CN113116878A - New use of 15S-HEPE for enhancing T cell mediated tumor immunotherapy - Google Patents
New use of 15S-HEPE for enhancing T cell mediated tumor immunotherapy Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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Abstract
The invention discloses a new application of a lipid derivative 15S-HEPE (high efficiency particulate antigen) as a T cell mediated anti-tumor immune activator for treating melanoma. The invention discovers that the 15S-HEPE can effectively inhibit the growth of the melanoma by improving the infiltration and activation level of T cells in the melanoma. The 15S-HEPE anti-tumor activity can be used for preparing anti-melanin tumor immunotherapy medicaments.
Description
Technical Field
The invention belongs to the technical field of medicine and immunotherapy, and particularly relates to a new application of a fatty acid derivative 15S-HEPE (high efficiency particulate E) produced by catalyzing polyunsaturated fatty acid EPA (EPA) by 15-lipoxygenase, namely the 15S-HEPE is used as a T cell-mediated anti-tumor immune activator and is used for the immunotherapy of tumors.
Technical Field
Melanoma is the most malignant skin-related tumor, and the incidence of melanoma has been on the rise over the past few decades. Melanoma-induced death accounts for a large portion of the skin-related tumor-induced deaths. Based on the different characteristics of melanoma, such as occurrence position, genetic background, mutation type and the like, various methods for treating melanoma, including surgery, radiotherapy, chemotherapy and targeted therapy, have been developed, and certain treatment effects are achieved. Nevertheless, these treatments are difficult to achieve with desirable results due to the wide variety of resistance mechanisms.
Tumor immunotherapy represents a major breakthrough in the treatment of melanoma. Immune checkpoints against CTLA-4, PD-1 and PD-L1 and the likeThe monoclonal blocking antibody of (a) shows a good effect in the treatment of melanoma. However, clinical studies have shown that only a fraction of patients exhibit a good response to treatment with immune checkpoint blocking antibodies. T cells play a central role in anti-tumor immunotherapy, and immune checkpoint blocking antibodies block T cell inhibitory signals to promote T cell activation and tumor killing. Therapeutic efficacy of immune checkpoint blocking antibodies such as CTLA-4 and pre-existing T cells in tumors, particularly CD8+The number and activation level of tumor infiltrating T cells are closely related. Thus, increase CD8 in the tumor microenvironment+The number and level of activation of T cells is important for the immunotherapy of melanoma.
The 15S-HEPE is a fatty acid derivative which is derived from polyunsaturated fatty acid EPA and is generated by catalysis of 15-lipoxygenase, and the structural formula is shown in the right picture:
the invention discovers that the 15S-HEPE can effectively inhibit the growth of melanoma by improving the infiltration and activation level of T cells in the melanoma.
Disclosure of Invention
The invention provides a new application of a fatty acid derivative 15S-HEPE generated by catalyzing polyunsaturated fatty acid EPA by 15-lipoxygenase.
15S-HEPE is used as a T cell mediated anti-tumor immune activator for tumor immunotherapy.
15S-HEPE was able to increase the number and level of activation of tumor infiltrating T cells.
Preferably, the tumor is melanoma.
The study of the invention finds that the 15S-HEPE treatment does not affect the growth of the tumor in a melanoma model cultured in vitro and inoculated in nude mice. In contrast, 15S-HEPE treatment significantly inhibited the growth of melanoma, which was inoculated into C57BL/6J mice, and prolonged the survival time of the mice. Flow detection shows that the number and activation level of tumor infiltrating T cells in the 15S-HEPE treatment group are obviously improved. Therefore, the 15S-HEPE can improve the infiltration of T cells in melanoma tumors, enhance the activation level of the infiltration T cells in tumors and inhibit the growth of the tumors. The 15S-HEPE anti-tumor activity can be used for preparing anti-tumor immunotherapy medicaments.
Drawings
FIG. 1 shows the results of the measurement of the effect of 15S-HEPE on the growth of B16-F1 cells cultured in vitro.
FIG. 2 shows the results of the examination of the growth of B16 melanoma by 15S-HEPE inoculated into nude mice. Wherein A: nude mouse tumor growth curve graph; b: the left graph is a nude mouse tumor real object graph, and the right graph is a nude mouse tumor weight statistical analysis result graph.
FIG. 3 is a graph showing that 15S-HEPE significantly inhibited the growth of B16 melanoma when inoculated into C57BL/6J mice. Wherein A: tumor growth profile; b: the left graph is a tumor real object graph, and the right graph is a tumor weight statistical analysis result; c: survival curves of tumor-bearing mice in the control group and the experimental group after receiving blank solvent and 15S-HEPE treatment respectively.
FIG. 4 is a graph of the results of 15S-HEPE significantly increasing the number and level of activation of infiltrating T cells in B16 melanoma inoculated in C57BL/6J mice. Wherein A: detecting the result of the tumor infiltration CD3e positive T cell flow cytometry; b: flow cytometry detection of tumor-infiltrating CD 4-positive T cells and CD 8-positive T cells CD44 and CD62L expression levels.
Detailed Description
The invention is further illustrated by the following specific examples, which are given for the purpose of illustration and are not to be construed as limiting the invention.
Example 1
15S-HEPE on growth of in vitro cultured B16-F1 cells
1. Experimental Material
Cell lines: mouse melanoma cell line B16-F1
Reagent consumables: RPMI-1640 culture medium (Wiesent), pancreatin cell digestive juice (Biyuntian), fetal calf serum (BI), 96-well plate (NEST), MTT (Biyuntian), DMSO (Shanghai national medicine)
The instrument comprises the following steps: cell culture case, superclean bench, enzyme-labeling instrument
2. The experimental method comprises the following steps:
collecting B16 cells of logarithmic growth phase, digesting with 0.25% pancreatin, collecting cells, counting cells, preparing single cell suspension with 20000/ml concentration, culturing in 96-well plate, 2000 cells per well (100 μ L), and culturing at 37 deg.C with 5% CO2Culturing, adding 1 μ L sterile physiological saline containing 0.1% ethanol into solvent control group (Vehicle), adding 1 μ L sterile physiological saline containing 0.1% ethanol into experimental group containing 15S-HEPE with corresponding concentration, and setting 15S-HEPE at final concentration of 1 μ M and 10 μ M; adding 10 μ L MTT solution (5mg/mL) on days 0, 1, 2 and 3 of cell culture, and further culturing for 4 h; the medium was aspirated off, 100. mu.L of DMSO was added to each well to dissolve the dark purple product formazan sufficiently, and absorbance was measured at 570 nm.
3. Results of the experiment
The results are shown in FIG. 1, and the growth rate and cell viability of B16 cells were not reduced by treating B16 cells cultured in vitro alone with 1. mu.M and 10. mu.M 15S-HEPE, compared to the control group. Thus, 1 μ M and 10 μ M15S-HEPE did not directly inhibit B16 cell growth and viability.
Example 2
Detection of growth effect of 15S-HEPE on B16 melanoma inoculated in nude mice
1. Experimental Material
Cell lines: mouse melanoma cell line B16-F1
Experimental animals: SPF level nude mouse
Reagent consumables: RPMI-1640 medium (Wiesent), pancreatin cell digest (Byunnan), fetal bovine serum (BI), 10cm petri dish (NEST), 1mL disposable sterile syringe, 100 μ L microsyringe,
the instrument comprises the following steps: cell culture box, super clean bench, SPF animal facility
2. Experimental methods
B16 cells were seeded in 10cm dishes at 37 ℃ with 5% CO2Culturing for 24-36 hours to enable the cells to be in a logarithmic phase; digesting cells with pancreatin cell digestive juice, collecting cells, counting cells, preparing 3.33 × 106Single cell suspension at individual/ml concentration; each nude mouse was inoculated to the outside of the right hind leg and injected intradermally with 30 μ L of single cell suspension; from inoculationFrom the next day, 15S-HEPE solution (dissolved in sterile normal saline containing 2% ethanol) is injected into the abdominal cavity, the dosage is 4 mug/kg of the body weight of the mouse, and the control group is injected into the abdominal cavity with the same volume of sterile normal saline containing 2% ethanol once a day until the experiment is finished; during the growth of the tumor, the minor axis a and major axis b of the tumor were measured with a vernier caliper, and the tumor volume V (V ═ 0.5 xa) was calculated2X b) and plotting a tumor growth curve; at the end of the experiment, mice were sacrificed, tumors were removed, tumor weights were weighed, and the difference in tumor weights between the treatment and control groups was statistically analyzed.
3. Results of the experiment
The results of the experiment are shown in FIG. 2. The experimental results show that, compared with the solvent control group, the intraperitoneal injection of 15S-HEPE (4 mug/kg per day) has no obvious inhibition effect on the growth of the tumor and the final weight of the tumor in the melanoma inoculated in the nude mice.
Example 3
Detection of growth Effect of 15S-HEPE on B16 melanoma inoculated in C57BL/6J mice
1. Experimental Material
Cell lines: mouse melanoma cell line B16-F1
Experimental animals: SPF grade C57BL/6J mice
Reagent consumables: RPMI-1640 medium (Wiesent), pancreatin cell digest (Byunnan), fetal bovine serum (BI), 10cm petri dish (NEST), 1mL disposable sterile syringe, 100. mu.L microsyringe.
The instrument comprises the following steps: cell culture box, super clean bench, SPF animal facility
2. Experimental methods
B16 cells were seeded in 10cm dishes at 37 ℃ with 5% CO2Culturing for 24-36 hours to enable the cells to be in a logarithmic phase; digesting cells with pancreatin cell digestive juice, collecting cells, counting cells, preparing 3.33 × 106Single cell suspension at individual/ml concentration; each C57BL/6J mouse was inoculated to the outside of the right hind leg and injected intradermally with 30 μ L of single cell suspension; from the day after inoculation, 15S-HEPE solution (dissolved in sterile physiological saline containing 2% ethanol) was intraperitoneally injected at a dose of 4 μ g/kg mouse body weight, and the control group wasInjecting an equal volume of sterile normal saline containing 2% ethanol into the abdominal cavity, and administering once a day until the experiment is finished; during the growth of the tumor, the minor axis a and major axis b of the tumor were measured with a vernier caliper, and the tumor volume V (V ═ 0.5 xa) was calculated2X b) and plotting a tumor growth curve; at the end of the experiment, mice were sacrificed, tumors were removed, tumor weights were weighed, and the difference in tumor weights between the treatment and control groups was statistically analyzed. The operation of the mouse survival rate detection experiment is the same as that of the experiment for measuring tumor growth, except that the experiment is carried out until all mice die, the death time of each mouse is recorded, and a mouse survival curve is drawn.
3. Results of the experiment
The results of the experiment are shown in FIG. 3. The experimental results show that, compared with the solvent control group, for melanoma inoculated in C57BL/6J mice, intraperitoneal injection of 15S-HEPE (4 mug/kg per day) obviously inhibits the growth of tumors, and the inhibition rate is 52.8%. Meanwhile, the survival time of the mice can be remarkably prolonged by treating the mice with 15S-HEPE (4 mug/kg per day). Thus, 15S-HEPE was able to inhibit melanoma growth in immunocompromised C57BL/6J mice.
Example 4
15S-HEPE increases T cell number and activation levels in B16 melanoma inoculated in C57BL/6J mice
1. Experimental Material
Cell lines: mouse melanoma cell line B16-F1
Experimental animals: SPF grade C57BL/6J mice
Reagent consumables: RPMI-1640 medium (wisent), pancreatin cell digest (petit clouds), fetal bovine serum (BI), collagenase type I (biosharp), collagenase type IV (biosharp), DNase I (biosharp), Hanks solution: 8.175g of NaCl, 0.403g of KCl and Na2PO4 0.114g，HEPES 5.958g，CaCl2 0.444g，MgSO40.197g, constant volume to 1000 mL; collagenase digestion solution: 100mL Hanks solution +50mg collagenase type I +50mg collagenase type IV +7.5mg DNase I; FACS buffer: PBS +25mM HEPES +1mM EDTA; 4% paraformaldehyde (bosch de); 10cm petri dish (NEST), 1mL disposable sterile syringe, 100 μ L microinjector, 100 mesh stainless steel sieve,a 100-micron pore size filter screen, a 40-micron pore size cell filter screen, 15mL and 50mL centrifuge tubes and a 5mL disposable sterile syringe; mouse Fc-receptor blocking reagent (Miltenyi Biotech), Mouse flow antibody: CD3e-PE (BD, clone 145-2C11), CD4-FITC (BD, clone GK1.5), CD8a-PerCP-Cy5.5(eBioscience, clone 53-6.7), CD44-APC (BD, clone IM7), CD62L-PE-Cy7(eBioscience, clone MEL-14).
The instrument comprises the following steps: cell culture box, super clean bench, SPF animal facility
2. Experimental methods
B16 cells were seeded in 10cm dishes at 37 ℃ with 5% CO2Culturing for 24-36 hours to enable the cells to be in a logarithmic phase; digesting cells with pancreatin cell digestive juice, collecting cells, counting cells, preparing 3.33 × 106Single cell suspension at individual/ml concentration; each C57BL/6J mouse was inoculated to the outside of the right hind leg and injected intradermally with 30 μ L of single cell suspension; from the day after inoculation, 15S-HEPE solution (dissolved in sterile normal saline containing 2% ethanol) is injected into the abdominal cavity, the dosage is 4 mug/kg of the body weight of the mouse, and a control group is injected into the abdominal cavity with equal volume of sterile normal saline containing 2% ethanol, and the administration is carried out once a day until the experiment is finished; at the end of the experiment, mice were sacrificed, tumors removed and tumor weights were weighed.
Taking out the tumor, placing the tumor in a culture dish of 10cm, and shearing the tumor into small blocks of 1-2 mm square by using scissors; adding a proper amount of collagenase digestive juice, placing the mixture in a table concentrator at 37 ℃ for digestion, wherein the rotating speed is 80rpm, and lightly blowing and uniformly mixing every 20-30 min; digesting for 1-1.5 h, filtering the digested cell suspension through a filter screen with the aperture of 100 mu m, placing the filtrate in a 50mL centrifuge tube, centrifuging for 5min at 4 ℃ and 1000rpm, and removing the supernatant; adding PBS into the cell sediment for washing once, centrifuging for 5min at 4 ℃ and 1000rpm, and removing the supernatant; adding a proper amount of preheated erythrocyte lysate (2-5 mL) into the cell sediment, incubating for 10min at 37 ℃, adding PBS with the same volume, centrifuging for 5min at 4 ℃ and 1000rpm, and removing the supernatant; adding a proper amount of PBS into the cell sediment for resuspending the cells, filtering the cell suspension by a filter screen of 40um to prepare single cell suspension, centrifuging at 4 ℃ and 1000rpm for 5min, and removing supernatant; adding a proper amount of FACS buffer into the cell sediment, and resuspending the cells to prepare a tumor tissue single cell suspension for later use.
Taking the prepared tumor tissue single cell suspension, detecting the cell density by using a blood counting chamber or a flow cytometer, and taking 1-2 multiplied by 107cells, resuspended in 100. mu.L FACS buffer; adding 1 mu L of mouse Fc receptor blocking reagent, and sealing for 15min at 4 ℃ in a dark place; adding 1uL of corresponding flow type antibody, reacting for 1h on ice in a dark place, and gently blowing and uniformly mixing once every 20min by using a pipettor; adding 900 μ L FACS buffer for resuspension, centrifuging at 4 deg.C and 1000rpm for 5min, and removing supernatant; adding 1mL of FACS buffer to resuspend the cells, centrifuging at 4 ℃ and 1000rpm for 5min, and removing supernatant; resuspending the cells with 200-400 μ L FACS buffer, followed by flow analysis;
if the sample is not immediately subjected to flow analysis, it can be fixed with 4% paraformaldehyde, as follows: after reacting with the flow antibody, diluting and cleaning the cell to be detected for 2 times by using FACS buffer, centrifuging for 5min at the temperature of 4 ℃ and 1000rpm, and removing the supernatant; adding 150 μ L of 4% paraformaldehyde for resuspension of cells, and fixing at room temperature for 30 min; add 450 u L FACS buffer directly to dilute paraformaldehyde to 1% final concentration, store at 4 ℃ in the dark until flow detection.
3. Results of the experiment
The results of the experiment are shown in FIG. 4. Treatment of B16 melanoma, inoculated in C57BL/6J mice, with 15S-HEPE increased the number of tumor infiltrating T cells and also the proportion of CD 44-positive activated T cell subsets among CD8T and CD 4T cells. Thus, 15S-HEPE can promote infiltration and activation of T cells in melanoma.
Claims (5)
1. The polyunsaturated fatty acid EPA is catalyzed by 15-lipoxygenase to produce the fatty acid derivative 15S-HEPE with anti-tumor activity and new application in T cell mediated tumor immunotherapy.
2. The anti-tumor activity of 15S-HEPE according to claim 1, characterized in that said anti-tumor activity of 15S-HEPE is anti-melanoma activity.
3. The anti-tumor activity of 15S-HEPE according to claim 1, characterized in that the anti-tumor activity of 15S-HEPE is inhibition of melanoma growth.
4. The new use according to claim 1, characterized by a new use of 15S-HEPE in melanoma immunotherapy.
5. The immunotherapy according to claims 1, 4, characterized in that 15S-HEPE increases the number and activation level of tumor infiltrating T cells.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107405324A (en) * | 2015-01-16 | 2017-11-28 | 艾菲穆恩有限公司 | Composition comprising 15 HEPE and its application method |
| US20180104207A1 (en) * | 2015-12-18 | 2018-04-19 | Afimmune Limited | Compositions comprising 15-hepe and methods of using the same |
| CN108025181A (en) * | 2015-07-21 | 2018-05-11 | 艾菲穆恩有限公司 | For treating or preventing the composition for including 15-HEPE of cancer and neurological disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107405324A (en) * | 2015-01-16 | 2017-11-28 | 艾菲穆恩有限公司 | Composition comprising 15 HEPE and its application method |
| CN108025181A (en) * | 2015-07-21 | 2018-05-11 | 艾菲穆恩有限公司 | For treating or preventing the composition for including 15-HEPE of cancer and neurological disease |
| US20180104207A1 (en) * | 2015-12-18 | 2018-04-19 | Afimmune Limited | Compositions comprising 15-hepe and methods of using the same |
| CN108697680A (en) * | 2015-12-18 | 2018-10-23 | 艾菲穆恩有限公司 | Include the composition and its application method of 15-HEPE |
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Application publication date: 20210716 |