CN114106200B

CN114106200B - Chimeric antigen receptor targeting CCR1 and application thereof

Info

Publication number: CN114106200B
Application number: CN202111394707.1A
Authority: CN
Inventors: 李光超; 罗敏; 王学俊; 刘劲松; 周兆
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-07-15
Anticipated expiration: 2041-11-23
Also published as: CN114106200A

Abstract

The invention relates to a chimeric antigen receptor targeting CCR1 and application thereof, wherein the chimeric antigen receptor comprises a single-chain antibody, 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 and target CCR 1; the single chain antibody targeting CCR1 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 shown as 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. The CAR-T cell constructed by the chimeric antigen receptor targeting CCR1 has a specific targeting effect on CCR1 positive tumor cells, has an obvious in-vivo and in-vitro killing effect, can effectively remove CCR1 positive tumor cells, and has important significance in the field of tumor treatment.

Description

Chimeric antigen receptor targeting CCR1 and uses thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a chimeric antigen receptor targeting CCR1 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, at present, chimeric antigen receptors with better tumor cell killing effect aiming at different tumor targets are still relatively lacking.

Disclosure of Invention

Based on this, there is a need to provide a chimeric antigen receptor that can target CCR1 and is highly effective in killing tumor cells.

A chimeric antigen receptor targeting CCR1, comprising a single chain antibody targeting CCR1, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain connected in sequence from amino terminus to carboxy terminus; the single-chain antibody targeting CCR1 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 shown 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.

The invention constructs a chimeric antigen receptor targeting CCR1, which comprises a single-chain antibody targeting CCR1, a hinge region, a transmembrane domain, a costimulatory domain and a signal transduction domain, wherein the amino acid sequences of a light chain variable region and a heavy chain variable region of the single-chain antibody targeting CCR1 are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The CAR-T cell constructed by the chimeric antigen receptor targeting CCR1 has a specific targeting effect on CCR1 positive tumor cells, and experiments prove that the T cell expressing the chimeric antigen receptor targeting CCR1 has a remarkable in-vivo and in-vitro killing effect, can effectively remove CCR1 positive tumor cells, and has important significance in the field of tumor treatment.

In one embodiment, 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

The transmembrane domain is selected from the transmembrane regions of at least one of the following proteins: a, β or ζ chain of a T cell receptor, CD epsilon, CD134, CD137, CD154, KIRDS, OX, CD, ICOS, GITR, CD, BAFFR, HVEM, SLAMF, NKp, CD160, CD, IL2 β, IL2 γ, IL7 α, 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, CEM, CRACAM, Ly, PSGL, CD100, SLAMF, SLAMB, BLAME, PAG, PLG, NKG, NKP, NKG2 bp, NKP, NKG2, NKP, NKG2 bp; 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-associated 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 ε, 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, 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: 5, respectively.

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 a chimeric antigen receptor CCR1 CAR in example 1 of the present invention;

FIG. 2 is a schematic structural diagram of an expression vector for CCR1 CAR in example 1 of the present invention;

FIG. 3 shows the result of detecting CCR1 expression by tumor cells in example 2 of the present invention;

FIG. 4 is flow assay CAR-T cell CAR expression data in example 4 of the invention;

FIG. 5 shows the results of the in vitro killing assay for CAR-T cells by ELISA in example 5 of the present invention;

FIG. 6 is a schematic diagram of the experimental design of mouse in example 6 of the present invention;

FIG. 7 is a graph of tumor growth in vivo in H929-luc tumor-bearing mice of example 6 of the present invention after treatment with various sets of CAR-T cells;

FIG. 8 is a graph of survival data for H929-luc tumor bearing mice of example 6 of the present invention before and after treatment with various groups of CAR-T.

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.

Interpretation of terms

"vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection such that the genetic material element it carries is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda bacteriophage or M13 bacteriophage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV 40).

"T cell" refers to T lymphocyte (T lymphocyte), which is a bone marrow-derived lymphoid stem cell that, after differentiation and maturation in the thymus, is distributed to immune organs and tissues throughout the body via lymph and blood circulation to exert an immune function. Mature T cells distribute to thymus dependent area of peripheral immune organ through blood stream to colonize, and can be recycled through lymphatic vessel, peripheral blood and tissue fluid, etc., to exert functions of cellular immunity and immunoregulation, etc. The recycling of T cells facilitates the widespread exposure to antigenic substances entering the body, enhancing the immune response and preserving immunological memory for a longer period of time. There are many different markers on the cell membrane of T cells, mainly surface antigens and surface receptors, which are giant protein molecules bound to the cell membrane.

The "single-chain antibody" is formed by connecting an antibody heavy chain variable domain (VH) and a light chain variable domain (VL) through a flexible short peptide (linker) consisting of 10 to 25 amino acids, and is the smallest recombinant antibody form (about 27 kDa). Essentially, scFv is a fusion protein and retains the specificity of the original immunoglobulin for the antigen. The scFv has small molecular size, brings the advantages of strong penetrating power in tumors, rapid degradation in blood, small negative feedback in human bodies and the like, and lays a foundation for clinical application of the scFv.

The chimeric antigen receptor targeting CCR1 of one embodiment of the present invention comprises a single chain antibody targeting CCR1, a hinge region, a transmembrane domain, a costimulatory domain, and a signal transduction domain connected in sequence from the amino terminus to the carboxy terminus; the single-chain antibody targeting CCR1 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 shown as 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.

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 having a seven-fold transmembrane structure. The ligands typically 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 CCL 16. CCL1 and its ligand-mediated signal transduction are key to the recruitment of effective immune cells to sites of inflammation, and play important roles 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 in the Bone Marrow (BM), malignant proliferation and induction of osteolysis. CCR1 is critical for 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) disease. 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 regulate osteoclast/osteoblast balance by inducing osteoclast differentiation and inhibiting osteoblast function, accelerating disease progression. The data show that after treatment with CCR1 specific antagonist drug, the phenomena 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 way 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.

In view of the fact that CCR1 participates in the occurrence and development processes of various blood cancers and solid tumors and can be used as a good target for treating various cancers, the invention innovatively constructs a specific chimeric antigen receptor aiming at CCR1, CAR-T cells modified by the chimeric antigen receptor can specifically and efficiently kill tumor cells in vitro and in a mouse body, and the CAR-T cells effectively eliminate CCR1 positive tumor cells, and have important significance in the field of tumor treatment.

In one particular example, the variable region of the light chain of the anti-CCR 1 antibody and the variable region of the heavy chain of the anti-CCR 1 antibody are linked by a linker peptide. Alternatively, the amino acid sequence of the linker peptide is as set forth in SEQ ID NO: 3, respectively.

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, CEM, CRACAM, Ly, PSGL, CD100, SLAMF, SLAMA, BLAME, PAG, PLG, NKG, NKP, NKG2, NKP 2, NKP, NKG, NKP, NKG, and IL2 beta.

In a specific example, the above-described 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-associated 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 ε, 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 particular example, the amino acid sequence of the chimeric antigen receptor is as set forth in SEQ ID NO: 4, 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 which encodes a chimeric antigen receptor targeting CCR1 as described above.

In one specific example, the nucleotide sequence of the nucleic acid is as set forth in SEQ ID NO: 5, respectively. 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 above 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 a CD3+ T cell, a CD3+ CD4+ T cell, or a CD3+ CD8+ T cell.

The pharmaceutical composition of an 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 one 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 one particular example, the antioxidant is selected from one or more of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, dibutylhydroxytoluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite.

The method for preparing 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 structure of the chimeric antigen receptor CCR1 CAR constructed in this example is shown in figure 1. Based on the amino acid sequence of the chimeric antigen receptor, the whole gene synthesis was performed in cantonese biotechnology limited, guangzhou, and constructed into a lentiviral expression vector, the vector structure of CCR1 CAR is shown in fig. 2, and the sequence information is shown below.

CCR1 CAR amino acid sequence:

MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCSASQGIRNYLNWYQQKPGKVPKLLIVYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYGKLPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDAWMDWVRQAPGKGLEWVGEIRSKVNNHETYYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARNDYFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

CCR1 CAR nucleotide sequence:

ATGGATATGAGAGTGCCTGCTCAACTGCTGGGTCTGCTCCTGCTGTGGCTGCGCGGTGCTCGGTGCGACATCCAGATGACTCAGTCTCCAAGCAGCCTGTCCGCATCAGTCGGCGATAGGGTCACCATCACATGCTCAGCATCTCAGGGAATTAGAAACTATCTGAACTGGTATCAGCAGAAACCCGGCAAAGTGCCTAAACTGCTCATCGTGTACACCTCCCGGCTGCACTCCGGAGTTCCCAGTAGATTTTCAGGCTCCGGCAGCGGAACTGACTTTACACTGACCATTAGCTCACTCCAACCCGAGGATGTTGCTACCTACTACTGCCAGCAATACGGCAAGCTGCCCTATACATTTGGCCAGGGAACAAAGCTGGAGATCAAGGGAGGTGGAGGCTCTGGCGGCGGAGGCAGCGGAGGAGGAGGCTCCGAAGTTCAACTCGTCGAATCAGGAGGAGGACTGGTGCAGCCCGGAGGAAGTCTCAGACTGAGTTGTGCTGCCAGCGGGTTCACTTTTAGCGATGCTTGGATGGATTGGGTGAGGCAAGCACCTGGCAAGGGCCTCGAATGGGTTGGCGAGATCAGGTCTAAAGTGAATAACCATGAGACTTACTACGCTGAGAGCGTTAAAGGGAGATTCACCATTAGTAGAGATGACTCCAAGAATAGCCTGTATCTCCAAATGAACTCTCTCAAGACTGAGGATACAGCAGTCTACTACTGTGCACGGAATGACTACTTTGACTACTGGGGTCAAGGAACAACCGTGACAGTGTCATCTACTACGACCCCTGCACCGCGGCCGCCTACTCCTGCACCTACAATCGCAAGTCAGCCACTGAGTCTCAGACCCGAAGCATGCCGCCCTGCTGCAGGCGGAGCTGTCCATACACGCGGACTGGACTTTGCATGCGATATATACATCTGGGCACCACTGGCCGGCACTTGCGGCGTGCTGCTCCTGTCCCTCGTGATTACCCTGTACTGCAAACGCGGCAGGAAGAAGCTCCTGTATATCTTTAAACAGCCCTTCATGAGGCCAGTGCAGACCACTCAAGAGGAAGACGGTTGTAGCTGCCGGTTTCCCGAGGAAGAAGAGGGAGGCTGCGAGCTCCGCGTGAAGTTCTCCCGCTCAGCCGATGCACCCGCCTATCAGCAAGGGCAGAACCAGCTGTACAATGAGCTCAACCTGGGAAGAAGGGAGGAATATGACGTTCTGGATAAACGGCGCGGTCGCGATCCCGAAATGGGTGGGAAGCCTCGCAGGAAGAATCCTCAGGAAGGGCTCTACAATGAGCTGCAGAAAGACAAAATGGCAGAGGCCTATTCTGAAATCGGCATGAAGGGCGAGCGCCGCAGAGGCAAAGGACACGACGGCCTGTACCAGGGCCTGTCTACAGCCACCAAGGACACCTATGACGCTCTCCACATGCAAGCCCTGCCACCAAGGTGA

example 2 flow assay of tumor cell surface expression of CCR1

After co-incubation of target cells with the APC anti-human CCR1 Antibody Antibody (biolegend brand), expression of CCR1 of U937, THP-1, HL60, KG1a, Jurkat, Daudi, RPMI8226, NCI-H929, Raji and K562 cells was flow-assayed. As shown in the results of FIG. 3, U937, THP-1, RPMI8226 and NCI-H929 were CCR1 positive cells, and HL60, KG1a, Jurkat, Daudi, Raji and K562 were CCR1 negative cells.

Example 3 preparation of anti-CCR 1 CAR-T cells

(1) Isolation of PBMC

Collecting 50mL of peripheral blood; respectively adding 15mL of lymphocyte separation solution into 2 50mL sterile centrifuge tubes in an ultra-clean 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 centrifuging 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 the supernatant, adding 20mL PBS to dilute the cell suspension, mixing, counting 20 μ L cell suspension, and centrifuging the rest suspension 500g for 10min for use.

(2) T cell proliferation

Resuspend PBMC by adding 10mL of T-activated medium comprising GT-T551H 3 medium, 2mL of inactivated autologous plasma, appropriate concentration of IL-2, etc.; counting 20 microliter of cell suspension; transferring the cell suspension into a cell culture bottle, and adjusting the cell density to (2-3) x 10 by using a T activation culture medium⁶Per mL at 37 deg.C,5％CO₂Culturing for 3-4 days in an incubator.

(3) Lentiviral packaging

The lentiviral expression vectors in the examples were each subjected to lentiviral packaging using a four plasmid system, the specific steps of which were as follows: the four-plasmid system respectively expresses gag/pol, Rev and envelope plasmids required by packaging of a lentiviral vector and the pBG-CCR1-CAR vector constructed by the invention: transient transfection is carried out on the plasmid on 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 cells, mixing uniformly and gently, and performing 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) Lentiviral 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 7 days of expansion culture, the cell suspension in the flask was transferred to a culture bag and added with T cell expansion medium at 37 ℃ with 5% CO₂Continuously culturing in an incubator; cells were collected for detection by culturing until day 10.

Example 4 flow assay for anti-CCR 1 CAR-T expression

The expression of the prepared anti-CCR 1 CAR-T cell surface CAR molecules was detected by flow cytometry, the T cell population was labeled with APC-anti-CD3 antibody, and the positive rate of CAR expression was then detected by FITC-Protein L (Acrobiosystems). Results as shown in figure 4, the CAR expression rates of anti-CCR 1 CAR-T cells were 51.40% and 38.99% for lentiviral infection continued in culture for 7 to 9 days, respectively.

Example 5 anti-CCR 1 CAR-T cell killing Capacity assessment

Respectively incubating anti-CCR 1 CAR-T cells with target cells THP-1-luc and K562-luc, and setting effective target ratio (E: T) to 1:1 and3:1, inoculating to a black 96-well plate, incubating for 4-6 h, and adding luciferase substrate (Promega, Bright-Glo)^TMLuciferase Assay System), taking 50 mu L of supernatant liquid from each well, and detecting IFN-gamma and IL-2 by enzyme-linked immunosorbent Assay, wherein the fluorescence value represents the corresponding killing rate, and the higher the value, the stronger the cell killing is.

The results are shown in FIG. 5, anti-CCR 1 CAR-T cells were able to kill CCR1 expressing THP-1 tumor cells strongly, producing large amounts of IFN-. gamma.and IL-2. Meanwhile, the anti-CCR 1 CAR-T cell does not obviously kill a K562 cell, which shows that the killing of the anti-CCR 1 CAR-T cell has better specificity.

Example 6 testing the in vivo efficacy of anti-CCR 1 CAR-T mice

As shown in FIG. 6, after obtaining H929-luc (luciferase-labeled human myeloma cell NCI-H929 cell) cells, using female B-NDG mice, based on the body weight measured 1 day (D-1) before administration, animals were divided into 3 groups at random by body weight segments using a computer system, and 1X 10 cells were inoculated into the tail vein of each group⁶H929-luc tumor cells. On day 2 after tumor cell inoculation, corresponding CAR-T cells (anti-CCR 1 CAR-T cells), positive control (anti-BCMA CAR-T) and non-transduced T cells (Mock T control), 3 × 10⁶Total cells/mouse. On

days

3, 9, 17, and 23 after receiving treatment, in vivo imaging was performed by intraperitoneal injection of the luminogenic substrate D-luciferin.

Results As shown in FIGS. 7 and 8, H929-luc tumor growth was significantly inhibited in mice 23 days after receiving anti-CCR 1 CAR-T cell therapy.

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 various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<210> 2

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Glu Ile Arg Ser Lys Val Asn Asn His Glu Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

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

85 90 95

Tyr Cys Ala Arg Asn Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 3

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 4

<211> 462

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Val Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Lys Leu Pro Tyr

85 90 95

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

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala Trp Met Asp Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Glu Ile Arg Ser Lys

165 170 175

Val Asn Asn His Glu Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn

195 200 205

Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Asp

210 215 220

Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Thr

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

<210> 5

<211> 1389

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gacatccaga tgactcagtc tccaagcagc ctgtccgcat cagtcggcga tagggtcacc 60

atcacatgct cagcatctca gggaattaga aactatctga actggtatca gcagaaaccc 120

ggcaaagtgc ctaaactgct catcgtgtac acctcccggc tgcactccgg agttcccagt 180

agattttcag gctccggcag cggaactgac tttacactga ccattagctc actccaaccc 240

gaggatgttg ctacctacta ctgccagcaa tacggcaagc tgccctatac atttggccag 300

ggaacaaagc tggagatcaa gggaggtgga ggctctggcg gcggaggcag cggaggagga 360

ggctccgaag ttcaactcgt cgaatcagga ggaggactgg tgcagcccgg aggaagtctc 420

agactgagtt gtgctgccag cgggttcact tttagcgatg cttggatgga ttgggtgagg 480

caagcacctg gcaagggcct cgaatgggtt ggcgagatca ggtctaaagt gaataaccat 540

gagacttact acgctgagag cgttaaaggg agattcacca ttagtagaga tgactccaag 600

aatagcctgt atctccaaat gaactctctc aagactgagg atacagcagt ctactactgt 660

gcacggaatg actactttga ctactggggt caaggaacaa ccgtgacagt gtcatctact 720

acgacccctg caccgcggcc gcctactcct gcacctacaa tcgcaagtca gccactgagt 780

ctcagacccg aagcatgccg ccctgctgca ggcggagctg tccatacacg cggactggac 840

tttgcatgcg atatatacat ctgggcacca ctggccggca cttgcggcgt gctgctcctg 900

tccctcgtga ttaccctgta ctgcaaacgc ggcaggaaga agctcctgta tatctttaaa 960

cagcccttca tgaggccagt gcagaccact caagaggaag acggttgtag ctgccggttt 1020

cccgaggaag aagagggagg ctgcgagctc cgcgtgaagt tctcccgctc agccgatgca 1080

cccgcctatc agcaagggca gaaccagctg tacaatgagc tcaacctggg aagaagggag 1140

gaatatgacg ttctggataa acggcgcggt cgcgatcccg aaatgggtgg gaagcctcgc 1200

aggaagaatc ctcaggaagg gctctacaat gagctgcaga aagacaaaat ggcagaggcc 1260

tattctgaaa tcggcatgaa gggcgagcgc cgcagaggca aaggacacga cggcctgtac 1320

cagggcctgt ctacagccac caaggacacc tatgacgctc tccacatgca agccctgcca 1380

ccaaggtga 1389

Claims

1. A chimeric antigen receptor targeting CCR1, comprising a CCR1 targeting single chain antibody, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain connected in sequence from an amino terminus to a carboxy terminus; the single-chain antibody targeting CCR1 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 shown as 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.

2. 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

The transmembrane domain is selected from 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.

3. 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, respectively.

4. The chimeric antigen receptor according to any one of claims 1 to 3, wherein the amino terminus of the chimeric antigen receptor further comprises a signal peptide.

5. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1 to 4.

6. The nucleic acid of claim 5, wherein the nucleotide sequence of the nucleic acid is as set forth in SEQ ID NO: 5, respectively.

7. A recombinant vector comprising the nucleic acid of claim 5 or 6.

8. A CAR-T cell comprising the nucleic acid of claim 5 or 6 or transformed with the recombinant vector of claim 7.

9. Use of the chimeric antigen receptor of any one of claims 1 to 4, the nucleic acid of claim 5 or 6, the recombinant vector of claim 7, or the CAR-T cell of claim 8 in the manufacture of a medicament for treating multiple myeloma or acute myeloid leukemia.

10. A pharmaceutical composition comprising the CAR-T cell of claim 8, and a pharmaceutically acceptable excipient.