Use of a combination of nk cells and a pd1 / pd-l1 inhibitor

Abstract

The present application relates to the technical field of tumor treatment, in particular to application of combined treatment of NK cells and PD1 / PD-L1 inhibitors, and proves that the combination of NK cells and PD1 / PD-L1 inhibitors has synergistic effect, can effectively improve the killing effect on tumor cells, and can be used for targeted treatment of various types of solid tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor treatment, in particular to the application of a combination of NK cells and PD1 / PD-L1 inhibitors. BACKGROUND

[0002] NK cells, also known as natural killer cells, are an important part of the human immune system, with multiple functions, especially in anti-tumor, immune regulation and anti-virus. NK cells mediate cytotoxicity through the secretion of interferon-gamma (IFN-γ) and the expression of factors such as T-bet and EOMES. They can recognize and kill target cells, a process that depends on the activation receptors and inhibitory receptors on the surface of NK cells, such as NKG2 and KIR families. NK cells also produce a series of cytokines and chemokines to regulate immune responses, promote the aggregation of dendritic cells (DCs) to solid tumors, and enhance the anti-tumor immune effects of CD8+ T cells.

[0003] PD-1 / PD-L1 inhibitors are an important class of immune checkpoint inhibitors that restore T cell immune surveillance and killing function of tumor cells by blocking the interaction between PD-1 and its ligand PD-L1. PD-1 / PD-L1 inhibitors show significant efficacy in various tumors, including malignant melanoma, non-small cell lung cancer, renal cancer, etc. For example, in classical Hodgkin's lymphoma (cHL), PD-1 inhibitors Nivolumab and Pembrolizumab show good efficacy.

[0004] However, research has found that the anti-tumor effect of the above-mentioned therapeutic agents needs to be improved. SUMMARY

[0005] In view of the technical problems existing in the prior art, the present application provides the application of a combination of NK cells and PD1 / PD-L1 inhibitors. The present application proves that the combination of NK cells and PD1 / PD-L1 inhibitors has a synergistic effect, which can effectively enhance the killing effect on tumor cells and can be used for targeted therapy of various types of solid tumors.

[0006] The present application first provides an antibody for targeting inhibition of PD1, which comprises a heavy chain variable region and a light chain variable region, the CDR1-3 of the heavy chain variable region are respectively as shown in SEQ ID NO. 1-3, and the CDR4-6 of the light chain variable region are respectively as shown in SEQ ID NO. 4-6.

[0007] Preferably, the heavy chain and light chain of the monoclonal antibody comprise the amino acids as shown in SEQ ID NO. 7 and 8.

[0008] The application also provides an antibody for targeting and inhibiting PD-L1, which comprises a heavy chain variable region and a light chain variable region, wherein the CDR1-3 of the heavy chain variable region are respectively shown as SEQ ID NO. 9-11, and the CDR4-6 of the light chain variable region are respectively shown as SEQ ID NO. 12-14.

[0009] Preferably, the heavy chain and the light chain of the monoclonal antibody comprise the amino acids shown as SEQ ID NO. 15 and 16.

[0010] The application also provides an NK cell, which is a CD3-CD56+CD16+CD160+ NK cell.

[0011] Preferably, the preparation method of the CD3-CD56+CD16+CD160+ NK cell comprises the following steps:

[0012] 1) NK cell separation: 20 mL of fresh healthy adult peripheral blood is extracted, anticoagulated with heparin, and separated by lymphocyte separation medium to obtain peripheral blood mononuclear cells, and the cells are resuspended with MACS buffer; anti-CD56 antibody, anti-CD56 antibody, anti-CD16 antibody and anti-CD160 antibody are sequentially selected and sorted by flow cytometry, and a cell suspension rich in CD3-CD56+CD16+CD160+ NK cells is collected;

[0013] 2) The NK cells obtained in step 1) are used to enrich CD3-CD56+CD16+CD160+ NK cells in vitro by using DMEM / F-12 medium, and are ready for use.

[0014] The application also provides the use of the combination of the NK cell and the PD1 / PD-L1 inhibitor in the preparation of an anti-tumor drug.

[0015] Preferably, the drug further comprises other tumor treatment agents, such as chemotherapy drugs, radiotherapy drugs, etc.

[0016] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0017] The application has the following advantages: the application proves that the combination of the NK cell and the PD1 / PD-L1 inhibitor has a synergistic effect, can effectively improve the killing effect on tumor cells, and can be used for targeted treatment of various types of solid tumors. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 . Different administration groups have specific killing effects on tumor cells. DETAILED DESCRIPTION

[0019] The application will be further described in connection with the following specific embodiments so that those skilled in the art can more clearly understand the application.

[0020] The following examples are used to illustrate the application and are not intended to limit the scope of the application. Based on the specific embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0021] In the embodiments of the application, all raw material components are commercially available products well known to those skilled in the art, unless otherwise specified. In the embodiments of the application, the technical means used are conventional means well known to those skilled in the art, unless otherwise specified.

[0022] Example 1

[0023] The application provides an antibody for targeting and inhibiting PD1, which comprises a heavy chain variable region and a light chain variable region, wherein CDR1-3 of the heavy chain variable region are respectively as shown in SEQ ID NO. 1-3, and CDR4-6 of the light chain variable region are respectively as shown in SEQ ID NO. 4-6.

[0024] Preferably, the heavy chain and the light chain of the monoclonal antibody comprise amino acids as shown in SEQ ID NO. 7 and 8.

[0025] The application also provides an antibody for targeting and inhibiting PD-L1, which comprises a heavy chain variable region and a light chain variable region, wherein CDR1-3 of the heavy chain variable region are respectively as shown in SEQ ID NO. 9-11, and CDR4-6 of the light chain variable region are respectively as shown in SEQ ID NO. 12-14.

[0026] Preferably, the heavy chain and the light chain of the monoclonal antibody comprise amino acids as shown in SEQ ID NO. 15 and 16.

[0027] The application also provides an NK cell, which is a CD3-CD56+CD16+CD160+ NK cell.

[0028] Preferably, the preparation method of the CD3-CD56+CD16+CD160+ NK cell comprises the following steps:

[0029] NK cell separation: 20 mL of fresh peripheral blood of healthy adults was extracted, anticoagulated with heparin, and peripheral blood mononuclear cells were obtained by lymphocyte separation medium, and the cells were resuspended with MACS buffer; anti-CD56 antibody, anti-CD56 antibody, anti-CD16 antibody and anti-CD160 antibody were sequentially selected and sorted by flow cytometry, and the cell suspension rich in CD3-CD56+CD16+CD160+NK cells was collected;

[0030] 2) The NK cells obtained in step 1) were enriched in CD3-CD56+CD16+CD160+NK cells in vitro using DMEM / F-12 medium, and were ready for use.

[0031] Example 2

[0032] NK cell combined with PD1 / PD-L1 inhibitor for specific killing of tumor cells

[0033] The NK cells prepared in Example 1 were prepared into a cell suspension of 1.0x10 8 The antibody targeting PD1 (100 μg / mL) or the antibody targeting PD-L1 (100 μg / mL) was slowly added into the cell suspension, and incubated for 60 min, with mixing every 10 min.

[0034] 1x10 6 The hepatocellular carcinoma HEPG2, lung cancer A549, breast cancer MCF7 and melanoma B16-F10 cells were taken at 1x10

[0035]

[0036] After mixing, the cells were cultured in a 37°C 5% CO2 incubator for 6 h, and the killing rate of the cells was analyzed according to the instructions of the LDH detection kit.

[0037] The results are shown in Table 1: Figure 1 The combination group has strong killing ability to different types of tumor cells, while the killing effect of the NK group and the antibody group is not good, and the combination of the two has a synergistic effect, which can effectively enhance the killing activity of NK cells and antibodies. Therefore, the NK cells combined with PD1 / PD-L1 inhibitor of the present application have good antitumor activity.

[0038] The above examples serve to illustrate the essential content of the present application, but do not limit the protection scope of the present application. Those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solutions of the present application.

Claims

1. An antibody that targets and inhibits PD1, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region, wherein CDR1-3 of the heavy chain variable region are shown as SEQ ID NO.1-3, and CDR4-6 of the light chain variable region are shown as SEQ ID NO.4-6.

2. The antibody as described in claim 1, characterized in that, The heavy chain of the antibody comprises the amino acid shown in SEQ ID NO.7, and the light chain of the antibody comprises the amino acid shown in SEQ ID NO.

8.

3. An antibody that targets and inhibits PD-L1, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region. The CDR1-3 of the heavy chain variable region are shown in SEQ ID NO.9-11, and the CDR4-6 of the light chain variable region are shown in SEQ ID NO.12-14.

4. The antibody as described in claim 3, characterized in that, The heavy chain of the antibody comprises amino acids as shown in SEQ ID NO.15, and the light chain of the antibody comprises amino acids as shown in SEQ ID NO.

16.

5. An NK cell, characterized in that, The NK cells are CD3. - CD56 + CD16 + CD160 + NK cells.

6. A method for preparing NK cells as described in claim 5, characterized in that, The method includes the following steps: 1) NK cell isolation: 20 mL of fresh peripheral blood was drawn from healthy adults, anticoagulated with heparin, and peripheral blood mononuclear cells were isolated using lymphocyte separation medium. The cells were resuspended in MACS buffer. Anti-CD56, anti-CD16, and anti-CD160 antibodies were sequentially used for sorting using flow cytometry, and cells rich in CD3 were collected. - CD56 + CD16 + CD160 + NK cell suspension; 2) The NK cells obtained in step 1) were enriched in vitro with CD3 using DMEM / F-12 medium. - CD56 + CD16 + CD160 + NK cells, ready for use.

7. Use of the combination of the NK cells according to any one of claims 5-6 and the antibody targeting and inhibiting PD1 according to any one of claims 1-2 in the preparation of an anti-solid tumor drug.

8. Use of the combination of the NK cells according to any one of claims 5-6 and the antibody targeting and inhibiting PD-L1 according to any one of claims 3-4 in the preparation of an anti-solid tumor drug.

9. The use as described in claim 7 or 8, characterized in that, The drug also includes other cancer treatment agents.

10. The use as described in claim 7 or 8, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

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