CN114106199A

CN114106199A - Chimeric antigen receptor targeting ADGRE2 and application thereof

Info

Publication number: CN114106199A
Application number: CN202111375226.6A
Authority: CN
Inventors: 李光超; 罗敏; 周兆
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-03-01
Anticipated expiration: 2041-11-19
Also published as: CN114106199B

Abstract

The invention relates to a chimeric antigen receptor targeting ADGRE2 and application thereof, comprising a single-chain antibody, a hinge region, a transmembrane domain, a costimulation domain and a signal transduction domain which are sequentially connected from an amino terminal to a carboxyl terminal and target ADGRE 2; the amino acid sequences of the light chain variable region and the heavy chain variable region of the single-chain antibody targeting ADGRE2 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The chimeric antigen receptor T cell of the target ADGRE2 constructed by the chimeric antigen receptor can specifically kill ADGRE2 positive tumor cells, thereby providing an effective cell treatment selection for treating ADGRE2 positive acute myeloid leukemia.

Description

Chimeric antigen receptor targeting ADGRE2 and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to an ADGRE 2-targeted chimeric antigen receptor and application thereof.

Background

Acute Myeloid Leukemia (AML) is the most common leukemia in adults, and the second most common leukemia in children. Standard chemotherapy-induced regimens have not changed substantially over the past few decades, with the majority of patients initially responding to chemotherapy and achieving a state of Complete Remission (CR), but only a very small percentage of people survive for a long time because chemotherapy resistance leads to disease recurrence. Studies have shown that patients who have never achieved disease remission or relapse within six months after CR are less likely to respond to any treatment.

In recent years, chimeric antigen receptor T cell (CAR T) therapy has shown very good efficacy in tumor therapy, especially CAR T cell therapy against CD 19. An ideal target for CARs should be highly expressed in tumor cells, especially tumor stem cells, and in most patients. For acute myeloid leukemia, multiple targets have been found to be useful for CAR T therapy, such as CD33, CD123, CLEC12A, LeY, CD70, CCR1, CD44v6, CD44, and CD 38. In addition, Fabiana Perna et al have been rigorously screened to obtain four potential therapeutic AML cell targets: ADGRE2, CCR1, CD70, and LILRB 2. The study found that ADGRE2 was highly expressed in AML cells and was detectable in 93% of patients; however, the protein is only expressed very little in the intestine, ovary and spleen. ADGRE2 is therefore a very promising target for cell therapy in AML.

Disclosure of Invention

Based on this, there is a need to provide a chimeric antigen receptor that can specifically kill ADGRE2 positive tumor cells.

A chimeric antigen receptor targeting ADGRE2, comprising a single chain antibody targeting ADGRE2, 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 amino acid sequences of the light chain variable region and the heavy chain variable region of the single-chain antibody targeting ADGRE2 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The invention constructs an ADGRE 2-targeted chimeric antigen receptor, which comprises a single-chain antibody, a hinge region, a transmembrane domain, a costimulatory domain and a signal transduction domain which are sequentially connected and targeted to ADGRE 2. The chimeric antigen receptor T cell of the target ADGRE2 constructed by the chimeric antigen receptor can specifically kill ADGRE2 positive tumor cells, thereby providing an effective cell treatment selection for treating ADGRE2 positive acute myeloid leukemia. ADGRE2 is also known as EMR2, and belongs to one of the Epidermal Growth Factor (EGF) -TM7 family of proteins, which also includes ADGRE1, F4/80 and ADGRE 5. Similar to AGDRE5, ADGRE2 also possesses a calcium binding EGF domain; however, unlike ADGRE5, ADGRE5 is widely expressed in a variety of cells, while ADGRE2 is restricted to monocytes/macrophages and granulocytes, and has limited expression in activated T cells and B cells, which is very suitable as a target for cell therapy for the treatment of acute myeloid leukemia.

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: 3, 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: 4, 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 the application of the chimeric antigen receptor, the nucleic acid, the recombinant vector or the CAR-T cell in preparing a medicament for treating acute myeloid leukemia.

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 view of a chimeric antigen receptor according to example 1 of the present invention;

FIG. 2 shows the data of ADGRE2-CAR T cell expansion assay in example 3 of the present invention;

FIG. 3 is data of ADGRE2-CAR T cells in example 4 of the present invention showing the expression of chimeric antigen receptors of Day3 and Day 11;

FIG. 4 is data of in vitro killing assay of target cells by ADGRE2-CAR T cells in example 5 of the present invention;

FIG. 5 is the data of ADGRE2-CAR T cell in example 6 of the present invention detecting the secretion of the cytokine IFN-gamma when killing target cells.

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, and the genetic material elements carried thereby are 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 phage or M13 phage, 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), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40).

"T cell" refers to a T lymphocyte (T lymphocyte) which is a bone marrow-derived lymphoid stem cell, and which is differentiated and matured in the thymus, and then distributed to immune organs and tissues throughout the body through 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 wide exposure to antigenic substances entering the body, enhances the immune response and maintains the immunological memory for a longer period. T cells have many different markers on their cell membranes, 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) by a flexible short peptide (linker) consisting of 10 to 25 amino acids, and is the smallest recombinant antibody (about 27 kDa). Essentially, scFv is a fusion protein and retains the specificity of the original immunoglobulin for the antigen. The small molecular size of scFv brings the advantages of strong penetrating power in tumor, rapid degradation in blood, small negative feedback in human body and the like, which lays a foundation for the clinical application of scFv.

"MOI" (Multiplicity of Infection) refers to the proportion of cells infected by the virus. The higher the infection efficiency of the virus and the expression level of the target protein, the lower the relative cell toxicity. For non-dividing cells, such as primary cells, the infection efficiency is low, and the MOI value may reach 200-300. Suitable MOIs can be selected for experiments by comparing the results of infection of cells with different MOIs (e.g., 0, 1, 5, 10, 50, 100, etc.): the cultured cells can be inoculated to a 96-well plate, then negative control viruses carrying reporter genes are used for infecting the cells according to different MOI value ratios, and MOI value exploration is carried out.

The chimeric antigen receptor targeting ADGRE2, provided by the embodiment of the invention, comprises a single-chain antibody targeting ADGRE2, 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 amino acid sequences of the light chain variable region and the heavy chain variable region of the single-chain antibody targeting ADGRE2 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2, 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, 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: 3, 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 of an embodiment of the invention that encodes a chimeric antigen receptor that targets ADGRE2 as described above.

In one specific example, the nucleotide sequence of the nucleic acid is as set forth in SEQ ID NO: 4, 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 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.

In one particular example, RetroNectin may also be used to increase the efficiency of infection of T cells by the virus at the time of infection.

In one specific example, the preparation method comprises the following steps:

peripheral blood mononuclear cells were sorted into CD3+ T cells using CD3 sorting magnetic beads, and cultured in GT-T551H 3 medium at a bead-to-cell ratio of 1: 1, adding CD3-CD28 to stimulate magnetic beads for stimulation, and carrying out virus infection after 48 hours;

coating retroNectin in a pore plate, and placing in a cell culture box at 37 ℃ for 2 h; absorbing RetroNectin, sealing the coated pore plate by using Hank's solution containing 2.5% BSA, and placing the coated pore plate in a 37 ℃ cell culture box for 0.5 h; sucking the confining liquid, washing a pore plate by using a Hank's solution containing 2% Hepes, adding a GT-T551H 3 culture medium, adding a proper amount of lentivirus solution, and centrifuging for 2 hours at 2000 g; discard the supernatant and add 1X 10⁶1000g for 10min at 37 ℃ in 5% CO₂And culturing in a cell culture box with certain humidity.

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

Example 1 vector construction

In this embodiment, a single chain antibody (scFv) of anti-human ADGRE2 antibody is used as an antigen binding domain, and a signal peptide, a hinge region, a transmembrane region, a costimulatory domain, and a CD3z intracellular domain are combined to construct a chimeric antigen receptor targeting ADGRE2, and a schematic diagram is shown in fig. 1. After obtaining the amino acid sequence of ADGRE2-CAR, the ADGRE2-CAR was subjected to whole-gene synthesis in Guangzhou Egyki Biotechnology Inc. and constructed into a lentiviral expression vector.

ADGRE2-CAR protein sequence (482 aa):

//MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASLGGKVTITCRASQDINKFISWYQHRPGKGP RLLIHYASTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLWTFGGGTKLEIRGGGGSGGGGSGGGG SEVQLQQSGPELVKPGASVRMSCKASGYTFTDYNMYWVKQSLGKSLEWIGYIYPNTCGATYNQKFKGKATLTVNKS SSTAFMELRSLTSEDSAVYCGRGGLGPFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*//

ADGRE2-CAR nucleic acid sequence (1449 bp):

//ATGGATATGAGGGTTCCTGCTCAACTCCTGGGTCTGCTCCTGCTGTGGCTCCGCGGAGCCAGGTGCGACATCCAGATGACACAGTCTCCCAGCTCACTGTCCGCAAGCCTGGGTGGCAAGGTTACAATTACATGTAGAGCTT CCCAGGACATTAACAAGTTCATCTCTTGGTATCAGCATAGGCCCGGCAAGGGCCCAAGGCTGCTGATTCATTATGC CAGTACACTGCAACCAGGGATTCCCTCACGGTTCTCAGGATCTGGCAGCGGACGCGATTACTCTTTCTCAATTAGT AATCTCGAACCAGAGGACATAGCCACTTATTACTGCCTGCAGTATGATAATCTGTGGACATTTGGCGGAGGCACAA AGCTGGAGATCAGAGGCGGAGGTGGGTCTGGAGGAGGAGGAAGTGGTGGCGGCGGTAGCGAAGTGCAGCTCCAGCA GAGCGGGCCAGAACTCGTCAAGCCAGGCGCCAGTGTCAGGATGTCCTGTAAGGCAAGTGGTTACACATTCACTGAC TACAACATGTACTGGGTCAAACAGAGCCTCGGTAAGTCACTGGAGTGGATTGGGTACATCTATCCCAATACTTGCG GAGCAACTTACAACCAAAAGTTTAAGGGCAAGGCCACTCTGACAGTTAACAAGTCCAGCTCCACCGCTTTTATGGA ACTGAGGTCACTCACATCAGAGGACAGTGCCGTCTACTGTGGTCGCGGAGGGCTCGGGCCTTTCGCCTACTGGGGC CAAGGGACACTGGTCACAGTGTCTGCTACTACGACCCCTGCACCGCGGCCGCCTACTCCTGCACCTACAATCGCAA GTCAGCCACTGAGTCTCAGACCCGAAGCATGCCGCCCTGCTGCAGGCGGAGCTGTCCATACACGCGGACTGGACTT TGCATGCGATATATACATCTGGGCACCACTGGCCGGCACTTGCGGCGTGCTGCTCCTGTCCCTCGTGATTACCCTG TACTGCAAACGCGGCAGGAAGAAGCTCCTGTATATCTTTAAACAGCCCTTCATGAGGCCAGTGCAGACCACTCAAG AGGAAGACGGTTGTAGCTGCCGGTTTCCCGAGGAAGAAGAGGGAGGCTGCGAGCTCCGCGTGAAGTTCTCCCGCTC AGCCGATGCACCCGCCTATCAGCAAGGGCAGAACCAGCTGTACAATGAGCTCAACCTGGGAAGAAGGGAGGAATAT GACGTTCTGGATAAACGGCGCGGTCGCGATCCCGAAATGGGTGGGAAGCCTCGCAGGAAGAATCCTCAGGAAGGGC TCTACAATGAGCTGCAGAAAGACAAAATGGCAGAGGCCTATTCTGAAATCGGCATGAAGGGCGAGCGCCGCAGAGG CAAAGGACACGACGGCCTGTACCAGGGCCTGTCTACAGCCACCAAGGACACCTATGACGCTCTCCACATGCAAGCC CTGCCACCAAGGTGA//

example 2 viral packaging

Plasmid transfection: placing plasmid, PEI and Opti-MEM culture medium at room temperature for 5 min; putting 436 mu L of Opti-MEM into a 1.5mL EP tube, adding 64 mu L of PEI, mixing uniformly, and standing for 5min at room temperature; adding pLP1, pLP2, pLP-VSVG and the target gene expression plasmid according to the ratio of 6:9:9:16, adding Opti-MEM to 500 mu L, and standing at room temperature for 5 min; adding the prepared PEI-Opti-MEM solution into the Opti-MEM containing the plasmid, and standing for 20min at room temperature; slowly dropping 1mL of DNA/PEI mixture into HEK 293T, gently mixing, incubating at 37 ℃ in an incubator, replacing fresh culture medium after 16-18 h, and adding 5% CO₂Incubation was continued in the incubator at 37 ℃.

Virus collection and concentration: after plasmid transfection for 48h, the supernatant was collected, centrifuged at 2820g at 4 ℃ for 20min to remove cell debris, and filtered through a 0.45 μm filter to obtain the supernatant; transferring the filtered virus supernatant into a super centrifuge tube, centrifuging for 2h at 50000g, removing the supernatant, adding a proper amount of culture medium to dissolve and precipitate to obtain virus liquid, subpackaging in a freezing tube, storing at-80 ℃, and taking 200 mu L of virus for titer determination.

And (3) virus titer detection: inoculation of 1X 10⁵HEK 293T cells in 24-well plates, different volumes of virus concentrate were added in 5% CO₂And culturing in an incubator at 37 ℃, staining by FITC-Protein L after 72 hours, and detecting the expression positive rate of the CAR of the cells so as to calculate the virus titer.

Example 3 preparation of ADGRE2-CAR T cells

Peripheral Blood Mononuclear Cells (PBMCs) were cultured using CD3 sorting magnetic beads (whirlpool), CD 3T cells sorted and transferred to GT-T551H 3 medium (300IU/mL of IL-2) at a bead to bead ratio of 1: 1, CD3-CD28 were added to stimulate magnetic beads (Thermo) and virus infection was carried out after 48 h.

The infection efficiency of lentivirus to T cells is improved by using retroNectin, 30 mu g of retroNectin is coated in a 6-hole plate and placed in a cell culture box at 37 ℃ for 2 h; absorbing RetroNectin, sealing the coated 6-well plate by using Hank's solution containing 2.5% BSA, and placing the plate in a 37 ℃ cell culture box for 0.5 h; sucking the confining liquid, washing a 6-well plate by using a Hank's solution containing 2% Hepes, adding a GT-T551H 3 culture medium, adding a proper amount of lentivirus solution, and centrifuging for 2 hours at 2000 g; discard the supernatant and add 1X 10⁶1000g for 10min at 37 ℃ in 5% CO₂And culturing in a cell culture box with certain humidity, and repeating the process the next day. The beads were removed on day 7. The medium GT-T551H 3 medium containing 300IU/mL was counted and replaced every two days, and the cell concentration was maintained at 0.5X 10⁶～1×10⁶and/mL. The proliferation of ADGRE2-CAR T cells was assessed by counting using a Countstar IC1000 automated cytometer in the United states.

The results are shown in figure 2, from which it can be seen that viral infection did not negatively affect T cell proliferation, and that both ADGRE2-CAR T and control T cells proliferated and expanded normally.

Example 4 expression assay of CAR-T cells

During the preparation of ADGRE2-CAR T cells, we sampled Day3 and Day11 (Day of viral infection, Day0) separately for flow assay. Cells were collected by centrifugation at 300g for 5min, washed once with PBS containing 1% BSA, then incubated with a solution containing FITC-Protein L for 40min at room temperature in the absence of light, washed once with PBS containing 1% BSA, then resuspended in 200. mu.L PBS, and finally subjected to flow-based detection of expression of ADGRE 2-CAR.

As shown in fig. 3, it can be seen that the positive rate of ADGRE2-CAR at Day3 was 47.4% to 51%, and the positive rate of CAR in the group with MOI (Multiplicity of Infection) of 10 was slightly higher than that in the group with MOI of 5. The positive rate of ADGRE2-CAR was almost the same as that of Day3 and was 47.4% when detected by Day 11. This suggests that ADGRE2-CAR can be stably expressed in T cells.

Example 5 in vitro killing function of ADGRE2-CAR T

Target cells stably expressing luciferase, HEK 293T-luc (negative for ADGRE2 expression) and U937-luc (positive for ADGRE2 expression), were seeded into black opaque 96-well plates, 2 ten thousand cells/well, and then expressed at an effective target ratio of 1: 1 and 3: 1 separately plating effector cells into wells, including untransduced control Mock T cells and ADGRE2-CAR T cells. And (3) co-incubating for 4-6 h in an inoculated incubator, adding an isovolumetric luciferase substrate (Promega), incubating for 5min at room temperature in a dark place, and detecting on a multifunctional microplate reader. The kill rate was calculated according to the formula: percent (1-RLU test set/RLUTarget only set) 100%.

The results are shown in FIG. 4, and it can be seen from the figure that all the ADGRE2-CAR T can specifically and efficiently kill the target cells U937-luc expressing positive ADGRE2 at different effective target ratios, and has no killing effect on HEK 293T-luc expressing negative, indicating that the ADGRE2-CAR T has specific killing capability.

Example 6 cytokine Release function of ADGRE2-CAR T

Target cells that stably expressed luciferase, HEK 293t-luc (negative for ADGRE2 expression) and U937-luc (positive for ADGRE2 expression), were seeded into round bottom 96-well plates, 2 ten thousand cells/well, and then expressed at an effective target ratio of 1: 1 and 3: 1 separately plating effector cells into wells, including control Mock T cells and ADGRE2-CAR T cells. Incubating for 24h in an incubator after inoculation, centrifuging for 15min at 500g, collecting supernatant, performing ELISA to detect the content of IFN-gamma, processing according to the instruction of an IFN-gamma detection kit (4A bio), and finally detecting on a multifunctional enzyme-labeling instrument.

The results are shown in figure 5, from which it can be seen that at different effective target ratios, ADGRE2-CAR T specifically released IFN- γ upon co-incubation with U937-luc cells positive for ADGRE 2; no significant release of IFN-. gamma.was observed upon incubation with HEK 293T-luc cells that were negative in expression. Specificity of ADGRE2-CAR T cell killing was also suggested.

In conclusion, the invention provides a chimeric antigen receptor targeting human ADGRE2, CAR-T cells constructed by the chimeric antigen receptor can specifically kill ADGRE2 positive tumor cells, and an effective cell treatment selection is provided for treating ADGRE2 positive acute myelogenous leukemia.

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.

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Lys Phe

20 25 30

Ile Ser Trp Tyr Gln His Arg Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Ala Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Asn Met Tyr Trp Val Lys Gln

145 150 155 160

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

165 170 175

Cys Gly Ala Thr Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr

180 185 190

Val Asn Lys Ser Ser Ser Thr Ala Phe Met Glu Leu Arg Ser Leu Thr

195 200 205

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

210 215 220

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Thr Thr

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

<210> 4

<211> 1383

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacatccaga tgacacagtc tcccagctca ctgtccgcaa gcctgggtgg caaggttaca 60

attacatgta gagcttccca ggacattaac aagttcatct cttggtatca gcataggccc 120

ggcaagggcc caaggctgct gattcattat gccagtacac tgcaaccagg gattccctca 180

cggttctcag gatctggcag cggacgcgat tactctttct caattagtaa tctcgaacca 240

gaggacatag ccacttatta ctgcctgcag tatgataatc tgtggacatt tggcggaggc 300

acaaagctgg agatcagagg cggaggtggg tctggaggag gaggaagtgg tggcggcggt 360

agcgaagtgc agctccagca gagcgggcca gaactcgtca agccaggcgc cagtgtcagg 420

atgtcctgta aggcaagtgg ttacacattc actgactaca acatgtactg ggtcaaacag 480

agcctcggta agtcactgga gtggattggg tacatctatc ccaatacttg cggagcaact 540

tacaaccaaa agtttaaggg caaggccact ctgacagtta acaagtccag ctccaccgct 600

tttatggaac tgaggtcact cacatcagag gacagtgccg tctactgtgg tcgcggaggg 660

ctcgggcctt tcgcctactg gggccaaggg acactggtca cagtgtctgc tactacgacc 720

cctgcaccgc ggccgcctac tcctgcacct acaatcgcaa gtcagccact gagtctcaga 780

cccgaagcat gccgccctgc tgcaggcgga gctgtccata cacgcggact ggactttgca 840

tgcgatatat acatctgggc accactggcc ggcacttgcg gcgtgctgct cctgtccctc 900

gtgattaccc tgtactgcaa acgcggcagg aagaagctcc tgtatatctt taaacagccc 960

ttcatgaggc cagtgcagac cactcaagag gaagacggtt gtagctgccg gtttcccgag 1020

gaagaagagg gaggctgcga gctccgcgtg aagttctccc gctcagccga tgcacccgcc 1080

tatcagcaag ggcagaacca gctgtacaat gagctcaacc tgggaagaag ggaggaatat 1140

gacgttctgg ataaacggcg cggtcgcgat cccgaaatgg gtgggaagcc tcgcaggaag 1200

aatcctcagg aagggctcta caatgagctg cagaaagaca aaatggcaga ggcctattct 1260

gaaatcggca tgaagggcga gcgccgcaga ggcaaaggac acgacggcct gtaccagggc 1320

ctgtctacag ccaccaagga cacctatgac gctctccaca tgcaagccct gccaccaagg 1380

tga 1383

Claims

1. A chimeric antigen receptor targeting ADGRE2, comprising a single chain antibody targeting ADGRE2, 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 amino acid sequences of the light chain variable region and the heavy chain variable region of the single-chain antibody targeting ADGRE2 are respectively shown in SEQ ID NO: 1 and 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

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: 3, 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: 4, 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 the treatment of acute myeloid leukemia.

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