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
- Publication number
- CN113116878A CN113116878A CN202010029581.7A CN202010029581A CN113116878A CN 113116878 A CN113116878 A CN 113116878A CN 202010029581 A CN202010029581 A CN 202010029581A CN 113116878 A CN113116878 A CN 113116878A
- Authority
- CN
- China
- Prior art keywords
- hepe
- tumor
- melanoma
- cells
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- WLKCSMCLEKGITB-DBVSHIMFSA-N 15(S)-HEPE Chemical compound CC\C=C/C[C@H](O)\C=C\C=C/C\C=C/C\C=C/CCCC(O)=O WLKCSMCLEKGITB-DBVSHIMFSA-N 0.000 title claims abstract description 45
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 44
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 19
- 238000009169 immunotherapy Methods 0.000 title claims abstract description 11
- 230000001404 mediated effect Effects 0.000 title claims abstract description 6
- 230000002708 enhancing effect Effects 0.000 title description 2
- 201000001441 melanoma Diseases 0.000 claims abstract description 27
- 230000012010 growth Effects 0.000 claims abstract description 17
- 230000004913 activation Effects 0.000 claims abstract description 11
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims description 7
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 claims description 5
- 102000009515 Arachidonate 15-Lipoxygenase Human genes 0.000 claims description 4
- 108010048907 Arachidonate 15-lipoxygenase Proteins 0.000 claims description 4
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 4
- 239000000194 fatty acid Substances 0.000 claims description 4
- 229930195729 fatty acid Natural products 0.000 claims description 4
- 150000004665 fatty acids Chemical class 0.000 claims description 4
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 claims description 4
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 230000003127 anti-melanomic effect Effects 0.000 claims 1
- 230000008595 infiltration Effects 0.000 abstract description 6
- 238000001764 infiltration Methods 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- 239000012190 activator Substances 0.000 abstract description 3
- 239000000427 antigen Substances 0.000 abstract 1
- 102000036639 antigens Human genes 0.000 abstract 1
- 108091007433 antigens Proteins 0.000 abstract 1
- 150000002632 lipids Chemical class 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 51
- 238000002474 experimental method Methods 0.000 description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 12
- 239000006285 cell suspension Substances 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 238000011580 nude mouse model Methods 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 108010019160 Pancreatin Proteins 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 8
- 229940055695 pancreatin Drugs 0.000 description 8
- 208000001382 Experimental Melanoma Diseases 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 102000029816 Collagenase Human genes 0.000 description 6
- 108060005980 Collagenase Proteins 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 229960002424 collagenase Drugs 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- 210000000683 abdominal cavity Anatomy 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 230000001079 digestive effect Effects 0.000 description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 4
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 239000012888 bovine serum Substances 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 210000003141 lower extremity Anatomy 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 238000011398 antitumor immunotherapy Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005206 flow analysis Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 241000283724 Bison bonasus Species 0.000 description 1
- 210000000195 CD3e-positive T cell Anatomy 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102100033467 L-selectin Human genes 0.000 description 1
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010029581.7A CN113116878A (en) | 2020-01-10 | 2020-01-10 | New use of 15S-HEPE for enhancing T cell mediated tumor immunotherapy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010029581.7A CN113116878A (en) | 2020-01-10 | 2020-01-10 | New use of 15S-HEPE for enhancing T cell mediated tumor immunotherapy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113116878A true CN113116878A (en) | 2021-07-16 |
Family
ID=76771035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010029581.7A Pending CN113116878A (en) | 2020-01-10 | 2020-01-10 | New use of 15S-HEPE for enhancing T cell mediated tumor immunotherapy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113116878A (en) |
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 |
-
2020
- 2020-01-10 CN CN202010029581.7A patent/CN113116878A/en active Pending
Patent Citations (4)
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105950560B (en) | Humanization PD-L1 tumor cell line and animal model and application with the cell line | |
| CN106222201B (en) | A kind of method for preparing CAR T cells and obtained CAR T cells and its application | |
| KR101419711B1 (en) | Method for activation treatment of antigen-presenting cell | |
| CN103756964B (en) | A kind of efficient amplification CD3 -cD56 +the method of natural killer cell culture systems | |
| CN101538554A (en) | Survivability-screened cancer stem cells, preparation method, application and kit as well as antigen composite thereof, preparation method, application and kit | |
| US11834677B2 (en) | Expansion and use of expanded NK cell fractions | |
| Sudarsanam et al. | Influence of culture conditions on ex vivo expansion of T lymphocytes and their function for therapy: Current insights and open questions | |
| JP3619853B2 (en) | Method of growing natural killer cells | |
| CN104894072B (en) | A kind of preparation method and applications of autologous natural killer cells propagation | |
| Kiaei et al. | Advances in natural killer cell therapies for breast cancer | |
| CN105969731B (en) | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions | |
| CN101560496A (en) | Dendritic cell carried by tumor stem cell antigen and subjected to tolerance screening, preparation method and application thereof, kit and vaccine comprising dendritic cell | |
| CN114717190B (en) | Human breast malignant phylliform tumor cell line BPT0713 and application thereof | |
| CN116144599A (en) | Immune cell for killing escape variant NK cells and application thereof | |
| CN102988415A (en) | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection | |
| US20220177839A1 (en) | Composition, culture medium and method for inducing and/or amplifying tscm in vitro | |
| CN110272871A (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN109957543A (en) | Utilize the method for Cord blood massive amplification Cord Blood Natural Killer Cells: Impact | |
| CN113116878A (en) | New use of 15S-HEPE for enhancing T cell mediated tumor immunotherapy | |
| Rossi et al. | EVIDENCE FOR TRANSFORMATION OF SPLEEN CELLS ONE DAY AFTER INFECTION OF MICE WITH FRIEND LEUKEMIA VIRUS: Autonomous Growth Potential and Expression of Hybrid-Resistance Genes | |
| CN116769723B (en) | GD2 chimeric antigen receptor modified T cell and application thereof | |
| CN101429496A (en) | Culture medium for human peripheral blood source hemopoietic stem cell | |
| CN101429495A (en) | Cultivation method for human peripheral blood source hemopoietic stem cell | |
| CN108690829A (en) | A kind of method of efficient amplification NK cells | |
| CN106047810A (en) | Novel DC-CTLs cell culture system and culture method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210716 |
|
| WD01 | Invention patent application deemed withdrawn after publication |