CN115212299A - Application of CAR-T and CAR-M combination in preparation of antitumor drugs - Google Patents

Abstract

The invention discloses an application of CAR-T and CAR-M in preparing antitumor drugs, wherein CAR-T is a T cell modified by a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor sequentially comprises from an amino terminal to a carboxyl terminal: a signal peptide, an NKG2D extracellular region, a hinge region, a transmembrane region, an intracellular costimulatory signal region, and an intracellular signal region; CAR-M is macrophage modified by chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor sequentially comprises from amino terminal to carboxyl terminal: a signal peptide, an NKG2D extracellular region, a hinge region, a transmembrane region, and an intracellular signal region; the NKG2D extracellular region is a human NKG2D extracellular domain, and the amino acid sequence is shown in SEQ ID NO. 2. The CAR-T and CAR-M are combined to combine the capability of the CAR-M in improving the tumor microenvironment with the capability of the CAR-T in directly killing tumor cells, so that the tumor cells can be killed strongly and efficiently, and the curative effect is greatly improved.

Description

Application of CAR-T and CAR-M combination in the preparation of antitumor drugs

technical field

The present invention relates to the field of tumor therapy, in particular to cellular immunotherapy of tumors, in particular to chimeric antigen receptor T cell (CAR-T) and chimeric antigen receptor macrophage (CAR-M) cellular immunotherapy, and more particularly to Application of CAR-T and CAR-M combination in the preparation of antitumor drugs.

Background technique

Tumors are a serious threat to the national health of our country, and cause a huge burden to the society and economy. Although with the rapid development of medical science and technology, more and more methods are used for tumor treatment, but it is far from enough, and there are still huge unmet clinical needs. Therefore, methods for developing new tumor treatments have been accumulated.

For a long time, the treatment effect of malignant tumor is insufficient and the cure rate is low, making it a major disease that seriously threatens human health. Traditional chemoradiotherapy aims to destroy tumors directly, but tumors often recur and metastasize, which in turn kills normal tissue cells. The goal of cellular immunotherapy of tumors is to promote or modify immune cells to attack cancer cells while maintaining the integrity of normal cells. Compared with traditional chemoradiotherapy, adoptive therapy of cells has good specificity, safety, efficacy and durability, ranking first in the top ten scientific and technological advances in 2013.

In recent years, adoptive cell therapy based on DCs, T cells, NK cells, etc. has achieved good results in tumor treatment. In particular, the immune cell therapy technology represented by Chimeric Antigen Receptor T-Cell (CAR-T) has shown strong efficacy and great development potential in the treatment of hematological tumors, and has become a human New hope against cancer. Chimeric antigen receptor (CAR) T-cell immunotherapy is a tumor adoptive cell immunotherapy. (MHC, major histocompatibility complex) kills tumor cells in a restricted manner. Although CAR T cells have achieved good results in clinical and clinical trials, they also have serious side effects, including cytokine release syndrome, neurotoxicity, and off-target effects. Moreover, the effectiveness of CAR-T cell therapy on tumors other than some hematological tumors (such as solid tumors) is not satisfactory in clinical trials and needs to be improved. In the treatment of solid tumors, CAR-T cell therapy has not achieved major breakthroughs for the following reasons: First, CAR-T cells must be transported and infiltrated into tumors, which is an area that requires extravasation, chemotaxis, and stromal tissue the process of penetration. CAR-T cells must traverse abnormal tumor vessels with reduced adhesion molecules, undergo chemokine/chemokine receptor mismatch, and must migrate through dense cellular and stromal barriers. After entering the tumor microenvironment (TME), effector cells encounter adverse conditions such as hypoxic and acidic environments, expression of immune checkpoint ligands, and a large number of immunosuppressive cells, such as tumor-associated macrophages (Tumor Associated Macrophages). , TAM), myeloid-derived suppressor cells (Myeloid DerivedSuppressor Cell, MDSC) and regulatory T cells (Regulatory Cells, T-regs). In addition, prolonged antigen exposure can lead to T cell exhaustion, thereby reducing the effector function of CAR T cells. Even if CAR-T cells survive in the TME, solid tumors often have heterogeneous surface antigen expression, which may lead to evasion of CAR T-cell detection, incomplete tumor clearance, and eventual growth of antigen-negative tumor cells. This was clearly demonstrated in the treatment of glioblastoma with EGFRvIII-targeting CAR T cells, where EGFRvIII expression decreased in 5/7 patients after treatment. Finally, CAR-T cells may recognize tumor-associated antigens in normal tissues and cells, leading to on-target/offtumor toxicity, which is another hurdle for solid tumor cell therapy. Therefore, combining CAR-T with other therapeutic methods may make up for the deficiency of CAR-T in this regard, resist the immunosuppressive influence of the local tumor microenvironment of solid tumors, and thus have a synergistic anti-tumor effect.

In recent years, the potential of macrophages to kill tumors has gradually attracted attention. CAR-modified macrophages (CAR-M) are considered a promising cell type. Macrophages activate lymphoid or other immune cells to respond to pathogens by engulfing cellular debris and pathogens in body fluids and tissues and presenting antigens. In the tumor microenvironment, macrophages are the innate immune cells with the highest infiltration rate and can interact with almost all cellular components in the TME to stimulate angiogenesis, increase tumor infiltration, and mediate immunosuppression. However, the effect of CAR-M in directly killing tumor cells is limited.

SUMMARY OF THE INVENTION

In order to solve the defects in the existing technology (1) CAR-T treatment of solid tumors: including tumor heterogeneity, which leads to the escape of CAR target-negative cells; CAR-T is difficult to infiltrate into the tumor, and the killing effect is insufficient; solid tumor immunosuppression The influence of the microenvironment leads to the depletion of CAR-T and the loss of killing function; the side effects of CAR-T itself, such as cytokine release syndrome, neurotoxicity and off-target effects, etc.; (2) CAR-M cells phagocytose or gnaw tumor cells To kill tumors, the efficiency of killing tumor cells is not high. The present invention aims to provide the application of the combination of CAR-T and CAR-M in the preparation of anti-tumor drugs.

Based on the comparative advantages of CAR-M and CAR-T: (1) CAR-M can improve the tumor immune microenvironment of solid tumors; it can present antigens as antigen-presenting cells after phagocytosing tumor cells; it is easier to infiltrate into tumors and cooperate with other immune systems (2) The ability of CAR-T to directly kill tumor cells, the present invention proposes a combination of CAR-T and CAR-M for tumor treatment. The specific plans are as follows:

The first aspect of the present invention provides the application of the combination of CAR-T and macrophages in the preparation of anti-tumor drugs, the CAR-T is a T cell modified by a chimeric antigen receptor, and the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region, intracellular costimulatory signal region and intracellular signal region;

The NKG2D extracellular domain is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2.

The second aspect of the present invention provides the application of the combined use of CAR-M and T cells in the preparation of anti-tumor drugs, the CAR-M is a macrophage modified with a chimeric antigen receptor, and the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region and intracellular signal region;

The NKG2D extracellular domain is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2.

The third aspect of the present invention provides the application of the combination of CAR-T and CAR-M in the preparation of anti-tumor drugs, the CAR-T is a T cell modified by a chimeric antigen receptor, and the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region, intracellular costimulatory signal region and intracellular signal region;

The CAR-M is a macrophage modified by a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor is from the amino terminus to the carboxyl terminus: signal peptide, NKG2D extracellular region, hinge region, transmembrane region and intracellular signaling region;

The chimeric antigen receptor NKG2D extracellular domain in the CAR-T and CAR-M is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2.

In the above application, the signal peptide is selected from CD8α signal peptide, CD28 signal peptide, CD4 signal peptide or GM-CSF signal peptide;

The hinge region is selected from the CD8α hinge region or the CD28 hinge region;

The transmembrane region is selected from the CD8α transmembrane region or the CD28 transmembrane region;

The intracellular signal region is selected from CD3ζ or FcRγ.

In the above application, the signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1;

The hinge region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.3;

The transmembrane region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.4;

The intracellular signal region is CD3ζ, and its amino acid sequence is shown in SEQ ID NO.5.

In the above application, the intracellular costimulatory signal region is selected from 4-1BB, CD28, CD27, OX40 or ICOS;

Preferably, the intracellular costimulatory signal region is selected from 4-1BB, and its amino acid sequence is shown in SEQ ID NO.11.

In the above application, the CAR-T construction method is: introducing the nucleic acid or vector of the CAR-T chimeric antigen receptor encoding gene into T cells;

The mode of introduction includes electroporation, transduction or transfection;

Preferably, the nucleic acids are located on different viral vectors; the viral vectors are lentiviral vectors, adenoviral vectors or retroviral vectors;

Preferably, the vector is a transposon or mRNA vector;

Preferably, a lentiviral vector comprising a gene encoding a CAR-T chimeric antigen receptor is introduced into T cells by transfection.

In the above application, the CAR-M construction method is: introducing the nucleic acid or vector of the CAR-M chimeric antigen receptor encoding gene into macrophage cells;

The mode of introduction includes electroporation, transduction or transfection;

Preferably, the nucleic acids are located on different viral vectors; the viral vectors are lentiviral vectors, adenoviral vectors or retroviral vectors;

Preferably, the vector is a transposon or mRNA vector;

Preferably, the adenoviral vector containing the gene encoding the CAR-M chimeric antigen receptor is introduced into macrophage cells by transfection.

In the above-mentioned applications, the macrophages are derived from a healthy human body or a cancer patient;

The macrophages are selected from autologous macrophages, allogeneic macrophages or iPSC-induced macrophages;

Preferably, the macrophages are primary macrophages;

The T cells are derived from a healthy human body or a cancer patient;

The T cells are selected from autologous T cells, allogeneic T cells or iPSC-induced T cells.

In the above application, the tumor is a tumor expressing NKG2D ligand;

the tumor is a hematological tumor or a solid tumor;

Preferably, the tumor is leukemia, multiple myeloma, malignant lymphoma, glioma, liver cancer, lung cancer, gastric cancer, colon cancer, pancreatic cancer or breast cancer.

The beneficial effects of the present invention are:

1. The present invention uses the natural sequence of the extracellular segment of NKG2D as the CAR recognition region, so that the chimeric antigen receptor has the advantages of low immunogenicity and easy expression, and can be expressed normally on macrophages and T cells. Provide the basis for chimeric antigen receptor macrophages (CAR-M) and chimeric antigen receptor T cells (CAR-T) that phagocytose tumor cells. Preferably, CD3ζ is used as the intracellular signal region, and the ITAM structure contained in CD3ζ endows CAR-M with better phagocytosis/killing effect on tumors.

2. The present invention can achieve synergistic synergy in anti-tumor and improve the effect of treating tumors through the combination of CAR-T and CAR-M, the combination of CAR-T and M cells, or the combination of CAR-M and T cells. In particular, the combination of CAR-T and CAR-M cells for tumor treatment combines the ability of CAR-M to improve the tumor microenvironment with the ability of CAR-T to directly kill tumor cells, which has a synergistic effect and can achieve a synergistic effect on tumor cells. The powerful and efficient killing of solid tumors (especially solid tumors) greatly improves the efficacy and solves the shortcomings of CAR-T alone in the treatment of solid tumors and the low efficiency of CAR-M alone in killing tumor cells. In addition, the combination regimen can reduce the amount of CAR-T cells used, thereby reducing the side effects of CAR-T treatment.

Description of drawings

Figure 1: Schematic representation of NKG2D CAR in NKG2D CAR-M.

Figure 2: Schematic representation of NKG2D CAR in NKG2D CAR-T.

Figure 3: CAR positive rate of NKG2D CAR-M cells.

Figure 4: CAR positive rate of NKG2D CAR-T cells.

Figure 5: Experiment of killing effect of human primary CAR-M and CAR-T cells on tumor cells, statistics of killing rate of each group after 48 hours of killing (effector cells: target cells=1).

Figure 6: Experiment of killing effect of human primary macrophages (M) and CAR-T cells on tumor cells, statistics of killing rate of each group after 48 hours of killing (effector cells: target cells=1).

Figure 7: Experiment of killing effect of human primary CAR-M and primary T cells on tumor cells, statistics of killing rate of each group after 48 hours of killing (effector cells: target cells=1).

Detailed ways

For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each original reagent material can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the instrument manufacturer.

Example 1

1. NKG2D chimeric antigen receptor used in NKG2D CAR-M

This example provides the NKG2D chimeric antigen receptor used in the NKG2D CAR-M, and its primary protein structure from the amino terminus to the carboxy terminus is: signal peptide (Signal pep), NKG2D extracellular domain (NKG2D ECD), Hinge region (Hinge), transmembrane region (TM) and intracellular signal region. Wherein, the signal peptide can be selected from the signal peptides of human CD8α, CD28, CD4, GM-CSF, etc.; the hinge region and the transmembrane region can be selected from the hinge region and the transmembrane region of human CD8α, CD28, etc.; the intracellular signal region can be selected from From CD3ζ, FcRγ, etc. In a specific embodiment (Figure 1), the signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; the NKG2D extracellular domain is the extracellular domain of human NKG2D (NM_007360.4), and its amino acid sequence is shown in Shown in SEQ ID NO.2; the hinge region and transmembrane region are derived from human CD8α, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.3, and the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.4; intracellular The signal region is CD3ζ, CD3ζ provides activation signal, and its amino acid sequence is shown in SEQ ID NO.5. Specific nucleotide sequences: CD8α signal peptide (SEQ ID NO.6), NKG2D extracellular region (SEQ ID NO.7), CD8α hinge region (SEQ ID NO.8), CD8α transmembrane region (SEQ ID NO.9) ) and CD3ζ intracellular signal region (SEQ ID NO. 10) were synthesized artificially to obtain the NKG2D chimeric antigen receptor sequence used in NKG2D CAR-M.

2. NKG2D chimeric antigen receptor used in NKG2D CAR-T

This example provides the NKG2D chimeric antigen receptor used in NKG2D CAR-T, and its primary protein structure from the amino terminus to the carboxy terminus is: signal peptide (Signal pep), NKG2D extracellular domain (NKG2D ECD), Hinge region (Hinge), transmembrane region (TM), intracellular costimulatory signaling region and intracellular signaling region. Wherein, the signal peptide can be selected from the signal peptides of human CD8α, CD28, CD4, GM-CSF, etc.; the hinge region and the transmembrane region can be selected from the hinge region and transmembrane region of human CD8α, CD28, etc.; the intracellular costimulatory signal region It can be selected from 41-BB, CD28, CD27, OX40 or ICOS; the intracellular signal region can be selected from CD3ζ, FcRγ and the like. In a specific embodiment (Figure 2), the signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; the NKG2D extracellular domain is the extracellular domain of human NKG2D (NM_007360.4), and its amino acid sequence is shown in Shown in SEQ ID NO.2; the hinge region and transmembrane region are derived from human CD8α, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO.3, and the amino acid sequence of the CD8α transmembrane region is shown in SEQ ID NO.4; intracellular The costimulatory signal region is selected from 41BB, and its amino acid sequence is shown in SEQ ID NO.11; the intracellular signal region is CD3ζ, which provides an activation signal, and its amino acid sequence is shown in SEQ ID NO.5. Specific nucleotide sequences: CD8α signal peptide (SEQ ID NO.6), NKG2D extracellular region (SEQ ID NO.7), CD8α hinge region (SEQ ID NO.8), CD8α transmembrane region (SEQ ID NO.9) ), 41BB intracellular costimulatory signal region (SEQ ID NO. 12), CD3ζ intracellular signal region (SEQ ID NO. 10) were artificially synthesized to obtain the NKG2D chimeric antigen receptor sequence used in NKG2D CAR-M.

3. Amino acid and nucleotide sequences in this example

CD8α signal peptide amino acid sequence (SEQ ID NO. 1):

MALPVTALLLPLALLLHAARP

NKG2D extracellular domain amino acid sequence (SEQ ID NO. 2):

IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

CD8α hinge region amino acid sequence (SEQ ID NO. 3):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

Amino acid sequence of CD8α transmembrane region (SEQ ID NO. 4):

IYIWAPLAGTCGVLLLSLVITLYC

Amino acid sequence of CD3ζ intracellular signal region (SEQ ID NO.5):

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD8α signal peptide nucleotide sequence (SEQ ID NO. 6):

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

NKG2D extracellular domain nucleotide sequence (SEQ ID NO. 7):

ATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTG

CD8α hinge region nucleotide sequence (SEQ ID NO. 8):

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

CD8α transmembrane region nucleotide sequence (SEQ ID NO. 9):

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

CD3ζ intracellular signal region nucleotide sequence (SEQ ID NO.10):

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

41BB intracellular co-stimulatory signal region amino acid sequence (SEQ ID NO. 11):

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

4-1BB intracellular costimulatory signal region nucleotide sequence (SEQ ID NO. 12):

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA

Example 2

1. Preparation of NKG2D CAR-M

1.1. Construction of recombinant adenovirus

The NKG2D CAR adenovirus is packaged using the pAdEasy system recombinant adenovirus packaging system, and the backbone vector used for the Ad5F35 adenovirus packaging is Ad5F35 Helper. Ad5F35-related packaging plasmids were transfected in HEK 293 cells using PEI, and placed in a CO ₂ incubator after mixing. After 14 days of culture, the cells were collected by centrifugation at 3500g, and the adenovirus cryopreserved solution was added to resuspend the pellet. The suspension was repeatedly freeze-thawed at -80°C and 37°C for four times. The collected virus supernatant was added dropwise to 10 10cm petri dishes on average, and then placed in a CO ₂ incubator after mixing. 2-3 days later, the supernatant and cells were collected by centrifugation. PEG8000 and NaCl were added to the virus supernatant. After mixing, the cells were placed upright at 4°C overnight. The supernatant was collected by centrifugation the next day. The solution was resuspended, freeze-thawed 4 times, and the supernatant was collected after high-speed centrifugation. The remaining cells were sonicated, and the sample supernatant was collected after centrifugation. After mixing all the collected samples, the virus was purified by iodixanol density gradient centrifugation. Finally, the purified virus solution was filtered through a 0.22-micron filter membrane and then stored in aliquots for cryogenic storage.

1.2 Preparation of chimeric antigen receptor M cells targeting NKG2D

a) Whole blood isolation of human peripheral blood mononuclear cells (PBMC)

Add 25mL of whole blood to a 50mL centrifuge tube, and then add 25mL of PBS to mix with it; add 15mL of FICOLL reagent to the 50mL centrifuge tube, and then slowly add 30mL of the above diluted blood along the wall; centrifuge at 20°C, 2110r/min, liters After centrifugation, the liquid is divided into four layers, the top layer of yellow liquid is removed, and the second layer of buffy coat layer is sucked by rotating circles. The obtained buffy coat layer liquid is divided into two tubes, and PBS is added to 40-45mL to mix well, and centrifuged Centrifuge at 20°C, 1800 r/min, lift 9-9, centrifuge for 8 min; remove the supernatant, add 40 mL of PBS to resuspend the two tubes into one tube, centrifuge at 20°C, 1200 r/min, lift 9-9, centrifuge for 8 min; Add 40 mL of PBS to resuspend and count the remaining cells at 20°C, 1200 r/min, rise by 9 to 9, and centrifuge for 8 min;

b) Isolation of human CD14 ⁺ cells

Put the recovered PBMC cells in a centrifuge at 300g for 5min, discard the supernatant; resuspend in about 4-5mL of MojoSort ^TM buffer. Filter the cells with a 70 μM cell mesh, centrifuge at 300 g for 5 min and remove the supernatant, then resuspend with an appropriate volume of MojoSort ^™ Buffer and adjust the cell concentration to 1×10 ⁸ cells/mL. 5 μL of Human TruStain FcX ^™ (Fc receptor blocking solution) was added to 100 μL of cell suspension (10 ⁷ cells), mixed well and incubated at room temperature for 10 min. If more cells are isolated, increase the amount of reagent proportionally. Add 10 μL Biotin-Antibody Cocktail, mix well and incubate on ice for 15 min. If more cells are isolated, increase the amount of reagent proportionally. Add 10 μL of Streptavidin Nanobeads resuspended by vortexing at maximum speed, mix well and incubate on ice for 15 min. If more cells are isolated, increase the amount of reagent proportionally. 4 mL of MojoSort ^TM buffer was added to wash the cells, placed in a centrifuge at 300 g for 5 min, and the supernatant was discarded. Add 2.5mL ^MojoSortTM buffer to resuspend the cells in a clean flow tube, and place them in a magnetic stand for 5min. Pour off and collect the liquid in a new 15mL centrifuge tube for later use. The remaining pellet was then resuspended with 2.5 mL of MojoSort ^™ buffer. The collected liquids were combined, placed in a centrifuge at 300 g for 5 min, and the prepared human primary macrophage medium (RPMI-1640 medium was added with 10% FBS, 1% PS and a final concentration of 20 ng/ mL of human GM-CSF) was resuspended and counted, and seeded in non-TC-treated 6-well plates for culture. An additional fraction of cells can be used for flow staining to check for purity.

c) Culture of human primary macrophages

The isolated human CD14 ⁺ cells were cultured with the prepared human primary macrophage medium in a non-TC-treated 6-well plate (3-8×10 ⁵ cells/mL) at 37°C in a 5% carbon dioxide incubator nourish. On the third day, the supernatant was gently aspirated and replaced with half of the medium, and the adherent cells obtained after the full amount of medium exchange on the fifth day were human primary macrophages.

d) Adenovirus infection of primary human macrophages

The isolated human CD14 ⁺ cells were counted and cultured in non-TC-treated 6-well plates (3-8×10 ⁵ cells/mL); 5 days later, adenovirus (control virus and NKG2D CAR adenovirus); after 24-48h, the medium was changed to a medium without virus; after 48h, the positive rate of CAR-M was detected: the digested CAR-M was subjected to flow staining to detect the expression of CAR.

The results of flow cytometry (Fig. 3) showed that the positive rate of NKG2D CAR-M was >50%. It showed that the human primary CAR-M cells targeting NKG2D (human primary CAR-M cells) were successfully prepared.

2. Preparation of NKG2D CAR-T

2.1. Construction of pWPXLd-CAR-NKG2D recombinant plasmid

The above-mentioned CAR-NKG2D coding gene was inserted into the pWPXLD vector between the BamHI and EcoRI restriction sites, and located after the elongation factor 1α (EF1α) of the pWPXLD vector, with EF1α as the promoter. When the encoding gene of the CAR-NKG2D is inserted into the pWPXLD vector, an initiation codon (such as ATG) can be added to the 5' end of the encoding gene of the CAR-NKG2D to connect with the BamHI restriction site in the pWPXLD vector, and the 3' A stop codon (such as TAA) can be added to the end to link with the EcoRI restriction site in the pWPXLD vector. Then it was transferred into E. coli competent cells DH5α, and the positive clones were identified by PCR and sequencing. The PCR product was detected by gel electrophoresis and sequenced to identify the size and sequence of the target fragment, and the recombinant plasmid pWPXLd-CAR-NKG2D was obtained.

2.2. Construction of recombinant lentivirus

The pWPXLd-CAR-NKG2D recombinant plasmid obtained above, the packaging plasmid psPAX2, and the envelope plasmid pMD2G were co-transfected into the cultured HEK293T cells by the lipofectamine3000 lipofection reagent. The virus-containing supernatant was harvested at 48h, filtered through a 0.45μm filter, and stored in an ultra-low temperature refrigerator at -80°C; the virus-containing supernatant was harvested twice at 72h, filtered with a 0.45μm filter, and the virus supernatant harvested at 48h After combining, put them into ultracentrifuge tubes together, and put them into the Beckman ultracentrifuge one by one. Set the centrifugation parameter to 25000rpm, the centrifugation time to 2h, and the centrifugation temperature to be controlled at 4°C; after the centrifugation, discard the supernatant and try to remove the residual Add the virus preservation solution to the liquid on the tube wall, and resuspend by gently pipetting repeatedly; after fully dissolving, centrifuge at high speed at 10,000 rpm for ⁵ min, and then take the supernatant to measure the titer by fluorescence method. Distributed in mL and stored in a -80°C ultra-low temperature refrigerator to obtain a recombinant lentivirus with the CAR-NKG2D encoding gene.

2.3 Preparation of chimeric antigen receptor T cells targeting NKG2D

a) Isolation of PBMCs (Peripheral Blood Mononuclear Cells)

PBMC are derived from autologous venous blood, autologous bone marrow, umbilical cord blood and placental blood. It is best to come from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.

The patient's blood was drawn and sent to the blood separation room; the peripheral blood mononuclear cells were collected, and the cells in the middle layer were obtained after Ficoll centrifugation; after washing with PBS, PBMCs were obtained.

b) Isolation of antigen-specific T lymphocytes by immunomagnetic beads

Take the above PBMC, add serum-free basal medium to form a cell suspension; according to the ratio of magnetic beads to cells 3:1, add CD3/CD28 immunomagnetic beads, incubate at room temperature for 1-2 hours; use a magnet to incubate well The cells with magnetic beads were screened; after washing with PBS, the immunomagnetic beads were removed to obtain CD3-positive T lymphocytes.

c) Preparation of antigen-specific T lymphocytes by virus transfection

Take the CD3-positive T lymphocytes obtained by the immunomagnetic bead separation method in b), and add the recombinant lentivirus described in 2.2 with the virus titer corresponding to the number of CD3-positive cells for culture.

On the 3rd day of culture, count the cells and change the medium, adjust the cell concentration to 1×10 ⁶ cells/mL, inoculate and culture; on the 5th day of culture, observe the cell state, if the cell density increases, the diluted cell concentration is 1×10 ⁶ cells/mL, detect cell viability, and continue to culture. The cells were collected from the 9th to 11th day of expansion and culture, and the expression of the chimeric antigen receptor CAR-NKG2D targeting NKG2D was detected by flow cytometry. The results are shown in Figure 4. After testing, the positive rate of CAR-NKG2D in T cells infected with the above recombinant lentivirus was about 70%, which indicated that CAR-T cells (CAR-T cells) targeting NKG2D were successfully obtained.

3. The killing effect of human primary CAR-M and CAR-T cells on tumor cells

The experiment was divided into 4 groups: Control group; CAR-M group; CAR-T group; CAR-T and CAR-M combination group; 50% CAR-T and 50% CAR-M group. The fluorescence intensity was collected and analyzed by a SPECTROstar Omega microplate reader for cell killing experiments. First, NKG2D CAR-M cells were plated at 2×10 ⁴ cells/well (CAR-M group; CAR-T and CAR-M combination group) or 1×10 ⁴ cells/well (50% CAR-T and 50% CAR). -M group) were inoculated into a light-tight 96-well cell culture plate, with 10 duplicate wells in each group, and allowed to stand for 24 hours. After 24 hours, PC-3 cells stably expressing luciferase were added at the ratio of effector cells:target cells=1:1, and 2×10 ⁴ cells/well (CAR-T group; CAR-T and CAR-M combination group) or The number of CAR-Ts at ¹ x 104/well (50% CAR-T and 50% CAR-M groups).

After co-cultivation for 48 hours, after culturing for 48 hours at 37°C in a 5% carbon dioxide incubator, add D-luciferin at a concentration of 100-200 μg/mL, potassium salt substrate to the opaque 96-well plate, and incubate at 37°C for 10 minutes in the dark. After that, the fluorescence intensity was collected and analyzed using the SPECTROstar Omega microplate reader, and the killing of PC-3 target cells by each immune cell group was calculated by the following formula:

% cell lysis (Lysis%)=[1-(fluorescence signal of co-cultured cells-background fluorescence signal)/

(Fluorescence signal of PC-3 cells cultured alone - background fluorescence signal)]*100

The killing results of PC-3 cells are shown in Figure 5: CAR-T group kills better than CAR-M group, 50% CAR-T and 50% CAR-M groups are significantly better than CAR-T group, CAR-T+ CAR-M has the strongest killing effect. In view of the fact that CAR-T cells have a stronger killing effect than CAR-M cells, in the 50% CAR-T and 50% CAR-M groups, the total number of cells is the same as the CAR-T group, which contains 50% CAR-T and 50% CAR-T cells. CAR-M cells, but the killing effect of 50% CAR-T and 50% CAR-M group was significantly better than that of CAR-T group, which indicates that CAR-T and CAR-M have a synergistic effect, 1+1>2.

4. The killing effect of human primary macrophages (M) and CAR-T cells on tumor cells

The experiment was divided into 4 groups: Control group; CAR-untransformed macrophage group (M group); CAR-T group; CAR-T and M combined group; 50% CAR-T and 50% M group. The fluorescence intensity was collected and analyzed by a SPECTROstar Omega microplate reader for cell killing experiments. First, macrophage (M) cells were plated at 2×10 ⁴ cells/well (M group; CAR-T and M combined group) or 1×10 ⁴ cells/well (50% CAR-T and 50% M group) The cells were inoculated into an opaque 96-well cell culture plate, with 10 duplicate wells in each group, and allowed to stand for 24 hours. After 24h, PC-3 cells stably expressing luciferase were added at the ratio of effector cells:target cells=1:1, and 2×10 ⁴ cells/well (CAR-T group; CAR-T and M combined group) or 1× ^The number of CAR-Ts at 104/well (50% CAR-T and 50% M groups).

After co-cultivation for 48 hours, after culturing for 48 hours at 37°C in a 5% carbon dioxide incubator, add D-luciferin at a concentration of 100-200 μg/mL, potassium salt substrate to the opaque 96-well plate, and incubate at 37°C for 10 minutes in the dark. After that, the fluorescence intensity was collected and analyzed using the SPECTROstar Omega microplate reader, and the killing of PC-3 target cells by each immune cell group was calculated by the following formula:

% cell lysis (Lysis%)=[1-(fluorescence signal of co-cultured cells-background fluorescence signal)/

(Fluorescence signal of PC-3 cells cultured alone - background fluorescence signal)]*100

The killing results of PC-3 cells are shown in Figure 6: CAR-T group kills better than M group, 50% CAR-T and 50% M groups are significantly better than CAR-T group, CAR-T+M kills the most powerful. Given that CAR-T cells have a stronger killing effect than M cells, in the 50% CAR-T and 50% M group, the total number of cells is the same as the CAR-T group, which contains 50% CAR-T and 50% M cells, but The killing effect of 50% CAR-T and 50% M group was significantly better than that of CAR-T group, which indicated that CAR-T and M had a synergistic effect, 1+1>2.

5. The killing effect of human primary CAR-M and primary T cells on tumor cells

The experiment was divided into 4 groups: Control group; CAR-M group; T cell group; T cell and CAR-M combination group; 50% T and 50% CAR-M group. The fluorescence intensity was collected and analyzed by a SPECTROstar Omega microplate reader for cell killing experiments. First, NKG2D CAR-M cells were plated at 2×10 ⁴ cells/well (CAR-M group; T and CAR-M combined group) or 1×10 ⁴ cells/well (50%T and 50% CAR-M group) The cells were inoculated into an opaque 96-well cell culture plate, with 10 duplicate wells in each group, and allowed to stand for 24 h. After 24h, PC-3 cells stably expressing luciferase were added at the ratio of effector cells:target cells=1:1, and 2×10 ⁴ cells/well (T group; T and CAR-M combination group) or 1×10 ⁴ cells Number of Ts/well (50%T and 50%CAR-M groups).

After co-cultivation for 48 hours, after culturing for 48 hours at 37°C in a 5% carbon dioxide incubator, add D-luciferin at a concentration of 100-200 μg/mL, potassium salt substrate to the opaque 96-well plate, and incubate at 37°C for 10 minutes in the dark. After that, the fluorescence intensity was collected and analyzed using the SPECTROstar Omega microplate reader, and the killing of PC-3 target cells by each immune cell group was calculated by the following formula:

% cell lysis (Lysis%)=[1-(fluorescence signal of co-cultured cells-background fluorescence signal)/

(Fluorescence signal of PC-3 cells cultured alone - background fluorescence signal)]*100

The killing results of PC-3 cells are shown in Figure 7: 50% T and 50% CAR-M groups were significantly better than T and CAR-M groups, and T+CAR-M had the strongest killing effect. In the 50% T and 50% CAR-M combination group, the total number of cells is the same as the T group and CAR-M group, which contains 50% T and 50% CAR-M cells, but 50% T and 50% CAR The killing effect of -M group was significantly better than that of T group and CAR-M, which indicated that CAR-T and CAR-M had a synergistic effect, 1+1>2.

Obviously, the above -mentioned examples are just a clear explanation of the examples, not the limitation of the embodiment. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Advanced Technology Research Institute

<120> Application of CAR-T and CAR-M combination in the preparation of antitumor drugs

<130> CP122010529C

<160> 12

<170> PatentIn version 3.3

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Claims (10)

1. The application of the combination of CAR-T and macrophages in the preparation of anti-tumor drugs, characterized in that the CAR-T is a T cell modified by a chimeric antigen receptor, and the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region, intracellular costimulatory signal region and intracellular signal region; The NKG2D extracellular domain is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2. 2. The application of the combined use of CAR-M and T cells in the preparation of anti-tumor drugs, wherein the CAR-M is a macrophage modified by a chimeric antigen receptor, and the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region and intracellular signal region; The NKG2D extracellular domain is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2. 3. The application of the combination of CAR-T and CAR-M in the preparation of anti-tumor drugs, characterized in that the CAR-T is a T cell modified by a chimeric antigen receptor, and the chimeric antigen receptor is a primary The protein structure from the amino terminus to the carboxyl terminus is: signal peptide, NKG2D extracellular region, hinge region, transmembrane region, intracellular costimulatory signal region and intracellular signal region; The CAR-M is a macrophage modified by a chimeric antigen receptor, and the primary protein structure of the chimeric antigen receptor is from the amino terminus to the carboxyl terminus: signal peptide, NKG2D extracellular region, hinge region, transmembrane region and intracellular signaling region; The chimeric antigen receptor NKG2D extracellular domain in the CAR-T and CAR-M is the extracellular domain of human NKG2D, and its amino acid sequence is shown in SEQ ID NO.2. 4. The use according to any one of claims 1-3, wherein the signal peptide is selected from CD8α signal peptide, CD28 signal peptide, CD4 signal peptide or GM-CSF signal peptide; The hinge region is selected from the CD8α hinge region or the CD28 hinge region; The transmembrane region is selected from the CD8α transmembrane region or the CD28 transmembrane region; The intracellular signal region is selected from CD3ζ or FcRγ. 5. The application according to any one of claims 1-3, wherein the signal peptide is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.1; The hinge region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.3; The transmembrane region is derived from human CD8α, and its amino acid sequence is shown in SEQ ID NO.4; The intracellular signal region is CD3ζ, and its amino acid sequence is shown in SEQ ID NO.5. 6. The use according to claim 1 or 3, wherein the intracellular costimulatory signal region is selected from 4-1BB, CD28, CD27, OX40 or ICOS; Preferably, the intracellular costimulatory signal region is selected from 4-1BB, and its amino acid sequence is shown in SEQ ID NO.11. 7. The application according to claim 1 or 3, wherein the CAR-T construction method is: introducing the nucleic acid or vector of the CAR-T chimeric antigen receptor encoding gene into T cells; The mode of introduction includes electroporation, transduction or transfection; Preferably, the nucleic acids are located on different viral vectors; the viral vectors are lentiviral vectors, adenoviral vectors or retroviral vectors; Preferably, the vector is a transposon or mRNA vector; Preferably, a lentiviral vector comprising a gene encoding a CAR-T chimeric antigen receptor is introduced into T cells by transfection. 8. The application according to claim 2 or 3, wherein the CAR-M construction method is: introducing the nucleic acid or vector of the CAR-M chimeric antigen receptor encoding gene into macrophage cells; The mode of introduction includes electroporation, transduction or transfection; Preferably, the nucleic acids are located on different viral vectors; the viral vectors are lentiviral vectors, adenoviral vectors or retroviral vectors; Preferably, the vector is a transposon or mRNA vector; Preferably, the adenoviral vector containing the gene encoding the CAR-M chimeric antigen receptor is introduced into macrophage cells by transfection. 9. The application according to any one of claims 1-3, wherein the macrophage is derived from a healthy human body or a cancer patient; The macrophages are selected from autologous macrophages, allogeneic macrophages or iPSC-induced macrophages; Preferably, the macrophages are primary macrophages; The T cells are derived from a healthy human body or a cancer patient; The T cells are selected from autologous T cells, allogeneic T cells or iPSC-induced T cells. 10. The application according to any one of claims 1-3, wherein the tumor is a tumor expressing NKG2D ligand; the tumor is a hematological tumor or a solid tumor; Preferably, the tumor is leukemia, multiple myeloma, malignant lymphoma, glioma, liver cancer, lung cancer, gastric cancer, colon cancer, pancreatic cancer or breast cancer.

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