CN115491355A

CN115491355A - Combined application of NK (natural killer) cells and PD1/PD-L1 inhibitor

Info

Publication number: CN115491355A
Application number: CN202211253684.7A
Authority: CN
Inventors: 黄园园; 张晓艳; 郭雷鸣; 杨月峰; 王立燕
Original assignee: Beijing Jingda Biotechnology Co ltd
Current assignee: Beijing Jingda Biotechnology Co ltd
Priority date: 2022-02-22
Filing date: 2022-10-13
Publication date: 2022-12-20
Anticipated expiration: 2042-10-13
Also published as: CN115369086B; CN115521913A; CN115369086A; CN115521913B; CN115491355B

Abstract

The invention relates to the field of cell therapy and antibody therapy, in particular to combined application of NK cells and a PD1/PD-L1 inhibitor. The NK cells are prepared by the following steps: 1) Preparing a mononuclear cell; 2) Pretreating the cell culture vessel with CD137, CD28, CD 3; 3) Inoculating 1) into 2), and culturing cells with a culture medium containing IL-2, IL-15, IL-12, CD137 and plasma; 4) Supplementing culture medium containing blood plasma, IL-2, IL-15, and IL-12; 5) The medium containing IL-2, IL-15 and IL-21 was supplemented.

Description

Combined application of NK cells and PD1/PD-L1 inhibitor

Technical Field

The invention relates to the field of cell therapy and antibody therapy, in particular to combined application of NK cells and a PD1/PD-L1 inhibitor.

Background

Natural Killer (NK) cells are derived from bone marrow lymphoid stem cells, have large cell bodies and large cytoplasm amount, and are also called large granular lymphocytes because dense granules are contained in the cells under the observation of an electron microscope, and the differentiation and development of the NK cells depend on the microenvironment of bone marrow and thymus and are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. Human NK cells account for approximately 10% -15% of peripheral blood lymphocytes and are surface marked by specific expression of CD56 and lack of CD3 expression. NK cells are an important component of the innate immune system of an organism and serve as a first defense line of host immunity, and natural killer cells can quickly respond to tumor and virus-infected target cells without being sensitized in advance, so that the NK cells play an important role in monitoring early immunity of the organism. Unlike traditional T cells, NK cells do not need to recognize antigens through gene rearrangement, and the material basis for their function is a series of receptors expressed on their surface, triggering self-activity through a dynamic equilibrium between activating and inhibitory receptors, thus initiating the process of killing target cells. NK cells can form synapses with target cells after being activated, release perforin and granzyme to lyse the target cells, and can promote killing of the target cells through interaction of Fas (CD 95) and FasL (CD 95L), binding of TNF and TNF receptors, secretion of IFN gamma and the like. In addition, NK cells can also influence the adaptive immune response function of the body by secreting effector cytokines.

The outcome of the interaction of NK cells with target cells is determined by the balance between NK cell inhibitory and activating signals. NK cell activating receptor recognition includes various cytokine receptors, integrins, killer receptors, recognition of certain inducible expression molecules of the receptor; NK cell inhibitory receptors include a variety of specific receptors that recognize MHC class i molecules. Normally, the NK cell inhibitory receptor recognizes self MHC class I molecules on the surface of a target cell, conducts inhibitory signals and inhibits the attack of NK cells on the target cell, and in a pathological state, the MHC class I molecules on the surface of the target cell are down-regulated, the inhibitory signals are weakened, the NK cells are activated and attack on the target cell is started.

The immune checkpoint inhibitor has a certain effect in various mouse cancer models, and experiments show that even if the mice have no anti-cancer T cells, the tumor of the mice is still reduced by the immune checkpoint inhibitor, which indicates that other immune cells respond under the action of the checkpoint inhibitor. However, when NK cells in mice are depleted, the anti-cancer effect of immune checkpoint inhibitors is greatly reduced. In addition, the NK cells have the same immune checkpoint receptor molecules as the T cells, so that the NK cells can directly respond to the action of an immune checkpoint inhibitor, and a brand-new choice is provided for immunotherapy of tumor patients.

Programmed cell death protein 1 (Programmed cell death protein 1, PD-1, CD279) is a type I transmembrane glycoprotein with molecular weight of 50-55KDa, is an important immune checkpoint receptor, and belongs to the CD28 family. PD-1 expression differs from restricted expression of other members of the CD28 family on T cells, but is broadly expressed on both thymic double negative α β T cells and γ δ T cells, as well as activated peripheral blood T and B cells. PD-1 belongs to a negative regulatory receptor in the CD28 family, and its ligands are PD-L1 (B7-H1) and PD-L2 (B7-DC). The extracellular domain of PD-1 is Ig V-type, and PD-1 exists in a monomeric form because it lacks the membrane proximal cysteine residue and cannot form homodimers like other members of the CD28 family. The intracellular juxtamembrane region of PD-1 has 2 tyrosine residues, respectively, belonging to immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based inhibitory motif (ITSM). When ligand stimulates, tyrosine residues in ITSM recruit protein tyrosine kinase SHP-2, dephosphorylate downstream effector molecules such as Syk and PI3K molecules in B cells, or ZAP70 and CD3 zeta molecules in T cells, and play a role in immune negative regulation, and PD-1 plays an important role in autoimmunity, tumor immunity, infection immunity, transplantation immunity, allergy and immune exemption due to the wide distribution of the ligand.

In tumor immunity, PD-1 and its ligand expressed on the surface of T cell, PD-L1 and PD-L2 expressed on the surface of tumor cell, after combining, will send out immunosuppressive signal to T cell, inhibit activation of T cell, prevent its killing to tumor cell, promote tumor cell to escape the immune surveillance of organism. PD-1 and PD-L1 inhibitors play an important role in tumor immunotherapy and achieve obvious curative effects in tumor immunotherapy. The current PD-1/PD-L1 monoclonal antibodies approved by FDA to be marketed are: pabulizumab (pembroglizumab, keytruda, K drug), nivolumab (Nivolumab, optivo, O drug), cimiciprizumab (cimipimab, libtayo), attentizumab (atezolimab, tecentip), duvatumumab (Imfinzi), avelumab (Avelumab, bavenco), wherein atizumab melilizumab, duvatumumab and Avelumab are PD-L1 mabs, and the remainder are PD-1 mabs. The K medicine is approved for treating 29 indications of 18 tumor species such as melanoma, lung cancer, head and neck squamous cancer, hodgkin lymphoma, urothelial cancer, MSI-H or dMMR solid tumor, gastric cancer, colorectal cancer, breast cancer, cervical cancer, B cell lymphoma, liver cancer, mercule cell cancer and the like, and the O medicine is approved for treating 24 indications of 11 tumor species such as melanoma, lung cancer, kidney cancer, hodgkin lymphoma, head and neck squamous cancer, urothelial cancer, colorectal cancer, stomach cancer, liver cancer and the like; the domestic approved PD-1/PD-L1 monoclonal antibody products comprise: terapril monoclonal antibody, cedilizumab, carrilizumab, tirelizumab, plenopril monoclonal antibody, cepalivizumab, envorlizumab and Sugurlizumab, wherein Envorlizumab and Sugurlizumab are PD-L1 monoclonal antibodies, and the balance is PD-1 monoclonal antibody. The use of domestic PD-1/PD-L1 monoclonal antibody relates to 11 cancer species, and has 44 indications, including non-small cell lung cancer, liver cancer, esophageal cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, head and neck cancer, urinary epithelial cancer, melanoma and Hodgkin's lymphoma.

The NK cells and the PD-1/PD-L1 inhibitor are used together, on one hand, the PD-1/PD-L1 can block the inhibition of tumor cells in a tumor microenvironment on the NK cells, ensure the activation of the NK cells and perform the nonspecific tumor killing function; on the other hand, the PD-1/PD-L1 inhibitor can restore the function of T cells in a tumor microenvironment, so that the T cells in an immune suppression state can exert the tumor killing effect again.

Disclosure of Invention

The invention provides a composition, which specifically comprises NK cells and a PD1/PD-L1 inhibitor; by using the composition, on one hand, the function of T cells in a tumor microenvironment can be recovered, and the anti-tumor effect of the T cells can be fully exerted; on the other hand, the NK cells can be ensured to play a non-specific killing function in an immunosuppression microenvironment. Meanwhile, the invention provides application of the composition in non-small cell lung cancer, liver cancer, esophageal cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, head and neck cancer, urothelial cancer, melanoma and Hodgkin's lymphoma.

NK cells in the composition are subjected to activation amplification culture, the method can amplify the NK cells with the characteristics of large quantity, high purity, strong cytotoxicity and the like, and the amplified and cultured NK cells are compounded with a PD1 or PDL1 inhibitor to form the composition which has a very good clinical application value.

In a first aspect of the invention, there is provided a composition comprising NK cells and a PD-L1/PD1 inhibitor, such as: pabulilizumab (Pembrolizumab, keytruda, K drug), nivolumab (Nivolumab, opdivo, O drug), cimiraprizumab (cemipimab, libtayo), teriprizumab, certilizumab, carpilizumab, tirelizumab, peraprizumab and seraprimab, attelizumab (atelizumab, tecentip), dutalizumab (durvalab, infinzi), avillumab (Avelumab, bavencio), engolizumab and sumatrizumab, the NK cells being prepared by the following method:

1) Preparing a mononuclear cell;

2) Pretreating the cell culture vessel with CD137, CD28, CD 3;

3) Inoculating 1) into 2), and culturing cells with a culture medium containing IL-2, IL-15, IL-12, CD137 and plasma;

4) Supplementing culture medium containing blood plasma, IL-2, IL-15, and IL-12; preferably, the fluid infusion is performed at least once, and specifically comprises: two, three or more times;

5) Supplementing a culture medium containing IL-2, IL-15 and IL-21; preferably, the culture medium may or may not contain plasma; preferably, the fluid infusion is performed at least once, and specifically comprises: two, three or more times;

preferably, in the method of the present invention: the volume can be changed into 2 times, 3 times, 4 times or more after each fluid infusion;

preferably, the basal medium of the culture medium of the present invention is a serum-free medium; serum-free basal media known in the art include: X-Vivo15, MEM medium, DMEM medium, IMDM medium, RPMI1640 medium, ham' F-12 medium, DMEM/F12 medium, M199 medium, and the like.

Preferably, as used in the specific embodiment of the present invention, the serum-free medium is X-Vivo15 (Lonza).

Preferably, the concentration of IL-2 in step 3) is 1000-10000IU/mL; more preferably, 2000IU/mL.

Preferably, the concentration of IL-15 in step 3) is 500-2000IU/mL; more preferably, 1000IU/mL.

Preferably, the concentration of IL-12 in step 3) is 50-500IU/mL; more preferably, 100IU/mL.

Preferably, the concentration of said CD137 in step 3) is 0-10 μm/ml; more preferably, 5. Mu.g/ml.

Preferably, the percentage of plasma in step 3) is 1-10%; more preferably, 5%.

Preferably, the concentration of IL-2 in the step 4) is 1000-10000IU/mL; more preferably, 2000IU/mL.

Preferably, the concentration of IL-15 in step 4) is 500-2000IU/mL; more preferably, 1000IU/mL.

Preferably, the concentration of IL-12 in step 4) is 50-500IU/mL; more preferably, 100IU/mL.

Preferably, the plasma volume in step 4) is 3-10%; preferably, 5%.

Preferably, the concentration of IL-2 in the step 5) is 1000-10000IU/mL; more preferably, 2000IU/mL.

Preferably, the concentration of IL-15 in step 5) is 500-2000IU/mL; more preferably, 1000IU/mL.

Preferably, the concentration of IL-21 in step 5) is 20-100IU/mL; more preferably, 50IU/mL.

Preferably, the plasma of the present invention is inactivated plasma or human serum albumin; more preferably, the plasma is autologous plasma, autologous inactivated plasma.

Preferably, the initial cell concentration of the culture is 1.0X 10 ⁶ Per ml-10.0X 10 ⁶ One per ml.

Preferably, the initial cell concentration of the culture is 1.0X 10 ⁶ Per ml-5.0X 10 ⁶ Per ml; specifically, it includes 1.0 × 10 ⁶ 1.5X 10 pieces/ml ⁶ 2.0X 10 pieces/ml ⁶ 2.5X 10 per ml ⁶ 3.0X 10 pieces/ml ⁶ Seed/ml, 3.5X 10 ⁶ Cell/ml, 4.0X 10 ⁶ 4.5X 10 pieces/ml ⁶ Seed/ml, 5.0X 10 ⁶ One per ml.

Such as the 2.0X 10 ⁶ Piece/ml was inoculated at the initial cell concentration.

Preferably, the mononuclear cells are derived from blood, cord blood, bone marrow;

preferably, the blood is peripheral blood.

Preferably, the mononuclear cells are peripheral blood derived mononuclear cells (PBMCs).

Preferably, the mononuclear cells are prepared by a Ficoll density gradient centrifugation method.

Preferably, the PD-1/PD-L1 inhibitor comprises pabulilizumab, nivolumab, cimiciprizumab, cetilizumab, covaliuzumab, avilumab, teriprizumab, certralizumab, carliximab, tirlizumab, perindopril mab, saprolimab, emparizumab, engelizumab, and sugrezumab.

Preferably, the PD-1/PD-L1 inhibitor is pabollizumab/acilizumab.

Preferably, a suitable concentration of the PD-1/PD-L1 inhibitor is 10-1000. Mu.g/mL.

The NK cells described herein include modified NK cells, such as CAR-NK cells, i.e. chimeric antigen receptor NK cells.

In some embodiments, the compositions of the present invention further comprise a pharmaceutically acceptable carrier.

The term "carrier" in the "pharmaceutically acceptable carrier" refers to diluents, adjuvants, excipients, etc., which can be administered to a patient with the active ingredient (i.e., the composition provided by the present invention). Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition may be formulated as a suppository, using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences of e.w. martin, which is hereby incorporated by reference.

A second aspect of the present invention provides a method for preparing the above composition, the method comprising preparing NK cells and mixing them with a PD1/PDL1 inhibitor, and incubating for 30-60 minutes; preferably, the oscillation is once every ten minutes.

The compositions of the present invention may be administered separately or in combination.

The compositions of the present invention are for simultaneous or sequential administration.

Preferably, the composition further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipient combinations.

Further, such combinations may include: buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

The compositions of matter disclosed herein can be formulated for oral, intravenous, topical, enteral and/or parenteral administration as desired.

A third aspect of the present invention provides the use of the above composition in the manufacture of a medicament for the treatment of cancer; the cancer comprises a solid tumor or a non-solid tumor;

preferably, the cancer is a solid tumor; the solid tumor includes melanoma, lung cancer, head and neck squamous carcinoma, urothelial cancer, MSI-H or dMMR solid tumor, gastric cancer, colorectal cancer, breast cancer, cervical cancer, liver cancer, etc.

More preferably, the cancer is lung cancer.

More preferably, the cancer is non-small cell lung cancer.

In a fourth aspect of the invention, there is provided the use of NK cells prepared according to the invention in combination with a PD-1/PD-L1 inhibitor to enhance the therapeutic effect.

Preferably, in the treatment of melanoma, lung cancer, head and neck squamous carcinoma, hodgkin's lymphoma, urothelial cancer, MSI-H or dMMR solid tumors, gastric cancer, colorectal cancer, breast cancer, cervical cancer, B-cell lymphoma, liver cancer, merckel cell carcinoma.

More preferably, an enhanced effect in the treatment of lung cancer.

More preferably, a synergistic effect in the treatment of non-small cell lung cancer.

In a fifth aspect of the invention, methods of treating cancer using the compositions of the invention are provided.

More preferably, the cancer is lung cancer.

More preferably, the cancer is non-small cell lung cancer.

In some embodiments, the presently disclosed methods of treatment comprise administering to a patient in need thereof a safe and effective amount of a compound of the present invention. Various embodiments disclosed herein include methods of treating a disease or condition described herein by administering a safe and effective amount of a composition of the invention to a patient in need thereof.

In some embodiments, the compositions disclosed herein may be administered once, or several times at different time intervals over a specified period of time, depending on the dosing regimen. For example, once, twice, three or four times weekly, once daily, etc. Suitable dosing regimens for the compositions disclosed herein depend on the pharmacokinetic properties of the composition, such as dilution, distribution and half-life, which can be determined by the skilled person.

In addition, the appropriate dosage regimen, including the duration of the regimen, of the composition of the invention will depend upon the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient being treated, the nature of concurrent therapy, the desired therapeutic effect, and other factors which are within the knowledge and experience of the skilled artisan. It will also be appreciated by those skilled in the art that adjustment of an appropriate dosage regimen may be required for the individual patient's response to the dosage regimen, or as the individual patient needs to change over time.

Drawings

FIG. 1 is a graph showing the results of flow assay of cell purity at different dates in the NK cell preparation method of the present invention.

FIG. 2 is a graph showing the results of the killing experiment.

FIG. 3 is a graph of statistical results of tumor volume changes in animal model experiments.

FIG. 4 is a graph of the statistical results of the change in animal body weight in an animal model experiment.

Detailed Description

The invention is described in detail below with reference to the drawings and examples so that those skilled in the art can understand and implement the invention and further appreciate the advantages of the invention. Unless defined otherwise in the present specification, all technical terms used herein are used in accordance with their customary definitions commonly used and understood by those of ordinary skill in the art. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The following description is only exemplary of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art may modify the present invention by applying the teachings set forth above in a manner equivalent to the above-described modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 preparation of NK cells

1.1 pretreatment of cell culture flasks

10mL of a physiological saline solution containing 8. Mu.g/mL of CD137, 8. Mu.g/mL of CD28 and 8. Mu.g/mL of CD3 was added to 75cm ² In a cell culture flask (Nunc) of basal area, andthe liquid was dispersed well at the bottom of the flask and kept flat overnight at 4 ℃.

1.2 isolation of Peripheral Blood Mononuclear Cells (PBMC)

For example, 50mL of peripheral blood can be adjusted accordingly if the blood volume is different. 50mL of sterilized peripheral blood of a patient subjected to plate sterilization is subjected to differential centrifugation (Hunan instrument) in a horizontal low-speed heart machine at room temperature for 30 minutes, and the speed is increased by 9 and decreased by 7 (namely, the speed reduction time from 1800rpm to 0rpm is 10 minutes) to separate blood plasma and blood cells.

Transferring the upper plasma layer into a centrifuge tube, inactivating at 56 deg.C for 30min, centrifuging at 2000rpm for 10min, and collecting the supernatant at 4 deg.C.

The blood cell pellet was mixed with an equal volume of physiological saline and Peripheral Blood Mononuclear Cells (PBMC) were separated by Ficoll density gradient centrifugation. Specifically, the mixture was carefully added to a 50mL centrifuge tube containing a Ficoll layer and subjected to differential centrifugation at room temperature for 20 minutes (up 9 down 0, i.e., 2000rmp to 0rmp, 30min). Sucking the PBMC layer, sucking the cell layer at the junction of the two liquid surfaces as far as possible, adding physiological saline, blowing, beating and uniformly mixing, and centrifuging at the room temperature of 1500rpm for 10 minutes. The cells were washed again with physiological saline.

After discarding the supernatant, the cells were resuspended in 10mL serum-free medium (X-VIVO 15) and the volume was reduced to 15mL. A small number of cells were aspirated for counting. Meanwhile, a small amount of cell suspension is taken for flow detection, and the proportion of NK (CD 3-CD56 +) is 15%.

1.3 inoculation

According to 2.0X 10 ⁶ (iii) cell concentration per mL, PBMC cells obtained in step 1.2 were seeded into coated flasks obtained in step 1.1 containing 2000IU/mL IL-2, 1000IU/mL IL-15, 100IU/mL IL-12, 5ug/mL CD137 and 10% 15mL of the patient inactivated plasma obtained in step 1.2 (X-VIVO 15). In an incubator (37 ℃, CO) ₂ Concentration 5%) were cultured.

1.4 first infusion of NK cells

On day 3 of culture, the flasks were supplemented with amplification medium (basal medium X-Vivo 15) containing 5% of patient inactivated plasma, IL-22000IU/mL, IL-15 1000IU/mL, and IL-12 100IU/mL, ensuring a final volume of 30mL. Note that do not blow the cells.

1.5 second infusion of NK cells and bottle rotation

On day 5, the flask was continuously supplemented with 120mL of amplification medium (basal medium X-Vivo 15) containing 5% patient inactivated plasma, IL-22000IU/mL, IL-15 1000IU/mL, and IL-12 100IU/mL, and the mixed cell suspension was transferred to a T175 cell flask, ensuring a final volume of 150mL of medium.

1.6 third infusion of NK cells and bagging thereof

On day 7, the cell concentration was measured to be 2.5X 10 ⁶ One per mL. The cells at the bottom of the flask were gently tapped (about 150 mL) and packed into a cell culture bag (GT-T610, from TAKARA) together with 300mL of X-VIVO serum-free cell culture medium containing IL-22000IU/mL, IL-15 1000IU/mL, IL-21 50IU/mL, and the remaining inactivated plasma (about 1.5%), to ensure a final volume of 450mL. And simultaneously counting the cells and detecting the production condition of the cells.

1.7 fourth fluid infusion

On day 9, the cell concentration was measured at 3.22X 10 ⁶ one/mL. The bags were removed from the cell culture chamber (37 ℃ C., CO) ₂ 5%) and the cell suspension was evenly distributed into 2 culture bags and supplemented with equal volume of expansion medium 2 (X-VIVO serum-free cell culture medium containing IL-22000IU/mL, IL-15 1000IU/mL, IL-21 50IU/mL) to ensure a final volume of 900mL. Placing the two bags of cells into an incubator to continue culturing. And simultaneously counting the cells to determine the production condition of the cells.

1.8 fifth infusion

On day 12, the cell concentration was 4.67X 10 ⁶ one/mL. The bags were removed from the cell culture chamber (37 ℃ C., CO) ₂ 5%) was added to each bag, and each bag was supplemented with 2 900mL of an amplification medium (X-VIVO serum-free cell culture medium containing IL-22000IU/mL, IL-15 1000IU/mL, and IL-21 50IU/mL), wherein the volume of each bag was 1350mL and the total volume was 2700mL. Placing two bags of cells intoAnd continuing culturing in the incubator. And simultaneously counting the cells to determine the production condition of the cells.

1.9 detecting bacteria

Cell concentration at 15 days of cell culture was 5.93X 10 ⁶ . And (4) carrying out bacteria detection and endotoxin detection on the cell suspension. The results showed that the endotoxin was less than 0.25EU/mL, and the endotoxin was sterile.

Results of the experiment

The number of cells was calculated from the cell concentration and the volume of the culture medium measured during the culture, and the change in the number of cells was as shown in FIG. 1 and Table 1.

TABLE 1 statistics of cell concentration changes

As can be seen, the concentration of NK cells gradually increased as the culture progressed (see Table 1), and the concentration of NK cells was increased to 2.00X 106/mL at day 0, to 3.11X 106/mL at day 7, to 4.79X 106/mL at day 12, and to 6.17X 106/mL at day 15. The purity of NK cells also gradually increased and exceeded 99% (see fig. 1), 18.37% at day 0, increased to 76.38% at day 7, reached 95.71% at day 11, and finally reached 99.26% at day 15.

Example 2: composition and combined application of killing activity on human non-small cell lung cancer cells

Preparation of cell composition: NK cells prepared in example 1 were prepared to 1.0X 10 ⁸ And (3) after each/mL of cell suspension, slowly adding a palbociclizumab (pembrolizumab, 100 mu g/mL) or azilizumab (100 mu g/mL) solution into the cell suspension, incubating the mixture in a carbon dioxide incubator in a water bath for 60min under an aseptic sealed environment, uniformly mixing every 10min, and ensuring aseptic operation in the whole process.

TABLE 2 details of the operation of each group in the cell experiments

Group of	Details of
		Composition set	1.0×10 ⁸ one/mL of a mixture suspension of NK cells and monoclonal antibody (100. Mu.g/mL)
Combination of NK cells and monoclonal antibodies	1.0×10 ⁸ NK cells per mL and monoclonal antibody (100. Mu.g/mL) were added separately
		Monoclonal antibody group	Pabolizumab (100. Mu.g/mL) or atilizumab (100. Mu.g/mL)
NK cell group	1.0×10 ⁸ NK cell suspension of one/mL

The specific experimental steps are as follows:

1. target cell processing

Non-small cell lung cancer cell line HCC827 Calcein-AM 5% CO at 37 ℃ ₂ Incubate in dark for 30min. After incubation, after washing with medium, the plates were plated and 5000-20000 cells/100. Mu.L were added to each well of a 96-well plate.

2. Adding NK cells or composition

And preparing the NK cells, the T cells, the monoclonal antibodies, the NK cells and the monoclonal antibody composition into required concentration by using a basic culture medium according to the designed effective target ratio, paving the plate according to the designed effective target ratio, and slightly shaking and uniformly mixing.

The experimental design was grouped as follows, with 3 wells per group.

1) Spontaneous release group: only 100. Mu.L of target cells were inoculated, and 100. Mu.L of basal medium was supplemented to volume.

2) Maximum release group: the target cells were inoculated in an amount of 100. Mu.L and the basal medium in an amount of 50. Mu.L, and after the co-culture was completed, triton X-100. Mu.L was added.

3) Experimental groups: dividing into 3 subgroups:

a group of: NK cell group: inoculating target cells and NK cells, wherein the effective target ratio is as follows: 0.5;

two groups are as follows: monoclonal antibody group: inoculating target cells and monoclonal antibody, not relating to the effective target ratio, and only 3 holes;

three groups: t cell group: inoculating target cells and T cells;

four groups: NK cell + T cell group: inoculating target cells, NK cells and T cells;

five groups are as follows: monoclonal antibody + T cell group; inoculating target cells, monoclonal antibodies and T cells;

six groups are as follows: NK cell + mab combination: inoculating target cells, NK cells and monoclonal antibodies;

seven groups are as follows: NK cell and mab composition groups: inoculating the target cells, the NK cells and the monoclonal antibody composition;

eight groups are as follows: NK cells in combination with mabs + T cell panel: inoculating target cells, NK cells, monoclonal antibodies and T cells, and the effect-target ratio is the same as above.

Nine groups: NK cell and mab composition + T cell group (composition group): the target cells, the NK cell and monoclonal antibody composition and the T cells are inoculated, and the effective-target ratio is the same as that of the target cells.

3. Co-cultivation and measurement

Mixing the above materials after inoculation, and adding 5% CO at 37 deg.C ₂ Co-culturing for 4h in an incubator, adding Triton X-100 into the maximum release group after co-culturing, slightly shaking and uniformly mixing, centrifuging for 5min at 400g, taking 150 mu L of supernatant from each well, transferring to a new 96-well enzyme-labeled plate, and measuring fluorescence by using an enzyme-labeled instrument, wherein the excitation wavelength and the emission wavelength are respectively Ex/Em =494nm/517nm.

4. Calculation of results

The data are expressed as Mean ± SEM, and the killing efficiency is calculated as the formula [ (experimental release-spontaneous release)/(maximum release-spontaneous release) ] -100.

The results of the killing experiment are shown in FIG. 2: killing activity of NK cells alone against non-small cell lung cancer cell line HCC827 at a potency-to-target ratio of 0.5, 1, 5; the killing efficiency of the palivizumab and the T cell used alone or the palivizumab and the T cell used together is 0.36 percent, 8.10 percent and 18.48 percent respectively; killing activity of NK cells when used in combination with T cells, 22.39%, 33.08%, 52.29%, 66.09% at a potency to target ratio of 0.5; the killing activity of the NK cells and the Pabolilizumab when used together with T cells is 36.24%, 47.96%, 67.82% and 85.69% when the effective-target ratio is 0.5; the killing activity of NK cells and the composition of palivizumab plus T cells when used was 35.42%, 50.75%, 70.13%, 84.39% at a potency to target ratio of 0.5. Therefore, the NK cell and the palivizumab are used in combination and then the T cell is used, and the NK cell and the palivizumab composition is used in combination and then the T cell, the killing activity to a non-small cell lung cancer cell line HCC827 is greater than the sum of the killing activities of the NK cell, the palivizumab and the T cell when the effective target ratio is 0.5; on the other hand, the PD-1/PD-L1 monoclonal antibody can restore the function of the T cell, so that the T cell in an immune suppression state can exert the tumor killing effect again, and the NK cell and the PD-1/PD-L1 are singly used in a combined way or form a composition for use, so that the combined application effect of the NK cell and the T cell can be indirectly achieved.

Example 3: composition and combined application on tumor animal model

The human non-small cell lung cancer cell HCC827 in exponential growth phase was prepared as a single cell suspension, which was subcutaneously inoculated into the lateral subcutaneous space of NCG mice in an amount of 1 × 10 ⁷ cells. Volume of tumor to be treatedUp to about 60-80mm ³ At the same time, the mice were divided into groups according to the tumor volume, each group had 6 mice with tumors, and the administration was started on the day of the division, and the division conditions and the administration schedule are detailed in table 3.

During the experiment, the length and width of the tumor were measured 1 time per week, and each mouse was weighed 1 time per week. The results are shown in FIGS. 3 and 4 and Table 4.

Table 3: grouping situation and dosing regimen

Table 4: tumor growth inhibition rate (TGI) of combination application on HCC827 tumor model

The tumor growth curve of the combination of NK cells and Pabolizumab and the combined application of NK cells and Pabolizumab on the human non-small cell lung cancer HCC827 tumor model is shown in figure 3, and the tumor growth inhibition rate is shown in table 4. As can be seen from the results, NK cells alone show certain anti-tumor effect on the human non-small cell lung cancer HCC827 tumor model, and the TGI of the NK cells is 40.18%, 33.41%, 33.35% and 25.76% respectively at 14 days, 21 days, 25 days and 35 days of the experiment. The TGI of the palivizumab used alone or the palivizumab used in combination with the T cells at 14 days of the experiment was 1.87%, 30.98% and 47.28%, respectively, and the TGI at 35 days of the experiment was 6.18%, 21.27% and 34.41%, respectively. The TGI of the NK cell and the Pabolizumab combined application + T cell group at 14 days, 21 days, 25 days and 35 days of the experiment is 63.09%, 56.00%, 55.25% and 58.76% respectively; the TGI of the NK cell and the Pabollizumab composition + T cell group at 14 days, 21 days, 25 days and 35 days of the experiment are 59.38%, 62.86%, 52.05 and 54.55% respectively. The combined application of the NK cells and the palivizumab and the composition of the NK cells and the palivizumab can effectively enhance the anti-tumor effect on a human non-small cell lung cancer HCC827 tumor model. The result of killing cells in vitro is further proved, on one hand, the PD-1/PD-L1 monoclonal antibody can block the immune check point inhibition of tumor cells on NK cells, and the killing effect of the NK cells is ensured; on the other hand, the PD-1/PD-L1 monoclonal antibody can restore the function of the T cell, so that the T cell in an immune suppression state can exert the tumor killing effect again, and a stronger anti-tumor effect can be exerted. The weight growth curve of the effect of the NK cell and the Pabolizumab composition on the human non-small cell lung cancer HCC827 tumor model when the NK cell and the Pabolizumab are combined is shown in figure 4, the weight mean value of each group of mice is not obviously abnormal, and the composition or the combined application is not obviously toxic.

Claims

1. A composition comprising NK cells and an antibody, said antibody comprising a PD-1/PD-L1 antibody, said NK cells having been produced by a method for producing NK cells comprising:

1) Preparing a mononuclear cell;

2) Pretreating the cell culture vessel with CD137, CD28, CD 3;

4) Supplementing culture medium containing blood plasma, IL-2, IL-15, and IL-12;

5) Supplementing a culture medium containing IL-2, IL-15 and IL-21;

preferably, the concentration of IL-2 in the step 3) is 1000-10000IU/mL; more preferably, 2000IU/mL;

preferably, the concentration of IL-15 in the step 3) is 500-2000IU/mL; more preferably, 1000IU/mL;

preferably, the concentration of IL-12 in step 3) is 50-500IU/mL; more preferably, 100IU/mL;

preferably, the concentration of the CD137 in the step 3) is 0-10 μm/ml; more preferably, 5 μ g/ml;

preferably, the percentage of plasma in step 3) is 1-10%; more preferably, 5%;

preferably, the concentration of IL-2 in the step 4) is 1000-10000IU/mL; more preferably, 2000IU/mL;

preferably, the concentration of IL-15 in step 4) is 500-2000IU/mL; more preferably, 1000IU/mL;

preferably, the concentration of IL-12 in step 4) is 50-500IU/mL; more preferably, 100IU/mL;

preferably, the plasma volume in step 4) is 3-10%; preferably, 5%;

preferably, the concentration of IL-2 in step 5) is 1000-10000IU/mL; more preferably, 2000IU/mL;

preferably, the concentration of IL-15 in step 5) is 500-2000IU/mL; more preferably, 1000IU/mL;

preferably, the concentration of IL-21 in step 5) is 20-100IU/mL; more preferably, 50IU/mL;

preferably, the plasma is inactivated plasma or human serum albumin; more preferably, the plasma is autologous plasma, autologous inactivated plasma.

2. The composition of claim 1, wherein the basal medium of the culture medium is a serum-free medium comprising X-Vivo15, MEM medium, DMEM medium, IMDM medium, RPMI1640 medium, ham' F-12 medium, DMEM/F12 medium, M199 medium;

preferably, the serum-free medium is X-Vivo15.

3. The composition of claim 1, wherein the mononuclear cells are derived from blood, cord blood, bone marrow;

preferably, the blood is peripheral blood;

4. The composition of claim 1, wherein the PD-1 antibody comprises pabulilizumab, nivolumab, cimetilizumab, terliplizumab, sediluzumab, carpulizumab, tiramizumab, peralizumab, peraprizumab, and sepilizumab;

preferably, the PD-L1 antibody comprises atilizumab, davuliuzumab, avilizumab, engolizumab, and sumulizumab;

preferably, the PD-1 antibody is pabollizumab;

preferably, the PD-L1 antibody is amituzumab;

preferably, the working concentration of the antibody is 50-1000 μ g/mL;

preferably, the working concentration of the antibody is 100. Mu.g/mL.

5. The composition of claim 1, wherein the NK cells comprise CAR-NK cells.

6. The composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient, wetting agent, emulsifier, pH buffering agent;

preferably, the carrier comprises sterile water, oil, saline solution, aqueous dextrose solution, or glycerol solution;

preferably, the excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk.

7. A method for preparing the composition of claim 1, comprising preparing NK cells according to the method for preparing NK cells of claim 1 and mixing them with an antibody, and incubating for 30-60 minutes;

preferably, the antibody comprises a PD-1/PD-L1 antibody;

preferably, the working concentration of the antibody is 50-1000 μ g/mL;

preferably, the working concentration of the antibody is 100. Mu.g/mL.

8. Use of a composition according to claim 1 for the manufacture of a medicament for the treatment of any one of the following cancers:

melanoma, lung cancer, head and neck squamous cancer, hodgkin lymphoma, urothelial cancer, MSI-H or dMMR solid tumor, gastric cancer, colorectal cancer, breast cancer, cervical cancer, B cell lymphoma, liver cancer, merckel cell carcinoma;

preferably, the lung cancer comprises non-small cell lung cancer and small cell lung cancer.

9. Use of NK cells of the composition of claim 1 for the preparation of a medicament for promoting the therapeutic effect of PD-1/PD-L1 antibodies.

10. The use of claim 9, wherein the therapeutic effect of the PD-1/PD-L1 antibody is included in the treatment of melanoma, lung cancer, head and neck squamous carcinoma, hodgkin's lymphoma, urothelial cancer, MSI-H or dMMR solid tumors, gastric cancer, colorectal cancer, breast cancer, cervical cancer, B-cell lymphoma, liver cancer, merkel cell carcinoma.