Application of calcium-dependent secretory activator protein 1 in NK cell therapy

Abstract

The present invention discloses the application of calcium ion-dependent secretory activation protein 1 in NK cell therapy. Through the study of tumor cells, the present invention found that the killing ability of NK cells overexpressing CAPS1 on tumor cells was significantly enhanced, indicating that CAPS1 has the effect of inhibiting tumor progression; multicolor immunohistochemistry found that CAPS1 molecules were lowly expressed in NK cells in the tumor microenvironment. In animal experiments of tumor modeling, the results showed that the tumor size of tumor cells co-cultured with NK cells of CAPS1 gene knockout mice was significantly larger than that of wild-type mice, and it was also concluded that CAPS1 has the effect of inhibiting tumor progression. The present invention proposes a new use of CAPS1 in tumor treatment, which provides good candidate drug targets and prognostic indicators for the clinical treatment of solid tumors such as liver cancer, lung cancer, colon cancer, melanoma, and blood system tumors such as B cell lymphoma and T cell lymphoma, and has good application prospects.

Description

Technical Field

[0001] The invention relates to application of calcium ion-dependent secretory activator protein 1 (CAPS1) in NK cell therapy, and belongs to the technical field of cell immunotherapy. Background Art

[0002] Primary liver cancer is the seventh most common malignant tumor in the world, and its tumor mortality rate ranks third. However, the current treatment methods for liver cancer are limited, and more effective treatment methods are urgently needed. In recent years, immunotherapy has become an important emerging therapy after surgery, radiotherapy and chemotherapy. It was named the world's annual scientific breakthrough in 2013 by Science magazine, including immune checkpoint inhibitors such as CTLA-4 and PD-1 and immune cell therapy such as CAR-T cells. CTLA-4 and PD-1 researchers won the 2018 Nobel Prize in Physiology or Medicine, which reflects the prospects and value of tumor immunotherapy. However, CAR-T cells are limited in clinical application due to many side effects. In contrast, the clinical application advantages of CAR-NK cells (Natural killer cells) are highlighted. Today, NK cells have become a safe and effective cell carrier for chimeric antigen receptors, and the efficacy of NK cell therapy in clinical anti-tumor treatment has gradually been recognized. NK cell therapy for liver cancer patients will not only not damage the normal immune function of the body, but also improve the body's immune function on the basis of killing tumor cells, thereby enhancing the efficacy of surgery and radiotherapy and prolonging survival time. However, the effect of tumor immunotherapy is unsatisfactory due to immune escape: tumor cells can escape the supervision of the immune system through self-modification and metabolic changes. In-depth research on the mechanism of tumor immune escape has important theoretical and practical significance for exploring effective treatments for liver cancer. As an important component of the liver cancer microenvironment and liver innate immunity, NK cells play a key role in tumor killing and immune surveillance, and their dysfunction is closely related to the immune escape of liver cancer cells. Research on the function and mechanism of NK cells can provide new means for immunotherapy of liver cancer.

[0003] Calcium-dependent secretion activator protein 1 (Ca 2+-dependent activator protein forsecretion, CAPS1) gene is located on chromosome 3p14.2. CAPS1 was originally identified as a protein that can reorganize calcium-dependent regulated secretion in pheochromocytoma 12 cells. It is composed of about 1400 amino acids and has a molecular weight of 145KDa. CAPS1 is more distributed in the pancreatic islets, spleen, stomach, and adrenal medulla. Previous studies have shown that CAPS1 is closely related to the exocytosis process of neurotransmitters or peptides / neuromodulators contained in nerve cells, neuroendocrine cells, and endocrine cells. Recent studies have found that CAPS1 is a SNARE auxiliary protein that promotes the formation of the SNARE complex through direct interaction with SNARE and other SNARE auxiliary proteins.

[0004] CAPS1 has high specificity (91.5%) and sensitivity (92.6%) for the diagnosis of liver cancer. CAPS1 is a highly conserved secretory protein in evolution, composed of dynamin-1 binding domain, C2 domain, PH domain, Munc-13 homology domain and dense core large vesicle LDCV binding domain. CAPS1 was discovered in the nervous system in 1992 and is a soluble protein necessary for reconstructive calcium-dependent secretion. As a phosphatidylinositol diphosphate (PIP2)-linked protein, CAPS1 participates in the calcium-regulated fusion process of large dense-core vesicles (LDCV) and membranes, and is closely related to the exocytosis of neurotransmitters or peptides / neuromodulators contained in nerve cells, neuroendocrine cells and endocrine cells. CAPS1 also acts on the upstream stage of LDCV fusion with membranes, plays a role in the formation of secretory vesicles and maintaining their stability, and is closely related to the secretory function of cells. In addition, CAPS1 can regulate the exocytosis of insulin-secreting vesicles in mouse pancreatic islet cells. When CAPS1 is reduced, vesicle stability and the ability to re-transport insulin are also reduced, leading to a decrease in insulin secretion.

[0005] At present, there are few studies on the expression changes and functional activities of CAPS1 in malignant tumors, especially the role of CAPS1 in tumor NK cell function. The endogenous ligands and agonists of CAPS1 have not yet been discovered, and the mechanism of signal transduction is not fully understood. At present, there are still great application limitations and ineffectiveness in tumor immunotherapy. In order to improve the early diagnosis and non-surgical treatment of tumors and improve the survival rate of tumor patients, it is urgent to find a new molecular target to improve the efficacy of tumor immunotherapy. Summary of the invention

[0006] The purpose of the present invention is to provide a novel molecular target calcium ion-dependent secretory activation protein 1 (Ca2+) in order to improve the early diagnosis and non-surgical treatment of tumors and the survival rate of tumor patients. 2+ -dependent activator protein for secretion, CAPS1) in NK cell therapy to improve the efficacy of tumor immunotherapy.

[0007] In order to achieve the above object, the present invention provides the use of an agent for promoting or activating CAPS1 gene expression or transcription in the preparation of an anti-tumor drug, wherein the tumor is a blood tumor or a solid tumor.

[0008] Preferably, the tumor is selected from any one of liver cancer, lung cancer, colon cancer, melanoma, B cell lymphoma and T cell lymphoma.

[0009] Preferably, the agent for promoting or activating CAPS1 gene expression or transcription includes any one of the following i) to iii):

[0010] i) Activating antibodies, nucleotides, lentiviruses or adenoviruses that use CAPS1 transcripts as target sequences and are capable of activating the expression of CAPS1 gene expression products or gene transcription;

[0011] ii) a construct containing a CAPS1 complementary sequence and capable of forming an activating molecule that promotes the expression of CAPS1 gene expression products or gene transcription after being introduced into the body;

[0012] iii) Immune-related cells, differentiated cells or constructs thereof after activation of the CAPS1 gene sequence.

[0013] The present invention also provides the use of a reagent for detecting the expression level of CAPS1 gene or protein in preparing a reagent for predicting the prognosis of liver cancer patients. The latter prediction includes survival prediction and recurrence prediction.

[0014] Preferably, the sample to be detected is NK cells in liver cancer tissue.

[0015] Through the study of tumor cells, the present invention found that the killing ability of NK cells overexpressing CAPS1 on tumor cells was significantly enhanced, indicating that CAPS1 has the effect of inhibiting tumor progression; multicolor immunohistochemistry found that CAPS1 molecules were lowly expressed in NK cells in the tumor microenvironment, and in animal experiments of tumor modeling, the results showed that the tumor size of tumor cells co-cultured with NK cells of CAPS1 gene knockout mice was significantly larger than that of wild-type mice, and it was also concluded that CAPS1 has the effect of inhibiting tumor progression and is expected to be applied to the comprehensive treatment of tumors.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes the application prospect of calcium ion-dependent secretion activator protein 1 (CAPS1) in tumor treatment. The experimental results of the present invention show that, on the one hand, CAPS1 is highly expressed and enhances the killing effect of NK cells on tumor cells, and on the other hand, CAPS1 deficiency promotes the growth of liver cancer in vivo. The present invention can provide good candidate drug targets and prognostic indicators for the clinical treatment of solid tumors such as liver cancer, lung cancer, colon cancer, melanoma, and blood system tumors such as B cell lymphoma and T cell lymphoma, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Based on the tissue specimen library platform of the Liver Cancer Research Institute of Zhongshan Hospital, the expression of CAPS1 in NK cells of cancer tissues of 210 patients with liver cancer was detected by multicolor immunofluorescence. It was found that: CAPS1 was mainly expressed in the cytoplasm of NK cells; compared with the paired adjacent cancer tissues, the expression of CAPS1 in NK cells of liver cancer tissues was significantly downregulated;

[0019] Figure 2 The effect of CAPS1 expression in NK cells of liver cancer tissue on the prognosis of liver cancer patients; the survival analysis of 210 liver cancer patients found that the decrease of CAPS1 expression in NK cells of liver cancer tissue was associated with the poor prognosis of liver cancer patients, which was manifested by the significantly shorter overall survival time (A) and time to recurrence (B) compared with those with positive expression;

[0020] Figure 3 The effect of NK cell CAPS1 on the killing ability of NK cells in vitro; NK-92 cells were used as effector cells and PLC / PRF liver cancer cells were used as target cells. The killing effect of NK cells overexpressing CAPS1 on liver cancer cells was studied at an effector-target ratio of 1:5. It was found that compared with NK-92 cells that did not overexpress CAPS1, CAPS1-CAR-NK cells showed a stronger killing effect on PLC / PRF / 5 cells;

[0021] Figure 4 The effect of NK cell CAPS1 gene deletion on the proliferation ability of liver cancer cells in vivo; primary NK cells from CAPS1 gene knockout mice and control wild-type mice were extracted and co-cultured with mouse liver cancer cells Hepa1-6, and then nude mouse tumor formation experiments were performed. It was found that compared with the control group, CAPS1 gene deletion in NK cells significantly promoted the growth of tumors in mice. DETAILED DESCRIPTION

[0022] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0023] Example 1 Detection of CAPS1 expression in NK cells of liver cancer tissues of patients with liver cancer

[0024] 1. Experimental Materials

[0025] Paraffin-embedded human liver cancer tissue samples. In this example, liver cancer tissues were randomly selected from 210 patients who met the following inclusion criteria among 1672 patients who underwent hepatocellular carcinoma resection in the Department of Liver Tumor Surgery, Zhongshan Hospital Affiliated to Fudan University between January 2008 and December 2009. Inclusion criteria: (1) Pathological diagnosis of hepatocellular carcinoma; (2) No distant metastasis was found in imaging; (3) The surgery was the first radical liver resection; (4) No anti-tumor treatment such as targeted drugs, intervention, radiofrequency or microwave before surgery; (5) Complete clinical pathological data and follow-up data. Clinical and follow-up data were obtained from the database of the Liver Cancer Research Institute of this hospital and verified by reviewing medical records. This study was approved by the Ethics Committee of Zhongshan Hospital Affiliated to Fudan University and the consent of the research population and relevant informed consent were obtained. Paraffin sections were all stained with HE, and corresponding pathological studies and immunofluorescence staining were performed. Postoperative specimens were fixed with formaldehyde and sliced ​​for later use.

[0026] 2. Experimental methods

[0027] 1) Baking: constant temperature oven 60-65 degrees Celsius for 30 minutes;

[0028] 2) Dewaxing and hydration: the sections were placed in xylene No. 1 for 10 min, xylene No. 2 for 8 min, xylene No. 3 for 8 min; anhydrous ethanol for 5 min, 90% ethanol for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min. ddH2O for 3 min × 3 times, TBST for 3 min × 3 times;

[0029] 3) Antigen repair: put the bath box + slice rack + designated antigen repair solution (sodium citrate buffer / pH 6.0) into the rice cooker and heat to boil. Put the slices into the slice rack and continue heating and boiling for 10 minutes. Cool naturally to room temperature; ddH2O 3 minutes × 3 times, TBST 3 minutes × 3 times;

[0030] 4) Immunohistochemistry pen circle: Take out the slices and shake dry. You can use absorbent paper to absorb the water droplets. Use a histochemistry pen to draw a circle around the tissue, making the circle complete;

[0031] 5) Place the slices in a wet box, drip 3% hydrogen peroxide into the circled tissue, incubate in the dark for 15 minutes, and wash the slices 3 times with TBST, 3 minutes each time;

[0032] 6) Dry the water, absorb the residual water, and block with blocking solution (about 30 minutes; the blocking solution in the Akoya kit is 10-15 minutes); no need to wash, just dry the water, absorb the residual water, and add the diluted primary antibody (1:1000);

[0033] 7) Primary antibody: Incubate at 4°C overnight;

[0034] 8) Wash the slides, rinse off the primary antibody with a rinse bottle, and then wash the slides 3 times with TBST, 3 minutes each time;

[0035] 9) Secondary antibody: Use the secondary antibody of the designated primary antibody, incubate for 45 min at room temperature in the dark; wash the slides 3 times with TBST, 3 min each time;

[0036] 10) Shake off the water, absorb the residual water, add opal reagent, incubate at room temperature in the dark for 10 minutes; wash the slides 3 times with TBST, 3 minutes each time;

[0037] 11) Put the bath box + slice rack + designated antigen repair solution into the microwave oven first, heat it on high for 2 minutes to make the antigen repair solution boil, add the slices, heat it on low for 10 minutes, take it out, and cool it naturally to room temperature; ddH2O 3min×2 times, TBST 3min×2 times;

[0038] 12) Dry the water, absorb the residual water, and block with blocking solution (about 30 minutes; the blocking solution in the Akoya kit is 10-15 minutes); no need to wash, just dry the water, absorb the residual water, and add the diluted primary antibody;

[0039] 13) Repeat the above steps until the last primary and secondary antibodies, opal and TBST 3min × 3 washes are completed;

[0040] 14) Add the DAPI diluent prepared in advance and incubate at room temperature in the dark for 5-10 minutes;

[0041] 15) Wash the slides with TBST for 3 min × 3 times; seal the slides with anti-extraction agent and store at 4 degrees Celsius away from light;

[0042] 3. Experimental results

[0043] The experimental results are shown in Figure 1 The results showed that compared with paired adjacent cancer tissues, the expression of CAPS1 in NK cells of liver cancer tissues was significantly downregulated.

[0044] Example 2 Survival analysis of CAPS1 expression in NK cells of liver cancer tissues of patients with liver cancer

[0045] 1. Experimental Materials

[0046] The above multicolor immunohistochemical wax blocks were taken from the patients. The follow-up started from the time of surgery, and the patients were reviewed every 3 months within 1 year after surgery, every 6 months in the second year, and once a year in the third to fifth years. The follow-up contents included routine blood tests, liver and kidney function, tumor markers (AFP, etc.) and various imaging examinations (such as B-ultrasound, CT or MRI). The end point of follow-up was the patient's death or the end of follow-up, and the survival time was in months.

[0047] 2. Experimental methods

[0048] Survival analysis: Survival curves and recurrence curves were drawn using the Kapalan-Meier method and compared between groups using the log-rank method. All statistical tests were two-sided, and P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 19.0 software.

[0049] 3. Experimental results

[0050] The experimental results are shown in Figure 2 The results showed that the decreased expression of CAPS1 in NK cells in liver cancer tissues was associated with a poor prognosis in patients with liver cancer, as evidenced by a significantly shorter overall survival time and time to recurrence than those with positive expression.

[0051] Example 3 The killing effect of CAPS1 on NK cells in vitro

[0052] 1. Experimental Materials

[0053] Cell line: Human hepatoma cells (PLC / PRF / 5 cells) were cultured in DMEM medium (containing 10% fetal bovine serum). NK-92 cells were cultured in a special culture medium (α-MEM 500ml+0.68ml Myo-inositol+1.24ml 2-mercaptoethanol+0.68ml Folic Acid+1ml recombinant IL-2+85ml 12.5% ​​HS+85ml12.5% ​​FBS+6.8ml 1% P / S).

[0054] 2. Experimental methods

[0055] NK-92 cell lines were cultured, and NK cells with CAPS1 overexpression were constructed by lentiviral infection. PLC / PRF / 5 hepatoma cells were co-cultured with NK-92 cells or CAPS1-CAR-NK cells at an effector-target ratio of 5:1. Tumor cells were stained with CFSE before the experiment and stained with 7AAD after co-culture to observe the killing effect of tumor cells.

[0056] 3. Experimental results

[0057] The experimental results are shown in Figure 3The results showed that compared with NK-92 cells that did not overexpress CAPS1, CAPS1-CAR-NK cells exhibited a stronger killing effect on PLC / PRF / 5 cells.

[0058] Example 4 Effect of NK cell CAPS1 on liver cancer growth in vivo

[0059] 1. Experimental Materials

[0060] (1) Cell line: Mouse hepatoma cell line (Hepa1-6 cells), cultured in DMEM medium (containing 10% fetal bovine serum).

[0061] (2) Experimental animals: 4-week-old SPF male Balb / c-nu / nu nude mice, weighing approximately 20 g, 5 mice per group.

[0062] 2. Experimental methods

[0063] (1) Cell culture: Cells were cultured in DMEM medium (containing 10% fetal bovine serum, pH 7.2) supplemented with 2 mmol / L glutamine and cultured in a cell culture incubator at 37° C. and 5% CO 2 .

[0064] (2) Animal experiments:

[0065] Primary NK cells extracted from CAPS1 knockout mice and control wild-type mice were co-cultured with mouse liver cancer cells Hepa1-6. 5 Hepa1-6 cells were injected into the right groin of mice with normal saline. When the tumor grew and could be measured, the tumor size was measured every two days (the long and short diameters of the tumor were measured, and the tumor volume = 1 / 2 × long diameter × short diameter). 2 ). The mice were sacrificed four weeks later.

[0066] 3. Experimental results

[0067] The experimental results are shown in Figure 4 , the results showed that the loss of CAPS1 gene in NK cells significantly promoted the growth of liver cancer in mice.

[0068] The above embodiments are only preferred embodiments of the present invention and are not any formal or substantial limitations of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the expression level of CAPS1 gene or protein in the preparation of a reagent for predicting the prognosis of liver cancer patients, characterized in that: The sample tested is NK cells from liver cancer tissue.

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