CN108392633B

CN108392633B - Application of PCSK9 inhibitor in malignant tumor immunotherapy

Info

Publication number: CN108392633B
Application number: CN201711463619.6A
Authority: CN
Inventors: 王红阳; 付静; 秦文昊; 杨知时; 陈瑶
Original assignee: Oriental Hepatobiliary Surgery Hospital Second Military Medical University Of Chinese Pla
Current assignee: Oriental Hepatobiliary Surgery Hospital Second Military Medical University Of Chinese Pla
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2021-08-24
Anticipated expiration: 2037-12-28
Also published as: CN108392633A

Abstract

The invention relates to application of a PCSK9 inhibitor in malignant tumor immunotherapy. The invention discloses that the PCSK9 inhibitor can effectively inhibit tumors. The PCSK9 inhibitor can enhance the activity of immune cells by increasing the expression of immune cell activation related genes, thereby inhibiting tumors. The invention also discloses the remarkable effects of the combined application of the PCSK9 inhibitor and the PD-1 inhibitor on the aspects of further improving the activity of immune cells and inhibiting tumors.

Description

Application of PCSK9 inhibitor in malignant tumor immunotherapy

Technical Field

The invention relates to the field of chemical medicines, in particular to application of a PCSK9 inhibitor in antitumor immunotherapy.

Background

The immunotherapy of tumor is the most promising new therapy which is emerging in recent years, and the therapy can enhance the reaction capability of the immune system and stimulate specific immune response, thereby achieving the purposes of delaying tumor progression, inhibiting tumor recurrence and metastasis, and even completely curing tumor.

Current immunotherapies for tumors can be divided into the following categories: (1) non-specific immunotherapy refers to the application of some immunomodulators to activate the anti-tumor immune response of the body by non-specifically enhancing the immune function of the body, so as to achieve the purpose of treating tumors. Currently, cytokines (1L-2, IFN, TNF, etc.), microorganisms and their products, vitamin K, Heat Shock Proteins (HSP), etc. are used in tumor therapy. (2) The research shows that the tumor cells can negatively regulate and control the immune cells in the tumor microenvironment through immune checkpoint molecules to inhibit the anti-tumor activity of the immune cells, and the effect of blocking the immune checkpoint can restore the killing capacity of the immune cells to inhibit the tumor development. Such as anti-PD 1 drugs that have been approved by the FDA for use in the treatment of malignancies, and anti-CTLA-4 drugs, among others. (3) Adoptive immunotherapy, which inputs immune cells with antitumor activity to malignant tumor patients to directly kill tumors or stimulate the antitumor immune effect of organisms, thereby achieving the purpose of treating malignant tumors.

Although the skilled person is working on anti-tumor studies and developing various means of tumor immunotherapy, there are still more technical difficulties, for example, there are some problems in immune checkpoint drugs: because the PD-1/PD-L1 signal channel only acts on the effect stage of T cells, the exertion of the anti-PD-1/PD-L1 drug effect requires the presentation of tumor antigens, and the drugs are often ineffective to treat about 70 percent of low-immunogenicity tumors clinically and need to be used together with chemotherapy, radiotherapy and other drugs; in addition, cancer cells sometimes achieve immune escape via a different pathway or pathways, and simply blocking the PD-1/PD-L1 pathway may not be effective for immune escape of some cancer cells. The antibody targeting CD47 has the functions of activating macrophages, enhancing T cell antigen presentation and the like, is complementary with anti-PD-1/PD-L1 in function, however, the expression of CD47 is not strictly tumor specificity, and the in vivo use of the CD47 antibody can cause adverse reactions such as anemia and the like.

Therefore, there is still a need to find a tumor immunotherapy method and a therapeutic drug which are significant in effect and easy to implement.

Disclosure of Invention

The invention aims to provide application of a PCSK9 inhibitor in malignant tumor immunotherapy.

In a first aspect of the invention, there is provided the use of a PCSK9 inhibitor in the manufacture of a medicament for inhibiting a tumour or enhancing immune cell activity.

In another aspect of the invention, the use of a PCSK9 inhibitor and a PD-1 inhibitor for the manufacture of a medicament for inhibiting a tumor or enhancing immune cell activity is provided.

In another preferred embodiment, the tumor is a tumor recognized by immune cells; preferably, the tumor includes (but is not limited to): leukemia, lymphoma, melanoma, lung cancer, liver cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, bile duct cancer, gallbladder cancer, etc.

In another preferred embodiment, the PCSK9 inhibitor or the PCSK9 inhibitor and the PD-1 inhibitor enhance tumor-inhibiting activity of immune cells by increasing expression of immune cell activation-related genes, thereby inhibiting tumors; preferably, the immune cell activation-related genes include: GZMB, IFN γ, TNF α, NOS2, NCR1, PRF, IL15, CCL3, CXCL10, CD8, NK1.1, FOXP 3.

In another preferred embodiment, the immune cell comprises: NK cells, T cells.

In another preferred embodiment, the PCSK9 inhibitor comprises: anti-PCSK 9 antibodies; an interfering molecule that interferes with the expression of the PCSK9 gene; a gene editing or gene silencing agent that specifically down-regulates PCSK 9; or PCSK9 small molecule inhibitors such as SBC-110736, SBC-115076, R-IMPP, available from MCE; or said PD-1 inhibitor comprises: an anti-PD-1 antibody, an interfering molecule that interferes with PD-1 gene expression, a gene editing or gene silencing agent that specifically down-regulates PD-1.

In another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting tumor, comprising: PCSK9 inhibitors and PD-1 inhibitors.

In another aspect of the invention, a kit for inhibiting a tumor is provided, the kit comprising a PCSK9 inhibitor and a PD-1 inhibitor; or the medicine box contains the medicine composition.

In another preferred embodiment, the kit further comprises one or more selected from the group consisting of: a tumor chemotherapeutic agent; a tumor radiotherapy medicament; instructions for use.

In another preferred embodiment, the pharmaceutical composition or the kit wherein the PCSK9 inhibitor comprises: an anti-PCSK 9 antibody, an interfering molecule that interferes with PCSK9 gene expression, a gene editing or gene silencing agent that specifically down-regulates PCSK 9; or PCSK9 small molecule inhibitors such as SBC-110736, SBC-115076, R-IMPP, available from MCE; or said PD-1 inhibitor comprises: an anti-PD-1 antibody, an interfering molecule that interferes with PD-1 gene expression, a gene editing or gene silencing agent that specifically down-regulates PD-1.

In another aspect of the present invention, there is provided a method of enhancing the activity of an immune cell, the method comprising: treating immune cells with an anti-PCSK 9 inhibitor; or treating the immune cells with a PCSK9 inhibitor in combination with a PD-1 inhibitor.

In a preferred embodiment, the method is a non-diagnostic and non-therapeutic method. For example, the method is an ex vivo method.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

Figure 1, control group, PCSK9 antibody group mouse melanoma cell B16F10 tumor-bearing tumor growth curves.

Figure 2, representative picture of mouse melanoma cells B16F10 subcutaneous tumor-bearing cells of PCSK9 antibody group of control group.

FIG. 3 is a representative picture of subcutaneous tumor-bearing cells of mice hepatoma cell Hepa1-6, lung carcinoma cell LLC and prostate cancer cell RM-1 in PCSK9 antibody group.

Figure 4, representative pictures of mouse melanoma cells B16 subcutaneous tumor-bearing cells of the control group (PBS), PCSK9 small molecule inhibitor group (SBC-110738).

FIG. 5 shows the relative blood cholesterol level of mice in the PCSK9 antibody group in the control group.

FIG. 6 is a photograph showing the expression level of each of the immunocyte markers in tumor tissues of mice belonging to a control group, PCSK9 antibody group.

FIG. 7, control group, PCSK9 antibody group tumor infiltrating NK cells and CD8⁺Cholesterol content of T cells.

FIG. 8, control, PCSK9 antibody group tumor infiltrating NK cells and CD8⁺The results of detection of T cell activation-associated genes, wherein FIG. 8A shows NK cell activation-associated genes, and FIG. 8B shows CD8⁺T cell activation related genes.

FIG. 9 shows tumor-bearing growth curves of mice in the control group, PD-1 antibody group, PCSK9 antibody + PD-1 antibody combination group, in which FIG. 9A shows the growth curve of melanoma B16, and FIG. 9B shows the tumor growth curve of lung cancer cell LLC (i.e., Lewis lung cancer cell, purchased from Chinese academy).

FIG. 10 is a graph showing a representative subcutaneous tumor in mice in a control group, a PD-1 antibody group, and a PCSK9 antibody + PD-1 antibody combination group. Wherein FIG. 10A is a representative picture of melanoma, and FIG. 10B is a representative picture of lung cancer cell LLC.

FIG. 11 shows flow-through detection of NK cells and CD8 in subcutaneous tumors of control group, PD-1 antibody group, PCSK9 antibody + PD-1 antibody combination group⁺Infiltration of T cells.

FIG. 12 shows tumor-infiltrating NK cells and CD8 in the control group, PD-1 antibody group, PCSK9 antibody + PD-1 antibody combination group⁺And (3) detection results of T cell activation related genes. Wherein FIG. 12A shows the result of detection of NK cell activation-associated gene, and FIG. 12B shows CD8⁺And (5) detecting T cell activation related genes.

Detailed Description

The present inventors have conducted extensive and intensive studies and have revealed for the first time that PCSK9 inhibitors (e.g., antibodies) are effective in inhibiting tumors. The PCSK9 inhibitor can enhance the activity of immune cells by increasing the expression of immune cell activation related genes, thereby inhibiting tumors. The invention also discloses a remarkable effect of the combined application of the PCSK9 inhibitor and the PD-1 inhibitor (such as an antibody) on the aspects of further improving the activity of immune cells and inhibiting tumors.

As used herein, a "tumor" is a "tumor" that is recognized by immune cells. The term "tumor" encompasses both solid and non-solid tumors; for example, including but not limited to: melanoma, lung cancer, leukemia, liver cancer, gastric cancer, esophageal cancer, bile duct cancer, gallbladder cancer, colorectal cancer, prostate cancer and breast cancer.

As used herein, the term "immune cell" includes: t cells, NK cells, NKT cells, Regulatory T cells (tregs).

PCSK9 inhibitors

PCSK9 is a hepatic secreted protein synthesized by hepatocytes, first synthesizes PCSK9 zymogen in the endoplasmic reticulum, which undergoes autocatalytic reaction in the endoplasmic reticulum or golgi apparatus, cleaves to release a propeptide, forms a mature protease, and is immediately secreted into the blood, binds to the extracellular domain of the Low Density Lipoprotein Receptor (LDLR) on the cell surface, mediates the internalization and degradation of LDLR, inhibits the uptake of low density lipoprotein cholesterol (LDL-C) by hepatocytes and peripheral cells, and increases LDL-C and total cholesterol levels in the blood. Studies have shown that PCSK9 levels are significantly associated with cholesterol, ox-LDL, triglycerides, etc. It not only can affect the level of plasma cholesterol and regulate the apoptosis of nerve cells, but also has a certain correlation with inflammatory reaction. At present, experiments and clinical researches are carried out on the cholesterol lowering effect of the anti-PCSK 9 antibody medicine, and no report is found on the regulation of immune cell function and the anti-tumor effect of the anti-PCSK 9 antibody medicine.

As used herein, reference to a "PCSK 9 inhibitor" includes nucleic acid inhibitors, antagonists, down-regulators, blockers, etc., as long as they are capable of lowering the level of expression of PCSK9, inhibiting the activity or function of PCSK 9. They may be chemical compounds, chemical small molecules, biological molecules. The biomolecule may be at the nucleic acid level (including DNA, RNA) or at the protein level.

The PCSK9 inhibitor refers to any substance which can reduce the activity of PCSK9, reduce the stability of PCSK9, down-regulate the expression of PCSK9 and reduce the effective action time of PCSK9, and the substances can be used for the invention, and can be used as substances which are useful for down-regulating PCSK9, thereby being used for inhibiting tumors. For example, the down-regulating agent is: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, compounds, nucleases, nucleic acid binding molecules, and the like, provided that they are capable of down-regulating the expression, inhibiting the activity or function of PCSK 9. Such nucleic acid inhibitors include, but are not limited to: an interfering molecule such as shRNA, antisense nucleic acid, small interfering RNA, microRNA or a construct capable of expressing or forming shRNA, antisense nucleic acid, small interfering RNA and microRNA by taking the PCSK9 gene or the transcript thereof as a target for inhibiting or silencing.

The PCSK9 inhibitor can also be some small molecule substances, such as small molecule compounds, which inhibit the activity of PCSK 9. Some small molecules in the art that inhibit PCSK9 activity may be used in the present invention to inhibit tumors or enhance immune cell activity. As a preferred mode of the invention, the PCSK9 inhibitor is SBC-110736, SBC-115076 or R-IMPP (purchased from MCE).

In addition, some gene editing means capable of directionally inhibiting or down-regulating gene expression by targeted gene editing exist, and the means and the reagent can also be applied to the invention for preparing the PCSK9 inhibitor.

In a preferred embodiment of the invention, the PCSK9 inhibitor is an anti-PCSK 9 antibody.

The antibody specifically inhibiting PCSK9 can be a monoclonal antibody or a polyclonal antibody. Polyclonal antibodies produced by animals, such as rabbit, mouse, rat, etc., can be immunized with the PCSK9 protein; various adjuvants may be used to enhance the immune response, including but not limited to Freund's adjuvant and the like. Similarly, cells expressing PCSK9 or an antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibody may also be a monoclonal antibody, and such monoclonal antibodies may be prepared using hybridoma technology.

PD-1 inhibitors

As used herein, the term "PD-1 inhibitor" includes nucleic acid inhibitors, antagonists, down-regulators, blockers, etc., as long as they are capable of down-regulating the expression level of PD-1, inhibiting the activity or function of PD-1. They may be chemical compounds, chemical small molecules, biological molecules. The biomolecule may be at the nucleic acid level (including DNA, RNA) or at the protein level.

The PD-1 inhibitor refers to any substance which can reduce the activity of PD-1, reduce the stability of PD-1, down-regulate the expression of PD-1 and reduce the effective action time of PD-1, and the substances can be used for the invention, and can be used as substances which are useful for down-regulating PD-1, thereby being used for inhibiting tumors. For example, the down-regulating agent is: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, compounds, nucleases, nucleic acid binding molecules, and the like, provided that they are capable of down-regulating the expression, inhibiting the activity or function of PD-1. Such nucleic acid inhibitors include, but are not limited to: interfering molecules such as shRNA, antisense nucleic acid, small interfering RNA, micro RNA or a construct capable of expressing or forming shRNA, antisense nucleic acid, small interfering RNA and micro RNA by taking PD-1 gene or transcript thereof as a target for inhibiting or silencing.

In addition, there are some gene editing means capable of directionally inhibiting or down-regulating gene expression by targeted gene editing, and such means and reagents can also be applied in the present invention for preparing PD-1 inhibitors.

In a preferred embodiment of the present invention, the PD-1 inhibitor is an anti-PD-1 antibody.

The antibody specifically inhibiting PD-1 may be a monoclonal antibody or a polyclonal antibody. Can be used for immunizing animals such as rabbit, mouse, rat, etc. with PD-1 protein to produce polyclonal antibody; various adjuvants may be used to enhance the immune response, including but not limited to Freund's adjuvant and the like. Similarly, cells expressing PD-1 or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibody may also be a monoclonal antibody, and such monoclonal antibodies may be prepared using hybridoma technology.

Applications of

In a preferred embodiment of the invention, the inventors have conducted experiments in a mouse tumor-bearing model using the PCSK9 antibody as a sole component. A mouse tumor-bearing model is established by subcutaneous injection of a melanoma cell line and a lung cancer cell line derived from a mouse, the growth condition of subcutaneous tumors of the mouse and the survival condition of the mouse are observed, the subcutaneous tumors of a control group grow rapidly, and the growth of the tumors of the mouse treated by the PCSK9 antibody is obviously slowed down. The blood total cholesterol level and the low-density lipoprotein cholesterol level of the mice are reduced by detecting the blood of the mice, and the CD8 is detected by PCR detection of tumor tissues⁺T cells and NK cells are infiltrated and increased, and immune cells are sorted by a magnetic bead sorting method to find that the cholesterol content is increased and the anti-tumor activity is obviously increased. The PCSK9 antibody and the PD1 antibody are combined, the tumor-bearing mice are treated, the combined treatment has better inhibition effect on tumor growth than the single application of the PD1 antibody, and the flow detection shows that the combined treatment can further improve NK cells and CD8⁺Infiltration of T cells, finding NK cells and CD8 by PCR detection⁺Secretion of anti-tumor cytokines by T cellsAnd is significantly increased.

Based on the above new findings of the present inventors, the present invention provides the use of a PCSK9 inhibitor for the preparation of a medicament for inhibiting tumor or enhancing immune cell activity. The PCSK9 inhibitor or the PCSK9 inhibitor and the PD-1 inhibitor enhance the tumor-inhibiting activity of immune cells by increasing the expression of immune cell activation-related genes, so that tumors are inhibited; preferably, the immune cell activation-related genes include: GZMB, IFN γ, TNF α, NOS2, NCR1, PRF, IL15, CCL3, CXCL10, CD8, NK1.1, FOXP 3.

Furthermore, the invention also provides application of the PCSK9 inhibitor and the PD-1 inhibitor as a medicine combination in preparing medicines for inhibiting tumors or enhancing the activity of immune cells.

Pharmaceutical composition and kit

The invention also provides a pharmaceutical composition, which contains an effective amount (such as 0.000001-50 wt%, preferably 0.00001-20 wt%, more preferably 0.0001-10 wt%) of the PCSK9 inhibitor and a pharmaceutically acceptable carrier.

The PCSK9 inhibitor can be used as a single component for preparing an antitumor drug, and can also be used as a single component for combined treatment of tumors with a PD1 inhibitor. Therefore, preferably, the pharmaceutical composition further comprises an effective amount (e.g., 0.000001-50 wt%, preferably 0.00001-20 wt%, more preferably 0.0001-10 wt%) of the PD-1 inhibitor. The pharmaceutical composition can be used for inhibiting tumors.

As used herein, the "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals.

As used herein, the "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration.

Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell transfection reagent.

Once the use of the PCSK9 inhibitors is known, the PCSK9 inhibitors or pharmaceutical compositions thereof can be administered to a mammal using a variety of methods well known in the art; preferably, the PD-1 inhibitor is also administered simultaneously. Modes of administration include, but are not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

The effective amount of the PCSK9 inhibitor and/or the PD-1 inhibitor, or pharmaceutical compositions containing the inhibitor ingredients, described herein may vary with the mode of administration and the severity of the condition to be treated, among other things. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life and the like of the PCSK9 inhibitor and/or the PD-1 inhibitor; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

The invention also provides a medicine composition containing the PCSK9 inhibitor and/or PD-1 inhibitor or a medicine kit directly containing the PCSK9 inhibitor and/or PD-1 inhibitor. In addition, the kit may further comprise one or more selected from the group consisting of: tumor chemotherapy drugs, tumor radiotherapy drugs, and instructions describing the methods of use of the drugs in the kit.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Primer probes for gene detection by qPCR are shown in table 1.

TABLE 1

Example 1: establishment of mouse tumor-bearing model

Firstly, in a tumor-bearing experiment of mice in a PCSK9 antibody (purchased from BPS Bioscience company), 20 male C57BL/6J mice with the age of 6-8 weeks are selected and inoculated with 2 multiplied by 10 for each mouse in a subcutaneous injection mode⁶Melanoma B16F10 cells, liver cancer cell Hepa1-6, lung cancer cell LLC, and prostate cancer cell RM-1 (purchased from Chinese academy of sciences) were observed after 8 days. 16 mice with similar tumor sizes were selected and then divided into a control group (8 mice) and a PCSK9 antibody group (8 mice).

Secondly, in the tumor-bearing experiment of PCSK9 antibody group mice, 30 male C57BL/6J mice with the age of 6-8 weeks are selected, and 2 multiplied by 10 are inoculated in each mouse by adopting a subcutaneous injection mode⁶The melanoma B16F10 cell or lung cancer cell LLC was observed after 8 days. 24 mice with similar tumor sizes were selected and then divided into a control group (8 mice), a PD-1 antibody group (8 mice) and a combination group (8 mice) of PCSK9 antibody + PD-1 antibody (purchased from Bio X Cell).

Example 2: effect of PCSK9 inhibitors on mouse tumor growth

1. Effect of PCSK9 antibodies on mouse tumor growth

After grouping mice, mice in the control group were given isotype control IgG, mice in the experimental group were given PCSK9 antibody (dissolved in physiological saline) at 10mg/Kg, every 3 days, i.p., or were given PD-1 antibody at 200. mu.g each (about 10mg/Kg), every 3 days, i.p., or a combination thereof. Subcutaneous tumor size was measured weekly for each group of mice after administration, and tumor volume (tumor volume ═ tumor major diameter × tumor minor diameter) was calculated²/2)。The maximum tumor volume of the mice reaches 2cm³On the left and right, each group of mice was sacrificed and subcutaneous tumors were removed and tumor growth curves were plotted.

As shown in FIGS. 1, 2 and 3, the subcutaneous tumors of the control mice grew more rapidly and increased in volume significantly. The experimental group given the PCSK9 antibody showed a significant decrease in the growth rate of subcutaneous tumors and a significant decrease in volume in mice. The experiments prove that the injection of the PCSK9 antibody can obviously inhibit the growth of various tumor cells in a mouse body.

As shown in fig. 9 and 10, the subcutaneous tumors of the control mice grew more rapidly and increased in volume significantly after transplantation of melanoma (fig. 9A and 10A) or lung cancer cells (fig. 9B and 10B). After the PD-1 antibody is given, the growth speed of subcutaneous tumor of the mouse is reduced, and the volume is reduced; following the combined administration of the PD-1 antibody and PCSK9 antibody, the mice further slowed the rate of subcutaneous tumor growth and further reduced the volume.

The above experiments demonstrate that the PCSK9 antibody can be used in conjunction with a PD-1 antibody for anti-tumor therapy.

2. Effect of PCSK9 Small molecule inhibitors on mouse tumor growth

After grouping, the mice in the control group were given PBS, and the mice in the experimental group were given PCSK9 small molecule inhibitor SBC-110736 (dissolved in PBS) 8mg/Kg once a day, i.e., intraperitoneal injection. The subcutaneous tumor size of the mice is observed every week after the administration, and the maximum tumor volume of the mice reaches 2cm³On the left and right, each group of mice was sacrificed and subcutaneous tumors were removed.

The results are shown in figure 4, and the subcutaneous tumor volume of the mice in the control group is obviously larger than that of the mice administered with the PCSK9 small-molecule inhibitor.

The results demonstrate that treatment with a small molecule inhibitor of PCSK9 significantly inhibits tumor cell growth in vivo.

Example 3: detection of mouse blood cholesterol related index

Blood is collected by an anticoagulation tube before each group of mice die, and supernatant is taken after centrifugation for detecting the total cholesterol content and the low density lipoprotein cholesterol content of the blood.

As shown in figure 5, the total cholesterol level in blood and the low density lipoprotein cholesterol level of the mice injected with the PCSK9 antibody are obviously reduced, while the triglyceride and the high density lipoprotein cholesterol are not obviously changed, which is consistent with the medicinal effect of the PCSK9 antibody.

Example 4: tumor-bearing tissue related detection of mice

Tumor-bearing tissues are collected after each group of mice die, RNA is extracted and then reverse transcription is carried out to obtain cDNA, and the expression level of each immune cell marker (CD4, CD8, NK1.1, CD19, CD209, CD68 and FOXP3) in the tumor-bearing tissues is detected by a qPCR method.

As shown in FIG. 6, the PCSK9 antibody-injected mice showed significantly increased expression levels of CD8 and NK1.1 in tumor-bearing tissues, indicating that CD8 is present in tumor tissues⁺Infiltration of T cells as well as NK cells was increased.

Example 5: mouse tumor infiltration NK and CD8+ T cell related detection

After the mice die, the tumor tissues are separated by a collagenase digestion method to obtain single cell suspension, a part of the suspension is used for detecting the proportion of immune cells in the tissues by a flow staining method, and the rest cells are used for obtaining NK cells and CD8 by a magnetic bead sorting method⁺T cells. The cholesterol level in two immune cells is detected by a cholesterol detection kit, RNA is extracted and subjected to reverse transcription to obtain cDNA, and the expression level of activation related genes (GZMB, IFN gamma, TNF alpha, NOS2, NCR1, NRG2D, PRF, IL15, CCL3 and CXCL10) is detected by a qPCR method.

As shown in FIG. 7, tumor infiltration of NK and CD8 following injection of mouse PCSK9 antibody⁺T cell cholesterol levels are all remarkably increased, which indicates that the PCSK9 antibody medicament has obvious effect on increasing the immune cell cholesterol levels.

As shown in FIG. 8, wherein FIG. 8A is NK cell activation-related gene and FIG. 8B is CD8⁺T cell activation related genes. Visible tumor infiltration of NK and CD8⁺The expression of T cell activation related genes is obviously increased in the PCSK9 group, which indicates that the antitumor activity of immune cells is also increased.

Flow assay for NK cells and CD8⁺T cell infiltration, as shown in FIG. 11, tumor-infiltrating NK and NK of the control group, PD-1 antibody group, PCSK9 antibody + PD-1 antibody combination group, were comparedCD8⁺The proportion of T cells was significantly increased, the NK cell proportion of the combined group was further increased than that of the PD-1 antibody group, and CD8⁺There was no significant difference in T cell ratios.

Tumor infiltrating NK and CD8 in activation-related gene detection, as shown in FIG. 12A-B, PD-1 antibody group, PCSK9 antibody + PD-1 antibody combination group⁺T cell activity was significantly increased compared to the control group. Moreover, the combination group has more obvious increase in the expression of IFN gamma, Perforin (PRF) and GZMB, which shows that the PCSK9 antibody and the PD-1 antibody can realize further synergistic action, further increase NK cells and CD8⁺Activity of T cells.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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<213> Artificial Sequence (Artificial Sequence)

<400> 23

gccacggcac agtcattga 19

<210> 24

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tgctgatggc ctgattgtct t 21

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tagtccttcc taccccaatt tcc 23

<210> 26

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ttggtcctta gccactcctt c 21

<210> 27

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aagcctgtag cccacgtcgt a 21

<210> 28

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggcaccacta gttggttgtc tttg 24

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

acatccatct cgtgctactt gt 22

<210> 30

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gcctctgttt tagggagacc t 21

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ttctctgtac catgacactc tgc 23

<210> 32

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

cgtggaatct tccggctgta g 21

<210> 33

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ccaagtgctg ccgtcatttt c 21

<210> 34

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ggctcgcagg gatgatttca a 21

Claims

1. A method of enhancing the activity of an immune cell, the method being non-diagnostic and non-therapeutic, the method comprising: treating immune cells with a PCSK9 inhibitor in combination with a PD-1 inhibitor; wherein the PCSK9 inhibitor is an anti-PCSK 9 antibody; the PD-1 inhibitor is an anti-PD-1 antibody; the immune cell is an NK cell.

2. The method of claim 1, wherein the PCSK9 inhibitor and the PD-1 inhibitor increase expression of immune cell activation-related genes that are: GZMB, IFN γ, TNF α, PRF.