Application of NK cell and PD1 / PD-L1 inhibitor in combined treatment

Abstract

The invention relates to the technical field of tumor treatment, in particular to application of combined treatment of NK cells and a PD1 / PD-L1 inhibitor. The invention proves that the NK cells and the PD1 / PD-L1 inhibitor are combined to have a synergistic effect, so that the killing effect on tumor cells can be effectively improved, and the NK cells and the PD1 / PD-L1 inhibitor can be used for targeted therapy of various types of solid tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to an application of combined treatment of NK cells and PD1 / PD-L1 inhibitors. Background Art

[0002] NK cells, or natural killer cells, are an important part of the human immune system and have multiple functions, especially playing an important role in anti-tumor, immunomodulation and anti-virus. NK cells mediate cytotoxicity by secreting interferon-γ (IFN-γ) and expressing factors such as T-bet and EOMES. They can recognize and kill target cells, a process that depends on activating and inhibitory receptors on the surface of NK cells, such as NKG2 and the KI R family. 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 effect of CD8+T cells.

[0003] PD-1 / PD-L1 inhibitors are an important class of immune checkpoint inhibitors that restore the immune surveillance and killing function of T cells against tumor cells by blocking the interaction between PD-1 and its ligand PD-L1. PD-1 / PD-L1 inhibitors have shown significant efficacy in a variety of 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 have shown good efficacy.

[0004] However, studies have shown that the anti-tumor effects of the above therapeutic agents need to be improved. Summary of the invention

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

[0006] The present invention first provides an antibody for targeted inhibition of PD1, wherein the antibody 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.

[0007] Preferably, the heavy chain and light chain of the monoclonal antibody comprise the amino acids shown in SEQ ID NOs. 7 and 8.

[0008] The present invention also provides an antibody for targeted inhibition of PD-L1, the antibody comprising a heavy chain variable region and a light chain variable region, CDR1-3 of the heavy chain variable region are respectively shown as: SEQ ID NO.9-11, and CDR4-6 of the light chain variable region are respectively shown as: SEQ ID NO.12-14.

[0009] Preferably, the heavy chain and light chain of the monoclonal antibody comprise the amino acids shown in SEQ ID NOs. 15 and 16.

[0010] The present invention also provides a NK cell, wherein the NK cell is a CD3-CD56+CD16+CD160+NK cell.

[0011] Preferably, the method for preparing CD3-CD56+CD16+CD160+NK cells comprises the following steps:

[0012] 1) NK cell separation: 20 mL of fresh healthy adult peripheral blood was extracted, anticoagulated with heparin, and peripheral blood mononuclear cells were separated using lymphocyte separation solution, and the cells were resuspended in MACS buffer; anti-CD56 antibody, anti-CD56 antibody, anti-CD16 antibody and anti-CD160 antibody were used in sequence for sorting using flow cytometry, and a cell suspension rich in CD3-CD56+CD16+CD160+NK cells was collected;

[0013] 2) The NK cells obtained in step 1) are enriched in vitro with DMEM / F-12 culture medium to obtain CD3-CD56+CD16+CD160+NK cells for later use.

[0014] The present invention also provides the use of a combination of NK cells and PD1 / PD-L1 inhibitors in the preparation of anti-tumor drugs.

[0015] Preferably, the drug also includes other tumor therapeutic agents, such as chemotherapy drugs, radiotherapy drugs, etc.

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

[0017] The advantages of the present invention are as follows: The present invention proves that the combination of NK cells and PD1 / PD-L1 inhibitors has a synergistic effect, can effectively enhance the killing effect on tumor cells, and can be used for targeted treatment of various types of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .Specific killing effect of different drug administration groups on tumor cells. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0020] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

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

[0022] Example 1

[0023] The present invention provides an antibody for targeted inhibition of PD1, the antibody comprising a heavy chain variable region and a light chain variable region, 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 light chain of the monoclonal antibody comprise the amino acids shown in SEQ ID NOs. 7 and 8.

[0025] The present invention also provides an antibody for targeted inhibition of PD-L1, the antibody comprising a heavy chain variable region and a light chain variable region, CDR1-3 of the heavy chain variable region are respectively shown as: SEQ ID NO.9-11, and CDR4-6 of the light chain variable region are respectively shown as: SEQ ID NO.12-14.

[0026] Preferably, the heavy chain and light chain of the monoclonal antibody comprise the amino acids shown in SEQ ID NOs. 15 and 16.

[0027] The present invention also provides a NK cell, wherein the NK cell is a CD3-CD56+CD16+CD160+NK cell.

[0028] Preferably, the method for preparing CD3-CD56+CD16+CD160+NK cells comprises the following steps:

[0029] NK cell separation: 20 mL of fresh healthy adult peripheral blood was drawn, anticoagulated with heparin, and peripheral blood mononuclear cells were separated using lymphocyte separation solution, and the cells were resuspended in MACS buffer; anti-CD56 antibody, anti-CD56 antibody, anti-CD16 antibody and anti-CD160 antibody were used in sequence for sorting using flow cytometry, and a cell suspension rich in CD3-CD56+CD16+CD160+NK cells was collected;

[0030] 2) The NK cells obtained in step 1) are enriched in vitro with DMEM / F-12 culture medium to obtain CD3-CD56+CD16+CD160+NK cells for later use.

[0031] Example 2

[0032] Specific killing effect of NK cells combined with PD1 / PD-L1 inhibitors on tumor cells

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

[0034] Take 1×10 6 Hepatocellular carcinoma cells HEPG2, lung cancer cells A549, breast cancer cells MCF7, and melanoma cells B16-F10 were added to the cell culture dish and allowed to adhere to the wall. Then, the drug administration was performed according to the following table:

[0035]

[0036] After the above inoculation is completed, mix well, and culture in a 37°C 5% CO2 incubator for 6 hours. Then, the cell killing rate is analyzed using the instructions of the LDH detection kit.

[0037] The results are as follows Figure 1 As shown: the combined group has a strong killing ability against different types of tumor cells, while the killing effect of the NK group and the monoclonal antibody group alone is poor. The combination of the two has a synergistic effect and can effectively enhance the killing activity of NK cells and antibodies. This shows that the NK cells combined with PD1 / PD-L1 inhibitors of the present invention have good anti-tumor activity.

[0038] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

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

2. The antibody according to claim 1, characterized in that The heavy chain and light chain of the monoclonal antibody include the amino acids shown in SEQ ID NOs. 7 and 8.

3. An antibody that targets and inhibits PD-L1, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, wherein CDR1-3 of the heavy chain variable region are respectively shown as SEQ ID NOs.9-11, and CDR4-6 of the light chain variable region are respectively shown as SEQ ID NOs.12-14.

4. The antibody according to claim 3, characterized in that The heavy chain and light chain of the monoclonal antibody include the amino acids shown in SEQ ID NOs. 15 and 16.

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

6. The NK cell according to claim 5, characterized in that The method for preparing CD3-CD56+CD16+CD160+NK cells comprises the following steps: 1) NK cell separation: 20 mL of fresh healthy adult peripheral blood was extracted, anticoagulated with heparin, and peripheral blood mononuclear cells were separated using lymphocyte separation solution, and the cells were resuspended in MACS buffer; anti-CD56 antibody, anti-CD56 antibody, anti-CD16 antibody and anti-CD160 antibody were used in sequence for sorting using flow cytometry, and a cell suspension rich in CD3-CD56+CD16+CD160+NK cells was collected; 2) The NK cells obtained in step 1) are enriched in vitro with DMEM / F-12 culture medium to obtain CD3-CD56+CD16+CD160+NK cells for later use.

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

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

9. The use according to claim 7 or 8, characterized in that The medicine also includes other tumor therapeutic agents.

10. The use according to claim 7 or 8, characterized in that The medicament further includes a pharmaceutically acceptable carrier or excipient.

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