A pharmaceutical composition comprising NK cells and its application in preparing a drug for treating tumors

Abstract

The present invention relates to a pharmaceutical composition comprising NK cells and its application in the preparation of a drug for treating tumors. The composition comprises NK cells and a PD-L1-targeted antibody; the pharmaceutical composition further comprises an immune cytokine, which is selected from one or more of human IL-2, IL-12, IL-15, IL-18 and IL-21. Targeted PD-L1 antibodies can enhance the anti-tumor effect of NK cells, increase the survival cycle of animals, promote the secretion of anti-tumor factors, and immunomodulatory factors such as IL-2 and IL-21 can promote the proliferation of NK cells, increase the degree of activation of NK cells, and further improve the anti-tumor effect of NK cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a pharmaceutical composition comprising NK cells and its application in the preparation of a drug for treating tumors. Background Art

[0002] Cancer has become the most important disease threatening human health, and traditional therapies are difficult to meet clinical needs. Immunotherapy drugs are the preferred drugs for systemic cancer treatment, especially antibody therapy, which has become the preferred treatment option for cancer because of its ability to specifically target molecules and low toxicity. However, in the complex pathogenesis of diseases, multiple factors can stimulate separate signaling pathways or promote repeated signaling cascades, which limits the effectiveness of treatment targeting a single molecule. Tumor cells are believed to express multiple receptors that interact to activate the unlimited proliferation, metastasis and inhibition of apoptosis of such tumor cells. Multiple monoclonal antibodies, bispecific antibodies and multispecific antibodies have been developed based on the relevant receptors.

[0003] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that can kill tumor or virus-infected cells with effector functions. It is worth noting that the higher the percentage of NK cell infiltration in solid tumors, the better the clinical treatment effect, which has been confirmed in breast cancer, 7 head and neck cancer, 8 and clear cell renal carcinoma (see Slattery K, Woods E, Zaiatz-Bittencourt V, et al. TGFβ drives NK cell metabolic dysfunction in human metastatic breast cancer. J Immunother Cancer. 2021; 9(2): e002044; Greene S, Robbins Y, Mydlarz WK, et al. Inhibition of MDSC trafficking with SX-682, aCXCR1 / 2inhibitor, enhances NK-Cell immunotherapy in head and neck cancer models. Clin Cancer Res. 2020; 26(6): 1420–31; Ziblat A, Iraolagoitia XLR, Nunez SY, et al. Circulating and tumor-infiltrating NK cells from clear cell renal cell carcinoma patients exhibit a predominantly inhibitory The phenotype is characterized by overexpression of CD85j, CD45, CD48 and PD-1. Front Immunol. 2021; 12: 681615). The composition and release of NK lytic granules filled with perforin and granzymes are comparable to those of cytotoxic T cell subsets, but without the robust cytokine release. Therefore, targeting NK cells through immunotherapy is an attractive anti-tumor strategy.

[0004] NK cells can express various activating and inhibitory receptors in response to tolerance or activation signals for cytotoxicity against target cells. When the total level of inhibitory receptor signaling exceeds the total level of activating receptor signaling, NK cell activation is hindered, thereby inducing cell tolerance. For example, NK cells usually upregulate stimulatory ligands of NK cell activating receptors, such as natural killer cell 2 (NKG2D), in response to viral infection or transformation (see Bryceson YT, March ME, Ljunggren HG, et al. Activation, coactivation, and costimulation of resting human natural killer cells. Immunol Rev. 2006; 214 (1): 73–91).

[0005] Combining NK cell immunotherapy with monoclonal antibodies (mAbs) that induce antibody-dependent cell-mediated cytotoxicity (ADCC) can overcome tumor-mediated immunosuppression and may offer new therapeutic prospects. ADCC is triggered when the NK cell activating receptor FcγRIIIa (also known as CD16a) binds to the Fc region of IgG1 / IgG3 antibodies directed against surface antigens of potential target cells. Two well-studied targets for ADCC in solid cancers are human epidermal growth factor receptor 2 (HER2; also known as ErbB-2) and programmed cell death protein ligand 1 (PD-L1; also known as B7-H1 or CD274). The monoclonal antibodies trastuzumab and avelumab, which target HER2 or PD-L1, respectively, are currently FDA-approved and have been shown to induce durable tumor remissions in various advanced solid cancers (see Quatrini L, Mariotti FR, Munari E, et al. The immune checkpoint PD-1 in natural killer cells: expression, function and targeting in tumour immunotherapy. Cancers (2020) 12(11): 3285; Nahta R, Esteva FJ. Herceptin: mechanisms of action and resistance. Cancer Lett (2006) 232(2): 123–38). However, immune checkpoint blockade targeting PD-L1 or HER2 monotherapy targeting trastuzumab have been shown to have limited activity in solid tumors. The ADCC-inducing ability and clinical efficacy of these antibodies have not been extensively studied in pancreatic cancer, and preliminary results on the ability of trastuzumab to induce ADCC exist, as it has been reported that the extent of ADCC induced by trastuzumab in vitro and in vivo is proportional to the level of HER-2 expression (see Gopal S, Kwon SJ, Ku B, et al. 3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity. Commun Biol (2021) 4(1): 1–14).A study showed that the use of a PD-1 blocking antibody (CT-011) can enhance NK cell target cell killing and cytokine production against primary multiple myeloma cells or myeloma cell lines expressing PD-L1 in vitro (see Benson DM Jr., Bakan CE, Mishra A, et al. The PD-1 / PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti–PD-1 antibody. Blood. 2010; 116(13): 2286–94).

[0006] In order to enhance the anti-tumor activity of NK cells and overcome the therapeutic limitations of monoclonal antibodies for solid tumors, the present invention provides a novel PD-L1-targeted antibody, which is combined with NK cells to treat solid tumors, and can limit and inhibit tumor growth in vitro and in vivo, and enhance tumor immunity. Summary of the invention

[0007] The first aspect of the present invention provides a pharmaceutical composition, comprising NK cells and an antibody targeting PD-L1, wherein the heavy chain variable region of the antibody targeting PD-L1 comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6.

[0008] The present invention uses a targeted PD-L1 antibody to contact the immune suppression of NK cells by the tumor microenvironment in the body and fight against the tumor escape mechanism, thereby helping to improve the anti-tumor activity, especially in the process of fighting solid tumors.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region of the antibody targeting PD-L1 is shown in SEQ ID NO:7.

[0010] Furthermore, the amino acid sequence of the light chain variable region of the PD-L1 targeting antibody is shown in SEQ ID NO:8.

[0011] Furthermore, the NK cell preparation method includes: taking peripheral blood, adding PBS in a 1:1 ratio and mixing, slowly adding to a centrifuge tube containing lymphocyte separation fluid, and centrifuging at 2000rpm at room temperature for 20min; after the centrifugation, slowly aspirating the buffy coat layer into a centrifuge tube pre-added with Hanks solution, and centrifuging at 1500rpm at room temperature for 5min to wash the cells once; using a magnetic bead method to separate the target cells; adding the obtained NK cells to a culture medium containing 100ng / ml IL-2 and 10% FBS for culturing, and after 7 days, using a culture medium containing 200ng / ml IL-2 and 10% FBS for culturing; after the 14th day of culture, collecting and obtaining NK cells.

[0012] Furthermore, the pharmaceutical composition also includes immune cytokines.

[0013] Furthermore, the immune cytokine is selected from one or more of human IL-2, IL-12, IL-15, IL-18 and IL-21.

[0014] Furthermore, the immune cytokines are IL-2 and IL-21.

[0015] Studies have shown that cytokine secretion is key to NK cell pre-activation (see Romee R., Rosario M., Berrien-Elliott MM, et al. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci. Transl. Med. 2016; 8(357): 123r–357r). Various cytokines, including IL-2, IL-12, IL-15, IL-18, and IL-21, can be used to enhance NK cell activation. In autologous NK cell transfer, IL-2 is often used for NK cell activation and expansion, and patients who receive NK cell infusions are often given IL-2 to promote in vivo expansion (see Mukherjee S., Jensen H., Stewart W., et al. In silico modeling identifies CD45 as a regulator of IL-2 synergy in the NKG2D-mediated activation of immature human NK cells. Sci. Signal. 2017; 10(485)). However, the anti-tumor effects of single IL-2, IL-12 or IL-18 are quite limited. In contrast, the combination of IL-12, IL-15 and IL-18 may induce considerable biological changes and memory NK cell populations, including enhanced response to IL-2, increased IFN-γ secretion and enhanced cytotoxicity. In addition, the combination of IL-21 and tumor-targeted monoclonal antibodies can enhance the activity of NK cells and improve their anti-tumor effects (see Mcmichael EL, Jaime-Ramirez AC, Guenterberg KD, et al. IL-21 enhances natural killer cell response to cetuximab-coated pancreatic tumor cells. Clin. Cancer Res. 2017; 23(2): 489–502). Studies have shown that among these cytokines, IL-15 is the most promising cytokine for activating NK cells.In clinical trials, infusion of IL-15 into patients with metastatic malignancies showed a significant increase in NK cell proliferation and activation (see Conlon KC, Lugli E., Welles HC, et al. Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during the first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer. J. Clin. Oncol. 2014; 33(1): 74–82).

[0016] The second aspect of the present invention provides a use of the pharmaceutical composition in preparing a drug for treating tumors.

[0017] Furthermore, the tumor is a solid tumor.

[0018] Furthermore, the tumor is liver cancer, breast cancer or pancreatic cancer.

[0019] Beneficial Effects

[0020] The present invention provides a pharmaceutical composition comprising NK cells and use thereof in preparing a drug for treating tumors, with the following specific beneficial effects:

[0021] (1) Provides a PD-L1-targeting antibody that can efficiently bind to the target antigen and has a high titer;

[0022] (2) Antibodies targeting PD-L1 can enhance the anti-tumor effect of NK cells, improve the survival period of animals, and promote the secretion of anti-tumor factors;

[0023] (3) Immunomodulatory factors such as IL-2 and IL-21 can promote NK cell proliferation, increase the degree of NK cell activation, and further improve the anti-tumor effect of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :The anti-tumor effect of NK cells;

[0025] Figure 2 :The effect of interleukin factors on promoting NK cell proliferation;

[0026] Figure 3 :Interleukin factors promote the secretion of perforin by NK cells;

[0027] Figure 4 : Animal life cycle;

[0028] Figure 5 : Expression of IFN-γ in serum;

[0029] Figure 6 :Expression of IL-1β in serum. DETAILED DESCRIPTION

[0030] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials and detection kits are all commercially available unless otherwise specified.

[0031] Example 1 Primary culture of NK cells

[0032] Peripheral blood was collected from healthy volunteers, placed in an anticoagulant collection tube, and PBS was added in a 1:1 ratio to mix, then slowly added to a centrifuge tube containing lymphocyte separation fluid, and centrifuged at 2000rpm for 20min at room temperature. After centrifugation, the buffy coat layer was slowly aspirated into a centrifuge tube pre-filled with Hanks solution, and the cells were washed once by centrifugation at 1500rpm for 5min at room temperature. Target NK cells were collected using an NK cell sorting kit (purchased from Miltenyi Biotec1). The obtained NK cells were cultured in a medium containing 100ng / ml IL-2 and 10% FBS, and cultured in a medium containing 200ng / ml IL-2 and 10% FBS after 7 days. After the 14th day of culture, NK cells were collected.

[0033] Example 2 Preparation of PD-L1 Targeting Antibody

[0034] Recombinant human PD-L1 protein (purchased from Jinan Protein Technology Co., Ltd.) was emulsified and mixed with an equal volume of Freund's complete adjuvant, and 6-week-old female BALB / c mice were immunized with multiple subcutaneous injections at the back of the neck, with 80 μg of protein per mouse. 14 days and 28 days after the first immunization, mice were immunized twice and three times with the same immunization dose and immunization route, respectively, using an equal volume of Freund's incomplete adjuvant for emulsification. Blood was collected 7 days after the third immunization, and the serum antibody titer was detected by indirect ELISA. The mice with the highest titer were selected for intraperitoneal injection of 100 μg of protein 3 days before fusion for booster immunization.

[0035] Feeder layer cells were prepared, SP2 / 0 cells in good growth condition were digested and centrifuged, and then fused with spleen cells in proportion under the action of PEG, and cultured with medium supplemented with HAT. Cell colonies were observed after 3 days, and half of the medium was replaced with fresh medium on the 7th day. Positive cell wells were screened by indirect ELISA on the 10th day. The cell lines preliminarily screened were subcloned 3 times by limiting dilution method until hybridoma cells proliferated from a single cell population that could stably secrete anti-PD-L1 protein antibodies were screened.

[0036] The obtained positive hybridoma cells were cultured continuously, and the antibody titer was detected by indirect ELISA method to screen the hybridoma cells that stably secreted antibodies. 8-10 week old BALB / c female mice were selected and each was intraperitoneally injected with 500 μL of paraffin oil. Three days later, 1×10 6 / cells per mouse, and the monoclonal hybridoma cells were intraperitoneally injected. The mouse ascites was collected after 7 days. The ascites was separated and purified to obtain the anti PD-L1 antibody. The anti PD-L1 antibody titer was measured by IFA method, and it was determined that the anti PD-L1 antibody titer could reach 1:2048000.

[0037] The amino acid sequence of the antibody was determined, and after bioinformatics analysis, the heavy chain variable region of the antibody included an amino acid sequence of HCDR1 as shown in SEQ ID NO: 1, an amino acid sequence of HCDR2 as shown in SEQ ID NO: 2, and an amino acid sequence of HCDR3 as shown in SEQ ID NO: 3, and the light chain variable region included an amino acid sequence of LCDR1 as shown in SEQ ID NO: 4, an amino acid sequence of LCDR2 as shown in SEQ ID NO: 5, and an amino acid sequence of LCDR3 as shown in SEQ ID NO: 6. Further, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0038] Example 3 Antibodies targeting PD-L1 improve the anti-tumor activity of NK cells

[0039] To verify the anti-tumor effects of PD-L1-targeted antibodies and NK cells, solid tumor cells such as liver cancer cell line Hep3B, breast cancer MDA-MB-435, and pancreatic cancer cell line AsPC-1 were used as the subjects to investigate the anti-tumor effects.

[0040] The tumor cells in good culture state were digested with trypsin and the cell density was adjusted to 5×10 4 / mL. The tumor cell suspension was inoculated into a 96-well plate, 100 μL per well. The 96-well plate was placed in a 5% CO2, 37°C cell culture incubator for 12 hours. NK cells in good culture state were used as effector cells and randomly divided into groups, in which NK cells and tumor cells were added to the NK group; NK cells and tumor cells were added to the combined group, and 100 ng / ml anti PD-L1 antibody was added at the same time; NK cells were not added to the control group. In the NK group and the combined group, the effector cells were added to the 96-well plate containing tumor cells at an effector-target ratio of E:T=5:1. There were 3 replicates in each group. The 96-well plate was placed in a 5% CO2, 37°C cell culture incubator for 12 hours. 20 μL of CCK8 solution was added to each well, and after incubation in a 5% CO2, 37°C cell culture incubator for 2-4 hours, the OD value at 450nm was measured using an enzyme reader. The NK cell killing activity was calculated according to the formula: killing activity % = [1-(ODT-ODE) / ODT] x 100%.

[0041] The results are as follows Figure 1 As shown in the results, in three tumor cells, the addition of anti-PD-L1 antibodies can effectively enhance the anti-tumor activity of NK cells, indicating that PD-L1 inhibitors are an effective means to improve the anti-tumor activity of NK cells.

[0042] Example 4 Immune factors regulate NK cell immune activity

[0043] 4.1 Immune factors promote NK cell proliferation

[0044] The proliferation ability of NK cells in the body is an important factor affecting its anti-tumor effect. If the proliferation ability of NK cells is severely impaired due to the influence of the tumor microenvironment, it is not enough to induce immune response and play a role. At present, interleukin family factors are known in the prior art, which have different degrees of promoting effects on NK cells. Therefore, this section examines the proliferation effects of different interleukin factors on NK cells.

[0045] NK cells were seeded in 96-well plates at 5000 cells per well and cultured in a 37°C, 5% CO2 incubator. Human IL-2, IL-12, IL-15, IL-18 and IL-21 (purchased from BD Biosciences) were added at a concentration of 100 ng / ml to treat the cells for 48 hours. A blank control group (including only cell culture medium) and a control group (NK cells, no other reagents) i.e., a DMSO group (concentration of 0.1%) were set up, and 3 replicates were set up in each group. 20 μL of CCK-8 solution was added to each well, and the culture was continued for 1 hour. The absorbance (OD) value at 450 nm was detected on an ELISA reader, and the cell proliferation rate was calculated according to the following formula: Proliferation rate (%) = (absorption value OD50 nm of the experimental group - absorption value OD450 nm of the blank control) / (absorption value OD450 nm of the control group - absorption value OD450 nm of the blank control) × 100%.

[0046] The results are as follows Figure 2 As shown, interleukin factors have different abilities to promote the proliferation of NK cells. Consistent with existing reports, IL-2 has the strongest ability to promote proliferation, followed by IL-18, IL-21, IL-15 and IL-12.

[0047] 4.2 Immune factors regulate NK cell activation

[0048] The anti-tumor effect of NK cells is not only related to their proliferation activity, but also to their activation degree. In this section, the perforin secretion ability of NK cells is detected to examine the activation degree of NK cells by different immune factors.

[0049] The cell treatment method was the same as in Section 4.1. After the cells were treated with immune factors for 48 h, the cell supernatant was collected by centrifugation and the Perforin concentration was detected using a Perforin ELISA kit (purchased from Wuxi Betech Scientific Instrument Co., Ltd.). The specific method was carried out according to the kit instructions.

[0050] The results are as follows Figure 3 As shown in the figure, among many interleukin factors, IL-21 has the strongest ability to promote the secretion of perforin by NK cells, which increases by nearly 1 times compared with NK cells alone, indicating that it can effectively promote the activation of NK cells.

[0051] The above experiments show that interleukin factors can effectively promote the proliferation and activation of NK cells, among which IL-2 and IL-21 have significant advantages in the proliferation and activation of NK cells. In addition, existing evidence has shown that the two have a synergistic effect on the activation of NK cells (Kresten Skak, Klaus Stensgaard Frederiksen, and Dorthe Lundsgaard. Interleukin-21 activates human natural killer cells and modulates their surface receptor expression, Immunology. 2008, 123 (4): 575-583.), so the present invention selects these two factors as NK cell activators.

[0052] Example 5 Antibodies targeting PD-L1 and immune factors jointly improve the anti-tumor effect of NK cells in vivo 5.1 Preparation and administration of pancreatic cancer animal model

[0053] C57BL / 6J mice aged 4-8 weeks were housed in an SPF animal room for 1 week to adapt to the feeding environment. AsPC-1 cells were cultured to the logarithmic growth phase and 5×10 6 The cells were injected subcutaneously under the armpit of the mouse, and tumor tissue could be palpated after 5 days. The mice were randomly divided into 4 groups, 10 mice in each group, namely, NK group: 1×10 cells were injected into the tail vein every week 6 NK cells were administered 4 times in total; in the NK+antibody group, 1×10 6 NK cells and 100 mg / kg anti-PD-L1 antibody were administered 4 times in total; in the NK+antibody+IL group, 1×10 6 NK cells and 100 mg / kg of anti-PD-L1 antibody were administered 4 times in total, and 50 mg / kg of IL-2 and IL-21 were injected every 2 days; the control group was not treated with drugs. The animals were treated for 4 weeks.

[0054] 5.2 Animal survival

[0055] The survival of mice was observed and recorded every day, and a survival curve was drawn. Figure 4 As mentioned above, although NK cells have a certain inhibitory effect on tumors in the body, their anti-tumor ability is weak; after the addition of anti PD-L1 antibodies, the anti-tumor activity was significantly enhanced, the animal survival period was effectively prolonged, and this effect was further enhanced after the addition of IL-2 and IL-21, indicating that the above-mentioned interleukin factors and anti PD-L1 antibodies can effectively improve the anti-tumor activity of NK cells.

[0056] 5.3 Secretion of immune factors in vivo

[0057] After 4 weeks of treatment, peripheral blood was collected from the experimental animals and centrifuged at 3000 rpm / min for 15 min to obtain serum. ELISA kits (purchased from Dakoway) were used to detect the secretion of IFN-γ and IL-1β in the serum. Figure 5 , Figure 6 As shown in the results, NK cells can promote the secretion of immune factors such as IFN-γ and IL-1β; the addition of anti-PD-L1 antibodies can significantly increase the expression levels of IFN-γ and IL-1β; the use of interleukins can increase the expression of immune factors to varying degrees, which is more obvious in IFN-γ.

[0058] It should be understood that although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A PD-L1-targeted antibody, wherein the heavy chain variable region of the PD-L1-targeted antibody comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO:

6.

2. A pharmaceutical composition comprising NK cells and an antibody targeting PD-L1, wherein the heavy chain variable region of the antibody targeting PD-L1 comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3, and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6, and the method for preparing the NK cells comprises: Peripheral blood was collected, PBS was added in a 1:1 ratio and mixed, then the mixture was slowly added to a centrifuge tube containing lymphocyte separation solution, and centrifuged at 2000 rpm at room temperature for 20 min; after centrifugation, the buffy coat layer was slowly aspirated into a centrifuge tube pre-filled with Hanks solution, and the cells were washed once by centrifugation at 1500 rpm at room temperature for 5 min; the target cells were separated using a magnetic bead method; the obtained NK cells were cultured in a medium containing 100 ng / ml IL-2 and 10% FBS, and cultured after 7 days using a medium containing 200 ng / ml IL-2 and 10% FBS; after the 14th day of culture, the NK cells were collected and obtained.

3. The pharmaceutical composition according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region of the antibody targeting PD-L1 is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region of the antibody targeting PD-L1 is shown in SEQ ID NO:

8.

4. The pharmaceutical composition according to any one of claims 2 to 3, characterized in that The pharmaceutical composition further comprises an immune cytokine, and the immune cytokine is selected from one or more of human IL-2, IL-12, IL-15, IL-18 and IL-21.

5. Use of the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a drug for treating tumors, wherein the tumor is a solid tumor.

6. The use according to claim 5, characterized in that: The tumor is liver cancer, breast cancer or pancreatic cancer.

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