CN114796469A - Rutin compound and CAR-T cell targeting NKG2D ligand are combined to prepare medicine for treating malignant tumor - Google Patents

Abstract

The invention provides application of a rutin compound and a CAR-T cell targeting an 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 rutin compound and the CAR-T cell targeting an NKG2D ligand. According to the invention, the CAR-T cell targeting the NKG2D ligand and the rutin compound are combined, so that the CAR-T cell and the rutin compound can generate a synergistic interaction effect, excellent curative effect on malignant tumors can be achieved, the dosage of the CAR-T cell is reduced, side effects caused by the CAR-T cell are reduced, and the treatment cost is reduced.

Description

Rutin compound and CAR-T cell targeting NKG2D ligand are combined to prepare medicine for treating malignant tumor

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a rutin 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 a C-type lectin-like receptor family, is a II-type transmembrane glycoprotein, expresses NKG2D ligand on tumor cells of almost all tissue sources of a human body to different degrees, rarely appears on the surface of healthy cells, so that CAR-T targeting NKG2D ligand can greatly improve the recognition and killing effect on the 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.

Rutin (Rutin) is a flavonol type flavonoid, also known as Rutin or vitamin P. Rutin is reported to have various pharmacological activities, including antioxidant, anti-inflammatory, antiviral, antithrombotic, blood vessel-protecting etc. At present, the combined report of rutin compounds and CAR-T cells targeting NKG2D ligands on the aspect of treating tumors is not found in the industry.

Disclosure of Invention

In view of the above, the invention provides a new application of a rutin compound and a CAR-T cell targeting an NKG2D ligand in preparation of a medicine for treating malignant tumor, and the combination of the rutin compound and the CAR-T cell can generate an obvious synergistic effect of killing tumor cells.

In a first aspect, the invention provides an application of a rutin compound and a chimeric antigen receptor T cell (CAR-T) targeting NKG2D ligand in combination in preparation of a medicament for treating malignant tumor.

The applicant of the application finds that the rutin 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 the malignant tumor cell, the combination 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 single CAR-T cell treatment can be reduced. In addition, the rutin 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 rutin compound can comprise rutin and derivatives thereof. The rutin derivatives include various derivatives which can modify and/or remove side chain groups on the skeleton by simple chemical methods (such as reduction, substitution and the like) and still retain the function of enhancing the killing of chimeric antigen receptor T cells, and also include pharmaceutically acceptable salts, isomers, metal complexes and the like of the above substances.

Optionally, the rutin compound includes one or more of rutin (CAS number is 153-18-4, structural formula is shown in formula (I)), troxerutin (CAS number is 7085-55-4, structural formula is shown in formula (II)), rutin chromium, quercetin (CAS number is 117-39-5, structural formula is shown in formula (II)), and the like, 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 rutin 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 rutin compounds include oral administration or injection and the like. For example, troxerutin is usually administered in the form of an injection thereof, and rutin, rutin chromium and quercetin may be administered in the form of an oral tablet.

When the rutin compound is combined with CAR-T cells targeting NKG2D ligand, the two can be administered sequentially or simultaneously, and the administration modes of the two can be the same or different. For example, the rutin compound is orally taken or injected firstly, and then the CAR-T cell targeting the NKG2D ligand is injected and administered, so that the rutin compound can stimulate a patient suffering from malignant tumor to generate more NKG2D ligand, and further enhance the recognition and treatment effects of the CAR-T cell targeting the NKG2D ligand on malignant tumor cells; 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 drug for treating malignant tumor, wherein the drug comprises rutin compound and CAR-T cells targeting NKG2D ligand.

Optionally, the administration of the NKG2D ligand-targeted CAR-T cells is by injection; common administration modes of the rutin compounds include oral administration or injection and the like. Optionally, in the medicament, the CAR-T cells targeting the NKG2D ligand and the rutin compound can be separately present or mixed together. 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. For example, when the CAR-T cells targeting NKG2D ligand and rutin compound are present in a mixed state, the drug may be an injectable formulation, and 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 in admixture with at least one of CAR-T cells and rutin-based compounds that target NKG2D ligands. 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 manner different from that of either or at least one of the rutin compound and the CAR-T cells targeting the NKG2D ligand.

According to the invention, the CAR-T cells targeting the NKG2D ligand and the rutin compounds 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 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 graph of the in vitro killing of Huh7 cells for each experimental group;

FIG. 3 is a statistical plot of the in vitro killing rate of Huh7 cells for each experimental group;

wherein Rutin represents Rutin, and NKG2D CAR-T represents CAR-T cell targeting NKG2D ligand.

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 a rutin compound and CAR-T cells of a targeted NKG2D ligand in preparation of a medicine for treating malignant tumor.

The applicant of the application finds that the rutin compound can up-regulate malignant tumor expression NKG2D ligand, further obviously enhance recognition and killing effects of the CAR-T cells targeting the NKG2D ligand on the malignant tumor cells, and the combination of the two shows a synergistic effect, so that a good tumor treatment effect can be achieved, the dosage of the CAR-T cells targeting the NKG2D ligand can be reduced, and side effects caused by single CAR-T cell treatment can be reduced. In addition, the rutin 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 rutin-based 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; the recombinant lentivirus is added into the cell culture medium for culture, so that CAR-T cells targeting NKG2D ligand are obtained and stored in a cell freezing medium special for back transfusion 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. Huh7 cells (human liver 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 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 RTCASP): 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 Huh7 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 Rutin group (Rutin), an NKG2D CAR-T cell and Rutin combined administration group. After Huh7 cells are inoculated for 24h, corresponding drugs are respectively added according to grouping conditions for treatment, wherein DMSO (dimethyl sulfoxide) is added in a control group and a NKG2D CAR-T cell group, DMSO solutions of Rutin are added in a Rutin group and a combined administration group, and the final concentration of the Rutin in corresponding holes is 0.2 mM.

4. After approximately 24h of dosing, either blank media or CAR-T cells targeting NKG2D ligand were added, respectively, depending on the grouping. Wherein, DMEM complete medium is added into the control group and the Rutin group, and DMEM complete medium containing 5000 CAR-T cells targeting NKG2D ligands is added into the NKG2D CAR-T cell group and the combined administration group.

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 Huh7 cells representing the survival, the worse the killing effect.

Fig. 2 and 3 summarize the results of killing Huh7 cells. From fig. 2-3, it can be known that rutin alone or the CAR-T cell targeting NKG2D ligand alone has weak killing effect on Huh7 tumor cells, but when the rutin and the CAR-T cell are combined, the killing effect on the tumor cells is obviously enhanced. The result shows that the CAR-T cells targeting the NKG2D ligand and the rutin compound are combined to achieve the effect of killing tumor cells synergistically.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood 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. The rutin compound and CAR-T cells targeting NKG2D ligand are combined to prepare the medicine for treating malignant tumor.

2. The use according to claim 1, wherein the rutin-based compound comprises one or more of rutin, troxerutin, chromium rutin, quercetin.

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 said NKG2D ligand-targeted CAR-T cells is by injection, and the administration of said rutin compound comprises oral administration or injection.

6. The use according to claim 5, wherein said rutin compound is administered prior to administration of said CAR-T cells targeting NKG2D ligand.

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. The drug for treating the malignant tumor is characterized by comprising a rutin compound and CAR-T cells targeting NKG2D ligand.

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, characterized in that said other active principles for the treatment of malignancies are present alone or in a mixed state with at least one of said rutin-like compound and said CAR-T cells targeted to NKG2D ligand.

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