CN114031690A

CN114031690A - Chimeric antigen receptor targeting CCR1 and NKG2D ligands and application thereof

Info

Publication number: CN114031690A
Application number: CN202111474258.1A
Authority: CN
Inventors: 李光超; 罗敏; 周兆; 王学俊
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-02-11
Anticipated expiration: 2041-12-03
Also published as: CN114031690B

Abstract

The invention relates to a chimeric antigen receptor targeting CCR1 and NKG2D ligands and application thereof, wherein the chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and a signal transduction domain which are sequentially connected from an amino terminal to a carboxyl terminal; the antigen binding domains include anti-CCR 1 single chain antibodies and NKG 2D. The CAR-T cell constructed by the chimeric antigen receptor simultaneously targeting the CCR1 and NKG2D ligands has a specific targeting effect on CCR1 and NKG2D ligand positive tumor cells, has an obvious in-vitro killing effect, can effectively remove CCR1 and NKG2D ligand single/double positive tumor cells, reduces antigen escape, and has important significance in the field of tumor treatment.

Description

Chimeric antigen receptor targeting CCR1 and NKG2D ligands and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a chimeric antigen receptor targeting CCR1 and NKG2D ligands and application thereof.

Background

In recent years, chimeric antigen receptor T cell (CAR T) therapy has exhibited very good efficacy in tumor therapy. The CAR-T therapy is called Chimeric antigen receptor T cell therapy, namely Chimeric antigen receptor T cell therapy, and the principle is to apply the T lymphocytes of a patient, reform the T lymphocytes in a laboratory, load receptors capable of recognizing tumor antigens and co-stimulation molecules, perform in-vitro amplification, and then re-infuse the receptors and co-stimulation molecules into the patient again, so as to recognize and attack the tumor cells of the patient. T cells, also called T lymphocytes, are a kind of human leukocyte, are derived from bone marrow hematopoietic stem cells, mature in thymus, and then colonize human blood, lymph and peripheral tissues and organs to exert immune function, which can resist and eliminate infection, tumor, foreign body, etc. In a laboratory, technicians activate T cells through a genetic engineering technology, install a positioning navigation device CAR (chimeric antigen receptor), modify the T cells into CAR-T cells, specifically recognize in vivo tumor cells by utilizing the chimeric antigen receptor, release a large amount of various effector factors through immune action, and can efficiently kill the tumor cells, thereby achieving the purpose of treating malignant tumors.

However, the existing chimeric antigen receptor has a large antigen escape problem, and the effect of tumor treatment is influenced.

Disclosure of Invention

Based on this, there is a need to provide a chimeric antigen receptor that can reduce antigen escape.

A chimeric antigen receptor targeting CCR1 and NKG2D ligands comprising, connected in sequence from amino terminus to carboxy terminus, an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain; the antigen binding domains include anti-CCR 1 single chain antibodies and NKG 2D.

The invention constructs a chimeric antigen receptor simultaneously targeting CCR1 and NKG2D ligands, which comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and a signal transduction domain, wherein the antigen binding domain comprises an anti-CCR 1 single-chain antibody and an NKG2D molecule. The CAR-T cell constructed by the chimeric antigen receptor simultaneously targeting the CCR1 and NKG2D ligands has a specific targeting effect on CCR1 and NKG2D ligand positive tumor cells, has an obvious in-vitro killing effect, can effectively remove CCR1 and NKG2D ligand single/double positive tumor cells, reduces antigen escape, and has important significance in the field of tumor treatment.

In one embodiment, the anti-CCR 1 single chain antibody comprises an anti-CCR 1 antibody light chain variable region and an anti-CCR 1 antibody heavy chain variable region, wherein the amino acid sequence of the anti-CCR 1 antibody light chain variable region is as set forth in SEQ ID NO: 1, the amino acid sequence of the heavy chain variable region of the anti-CCR 1 antibody is shown as SEQ ID NO: 2, respectively.

In one embodiment, the amino acid sequence of NKG2D is as set forth in SEQ ID NO: 3, respectively.

In one embodiment, the hinge region is selected from the hinge regions of at least one of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD 154; and/or

The transmembrane domain is selected from the transmembrane regions of at least one of the following proteins: CD, CD epsilon, CD160, CD, IL2 beta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD, ITGB, TNFR, DNAM, CD, CEACAM, CRTAM, Ly, PSGL, CD100, SLAMF, SLAMG, BLAM, BLAMG, PAG, NKG2, NKP, NKG2, NKG, CD, BAFFR, HVEM, SLAMF, NKp, CD160, CD, IL2 beta, IL2 gamma, ITGAL 1, ITGAL, CD11, ITGAX, ITGA, ITGB, TNFR, TNAG, NKGA 2, NKGA, NKG, NKGA 2, NKGA, NKG, and NKG 2B; and/or

The co-stimulatory domain is selected from the intracellular domains of at least one of the following proteins: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-related antigen-1, CD2, CD7, LIGHT, NKG2C and B7-H3; and/or

The signal transduction domain is selected from the intracellular domains of at least one of the following proteins: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66 d.

In one embodiment, the amino acid sequence of the chimeric antigen receptor is as set forth in SEQ ID NO: 4 or SEQ ID NO: 5, respectively.

In one embodiment, the amino terminus of the chimeric antigen receptor further comprises a signal peptide.

The invention also provides a nucleic acid encoding a chimeric antigen receptor as described above.

In one embodiment, the nucleotide sequence of the nucleic acid is as set forth in SEQ ID NO: 6 or SEQ ID NO: shown at 7.

The invention also provides a recombinant vector containing the nucleic acid.

The invention also provides a CAR-T cell containing a nucleic acid as described above or transformed with a recombinant vector as described above.

The invention also provides application of the chimeric antigen receptor, the nucleic acid, the recombinant vector or the CAR-T cell in preparation of medicines for treating cancers, such as multiple myeloma, acute myelogenous leukemia, various solid tumors and the like.

The invention also provides a pharmaceutical composition, which comprises the CAR-T cell and pharmaceutically acceptable auxiliary materials.

Drawings

FIG. 1 is a schematic structural diagram of two chimeric antigen receptors in example 1 of the present invention, wherein A is NKG2D-CCR1CAR, and B is CCR1-NKG2D CAR;

FIG. 2 is a schematic structural diagram of an expression vector for NKG2D-CCR1CAR in example 1 of the present invention;

FIG. 3 is a schematic structural diagram of an expression vector for CCR1-NKG2D CAR in example 1 of the present invention;

FIG. 4 shows the result of detecting CCR1 expression by tumor cells in example 2 of the present invention;

FIG. 5 shows the results of the assay of NCI-H929 cells expressing NKG2D ligand in example 2 of the present invention;

FIG. 6 shows the results of the measurement of NKG2D ligand expression by K562 and U937 cells in example 2;

FIG. 7 is data of the flow-based assay of CAR expression in sets of CAR-T cells in example 4 of the present invention;

FIG. 8 shows data of the detection of various sets of CAR-T cell killing tumor target cells by ELISA in example 5 of the present invention.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The chimeric antigen receptor targeting the CCR1 and NKG2D ligands of one embodiment of the present invention comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain connected in sequence from the amino terminus to the carboxy terminus; such antigen binding domains include anti-CCR 1 single chain antibodies and NKG 2D.

CCR1 belongs to a member of the β chemokine receptor family, is composed of 355 amino acids in total length, and is a G protein-coupled receptor with a seven-fold transmembrane-type structure. The ligands thereof generally include human macrophage inflammatory protein 1 alpha (CCL3), activated normal T expression and secretion protein (CCL5), monocyte chemotactic protein 2(CCL8), myeloid progenitor cell inhibitory factor 1, CCL14, CCL15 and CCL16, etc. CCL1 and its ligand-mediated signal transduction are key to efficient immune cell recruitment to inflammatory sites, playing an important role in cancer progression, host anti-inflammatory responses, immune responses, and the like.

For example, Multiple Myeloma (MM) is a plasma cell malignancy characterized by abnormal localization of MM cells to the Bone Marrow (BM), malignant proliferation and induction of osteolysis. CCR1 is critical for the homing or migration of MM cells to the BM to receive the necessary survival signals and is therefore often an important target for the treatment of Multiple Myeloma (MM) diseases. Among them, CCR1 and CCL3 play a central role in the pathogenesis of MM and MM-induced osteolytic bone disease, which in combination directly and indirectly stimulate tumor cell growth by up-regulating cell adhesion and cytokine secretion; in addition, they can also regulate osteoclast/osteoblast balance by inducing osteoclast differentiation and inhibiting osteoblast function, accelerating disease progression. The data show that after being treated by CCR1 specific antagonist drugs, the phenomenon of osteoclast generation and osteoclast absorption caused by MM can be obviously reduced, and the osteolytic lesion caused by MM can be obviously reduced. In addition, Acute Myeloid Leukemia (AML) cells typically exhibit constitutive release of multiple chemokines and have multiple chemokine receptors on the cell surface, and these chemokine/chemokine receptor expression patterns may be critical for leukemia development and response to therapy, with significant upregulation of chemokines CCR1, CXCR4, CCR2, etc., in AML patient samples compared to CD34+ normal cells. The chemokine CCL23 and the corresponding receptor CCR1 are highly up-regulated in KMT2A-MLLT3(KM3) AML, and CCL23/CCR1 axis signals can activate ERK1/2 in an AML cell line through a beta-repressor mediated pathway to promote the development of AML diseases; meanwhile, researches find that a channel composed of CCR1-CCL3 can also directly promote the proliferation and colony formation of AML cells, and when the CCR1-CCL3 channel is specifically blocked, the growth of the AML cells can be inhibited, and the attack of AML is delayed. In addition, the chemokine receptor CCR1 and its ligand also play an important role in the processes of malignant proliferation and metastasis of various solid tumors such as liver cancer, rectal cancer, lung cancer and breast cancer, tumor microenvironment inhibition and the like.

NKG2D is an important activated C-type lectin receptor in the innate immune system, which exerts a killing effect on target cells by recognizing ligands induced on the surface of the target cells to transmit activation signals and activate the immune system. All NK cells, CD8 positive T cells, most NKT cells, macrophages, and γ δ T cells express NKG 2D. The ligands for NKG2D are mainly of two types: one is MHC class I chain-related A/B molecules (MICA/B), and the other is UL16 Binding protein (UL16 Binding Proteins, ULBPs). The NKG2D ligand is mainly expressed on most epithelial tumor cells, such as ovarian cancer, colon cancer, leukemia and the like, and rarely expressed on normal cells. In general, the ligand of NKG2D is not expressed or is expressed in a very small amount in normal cells, but when the cells are infected or cancerated, the expression amount of the ligand is greatly increased, so that the ligand of NKG2D becomes a potential target for CAR-T treatment.

In view of the fact that CCR1 and NKG2D ligands participate in the occurrence and progress processes of various blood cancers and solid tumors and can be used as good targets for treating various cancers, the invention innovatively constructs a bispecific CAR structure capable of simultaneously targeting CCR1 and NKG2D ligands, the modified chimeric antigen receptor and the corresponding CAR-T cell can specifically and efficiently kill tumor cells, the single/double positive tumor cells of CCR1 and NKG2D ligands are effectively eliminated, antigen escape is reduced, tumors can be more effectively and comprehensively covered, most of blood and solid tumors are eliminated, the risk of tumor recurrence is reduced, and the invention has important significance in the field of tumor treatment.

In one particular example, the anti-CCR 1 single chain antibody comprises an anti-CCR 1 antibody light chain variable region and an anti-CCR 1 antibody heavy chain variable region, the amino acid sequence of the anti-CCR 1 antibody light chain variable region being as set forth in SEQ ID NO: 1, the amino acid sequence of the heavy chain variable region of the anti-CCR 1 antibody is shown as SEQ ID NO: 2, respectively.

In a specific example, the amino acid sequence of NKG2D is as set forth in SEQ ID NO: 3, respectively.

In one specific example, the variable region of the light chain of the anti-CCR 1 antibody, the variable region of the heavy chain of the anti-CCR 1 antibody and NKG2D are linked by a linker peptide. Alternatively, the linker peptide is selected from (G4S)₃And GSTSGSGKPGSGEGSTKGAnd (4) a plurality of.

In one particular example, the antigen binding domain comprises, in order from amino terminus to carboxy terminus, the anti-CCR 1 antibody light chain variable region described above, anti-CCR 1 antibody heavy chain variable region, and NKG 2D. In a specific example, the antigen binding domain comprises, in order from amino terminus to carboxy terminus, the NKG2D, anti-CCR 1 antibody light chain variable region, and anti-CCR 1 antibody heavy chain variable region described above.

In a specific example, the hinge region is selected from the hinge region of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD 154.

In a specific example, the transmembrane domain is selected from the transmembrane regions of: CD, CD epsilon, CD160, CD, IL2 beta, IL2 gamma, IL7 alpha, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD, ITGB, TNFR, DNAM, CD, CEACAM, CRTAM, Ly, PSGL, CD100, SLAMF, SLAMG, BLAM, BLAMG, PAG, NKPLG, NKG2, NKP, NKG2, and LTbp of T cell receptors.

In a specific example, the co-stimulatory domain is selected from the intracellular domains of the following proteins: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-related antigen-1, CD2, CD7, LIGHT, NKG2C and B7-H3.

In a specific example, the signal transduction domain is selected from the intracellular domains of the following proteins: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CD3 ζ, CD22, CD79a, CD79b, and CD66 d.

Preferably, the hinge region is the hinge region of human CD8 α. Preferably, the transmembrane domain is the transmembrane region of human CD 8. Preferably, the co-stimulatory domain is the intracellular domain of human 4-1 BB. Preferably, the signal transduction domain is an intracellular domain of human CD3 ζ.

In one embodiment, the chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 5, respectively.

It is understood that the polypeptide fragments of the present invention (e.g., signal peptide, single chain antibody, hinge region, transmembrane domain, co-stimulatory domain and signal transduction domain) may be independently selected from the same or different species sources, such as murine (mouse, rat), rabbit, sheep, goat, horse, chicken, bovine, canine and human, preferably the species source is human.

A nucleic acid according to one embodiment of the invention encoding a chimeric antigen receptor targeting a CCR1 and NKG2D ligand as described above.

In one specific example, the nucleotide sequence of the nucleic acid is as set forth in SEQ ID NO: 6 or SEQ ID NO: shown at 7. It is understood that nucleic acid sequences capable of expressing the same protein have various forms due to degeneracy of codons, which are codon-optimized nucleic acid sequences for achieving high expression of a protein of interest, but the specific sequence form is not limited thereto.

The recombinant vector of one embodiment of the present invention contains the nucleic acid as described above. It is understood that the nucleic acid may be DNA or RNA.

In a specific example, the recombinant vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a CRISPR/CAS plasmid. Preferably, the recombinant vector is a lentiviral vector. The lentivirus vector can effectively integrate the exogenous gene onto the host chromosome, thereby achieving the effect of persistently expressing the target sequence. Can effectively infect various cells such as neuron cells, liver cells, cardiac muscle cells, tumor cells, endothelial cells, stem cells and the like in the aspect of infection capacity, thereby achieving good gene therapy effect. For some cells which are difficult to transfect, such as primary cells, stem cells, undifferentiated cells and the like, the lentiviral vector is used, so that the transduction efficiency of the target gene can be greatly improved, the probability of integrating the target gene into the host cell genome is greatly increased, and the long-term and stable expression of the target gene can be conveniently and quickly realized. It will be appreciated that the type of carrier is not limited thereto and may be adjusted according to specific needs. In addition, the vector may contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, etc., and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

The CAR-T cell of an embodiment of the invention comprises a nucleic acid as described above or is transformed with a recombinant vector as described above.

In a specific example, the CAR-T cell is any one of helper T cell, cytotoxic T cell, memory T cell, regulatory T cell, MAIT cell, and γ δ T cell. Optionally, the CAR-T cell is of the type CD3+ T cell, CD3+ CD4+ T cell, or CD3+ CD8+ T cell.

The pharmaceutical composition of one embodiment of the invention comprises the CAR-T cell as described above, and a pharmaceutically acceptable excipient.

In one particular example, the adjuvant comprises one or more of a diluent, a preservative, a buffer, a disintegrant, an antioxidant, a suspending agent, a colorant, and an excipient.

In one particular example, the diluent is selected from one or more of polyethylene glycol, propylene glycol, vegetable oil and mineral oil. In a specific example, the preservative is selected from one or more of sorbic acid, methyl sorbate, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzyl paraben, sodium methyl paraben, benzoic acid, and benzyl alcohol. In a particular example, the buffering agent is selected from one or more of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium tartrate, and sodium acetate. In a specific example, the antioxidant is selected from one or more of ethylenediaminetetraacetic acid, disodium salts of ethylenediaminetetraacetic acid, dibutylhydroxytoluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite.

The preparation method of the CAR-T cell comprises the following steps: collecting peripheral blood mononuclear cells; separating T cells such as CD3+ T cells from the peripheral blood mononuclear cells; t cells are cultured in an activated state, infected with a virus containing the above-mentioned nucleic acid, and then cultured in an amplified state.

Embodiments of the present invention will be described in detail below with reference to specific examples and the accompanying drawings.

Example 1 vector construction

The structures of two chimeric antigen receptors constructed in this example are shown in FIG. 1, where A is NKG2D-CCR1CAR and B is CCR1-NKG2D CAR. Based on the amino acid sequence of the chimeric antigen receptor, the entire gene synthesis was performed in Egyptian technologies, Inc., Guangzhou, and constructed into lentiviral expression vectors, and the vector structures of NKG2D-CCR1CAR and CCR1-NKG2D CAR are shown in FIGS. 2 and 3, respectively, and the sequence information is shown below.

NKG2D-CCR1 CAR(sp-NKG2D-Linker-antiCCR1 VL-(G4S)_3-VH-CD8hinge-CD8TM-4-1BB-CD3Z) amino acid sequence: MDMRVPAQLLGLLLLWLRGARCLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGSTSGSGKPGSGEGSTKGDVVMTQTPRSLPVSLGDQASISCRSRQSLIHSNGITFLHWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGVYFCSQGTHVPPTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLNNYGVHWVRQPPGKGLEWLGVIWSAGTTVYNAAFISRLSISKDDSKSQVFFKMNSLQAGDTAIYYCAKDGSRYYTAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

NKG2D-CCR1CAR nucleotide sequence:ATGGATATGCGCGTTCCTGCTCAACTCCTCGGTCTGCTCCTGC TGTGGCTCAGAGGAGCCCGCTGCCTCTTTAACCAGGAGGTGCAAATTCCACTGACTGAATCCTATTGCGGCCCTTGTCCCAAGAACTGGATTTGTTATAAGAACAACTGTTATCAATTCTTCGATGAGTCTAAGAACTGGTATGAAAGCCAAGCATCCTGTATGTCCCAGAACGCTTCACTCCTGAAGGTCTACAGCAAAGAAGATCAGGACCTCCTCAAGCTGGTGAAATCATATCACTGGATGGGACTGGTGCACATCCCTACAAACGGCTCATGGCAGTGGGAGGACGGCTCCATCCTGAGCCCAAATCTGCTCACAATCATTGAAATGCAGAAGGGTGATTGCGCTCTCTACGCAAGCTCTTTTAAAGGCTACATAGAGAACTGCTCCACTCCCAATACATACATTTGTATGCAGCGCACTGTGGGATCAACCAGCGGAAGCGGGAAGCCTGGTTCCGGAGAAGGATCAACTAAAGGCGATGTGGTCATGACTCAGACTCCAAGGAGCCTGCCCGTGTCCCTCGGAGATCAGGCAAGCATCAGCTGCCGGAGCAGGCAGAGTCTGATTCACAGTAATGGAATAACTTTTCTCCACTGGTACCTGCAGAAGGCCGGTCAGAGTCCTAAGCTCCTGATATACAAAGTGAGTAACAGATTCAGTGGAGTGCCTGACAGATTCTCAGGATCCGGCTCAGGGACCGACTTCACCCTGCGGATTAGCAGAGTGGAGGCAGAAGACCTCGGCGTCTACTTTTGTAGTCAAGGGACTCACGTGCCACCTACATTTGGTGGCGGGACTAAACTGGAAATCAAGGGCGGAGGCGGTTCAGGCGGAGGCGGCTCAGGCGGTGGCGGTTCACAAGTGCAGCTCAAGCAGTCAGGGCCAGGCCTCGTCCAGCCAAGTCAAAGTCTGAGCATTACCTGCACTGTGAGTGGTTTTAGCCTGAACAATTATGGAGTGCATTGGGTTAGGCAGCCACCTGGAAAGGGCCTGGAATGGCTGGGCGTGATTTGGTCTGCCGGCACTACTGTGTACAACGCCGCATTCATCTCCAGACTCAGCATCAGTAAAGACGACTCCAAATCCCAGGTGTTCTTCAAAATGAACTCACTGCAAGCTGGAGATACAGCAATCTACTACTGCGCTAAAGACGGCTCTCGGTATTACACTGCAATGGACTACTGGGGGCAAGGGACATCCGTGACCGTGAGCTCAACTACGACCCCTGCACCGCGGCCGCCTACTCCTGCACCTACAATCGCAAGTCAGCCACTGAGTCTCAGACCCGAAGCATGCCGCCCTGCTGCAGGCGGAGCTGTCCATACACGCGGACTGGACTTTGCATGCGATATATACATCTGGGCACCACTGGCCGGCACTTGCGGCGTGCTGCTCCTGTCCCTCGTGATTACCCTGTACTGCAAACGCGGCAGGAAGAAGCTCCTGTATATCTTTAAACAGCCCTTCATGAGGCCAGTGCAGACCACTCAAGAGGAAGACGGTTGTAGCTGCCGGTTTCCCGAGGAAGAAGAGGGAGGCTGCGAGCTCCGCGTGAAGTTCTCCCGCTCAGCCGATGCACCCGCCTATCAGCAAGGGCAGAACCAGCTGTACAATGAGCTCAACCTGGGAAGAAGGGAGGAATATGACGTTCTGGATAAACGGCGCGGTCGCGATCCCGAAATGGGTGGGAAGCCTCGCAGGAAGAATCCTCAGGAAGGGCTCTACAATGAGCTGCAGAAAGACAAAATGGCAGAGGCCTATTCTGAAATCGGCATGAAGGGCGAGCGCCGCAGAGGCAAAGGACACGACGGCCTGTACCAGGGCCTGTCTACAGCCACCAAGGACACCTATGACGCTCTCCACATGCAAGCCCTGCCACCAAGGTGA

CCR1-NKG2D CAR(sp-antiCCR1 VL-(G4S)_3-VH-Linker-NKG2D- -CD8hinge-CD8TM-4-1BB-CD3 ζ) amino acid sequence: MDMRVPAQLLGLLLLWLRGARCDVVMTQTPRSLPVSLGDQASISCRSRQSLIHSNGITFLHWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGVYFCSQGTHVPPTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLNNYGVHWVRQPPGKGLEWLGVIWSAGTTVYNAAFISRLSISKDDSKSQVFFKMNSLQAGDTAIYYCAKDGSRYYTAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

CCR1-NKG2D CAR nucleotide sequence: ATGGATATGCGCGTTCCTGCTCAACTCCTCGGTCTGCTCCTGCTGTGGCTCAGAGGAGCCCGCTGCGATGTGGTCATGACTCAGACTCCAAGGAGCCTGCCCGTGTCCCTCGGAGATCAGGCAAGCATCAGCTGCCGGAGCAGGCAGAGTCTGATTCACAGTAATGGAATAACTTTTCTCCACTGGTACCTGCAGAAGGCCGGTCAGAGTCCTAAGCTCCTGATATACAAAGTGAGTAACAGATTCAGTGGAGTGCCTGACAGATTCTCAGGATCCGGCTCAGGGACCGACTTCACCCTGCGGATTAGCAGAGTGGAGGCAGAAGACCTCGGCGTCTACTTTTGTAGTCAAGGGACTCACGTGCCACCTACATTTGGTGGCGGGACTAAACTGGAAATCAAGGGCGGAGGCGGTTCAGGCGGAGGCGGCTCAGGCGGTGGCGGTTCACAAGTGCAGCTCAAGCAGTCAGGGCCAGGCCTCGTCCAGCCAAGTCAAAGTCTGAGCATTACCTGCACTGTGAGTGGTTTTAGCCTGAACAATTATGGAGTGCATTGGGTTAGGCAGCCACCTGGAAAGGGCCTGGAATGGCTGGGCGTGATTTGGTCTGCCGGCACTACTGTGTACAACGCCGCATTCATCTCCAGACTCAGCATCAGTAAAGACGACTCCAAATCCCAGGTGTTCTTCAAAATGAACTCACTGCAAGCTGGAGATACAGCAATCTACTACTGCGCTAAAGACGGCTCTCGGTATTACACTGCAATGGACTACTGGGGGCAAGGGACATCCGTGACCGTGAGCTCAGGATCAACCAGCGGAAGCGGGAAGCCTGGTTCCGGAGAAGGATCAACTAAAGGCCTCTTTAACCAGGAGGTGCAAATTCCACTGACTGAATCCTATTGCGGCCCTTGTCCCAAGAACTGGATTTGTTATAAGAACAACTGTTATCAATTCTTCGATGAGTCTAAGAACTGGTATGAAAGCCAAGCATCCTGTATGTCCCAGAACGCTTCACTCCTGAAGGTCTACAGCAAAGAAGATCAGGACCTCCTCAAGCTGGTGAAATCATATCACTGGATGGGACTGGTGCACATCCCTACAAACGGCTCATGGCAGTGGGAGGACGGCTCCATCCTGAGCCCAAATCTGCTCACAATCATTGAAATGCAGAAGGGTGATTGCGCTCTCTACGCAAGCTCTTTTAAAGGCTACATAGAGAACTGCTCCACTCCCAATACATACATTTGTATGCAGCGCACTGTGACTACGACCCCTGCACCGCGGCCGCCTACTCCTGCACCTACAATCGCAAGTCAGCCACTGAGTCTCAGACCCGAAGCATGCCGCCCTGCTGCAGGCGGAGCTGTCCATACACGCGGACTGGACTTTGCATGCGATATATACATCTGGGCACCACTGGCCGGCACTTGCGGCGTGCTGCTCCTGTCCCTCGTGATTACCCTGTACTGCAAACGCGGCAGGAAGAAGCTCCTGTATATCTTTAAACAGCCCTTCATGAGGCCAGTGCAGACCACTCAAGAGGAAGACGGTTGTAGCTGCCGGTTTCCCGAGGAAGAAGAGGGAGGCTGCGAGCTCCGCGTGAAGTTCTCCCGCTCAGCCGATGCACCCGCCTATCAGCAAGGGCAGAACCAGCTGTACAATGAGCTCAACCTGGGAAGAAGGGAGGAATATGACGTTCTGGATAAACGGCGCGGTCGCGATCCCGAAATGGGTGGGAAGCCTCGCAGGAAGAATCCTCAGGAAGGGCTCTACAATGAGCTGCAGAAAGACAAAATGGCAGAGGCCTATTCTGAAATCGGCATGAAGGGCGAGCGCCGCAGAGGCAAAGGACACGACGGCCTGTACCAGGGCCTGTCTACAGCCACCAAGGACACCTATGACGCTCTCCACATGCAAGCCCTGCCACCAAGGTGA

Example 2 detection of expression of CCR1 and NKG2D ligands by tumor cells

First, after co-incubation of target cells with APC anti-human CCR1 Antibody (biolegend brand), expression of CCR1 was flow-assayed for U937, THP-1, HL60, KG1a, Jurkat, Daudi, RPMI8226, NCI-H929, Raji, and K562 cells. As shown in FIG. 4, U937, THP-1, RPMI8226 and NCI-H929 were CCR1 positive cells, and HL60, KG1a, Jurkat, Daudi, Raji and K562 were CCR1 negative cells.

Antigen expression of target cells K562, NCI-H929 and U937 was detected by anti-NKG2D ligand antibody (PE-anti-NKG2DL-Fc) (Acro brand), APC-anti-MICA (APC-anti-MICA) and PE-anti-ULBP2/5/6 (R & D systems brand). The results are shown in FIG. 5 and FIG. 6, wherein K562 cells are negatively expressed in CCR1 and are positively expressed in NKG2DL, MICA and ULBP 2/5/6; NCI-H929 cell CCR1 is strongly positively expressed, and NKG2DL, MICA and ULBP2/5/6 are negatively expressed; u937 cells are positively expressed by CCR1, positively expressed by NKG2DL and ULBP2/5/6, and negatively expressed by MICA.

TABLE 1 expression of CCR1 and NKG2D ligands (NKG2DL) in tumor target cells

	K562	NCI-H929	U937	Detection antibody used
					NKG2DL	++	-	+++	PE-NKG2D-Fc
MICA	+	-	-	APC-anti-MICA
					ULBP2/5/6	+	-	+++	PE-anti-ULBP2/5/6
CCR1	-	+++	+	anti-CCR1

Example 3 preparation of anti-CCR 1, NKG2D Dual-target CAR-T cells

(1) Isolation of PBMC

Collecting 50mL of peripheral blood; respectively adding 15mL of lymphocyte separation solution into 2 sterilized 50mL centrifuge tubes in an ultraclean workbench, slowly injecting 25-30 mL of peripheral blood into the centrifuge tube containing the lymphocyte separation solution, centrifuging the centrifuge tube at 700g for 20min at room temperature according to the use instruction of the lymphocyte separation solution, increasing the speed to 1, decreasing the speed to 2, and increasing the centrifugal time to 30min if the blood is stored for more than 2 hours;

after centrifugation, the blood is divided into 4 layers, which are composed of plasma (upper layer), mononuclear cells (layer 2) between the plasma and the separating medium, the separating medium (layer 3) and red blood cells (bottom layer), the mononuclear cells at the layer 2 are collected into a new centrifugal tube by a straw, 20mL of PBS is added to dilute the cell suspension, and 500g of the cell suspension is centrifuged for 10 min; removing supernatant, adding 20mL PBS to dilute cell suspension, mixing, counting 20 μ L cell suspension, and centrifuging the rest suspension 500g for 10 min.

(2) T cell proliferation

Suspending PBMC by adding 10mL of T activation medium, wherein the T activation medium comprises GT-T551H 3 medium, 2mL of inactivated autologous plasma, appropriate concentration of IL-2 and the like; counting 20 microliter of cell suspension; transferring the cell suspension into a cell culture bottle, and adjusting the cell density to (2-3) × 10 with a T-activated culture medium⁶Per mL, 37 ℃, 5% CO₂Culturing for 3-4 days in an incubator.

(3) Lentiviral packaging

The lentiviral expression vectors in the foregoing examples were each subjected to lentiviral packaging using a four-plasmid system, comprising the following specific steps: the four-plasmid system respectively expresses gag/pol, Rev and envelope plasmids required by packaging of a lentiviral vector, and pBG-NKG2D-CCR 1CAR and pBG-CCR1-NKG2D CAR vectors constructed by the invention; transient transfection is carried out on the plasmid for 293T cells, and the DNA content is 2 mug/mL; mixing the plasmid and PEI transfection reagent, adding into serum-free DMEM with a certain volume, standing for 15 minutes after mixing uniformly, adding the mixed solution into a T75 culture bottle paved with 293T cell cells, mixing uniformly and gently, and carrying out 5% CO treatment at 37 DEG C₂Culturing for 6h in a cell culture box; after 6h, the culture medium was replaced with fresh medium and the culture was continued, and 10mM sodium butyrate solution was added, and after 72 hours, culture supernatant of lentivirus was collected for purification and examination.

(4) Lentivirus infection and expansion culture

After 3-4 days of T cell activation, the virus containing the CAR gene is infected overnight; after infection, the T cell amplification medium (containing 10% serum, 500IU/mL IL-2, etc.) is replaced, and the cell density is adjusted to (1-2) x 10⁶Per mL; observing the growth state of the cells every 2-3 days; after the enlargement culture for 7 days, the culture flask was filled withTransferring the cell suspension into a culture bag, adding a T cell amplification culture medium, and continuously culturing in a 5% CO2 incubator at 37 ℃; cells were collected for detection by culturing until day 10.

Example 4 flow assay of anti-CCR 1, NKG2D Dual-target CAR-T expression

Expression of the prepared CCR1CAR-T, NKG2D-CCR1CAR-T, CCR1-NKG2D CAR-T cell surface CAR molecules was detected by flow cytometry, the T cell population was labeled with an APC-anti-CD3 antibody, and then the positive rate of CAR expression was detected with FITC-Protein L (Acrobiosystems, Inc.). Results as shown in figure 7, the lentiviral infection was continued for 3 days with a CAR positive rate of 25.2% for CCR1CAR-T cells; the CAR expression rates of NKG2D-CCR1CAR-T cells, CCR1-NKG2D CAR-T cells were 18.30% and 64.30%, respectively.

Example 5ELISA detection of IFN gamma and IL2 secretion when anti-CCR 1, NKG2D double-target CAR-T kill tumor target cells

Respectively co-incubating non-transduced T cells (TM), CCR1CAR-T, NKG2D-CCR1CAR-T, CCR1-NKG2D CAR-T cells and target cells U937-luc, THP-1-luc, Jurkat-luc, H929-luc and K562-luc, respectively, setting effective target ratios (E: T) to be 1:3 and 1:1, inoculating the cells to a black 96-well plate, adding a luciferase substrate (Promega, Bright-GloTMLuciferase Assay System) after co-incubating for 4-6H, taking 50 mu L of supernatant per well for detecting IFN-gamma and IL-2 by an enzyme-linked immunosorbent Assay, wherein the higher the fluorescence value represents the corresponding killing rate, and the higher the killing rate represents the stronger cell killing rate.

Results As shown in FIG. 8, CCR1CAR-T was effective in killing U937-luc, THP-1-luc and H929-luc cells positively expressed for CCR 1; but has no killing effect on Jurkat-luc and K562-luc cells which are negatively expressed by CCR 1. Two dual-target CAR-T (CCR1-NKG2D CAR-T and NKG2D-CCR1CAR-T) cells were able to kill U937-luc, THP-1-luc and H929-luc cells; in addition, it also kills CCR 1-negative K562-luc cells (expressing NKG2D ligand). The result shows that the CCR1 and NKG2D double-target CAR-T cells also have a remarkable killing effect on CCR1 negative target cells, and can effectively eliminate CCR1 and/or NKG2D ligand positive tumor cells, so that antigen escape is reduced.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Guangzhou Bai-and-Gen-Tech Co Ltd

<120> chimeric antigen receptor targeting CCR1 and NKG2D ligands and uses thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Val Val Met Thr Gln Thr Pro Arg Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Leu Ile His Ser

20 25 30

Asn Gly Ile Thr Phe Leu His Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Gly

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 2

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Ala Gly Thr Thr Val Tyr Asn Ala Ala Phe Ile

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asp Ser Lys Ser Gln Val Phe Phe

65 70 75 80

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

85 90 95

Lys Asp Gly Ser Arg Tyr Tyr Thr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 3

<211> 135

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ile Cys Met Gln Arg Thr Val

130 135

<210> 4

<211> 622

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asp Val Val Met Thr Gln Thr Pro Arg Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Leu Ile His Ser

20 25 30

Asn Gly Ile Thr Phe Leu His Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Gly

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser

130 135 140

Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Asn Tyr Gly

145 150 155 160

Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly

165 170 175

Val Ile Trp Ser Ala Gly Thr Thr Val Tyr Asn Ala Ala Phe Ile Ser

180 185 190

Arg Leu Ser Ile Ser Lys Asp Asp Ser Lys Ser Gln Val Phe Phe Lys

195 200 205

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

210 215 220

Asp Gly Ser Arg Tyr Tyr Thr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly

245 250 255

Ser Gly Glu Gly Ser Thr Lys Gly Leu Phe Asn Gln Glu Val Gln Ile

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

<210> 5

<211> 622

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ile Cys Met Gln Arg Thr Val Gly Ser Thr Ser Gly Ser Gly Lys Pro

130 135 140

Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Val Val Met Thr Gln Thr

145 150 155 160

Pro Arg Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys

165 170 175

Arg Ser Arg Gln Ser Leu Ile His Ser Asn Gly Ile Thr Phe Leu His

180 185 190

Trp Tyr Leu Gln Lys Ala Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys

195 200 205

Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp

225 230 235 240

Leu Gly Val Tyr Phe Cys Ser Gln Gly Thr His Val Pro Pro Thr Phe

245 250 255

Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser Gly

275 280 285

Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val

290 295 300

Ser Gly Phe Ser Leu Asn Asn Tyr Gly Val His Trp Val Arg Gln Pro

305 310 315 320

Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Ala Gly Thr

325 330 335

Thr Val Tyr Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile Ser Lys Asp

340 345 350

Asp Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ala Gly

355 360 365

Asp Thr Ala Ile Tyr Tyr Cys Ala Lys Asp Gly Ser Arg Tyr Tyr Thr

370 375 380

Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

<210> 6

<211> 1869

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatgtggtca tgactcagac tccaaggagc ctgcccgtgt ccctcggaga tcaggcaagc 60

atcagctgcc ggagcaggca gagtctgatt cacagtaatg gaataacttt tctccactgg 120

tacctgcaga aggccggtca gagtcctaag ctcctgatat acaaagtgag taacagattc 180

agtggagtgc ctgacagatt ctcaggatcc ggctcaggga ccgacttcac cctgcggatt 240

agcagagtgg aggcagaaga cctcggcgtc tacttttgta gtcaagggac tcacgtgcca 300

cctacatttg gtggcgggac taaactggaa atcaagggcg gaggcggttc aggcggaggc 360

ggctcaggcg gtggcggttc acaagtgcag ctcaagcagt cagggccagg cctcgtccag 420

ccaagtcaaa gtctgagcat tacctgcact gtgagtggtt ttagcctgaa caattatgga 480

gtgcattggg ttaggcagcc acctggaaag ggcctggaat ggctgggcgt gatttggtct 540

gccggcacta ctgtgtacaa cgccgcattc atctccagac tcagcatcag taaagacgac 600

tccaaatccc aggtgttctt caaaatgaac tcactgcaag ctggagatac agcaatctac 660

tactgcgcta aagacggctc tcggtattac actgcaatgg actactgggg gcaagggaca 720

tccgtgaccg tgagctcagg atcaaccagc ggaagcggga agcctggttc cggagaagga 780

tcaactaaag gcctctttaa ccaggaggtg caaattccac tgactgaatc ctattgcggc 840

ccttgtccca agaactggat ttgttataag aacaactgtt atcaattctt cgatgagtct 900

aagaactggt atgaaagcca agcatcctgt atgtcccaga acgcttcact cctgaaggtc 960

tacagcaaag aagatcagga cctcctcaag ctggtgaaat catatcactg gatgggactg 1020

gtgcacatcc ctacaaacgg ctcatggcag tgggaggacg gctccatcct gagcccaaat 1080

ctgctcacaa tcattgaaat gcagaagggt gattgcgctc tctacgcaag ctcttttaaa 1140

ggctacatag agaactgctc cactcccaat acatacattt gtatgcagcg cactgtgact 1200

acgacccctg caccgcggcc gcctactcct gcacctacaa tcgcaagtca gccactgagt 1260

ctcagacccg aagcatgccg ccctgctgca ggcggagctg tccatacacg cggactggac 1320

tttgcatgcg atatatacat ctgggcacca ctggccggca cttgcggcgt gctgctcctg 1380

tccctcgtga ttaccctgta ctgcaaacgc ggcaggaaga agctcctgta tatctttaaa 1440

cagcccttca tgaggccagt gcagaccact caagaggaag acggttgtag ctgccggttt 1500

cccgaggaag aagagggagg ctgcgagctc cgcgtgaagt tctcccgctc agccgatgca 1560

cccgcctatc agcaagggca gaaccagctg tacaatgagc tcaacctggg aagaagggag 1620

gaatatgacg ttctggataa acggcgcggt cgcgatcccg aaatgggtgg gaagcctcgc 1680

aggaagaatc ctcaggaagg gctctacaat gagctgcaga aagacaaaat ggcagaggcc 1740

tattctgaaa tcggcatgaa gggcgagcgc cgcagaggca aaggacacga cggcctgtac 1800

cagggcctgt ctacagccac caaggacacc tatgacgctc tccacatgca agccctgcca 1860

ccaaggtga 1869

<210> 7

<211> 1869

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctctttaacc aggaggtgca aattccactg actgaatcct attgcggccc ttgtcccaag 60

aactggattt gttataagaa caactgttat caattcttcg atgagtctaa gaactggtat 120

gaaagccaag catcctgtat gtcccagaac gcttcactcc tgaaggtcta cagcaaagaa 180

gatcaggacc tcctcaagct ggtgaaatca tatcactgga tgggactggt gcacatccct 240

acaaacggct catggcagtg ggaggacggc tccatcctga gcccaaatct gctcacaatc 300

attgaaatgc agaagggtga ttgcgctctc tacgcaagct cttttaaagg ctacatagag 360

aactgctcca ctcccaatac atacatttgt atgcagcgca ctgtgggatc aaccagcgga 420

agcgggaagc ctggttccgg agaaggatca actaaaggcg atgtggtcat gactcagact 480

ccaaggagcc tgcccgtgtc cctcggagat caggcaagca tcagctgccg gagcaggcag 540

agtctgattc acagtaatgg aataactttt ctccactggt acctgcagaa ggccggtcag 600

agtcctaagc tcctgatata caaagtgagt aacagattca gtggagtgcc tgacagattc 660

tcaggatccg gctcagggac cgacttcacc ctgcggatta gcagagtgga ggcagaagac 720

ctcggcgtct acttttgtag tcaagggact cacgtgccac ctacatttgg tggcgggact 780

aaactggaaa tcaagggcgg aggcggttca ggcggaggcg gctcaggcgg tggcggttca 840

caagtgcagc tcaagcagtc agggccaggc ctcgtccagc caagtcaaag tctgagcatt 900

acctgcactg tgagtggttt tagcctgaac aattatggag tgcattgggt taggcagcca 960

cctggaaagg gcctggaatg gctgggcgtg atttggtctg ccggcactac tgtgtacaac 1020

gccgcattca tctccagact cagcatcagt aaagacgact ccaaatccca ggtgttcttc 1080

aaaatgaact cactgcaagc tggagataca gcaatctact actgcgctaa agacggctct 1140

cggtattaca ctgcaatgga ctactggggg caagggacat ccgtgaccgt gagctcaact 1200

acgacccctg caccgcggcc gcctactcct gcacctacaa tcgcaagtca gccactgagt 1260

ctcagacccg aagcatgccg ccctgctgca ggcggagctg tccatacacg cggactggac 1320

tttgcatgcg atatatacat ctgggcacca ctggccggca cttgcggcgt gctgctcctg 1380

tccctcgtga ttaccctgta ctgcaaacgc ggcaggaaga agctcctgta tatctttaaa 1440

cagcccttca tgaggccagt gcagaccact caagaggaag acggttgtag ctgccggttt 1500

cccgaggaag aagagggagg ctgcgagctc cgcgtgaagt tctcccgctc agccgatgca 1560

cccgcctatc agcaagggca gaaccagctg tacaatgagc tcaacctggg aagaagggag 1620

gaatatgacg ttctggataa acggcgcggt cgcgatcccg aaatgggtgg gaagcctcgc 1680

aggaagaatc ctcaggaagg gctctacaat gagctgcaga aagacaaaat ggcagaggcc 1740

tattctgaaa tcggcatgaa gggcgagcgc cgcagaggca aaggacacga cggcctgtac 1800

cagggcctgt ctacagccac caaggacacc tatgacgctc tccacatgca agccctgcca 1860

ccaaggtga 1869

Claims

1. A chimeric antigen receptor targeting CCR1 and NKG2D ligands, comprising an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain connected in sequence from amino terminus to carboxy terminus; the antigen binding domains include anti-CCR 1 single chain antibodies and NKG 2D.

2. The chimeric antigen receptor according to claim 1, wherein the anti-CCR 1 single chain antibody comprises an anti-CCR 1 antibody light chain variable region and an anti-CCR 1 antibody heavy chain variable region, wherein the amino acid sequence of the anti-CCR 1 antibody light chain variable region is as set forth in SEQ ID NO: 1, the amino acid sequence of the heavy chain variable region of the anti-CCR 1 antibody is shown as SEQ ID NO: 2, respectively.

3. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of NKG2D is as set forth in SEQ ID NO: 3, respectively.

4. The chimeric antigen receptor according to claim 1, wherein the hinge region is selected from the hinge region of at least one of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD 154; and/or

5. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is as set forth in SEQ ID NO: 4 or SEQ ID NO: 5, respectively.

6. The chimeric antigen receptor according to any one of claims 1 to 5, wherein the amino terminus of the chimeric antigen receptor further comprises a signal peptide.

7. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1 to 6.

8. The nucleic acid of claim 7, wherein the nucleotide sequence of the nucleic acid is as set forth in SEQ ID NO: 6 or SEQ ID NO: shown at 7.

9. A recombinant vector comprising the nucleic acid of claim 7 or 8.

10. A CAR-T cell comprising the nucleic acid of claim 7 or 8 or transformed with the recombinant vector of claim 9.

11. Use of the chimeric antigen receptor of any one of claims 1 to 6, the nucleic acid of claim 7 or 8, the recombinant vector of claim 9, or the CAR-T cell of claim 10 in the preparation of a medicament for the treatment of cancer.

12. A pharmaceutical composition comprising the CAR-T cell of claim 10, and a pharmaceutically acceptable excipient.