, KD-025 cells and KD-275 effector cells; then placing the cells in an incubator at 37 ℃ for culturing for 24 h; finally, the killing of the target cell line by KD-275CAR-T cells was evaluated using the 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, the graphs A and B are the killing results of NUGC4 and Bxpc3 cells respectively, and KD-025 and KD-275 have certain killing effects on NUGC4 and Bxpc 3; after TGF-beta 1 is added, KD-275 has higher killing effect on NUGC4 and Bxpc3 than KD-025, which shows that TGF-beta RII secreted by KD-275 neutralizes the immunosuppressive effect of TGF-beta 1 on T cells.

Example 7: KD-275CAR-T cell in vitro cytokine release assay

Experiment set up one experimental group and 3 control groups, wherein, the experimental group adds the cell suspension of the CAR-T cell which is provided with the specificity targeting human NKG2DL and targets human TGF-beta obtained in the 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 added unrelated CAR-T cells targeting CD 19; the KD-025 control group added CAR-T cells targeted to human NKG2 DL.

First, KD-019, KD-025, and KD-275CAR-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 into 96-well plates (tumor cells were resuspended in 2 groups on average, T cell medium for group A, T cell medium +250pg/mL TGF-. beta.1 for group B (purchased from R)&Company D, product model DY 240); according toThe effective target ratio is 5: 1 adding T, KD-019 effector cells, KD-025 cells and KD-275 effector cells; then placing the cells in an incubator at 37 ℃ for culturing for 24 h; finally, the release of killer cytokines from KD-275CAR-T cells on the above 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 co-culturing KD-025 and KD-275CAR-T cells with tumor cells Bxpc3, INF- γ was significantly increased compared to the control group, whereas the release of INF- γ was significantly reduced after addition of TGF- β, but the release of INF- γ was significantly higher in the KD-275 group than in the KD-025 group, suggesting that TGF- β RII secreted by KD-275 neutralizes the immunosuppressive effects of TGF- β 1 on T cells.

Example 8: in vivo safety experiments in KD-275CAR-T mice

First, KD-275CAR-T cells were infected according to the method of example 4, and the effect of KD-275CAR-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-275CAR-T cells do not cause inflammation, edema and necrosis of the major organs such as heart, liver, lung and kidney of the mice (FIG. A), and do not have any negative effect on the survival cycle of the mice (FIG. B).

In a word, the virus vector of the chimeric antigen receptor of the armed targeting TGF-beta specifically targeting human NKG2DL and the modified engineering immune cell can be applied to treating various tumors, including liver cancer, glioma, lung cancer, acute leukemia and the like.

Sequence listing

<110> Nanjing Kaidi Biotech Co., Ltd

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

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Tyr Ile Cys Met Gln Arg Thr Val

130 135

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

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

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

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

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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> 9

<211> 24

<212> PRT

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

<400> 9

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> 10

<211> 27

<212> PRT

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

<400> 10

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> 11

<211> 42

<212> PRT

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

<400> 11

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> 12

<211> 41

<212> PRT

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

<400> 12

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> 13

<211> 36

<212> PRT

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

<400> 13

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> 14

<211> 112

<212> PRT

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

<400> 14

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> 15

<211> 26

<212> PRT

<213> Furin-2A(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 15

Arg Ala Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr

1 5 10 15

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25

<210> 16

<211> 137

<212> PRT

<213> TGF. beta. RII sequence 1(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 16

Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 17

<211> 137

<212> PRT

<213> TGF. beta. RII sequence 2(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 17

Thr Ile Pro Pro His Val Lys Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Lys Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 18

<211> 137

<212> PRT

<213> TGF-. beta.RII sequence 3(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 18

Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Lys Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Lys Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 19

<211> 137

<212> PRT

<213> TGF-. beta.RII sequence 4(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 19

Thr Ile Pro Pro His Val Arg Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Arg Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 20

<211> 137

<212> PRT

<213> TGF-. beta.RII sequence 5(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 20

Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Arg Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Lys Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 21

<211> 137

<212> PRT

<213> TGF-. beta.RII sequence 6(2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 21

Thr Ile Pro Pro His Val Lys Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Arg Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 22

<211> 563

<212> PRT

<213> KD-275 CAR(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 22

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 Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu

20 25 30

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

35 40 45

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

50 55 60

Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser

65 70 75 80

Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met

85 90 95

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

100 105 110

Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly

115 120 125

Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys

130 135 140

Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Thr Thr Thr

145 150 155 160

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

165 170 175

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

180 185 190

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

195 200 205

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

210 215 220

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

225 230 235 240

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

245 250 255

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

260 265 270

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

275 280 285

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

290 295 300

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

305 310 315 320

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

385 390 395 400

Glu Glu Asn Pro Gly Pro Met Tyr Arg Met Gln Leu Leu Ser Cys Ile

405 410 415

Ala Leu Ser Leu Ala Leu Val Thr Asn Ser Thr Ile Pro Pro His Val

420 425 430

Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala

435 440 445

Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr

450 455 460

Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile

465 470 475 480

Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp

485 490 495

Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr

500 505 510

His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys

515 520 525

Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser

530 535 540

Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser

545 550 555 560

Asn Pro Asp

<210> 23

<211> 1689

<212> DNA

<213> KD-275CAR nucleotides (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 23

atggcgttgc cggtgaccgc gctccttctt cccctggcat tgttgctgca cgccgcgaga 60

ccgtcactct ttaatcagga agtacagatt ccgctcactg agtcttattg tggcccctgt 120

cctaaaaatt ggatctgtta caaaaataac tgttatcaat tcttcgatga aagtaagaat 180

tggtacgagt cccaggcatc ctgcatgagt cagaacgcga gtttgctcaa agtctacagt 240

aaggaagatc aggatctgct taaactggtt aagagttatc actggatggg attggtacat 300

attccgacga atggtagttg gcaatgggaa gacggatcaa ttttgtcccc caaccttctt 360

actataatcg agatgcagaa gggggactgc gccctctacg catccagctt caagggatat 420

attgagaact gctcaacgcc taacacgtac atttgcatgc aaagaacggt tactacaact 480

ccggcccccc ggccaccaac gcccgcccca accatagcgt cccagccttt gagtcttcga 540

ccggaagcat gccgcccggc agcaggagga gcagttcata cgcgcggact ggattttgcg 600

tgcgacattt acatctgggc tccattggcg ggaacttgtg gagttctcct cctgtcactt 660

gtgatcactt tgtattgtaa gcgaggacga aagaaactcc tttatatatt caaacagccc 720

ttcatgagac cagtgcaaac gacacaggag gaggatggtt gttcatgtcg gtttcctgag 780

gaggaggagg gggggtgcga gcttcgcgta aagtttagcc ggtctgccga cgccccagcc 840

tatcaacaag gccaaaacca attgtacaat gagttgaact tgggtcgaag agaggaatat 900

gatgtattgg acaagaggcg aggaagggat ccagagatgg gagggaagcc aagacgcaag 960

aatccgcaag aaggactcta taacgaattg cagaaagaca aaatggccga ggcttattca 1020

gagattggaa tgaagggcga gcgaagacgc ggcaagggtc atgacgggct gtatcaaggt 1080

ttgtctacgg caactaaaga cacatatgat gcgctccaca tgcaggcctt gcctcctagg 1140

agagcgaagc gatctggtag tggggaaggt cgagggtctc ttctgacgtg tggtgatgtc 1200

gaagagaacc caggtccgat gtatcgcatg cagcttctct catgtatagc gcttagcttg 1260

gccttggtca cgaattccac gattccgccc cacgtacaga agtccgtaaa taatgacatg 1320

atcgtgacgg ataataacgg cgcagtaaag tttccgcaac tgtgcaagtt ttgcgatgtc 1380

cgattcagta cttgtgacaa ccagaagagt tgcatgtcaa actgcagtat tactagcatt 1440

tgcgagaaac cacaggaggt gtgcgtagcg gtgtggcgaa aaaatgacga gaacatcact 1500

cttgaaaccg tatgccatga ccctaaactc ccgtaccatg atttcatact cgaggacgcc 1560

gccagcccta aatgtattat gaaggaaaag aaaaagcctg gagaaacatt ttttatgtgc 1620

tcctgttcct cagacgagtg taacgataat ataatattta gcgaagaata taataccagt 1680

aatcccgat 1689

Claims (7)

1. A chimeric antigen receptor specifically targeting human NKG2DL armed with targeting human TGF- β characterized by: from the amino terminal to the carboxyl terminal, the amino acid sequence of the chimeric antigen receptor is formed by connecting a leader peptide shown as SEQ ID No.1, an amino acid sequence of human NKG2D shown as SEQ ID No.2, an amino acid sequence of a transmembrane region of human CD8 shown as SEQ ID No.9, an amino acid sequence of a human 4-1BB intracellular domain shown as SEQ ID No.11, an amino acid sequence of a human CD3 zeta domain shown as SEQ ID No.14, an amino acid sequence of Furin-2A shown as SEQ ID No.15 and an amino acid sequence of a targeted human TGF-beta shown as SEQ ID No.16 in series in sequence.

2. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1, wherein: the nucleotide sequence is SEQ ID No. 23.

3. A recombinant vector armed with a chimeric antigen receptor targeting TGF- β specifically to human NKG2DL, comprising the nucleic acid molecule of claim 2.

4. A recombinant virus comprising the recombinant vector of claim 3; the virus includes lentivirus, adenovirus, adeno-associated virus or retrovirus.

5. An isolated genetically modified functionalized immunoresponsive cell obtained by infecting an immune effector cell with the recombinant virus of claim 4, which is a functionalized immunoresponsive cell modified with a chimeric antigen receptor specifically targeting human NKG2DL armed with targeting TGF- β; the immune effector cells include cytotoxic T lymphocytes, NK cells, NKT cells, helper T cells, or macrophages.

6. A biological product characterized by comprising the amino acid sequence of claim 1; or a nucleotide sequence according to claim 2; or a recombinant vector according to claim 3; or a recombinant virus according to claim 4; or comprising an immune cell according to claim 5.

7. Use of a biological product according to claim 6 in the manufacture of a medicament for the treatment of cancer; the cancer is pancreatic cancer, gastric cancer, liver cancer, brain cancer, prostatic cancer, lymph cancer, leukemia, intestinal cancer, lung cancer or breast cancer expressing NKG2 DL.

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