CN114796467A

CN114796467A - Sodium valproate compound and CAR-T cell targeting NKG2D ligand combined preparation of medicine for treating malignant tumor

Info

Publication number: CN114796467A
Application number: CN202110119999.1A
Authority: CN
Inventors: 刘茂玄; 许晨光; 戴昆
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 a sodium valproate 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 sodium valproate compound and the CAR-T cell targeting an NKG2D ligand. According to the invention, the CAR-T cell targeting the NKG2D ligand and the sodium valproate compound are combined, so that the CAR-T cell and the sodium valproate compound can generate a synergistic effect, an excellent curative effect on malignant tumors can be generated, 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

Sodium valproate 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 an application of a sodium valproate 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.

Sodium Valproate (Sodium Valproate, abbreviated as VPA) is a nitrogen-free broad-spectrum antiepileptic drug, and has also been reported to have an antitumor effect in recent years. However, no combination report of sodium valproate compounds and CAR-T cells targeting NKG2D ligand in tumor treatment is found in the industry at present.

Disclosure of Invention

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

In a first aspect, the invention provides the use of a sodium valproate 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 sodium valproate compound can up-regulate malignant tumor expression NKG2D ligand, 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 synergistic effect is shown by the combination of the two, the 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 independent CAR-T cell treatment can be reduced. In addition, the sodium valproate compound is a small molecular organic compound, is low in price, and can reduce the immunotherapy cost of the CAR-T cells.

In the present invention, the sodium valproate compound may include sodium valproate and derivatives thereof. The sodium valproate derivative includes 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 isomers, hydrates and the like of the above substances.

Optionally, the sodium valproate compound comprises one or more of sodium valproate (CAS number 1069-66-5), sodium butyrate, sodium methyl valerate, 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 sodium valproate 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 sodium valproate compound comprise oral administration, injection and the like.

When the sodium valproate compound is used in combination with a CAR-T cell targeting an NKG2D ligand, the two can be administered sequentially or simultaneously, and the administration can be the same or different. For example, the sodium valproate compound is administered orally or by injection, and then the CAR-T cell targeting the NKG2D ligand is administered by injection, so that the sodium valproate 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 the malignant tumor cell 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 in 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 tumor, which comprises a sodium valproate compound and CAR-T cells targeting NKG2D ligand.

Alternatively, in the medicament, the CAR-T cells targeting the NKG2D ligand and the sodium valproate compound may be present separately or mixed together. Wherein the single separated existence can exert better effect of treating malignant tumor. When the NKG2D ligand-targeted CAR-T cells and the sodium valproate compound exist separately, the administration modes of the CAR-T cells and the sodium valproate compound can be the same or different, and the CAR-T cells and the sodium valproate compound can be administered simultaneously or sequentially and respectively. When the NKG2D ligand-targeted CAR-T cells and the sodium valproate 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 medicine also comprises other active ingredients for treating malignant tumor. 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.

Further 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 a sodium valproate compound. 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 both the sodium valproate compound and the CAR-T cell targeting NKG2D ligand, or at least one of the two. For another example, the other active ingredients for treating malignant tumors may be administered simultaneously or sequentially with the valproate sodium compound. Preferably, in the medicament, the CAR-T cells targeting the NKG2D ligand are administered last.

According to the invention, the CAR-T cells targeting the NKG2D ligand and the sodium valproate compound with 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 statistical graph of the in vitro killing rate of U251 cells for each experimental group;

wherein VPA represents sodium valproate and NKG2D CAR-T represents CAR-T cells 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 an application of a sodium valproate compound and CAR-T cells targeting NKG2D ligand in preparation of a medicine for treating malignant tumor.

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

The synergy between CAR-T cells targeting NKG2D ligands and valproate sodium 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. The frozen U251 cells (human glioma cells) in liquid nitrogen were thawed and subcultured in DMEM complete medium containing serum, using them as target cells. 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 4000U 251 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 sodium valproate group (VPA), an NKG2D CAR-T cell and sodium valproate combined administration group. After the U251 cells are inoculated for 24 hours, corresponding medicines are respectively added according to grouping conditions for treatment, wherein PBS buffer solution is added into the control group and the NKG2D CAR-T cell group, PBS buffer solution is added into the VPA group and the combined administration group, and the final concentration of sodium valproate in corresponding wells is 3.2 mM.

4. After approximately 48h 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 VPA 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 1000 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 killing results for U251 cells. From fig. 2, it can be known that sodium valproate alone or CAR-T cells targeting NKG2D alone both have weak killing effect on U251 tumor cells, but when the two are combined, the killing effect on tumor cells is significantly enhanced. This indicates that the CAR-T cells targeting NKG2D ligand and sodium valproate 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 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 should be subject to the appended claims.

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Claims

1. The application of the sodium valproate compound and the CAR-T cell targeted to NKG2D ligand in preparing the medicine for treating malignant tumor is disclosed.

2. Use according to claim 1, wherein the sodium valproate-like compound comprises one or more of sodium valproate, sodium butyrate, sodium methyl valerate.

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 sodium valproate-like compound comprises oral administration and/or injection.

6. The use according to claim 5, wherein the valproate sodium 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 a malignant tumor, comprising a sodium valproate compound and CAR-T cells targeting an NKG2D ligand.

9. The medicament of claim 8, wherein said NKG2D ligand-targeted CAR-T cells are present separately from said sodium valproate compound.

10. 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.