CN114796471A

CN114796471A - Artemisinin compound and CAR-T cell targeting NKG2D ligand combined preparation of medicine for treating malignant tumor

Info

Publication number: CN114796471A
Application number: CN202110134104.1A
Authority: CN
Inventors: 刘茂玄; 许晨光; 黎琴子
Original assignee: Shenzhen Bindebiotech Co ltd
Current assignee: Shenzhen Bindebiotech Co ltd
Priority date: 2021-01-28
Filing date: 2021-01-28
Publication date: 2022-07-29

Abstract

The invention provides an application of an artemisinin compound and a CAR-T cell targeting NKG2D ligand in combination in preparation of a medicine for treating malignant tumor, and also provides a medicine for treating malignant tumor, which comprises the artemisinin compound and the CAR-T cell targeting NKG2D ligand. According to the invention, the CAR-T cell targeting the NKG2D ligand and the artemisinin compound are combined, so that the CAR-T cell and the artemisinin compound can generate a synergistic effect, excellent curative effect on malignant tumors can be generated, the dosage of the CAR-T cell is reduced so as to reduce side effects caused by the CAR-T cell, and the treatment cost is reduced.

Description

Artemisinin compound and CAR-T cell targeting NKG2D ligand combined preparation of medicine for treating malignant tumor

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of an artemisinin compound and CAR-T cells targeting NKG2D ligand in preparation of a medicine for treating malignant tumors.

Background

The chimeric antigen receptor T (CAR-T) cell technology is considered to be one of the most effective treatment modes of malignant tumors at present, wherein malignant diseases such as cancer seriously harm human health, and the chimeric antigen receptor T (CAR-T) cell technology is used for infusing genetically modified T lymphocytes (capable of expressing specific chimeric antigen receptors) back to a human body, activating the autoimmune system of the human body and further continuously killing malignant tumor cells. And NKG2D belongs to C-type lectin-like receptor family, is a II-type transmembrane glycoprotein, has different degrees of expression of NK G2D ligand on tumor cells of almost all tissue sources of human bodies, rarely appears on the surface of healthy cells, so that CAR-T targeting NKG2D ligand can greatly improve the recognition and killing effect on tumor cells.

Although CAR-T cell therapy has achieved good results in some clinical trials, it has significant side effects (e.g., cytokine release syndrome, neurotoxicity, off-target effects, etc.) during treatment and does not kill target cells sufficiently. In addition, cancer patients often have a weak immune system, a low number of immune cells in the body, and an unexpectedly strong ability to kill tumor cells. Therefore, there is a need to further investigate CAR-T cell therapy to enhance its killing effect on tumor cells.

Artemisinin is a flavonol type flavonoid compound, can be used for treating malaria drug resistance, and also has antitumor, antiviral, antiinflammatory, and antibacterial effects. At present, the combined report of artemisinin compounds and CAR-T cells targeting NKG2D ligands on the aspect of tumor treatment is not found in the industry.

Disclosure of Invention

In view of the above, the invention provides a new application of the combination of an artemisinin compound and CAR-T cells targeting NKG2D ligand in the preparation of a medicament for treating malignant tumors, and the combination of the artemisinin compound and the CAR-T cells can achieve a remarkable synergistic effect of killing tumor cells.

In a first aspect, the invention provides the use of an artemisinin compound in combination with chimeric antigen receptor T cells (CAR-T) targeting a NKG2D ligand in the preparation of a medicament for the treatment of a malignant tumor.

The applicant of the application finds that the artemisinin compound can up-regulate the expression of the NKG2D ligand of the malignant tumor, so that the recognition and killing effects of the CAR-T cell targeting the NKG2D ligand on the malignant tumor cell are obviously enhanced, the combined use of the two shows a synergistic effect, a good tumor treatment effect can be achieved, the dosage of the CAR-T cell targeting the NKG2D ligand can be reduced, and the side effect caused by the single CAR-T cell treatment can be reduced. In addition, the artemisinin compound is a small-molecule organic compound, is low in price, and can reduce the immunotherapy cost of the CAR-T cell.

In the invention, the artemisinin compound can comprise artemisinin and derivatives thereof. The artemisinin derivative comprises various derivatives which can modify and/or remove side chain groups on a skeleton by simple chemical methods (such as reduction, substitution and the like) and still retain the function of enhancing the T cell killing function of the chimeric antigen receptor, and also comprises pharmaceutically acceptable salts, isomers, hydrates and the like of the above substances.

Optionally, the artemisinin compound comprises one or more of artemisinin (structural formula is shown in the following formula (I)), dihydroartemisinin (structural formula is shown in the following formula (II)), artemether (structural formula is shown in the following formula (III)), arteether (structural formula is shown in the following formula (IV)) and artesunate (structural formula is shown in the following formula (V)). In some embodiments of the invention, the artemisinin compound is dihydroartemisinin.

Wherein the malignant tumor comprises one or more of leukemia, multiple myeloma, malignant lymphoma, brain glioma, colon cancer, gastric cancer, esophageal cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer and prostate cancer, but is not limited thereto, and only tumors capable of expressing NKG2D ligand are required. After the artemisinin compound is combined with CAR-T cells targeting NKG2D ligand, the therapeutic effect on gastric cancer, esophageal cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer, prostate cancer and other solid tumors is more obvious.

Optionally, the CAR-T cells targeted to NKG2D ligands are administered by injection, including specifically, for example, intravenous, arterial, subcutaneous, intradermal, intrathecal, or intramuscular injection. The CAR-T cells can be dissolved or suspended in a solution suitable for injectable administration in an acceptable carrier, including isotonic sterile injectable aqueous and non-aqueous solutions. Common administration modes of the artemisinin compounds include oral administration or injection and the like. For example, artesunate is usually administered in the form of an injection thereof, and artemisinin or dihydroartemisinin may be administered orally.

When an artemisinin compound is used in combination with a CAR-T cell targeted to an NKG2D ligand, the two may be administered sequentially or simultaneously, and the administration may be the same or different. For example, the artemisinin compound is administered orally or by injection, and then the CAR-T cell targeting the NKG2D ligand is administered by injection, so that the artemisinin compound can stimulate a patient suffering from malignant tumor to generate more NKG2D ligand, and the recognition and treatment effects of the CAR-T cell targeting the NKG2D ligand on malignant tumor cells are further enhanced; and does not affect the cellular activity of CAR-T cells themselves that target NKG2D ligands.

In some embodiments of the present application, the NKG2D ligand-targeted chimeric antigen receptor T cell has a NKG2D ligand-targeted chimeric antigen receptor CAR-NKG2D, wherein the CAR-NKG2D comprises, sequentially joined from amino-terminus to carboxy-terminus, an amino acid sequence of a single-chain antibody targeting a NKG2D ligand, an extracellular hinge region, a transmembrane region, and an intracellular signal region; the amino acid sequence of the single-chain antibody targeting the NKG2D ligand comprises the amino acid sequence as set forth in SEQ ID NO: 1.

The single-chain antibody targeting the NKG2D ligand is a humanized single-chain antibody, can avoid causing immune reaction of a human body, and has high safety. Alternatively, the gene encoding the NKG2D ligand-targeted single chain antibody comprises the amino acid sequence as set forth in SEQ ID NO: 5.

In one embodiment of the invention, the extracellular hinge region is a CD8 a hinge region; the transmembrane region is a CD8 transmembrane region; the intracellular signal region comprises a 4-1BB signal region and a CD3 zeta signal region which are sequentially connected from an amino terminal to a carboxyl terminal. That is, the amino acid sequence of CAR-NKG2D includes the amino acid sequence of a single chain antibody targeting NKG2D ligand, CD8 a hinge region, CD8 transmembrane region, 4-1BB signal region, and CD3 zeta signal region, sequentially linked from amino terminus to carboxy terminus. Alternatively, the amino acid sequence of CAR-NKG2D comprises the amino acid sequence set forth as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. Further optionally, the gene encoding CAR-NKG2D comprises the amino acid sequence set forth in SEQ ID NO: 3.

Further, the genes encoding CAR-NKG2D include a gene encoding a signal peptide, a gene encoding a single-chain antibody targeting an NKG2D ligand, a gene encoding an extracellular hinge region, a gene encoding a transmembrane region, and a gene encoding an intracellular signal region, which are connected in sequence from the 5 'end to the 3' end. In this case, the gene encoding CAR-NKG2D may include the sequence as set forth in SEQ ID NO: 4. And SEQ ID NO: 3, compared with the nucleotide sequence shown in SEQ ID NO: 4, the coding gene of the signal peptide is added in the nucleotide sequence shown in the specification. Wherein, the coding gene of the signal peptide can better guide the expression of CAR-NKG2D to the surface of a T cell, but when the expression of CAR-NKG2D reaches the surface of the T cell, the signal peptide is cut by the signal peptidase in the process of protein translation maturation.

Optionally, the T cells in the NKG2D ligand-targeted CAR-T cells are from autologous T cells, allogeneic T cells, or iPSC-induced T cells. Preferably autologous T cells from patients suffering from malignancies, to minimize the immune response when CAR-T cells targeting NKG2D ligands are returned to the patient.

In a second aspect, the invention also provides a medicament for treating malignant tumors, which comprises an artemisinin compound and CAR-T cells targeting NKG2D ligands.

Optionally, the administration of the NKG2D ligand-targeted CAR-T cells is by injection; common administration modes of the artemisinin compounds include oral administration or injection and the like. Alternatively, in the medicament, the CAR-T cell targeting the NKG2D ligand and the artemisinin compound may be present separately or mixed together. Wherein, when the CAR-T cell targeting the NKG2D ligand and the artemisinin compound exist separately, the administration modes of the CAR-T cell and the artemisinin compound can be the same or different, and the CAR-T cell and the artemisinin compound can be administered simultaneously or sequentially and respectively. For example, when CAR-T cells targeting NKG2D ligands are mixed together with artemisinin compounds, the drugs may be in an injectable formulation, administered simultaneously by injection. In this case, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, such as at least one diluent, e.g., water, physiological saline, aqueous dextrose, and other non-aqueous solvents, and at least one stabilizer, but is not limited thereto.

Furthermore, the medicine also comprises other active ingredients for treating malignant tumors. The other active ingredients for treating malignant tumor include at least one of chemotherapeutic agent and immune checkpoint inhibitor. Among them, as the immune checkpoint inhibitor, one or more of an anti-PD-1 antibody, an anti-PDL 1 antibody, an anti-TIM antibody, an anti-CTLA-4 antibody, and the like can be exemplified. Immune checkpoint inhibitors can enhance their anti-tumor effect with CAR-T cells by relieving immune suppression. The chemotherapeutic agent may include at least one of alkylating agent (such as cyclophosphamide, moxidectin, etc.), antimetabolite (such as methotrexate, fluorouracil, etc.), antitumor antibiotic (such as adriamycin, etc.), antitumor animal and plant component drug (such as vincristine, taxol, etc.), antitumor hormone (such as dexamethasone, testosterone propionate, etc.), platinum chemotherapeutic drug (such as cisplatin, carboplatin, etc.), etc. Chemotherapeutic agents may be selected for a particular malignancy.

Alternatively, the additional active ingredient for treating malignancy may be present alone or mixed with at least one of CAR-T cells targeting NKG2D ligand and artemisinin compounds. The other active ingredient for treating malignant tumor can be administered orally or by injection (e.g., intravenous, subcutaneous, or intramuscular injection). The mode of administration, order of administration, etc. may be selected according to the particular other active ingredients and distribution of the active ingredients for treating malignant tumors. For example, when the other active ingredient for treating malignancy is present alone, it may be administered in a different manner than both the artemisinin compound and the CAR-T cells targeting the NKG2D ligand, or at least one of the two.

According to the invention, the CAR-T cells targeting the NKG2D ligand and the artemisinin compounds having synergistic effect with the same are combined, so that the side effects caused by the CAR-T cells can be reduced by reducing the dose of the CAR-T cells on the premise of ensuring the curative effect, and meanwhile, the treatment effect of the CAR-T cell therapy can be enhanced.

Drawings

FIG. 1 is a diagram showing the results of flow-based assay of the chimeric antigen receptor CAR-NKG2D expressed after a recombinant lentivirus carrying the gene encoding CAR-NKG2D infects T cells according to an embodiment of the present invention;

FIG. 2 is a statistical plot of the in vitro killing rates of PC-3 cells for each experimental group;

FIG. 3 is a diagram of the tumor inhibition effect of each experimental group on tumor-bearing mice;

wherein, DHA represents artemisinin, NKG2D CAR-T represents CAR-T cells targeting NKG2D ligand, UTD represents T cells not transduced by virus.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

The invention provides an application of artemisinin compounds and CAR-T cells targeting NKG2D ligands in preparation of a medicine for treating malignant tumors.

The synergy between CAR-T cells targeting NKG2D ligands and artemisinin compounds is demonstrated below by specific examples.

First, CAR-T cells targeting NKG2D ligands were prepared comprising the following steps:

(1) construction of pWPXld-CAR-NKG2D recombinant plasmid

The sequence is shown as SEQ ID NO: 4, the coding gene of CAR-NKG2D is inserted between BamHI and EcoRI sites of pWPXLD vector, and is positioned behind elongation factor 1 alpha (EF1 alpha) of pWPXLD vector, and EF1 alpha is used as promoter. When the coding gene of CAR-NKG2D is inserted into pWPXLD vector, the 5 'end of the coding gene of CAR-NKG2D can be added with initiation codon (such as ATG) to be connected with BamHI enzyme cutting site in pWPXLD vector, and the 3' end can be added with termination codon (such as TAA) to be connected with EcoRI enzyme cutting site in pWPXLD vector. Then transferred into escherichia coli competent cell DH5 alpha, and positive clone PCR identification and sequencing identification are carried out. And (3) successfully constructing the pWPXld-CAR-NKG2D recombinant plasmid by detecting PCR product gel electrophoresis and sequencing to identify the size and the sequence of the fragment which accord with the target.

(2) Recombinant lentivirus construction

The pWPXLD-CAR-NKG2D recombinant plasmid obtained in the above way, a packaging plasmid psPAX2 and an envelope plasmid pMD2G are subjected to cotransfection with a liposome transfection reagent Lipofectamine3000 to cultured HEK293T cells, and after 48 hours, the recombinant lentivirus with the CAR-NKG2D coding gene is obtained through centrifugation.

(3) Preparation of chimeric antigen receptor T cells targeting NKG2D ligand

Collecting and separating Peripheral Blood Mononuclear Cells (PBMC) of healthy people, adding CD3/CD28 immunomagnetic beads for incubation, and screening to obtain CD3 positive T lymphocytes; adding the recombinant lentivirus into the cell culture medium for culture to obtain CAR-T cells targeting NKG2D ligand, and storing the CAR-T cells in cell frozen stock solution special for reinfusion for later use.

The expression of the chimeric antigen receptor CAR-NKG2D targeting the NKG2D ligand was also detected by flow cytometry, and the results are shown in figure 1. Through detection, the positive rate of CAR-NK G2D in T cells infected by the recombinant lentivirus is about 39%. This indicates that CAR-T cells targeted to NKG2D ligands were successfully produced and stored in cell stocks dedicated for reinfusion for patient reinfusion.

In vitro tumor cell killing assay

1. PC-3 cells (human prostate cancer cells) frozen in liquid nitrogen were recovered and used as target cells, which were subcultured in serum-containing DMEM complete medium. After thawing cryopreserved C AR-T cells targeting NKG2D ligand, the positive rate of CAR-NKG2D was approximately 39% (see FIG. 1, UTD stands for untreated PBMC cells) and they were subcultured in 581 medium with IL-2(50 IU/mL).

2. Tumor cell killing experiments were performed using a real-time cell function analyzer (xcelligene RTCA SP): first, 50. mu.L of DMEM complete medium was added to the E-Plate equipped with the apparatus, and the E-Plate was placed in the apparatus for Blank scanning, and then 50. mu.L of DMEM complete medium containing 5000 PC-3 cells was added to each well of the E-Plate.

3. The experiments were divided into 4 groups: a control group, an NKG2D CAR-T cell group, a dihydroartemisinin group (DHA), an NKG2D CAR-T cell and dihydroartemisinin combined administration group. After the PC-3 cells are inoculated for 24h, the corresponding drugs are respectively added according to grouping conditions for treatment, wherein DMSO (dimethyl sulfoxide) is added in the control group and the NKG2D CAR-T cell group, DMSO solutions of dihydroartemisinin are added in the DHA group and the combined administration group, and the final concentration of the dihydroartemisinin in each corresponding hole is 1 muM.

4. After approximately 24h of dosing, either blank media or CAR-T cells targeting NKG2D ligand were added, respectively, depending on the grouping. Wherein, the control group and the DHA group are both added with DMEM complete medium, and the NK G2D CAR-T cell group and the combined administration group are both added with DMEM complete medium containing 10000 CA R-T cells targeting NKG2D ligand.

5. And stopping the experiment after the CAR-T cells which target the NKG2D ligand are added to kill for 48 hours, and analyzing the killing effect according to the cell index CI value of a real-time cell function analyzer. Wherein the killing rate of each test group is the ratio of the difference between the CI value of each test group and the CI value of the control group to the CI value of the control group.

FIG. 2 summarizes the results of killing PC-3 cells. From FIG. 2, it can be known that dihydroartemisinin (1 μ M DHA) alone or CAR-T cells targeting NKG2D ligand both have weak killing effect on PC-3 tumor cells, but when the two are combined, the killing effect on tumor cells is remarkably enhanced.

In vivo evaluation experiments were as follows:

in vivo averaging experiments were performed using immunodeficient mouse B-NDG. Each mouse was inoculated with 2X 10 PC-3 cells ⁶ One (mixed with Matrigel in equal volume) to the subcutaneous part, and the subcutaneous tumor grows to about 150mm after about 10 days ³ Then randomly divided into 4 groups: UTD group (untreated group), DHA-administered group (DHA), CAR-T group targeting NKG2D ligand, CAR-T and DHA combination group targeting NKG2D ligand (CA R-T + DHA). The DHA group and the CAR-T + DHA group are respectively administered with 1 mg/DHA per abdominal cavity every day for 7 days; UTD and CAR-T groups were each administered intraperitoneally daily for 7 consecutive days with a solvent control. After 7 days of administration, CAR-T cell administration treatment was performed. UTD group mouse tail vein injection 1X 10 ⁶ UTD cells (T cells not virus-transduced), DHA group caudal intravenous injection of equal volume of PBS, CAR-T group and CAR-T + DHA group mice were intravenous injection of 1X 10 ⁶ A CAR-T cell targeted to an NKG2D ligand. Mice were well conditioned throughout the evaluation period after dosing and tumor volumes were measured and the results are shown in fig. 3.

As can be seen from FIG. 3, the CAR-T group and the DHA group had no significant tumor-inhibiting effect compared to the UTD group, but the CAR-T and DHA group had significant difference (P <0.01), showing stronger tumor-inhibiting effect. The results show that the CAR-T cell targeting the NKG2D ligand and dihydroartemisinin have strong synergistic tumor inhibition effect.

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 present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims

1. Use of an artemisinin compound in combination with a CAR-T cell targeting an NKG2D ligand for the preparation of a medicament for the treatment of a malignant tumor.

2. The use according to claim 1, wherein the artemisinin compound comprises one or more of artemisinin, dihydroartemisinin, artemether, arteether and artesunate.

3. The use of claim 1, wherein said NKG2D ligand-targeted chimeric antigen receptor T-cell has a NKG2D ligand-targeted chimeric antigen receptor CAR-NKG2D, wherein said CAR-NKG2D comprises, connected sequentially from amino-terminus to carboxy-terminus, the amino acid sequence of a NKG2D ligand-targeted single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signal region; the amino acid sequence of the single-chain antibody targeting the NKG2D ligand includes the amino acid sequence set forth in SEQ ID NO: 1.

4. The use according to claim 1, characterized in that the T cells used in the chimeric CAR-T cells targeting the NKG2D ligand are derived from autologous T cells, allogeneic T cells or iPSC-induced T cells.

5. The use according to claim 1, wherein the administration of the NKG2D ligand-targeted CAR-T cells is by injection and the administration of the artemisinin compound comprises oral administration or injection.

6. The use according to claim 5, wherein the artemisinin compound is administered first and the CAR-T cells targeting NKG2D ligand are administered later.

7. The use of claim 1, wherein the malignancy comprises one or more of leukemia, multiple myeloma, malignant lymphoma, brain glioma, colon cancer, gastric cancer, esophageal cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer, and prostate cancer.

8. A medicament for treating malignant tumors, which comprises an artemisinin compound and CAR-T cells targeting NKG2D ligands.

9. The medicament of claim 8, wherein the NKG2D ligand-targeted CAR-T cells and the artemisinin compound are present alone or in a mixture.

10. The medicament of claim 9, further comprising an additional active component for treating malignancy, said additional active component for treating malignancy being present separately or in admixture with at least one of the artemisinin compound and the NKG2D ligand-targeted CAR-T cells.