CN114796470A - Resveratrol compound and CAR-T cell targeting NKG2D ligand combined preparation of medicine for treating malignant tumor - Google Patents

Abstract

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

Description

Resveratrol 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, and in particular relates to application of resveratrol compound and CAR-T cell combination targeting NKG2D ligand in preparation of a medicine for treating malignant tumor.

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 well. 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.

Resveratrol (Res) is a non-flavonoid polyphenol compound with antioxidant, antiinflammatory, anticancer and cardiovascular protecting effects. At present, the industry has not reported the combination of resveratrol and CAR-T cells targeting NKG2D ligand in the aspect of tumor treatment.

Disclosure of Invention

In view of the above, the invention provides a new application of resveratrol compound and CAR-T cell targeting NKG2D ligand in preparing a medicine for treating malignant tumor, and the combination of resveratrol compound and CAR-T cell can produce significant synergistic effect of killing tumor cells.

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

The applicant of the application finds that the resveratrol compound can up-regulate malignant tumor expression NKG2D ligand, further obviously enhance the recognition and killing effects of the CAR-T cell targeting the NKG2D ligand on malignant tumor cells, and the combination of the resveratrol compound and the CAR-T cell presents a synergistic effect, so that a good tumor treatment effect can be achieved, the using amount of the CAR-T cell targeting the NKG2D ligand can be reduced, and the side effect caused by single CAR-T cell treatment can be reduced. In addition, the resveratrol compound is a small-molecular organic compound, is low in price and can reduce the immunotherapy cost of CAR-T cells.

In the invention, the resveratrol compound can comprise one or more of resveratrol, resveratrol derivatives and prodrugs, isomers and pharmaceutically acceptable salts thereof, which are all reserved to enhance the killing function of CAR-T cells. Wherein the resveratrol derivative comprises a compound which takes Res as a framework and is obtained by changing substituents on two benzene rings of Res or changing a cis-trans structure and the like. In particular, the amount of the solvent to be used,

the CAS number of resveratrol is 501-36-0, and the structural formula is shown as the following formula (I):

the resveratrol compound may include one or more of 3' -hydroxyresveratrol (piceatannol), 3,4,5, 4' -tetrahydroxystilbene (4-HR), trans-3, 5,4' -Trimethoxystilbene (TMB) obtained by methylating all the hydroxyl groups of Res), 3,4,5, 4' -tetramethoxystilbene (MR-4), 3,4, 5-trimethoxy-4 ' -amino-stilbene, 4' -acetamido-3, 4, 5-trimethoxy-stilbene, 3,5,4' -trimethoxystilbene (resveratrol trimethy ether), etc. having antitumor activity, but is not limited thereto.

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 resveratrol 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 resveratrol compounds include oral administration, injection and the like.

When resveratrol is used in combination with CAR-T cells targeting NKG2D ligands, the two may be administered sequentially or simultaneously. For example, the resveratrol compound is orally or injected firstly, and then the CAR-T cell targeting the NKG2D ligand is injected and administered, so that the resveratrol 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 CAR-NKG 2D-encoding gene includes a signal peptide-encoding gene, a single-chain antibody-targeting NKG2D ligand-encoding gene, an extracellular hinge region-encoding gene, a transmembrane region-encoding gene and an intracellular signal region-encoding gene, which are sequentially linked 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, and 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 a resveratrol compound and CAR-T cells targeting NKG2D ligand.

Optionally, in the medicament, the CAR-T cells targeting the NKG2D ligand and the rutin compound can be separately present or mixed together. Wherein the single separated existence can exert better effect of treating malignant tumor. When the CAR-T cell targeting the NKG2D ligand and the rutin compound exist separately, the administration modes of the CAR-T cell and the rutin compound can be the same or different, and the CAR-T cell and the rutin compound can be administered simultaneously or sequentially and respectively. When the CAR-T cells targeting the NKG2D ligand and the rutin compound exist in a mixed state, the medicine can be an injection preparation and can be simultaneously administered 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.

Optionally, the medicament for treating malignant tumor further comprises other active ingredients for treating malignant tumor. Wherein the other active ingredients for treating malignant tumor comprise 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 immunosuppression. 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.

Further, the additional active ingredient for treating malignancy may be present alone or in admixture with at least one of a CAR-T cell targeting NKG2D ligand and a resveratrol compound. The other active ingredients for treating malignant tumor can be administered orally or by injection. The mode of administration, order of administration, etc. may be selected according to the particular other active ingredient being used to treat the malignancy. For example, when other active ingredients for treating malignancies are present alone, they can be administered in a manner different from, or at least the same as, the manner in which both the resveratrol compound and the CAR-T cells targeting the NKG2D ligand are administered.

According to the invention, the CAR-T cells targeting the NKG2D ligand and the resveratrol compound having a synergistic effect 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 curative 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 graph showing the killing of PC-3 cells by each experimental group;

FIG. 3 is a statistical chart of the killing rate of PC-3 cells after 24 hours of killing for each experimental group;

wherein Res represents resveratrol and NKG2D CAR-T represents CAR-T cells targeted to NKG2D ligands.

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 application of resveratrol compound and CAR-T cell of targeting NKG2D ligand in preparation of drugs for treating malignant tumor.

The applicant of the application finds that the resveratrol compound can up-regulate malignant tumor expression NKG2D ligand, further obviously enhance the recognition and killing effects of the CAR-T cell targeting the NKG2D ligand on malignant tumor cells, and the combination of the resveratrol compound and the CAR-T cell presents a synergistic effect, so that a good tumor treatment effect can be achieved, the using amount of the CAR-T cell targeting the NKG2D ligand can be reduced, and the side effect caused by single CAR-T cell treatment can be reduced. In addition, the resveratrol compound is a small-molecular organic compound, is low in price and can reduce the immunotherapy cost of CAR-T cells.

The synergy between CAR-T cells targeting NKG2D ligands and resveratrol 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. The positive rate of CAR-NKG2D 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. The frozen PC-3 cells (human prostate cancer cells) were recovered from liquid nitrogen, and used as target cells, which were subcultured in serum-containing DMEM complete medium. After thawing cryopreserved NKG2D ligand-targeted CAR-T cells, the positive rate of CAR-NKG2D was approximately 39% (see fig. 1, UTD stands for untreated PBMC cells), which 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 an E-Plate equipped with the apparatus, and the apparatus was set to perform 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 resveratrol group (Res), an NKG2D CAR-T cell and resveratrol combined administration group. After the PC-3 cells are inoculated for 24 hours, corresponding medicines are respectively added according to grouping conditions for treatment, wherein DMSO (dimethyl sulfoxide) is added into the control group and the NKG2D CAR-T cell group, DMSO solutions of resveratrol are added into the Res group and the combined administration group, and the final concentration of the resveratrol in corresponding wells is 30 mu M.

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 Res group are added with DMEM complete medium, and the NKG2D CAR-T cell group and the combined administration group are added with DMEM complete medium containing 10000 CAR-T cells targeting NKG2D ligands.

5. After the CAR-T cells which target NKG2D ligand are added to kill for 30h, the experiment is stopped, and the killing effect is analyzed according to the cell index CI value of a real-time cell function analyzer. Wherein, the larger the CI value, the larger the number of the PC-3 cells representing the survival, the worse the killing effect.

FIGS. 2 and 3 show the results of killing PC-3 cells. From fig. 2-3, it can be known that the killing effect of the resveratrol alone or the CAR-T cell targeting NKG2D ligand on the PC-3 tumor cell is weak, but when the resveratrol and the CAR-T cell are combined, the killing effect on the tumor cell is obviously enhanced. This indicates that the CAR-T cells targeting NKG2D ligand in combination with resveratrol compounds can achieve synergistic killing of tumor cells.

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.

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Claims (10)

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

2. The use according to claim 1, wherein the resveratrol compound comprises one or more of resveratrol, piceatannol, 3,4,5,4 '-tetrahydroxystilbene, trans-3, 5,4' -trimethoxystilbene, 3,4,5,4 '-tetramethoxystilbene, 3,4, 5-trimethoxy-4' -amino-stilbene, 4 '-acetamido-3, 4, 5-trimethoxy-stilbene, 3,5,4' -trimethoxystilbene and prodrugs, pharmaceutically acceptable salts thereof.

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 comprises the amino acid sequence as 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 resveratrol-based compound comprises oral administration and/or injection.

6. The use according to claim 5, wherein the resveratrol 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 resveratrol compounds and CAR-T cells targeting NKG2D ligands.

9. The medicament of claim 8, further comprising an additional active component for treating malignancy, the additional active component for treating malignancy comprising at least one of a chemotherapeutic agent and an immune checkpoint inhibitor.

10. The medicament according to claim 9, wherein the other active ingredients for the treatment of malignancies are present in a mixed state or separately from at least one of the resveratrol compounds and the NKG2D ligand-targeted CAR-T cells.

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