CN110358729A - The natural killer cells of cancer of bile ducts treatment - Google Patents
The natural killer cells of cancer of bile ducts treatment Download PDFInfo
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- CN110358729A CN110358729A CN201811004930.9A CN201811004930A CN110358729A CN 110358729 A CN110358729 A CN 110358729A CN 201811004930 A CN201811004930 A CN 201811004930A CN 110358729 A CN110358729 A CN 110358729A
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- 235000010981 methylcellulose Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229940126701 oral medication Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- CLSUSRZJUQMOHH-UHFFFAOYSA-L platinum dichloride Chemical compound Cl[Pt]Cl CLSUSRZJUQMOHH-UHFFFAOYSA-L 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000001562 sternum Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000007940 sugar coated tablet Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
Abstract
The present invention relates to can effectively prevent or treat the natural killer cells of cancer of bile ducts, the prevention or treatment pharmaceutical composition of its abductive approach and the cancer of bile ducts comprising the natural killer cells.The natural killer cells induced in the present invention especially has significant killing ability to biliary tract cancer cell in liver, can effectively prevent or treat cancer of bile ducts in liver in biliary tract cancer cell.
Description
Technical field
The present invention relates to the natural killer cells for effectively preventing or treating cancer of bile ducts, the method for inducing the cell, and
The pharmaceutical composition for being used to prevent or treat cancer of bile ducts comprising above-mentioned natural killer cells.
Background technique
Cancer of bile ducts is the cancer generated in bile duct, and is considered that (Sell and occurs by the identical stem cell of liver cancer
Dunsford Am J.Pathol.134:1347-1363,1989).Although the disease incidence of cancer of bile ducts is far below liver cancer on the whole world,
But in the disease incidence of south east asia much higher than Europe or North America.Since the recurrence rate of cancer of bile ducts is higher, there are surgeries
The effect of excising operation is low, the bad problem of the effect of the common chemotherapy and radiotherapy (Cancer such as Pederson
Res.4325-4332,1997).It is reported that cancer of bile ducts diagnosis is not easy, the sense of bacterium or helminth of the possible morbidity to bile pipe
The inflammation of the chronic bile pipe of chronic inflammation caused by contaminating leads to the tendency of cancer of bile ducts.(Roberts etc.,
Gastroenterology 112:269-279,1997)。
Nevertheless, the pathogenesis to cancer of bile ducts is unclear, the targeting point for treating cancer of bile ducts is not disclosed yet
Son.Furthermore, it was recently reported that the cytogene result of study of some cancer of bile ducts, the cell strain (Yamaguchi for only establishing only a few
Deng J.Natl Cancer Inst 75:29-35,1985；Ding etc., Br J Cancer 67:1007-1010,1993), and
The not publicly method of the antibody using cell strain preparation in conjunction with cancer of bile ducts.
It is reported that natural killer cells (natural killer cell, NK cell) participates in removal pathogen and cancer cell
Congenital immunity (innate immunity), to secrete interferon (IFN-γ), tumor necrosis factor-alpha (TNF-
α), -1 β of macrophage inflammatory protein (MIP-1 β) and the substance of other interventions adaptive immunity (adaptive immunity).
If NK cell is contacted with other cells, acted on by the MHC molecule of NK cell, for as cancer cell without MHC
Class 1, or for as be infected virus cell MHC Class 1 form it is abnormal when, to NK cell interior
Signal is issued, these abnormal cells are attacked.But it has been reported that, function and differentiation potency of the kinds of tumors in these NK cells
Existing defects in power, therefore the existence of known cancer cell and the activity of NK cell have substantial connection.Although in order to which cancer immunity is controlled
Treat, diversified forms carry out NK cell quantity or it is active increase relevant research, but so far without obtain satisfactorily at
Fruit.
Summary of the invention
Technical task
One of the objects of the present invention is to provide the natural killer cells of a kind of prevention that can be used in cancer of bile ducts or treatment
Abductive approach, and by this method induce natural killer cells.
Another object of the present invention is that providing a kind of including the gallbladder for the natural killer cells being induced in the present invention
The prevention or treatment pharmaceutical composition of road cancer.
It is further clarified by following detailed description of the invention, the range of claims and attached drawing of the invention another
One purpose and advantage.
Technical solution
Other than being in addition defined in the present invention, all technologies as used in this specification and scientific word with
The meaning that those skilled in the art in the invention are understood is identical.It can be used in test of the invention and remember with this specification
The similar or equivalent any means and material carried, but this specification describes preferably materials and method.Of the invention
When recording and being claimed, using such as giving a definition.The wooden language range of profession as used in this specification is unrestricted, instead may be used
It is understood to be used for the purpose of specific embodiment in the present specification.Article " one " or " one in this specification
It is a " one or meaning more than one (that is, at least one or more than one) of the expression as phraseological target language.
The research that immunological diseases are treated using natural killer cells was carried out in the past, but not yet acquirement makes us full so far
The achievement of meaning.In particular, it is known that the treatment of cancer of bile ducts is more difficult in cancer, do not reported so far using natural killer cells come at
Treat to function the result of study of cancer of bile ducts.
In order to develop cancer of bile ducts therapeutic agent, the present inventor is by concentrating on studies, as a result, it has been found that using according to this hair
The natural killer cells of bright following methods induction is very prominent to cancer of bile ducts therapeutic effect, so as to complete the present invention
The present invention relates to a kind of abductive approach of natural killer cells, implement the step that blood is obtained from the people of health first
Suddenly, the blood is whole blood, Cord blood, marrow or peripheral blood, preferably peripheral blood.
The monocyte of the implementable isolated peripheral blood of peripheral blood obtained from above-mentioned steps in the present invention
The step of (peripheral blood mononuclear cell, PBMC).
In the present invention, the monocyte of the peripheral blood (peripheral blood mononuclear cells,
PBMC) for from mammal, the monocyte preferably separated from the peripheral blood of the mankind, it is main include as B cell, T cell,
The immunocytes such as natural killer cells, and with basocyte (basophil), acidophil (eosinophil), neutrophil(e) granule
The granulocytes such as cell (neutrophil).The peripheral blood that above-mentioned PBMC can be obtained from organism passes through common manufacturer
Method prepares.The PBMC is preferably separated from peripheral blood by using the density-gradient centrifugation method of Ficoll.
It is implementable to remove CD3 from the monocyte of above-mentioned isolated peripheral blood in the present invention+The step of T cell.This hair
CD3 is removed in bright+The method of T cell is unrestricted, but immunomagnetic isolation product available on the market can be used and carry out,
Such as MACSxpress (Mei Tian Ni Bioisystech Co., Ltd (Milteyi Biotec), Germany) etc..
It is implementable to be removed CD3 for described in the present invention+The peripheral blood mononuclear cells of T cell include 500 to
The step of culture medium of the cell factor (cytokine) of 1500IU/ml is cultivated.
In the present invention, the culture medium may include the sodium chloride (NaCl) of 0.5~0.6 weight %, 0.4~0.5 weight %
D-Glucose (D-glucose), the amino acid of 0.2~0.3 weight %, 0.15~0.25 weight % NaHCO3And 0.15
HEPES (4- (2- ethoxy) -1- piperazine ethanesulfonic acid (HEPES (4- (2-hydroxyethyl) -1- of~0.25 weight %
Piperazineethanesulfonic acid) but not limited to this.It is limited that cell science & technical research institute usually can be used
The AlyS505NK-EX of company (Cell Science& Technology Institute inc.) (CSTI), but not limited to this.
In the present invention, the cell factor (cytokine), which refers to, can be used for inducing peripheral blood mononuclear cells to nature
Kill the immunologically activated cell factor of cell.It in one embodiment of the invention, may include IL as such cell factor
(interleukins) -2, IL-15, IL-21, Flt3-L, SCF, IL-7, IL-12, IL18 or can it is two or more mixing make
With.In particular, it is known that IL-2, IL-15 or IL-21 cell factor have brilliance for the differentiation and breeding of natural killer cells
Effect most preferably can be IL-2 therefore it is preferable to use these cell factors.
For the purposes of the present invention, the amount of the cell factor preferably 500~1500IU/ml, more preferable 800~
1200IU/ml。
In addition, in the present invention, implementable 10~25 days of the culture will preferably eliminate CD3+The peripheral blood of T cell
Monocyte is cultivated 5~7 days in the T75 flask comprising 10~30ml culture solution, is moved on to T175 flask and is further cultured for 7~14 days.
In the present invention, static gas wave refrigerator (stationary culture) is can be used or the culture that suspends in the culture
(suspension culture) method, static gas wave refrigerator refer on incubator not by stirring (agitating) or shake
Dynamic (shaking) come the method cultivated, the culture that suspends refers to making by ventilation (aeration) or stirring etc. thin
The method that born of the same parents are cultivated on the lower part or side surface part for be not attached to reactor and on suspended state.In addition, static gas wave refrigerator
Reactor and suspend culture reactor can be likewise, being also possible to different.Such as the reaction of static gas wave refrigerator
If if device and the reactor for the culture that suspends are same, after completing static gas wave refrigerator on same reactor, additional supply packet
The culture solution of nutritional ingredient needed for including cell factor (cytokine) etc., is cultivated with suspension training method, if using
If different reactors, after completing static gas wave refrigerator, culture material is moved on to and carries out suspension training in suspension cultivation reactor
It supports.
In the present invention, there is CD3 phenotype by the natural killer cells that method as described above is induced, can also have
There is CD56brightOr CD56dimPhenotype.
In addition, the natural killer cells being induced as described above in the present invention is to biliary tract cancer cell, especially selectively
There is very strong cytotoxicity to (intrahepatic) biliary tract cancer cell in liver, can effectively prevent or treat liver liner
Road cancer.
Further embodiments according to the present invention are related to a kind of natural kill comprising obtaining by means of the present invention
Cell as effective component, the pharmaceutical composition of prevention or treatment for cancer of bile ducts.
In the present invention, described pharmaceutical composition refers to " cellular therapeutic agent (cellulartherapeutic agent) ".
Term " cellular therapeutic agent (cellulartherapeutic agent) " of the invention refer to by using from individual separation, training
Cell and tissue made from feeding and special operation, as pharmaceuticals (beauty used for the purpose for the treatment of, diagnosis and prevention
State FDA regulation), in order to restore the function of cell or tissue, by by it is lived self, of the same race or heterogenous cell in vitro
The a series of behavior such as Multiplying culture or the biological characteristics for changing cell with other methods, for treatment, diagnosis and pre-
Anti- purpose come using pharmaceuticals.
In the present invention, the natural killer cells that described pharmaceutical composition is included is to biliary tract cancer cell, especially in liver
Biliary tract cancer cell selectivity cytotoxicity is very strong, can effectively prevent or treat cancer of bile ducts in liver.
Pharmaceutical composition of the invention be may include 1 Х 104A above, 1 Х 105A above, 3 Х 105A above, 1 Х 106
A above, 3 Х 106A above, 6 Х 106A above or 1 Х 107More than a, and 1 Х 1010A natural kill below is thin
Born of the same parents.
Term used in the present invention " prevention " refers to the administration by described pharmaceutical composition, inhibits or postpone gallbladder
All behaviors of road cancer disease.
In addition, term used in the present invention " treatment " refers to the administration by described pharmaceutical composition, improves or fully recover from an illness
More all behaviors of cancer of bile ducts disease symptoms.
In the present invention, the feature of described pharmaceutical composition can for capsule, pastille, particle, injection, paste, pulvis or
The feature of drink form, described pharmaceutical composition can be people for its object.
Pharmaceutical composition of the invention includes according to usual way with powder, granule, capsule, pastille, aqueous suspension
The forms such as the oral types such as liquid dosage form, external preparation, suppository and sterile injectable preparation.Pharmaceutical composition of the invention is included in pharmacy
The carrier of upper permission.Bonding agent, lubricant, disintegrating agent, tax can be used when being administered orally in the carrier allowed in pharmacy
Shape agent, solubilizer, dispersing agent, stabilizer, suspending agent, pigment, fragrance etc., as injection Shi Keyu buffer, antistaling agent,
Analgesic, solubilizer, isotonic agent, stabilizer etc. are used in mixed way, can be used when local administration air entraining agent, excipient, lubricant,
Antistaling agent etc..As described above, pharmaceutical composition of the invention can with allow in above-mentioned pharmacy using carrier mix and more
Kind mode manufactures.Such as it can be in the form of pastille, tablet, capsule, elixir, suspension, syrup, chip etc. when oral medication
It is made, can be manufactured in the form of single medication essence or multiple medication when as injection.It can be with solution, suspension
The dosage forms such as liquid, pastille, capsule, slow release type preparation are manufacture.
Suitable for formulation carrier, suspending agent and diluent include lactose, glucose, sucrose, D-sorbite, mannitol,
Xylitol, erythritol, maltitol, starch, Arabic gum, alginates, gelatin, calcium phosphate, calcium silicates, cellulose, methyl
Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, nipasol, talcum, tristearin
Sour magnesium or mineral oil.It simultaneously also include filler, anti-coagulants, lubricant, wetting agent, fragrance, emulsifier, preservative etc..
The administration route of pharmaceutical composition of the invention include in oral cavity, intravenous, intramuscular, intra-arterial, marrow,
In diaphragm, heart is interior, percutaneous, subcutaneous, intraperitoneal, nasal cavity is interior, intestinal tube, local, sublingual or rectum, but not limited to this.It is preferred that
It is oral or non-oral.
In the present invention, " non-oral " be include subcutaneous, intradermal, intravenous, intramuscular, in liquid capsule intra-articular, living, breastbone
It is interior, diaphragm is interior, intralesional and head intraosseous injection or injection technique.Meanwhile pharmaceutical composition of the invention can be with the shape of suppository
State rectally.
Pharmaceutical composition of the invention according to include the activity of used particular chemicals, it is the age, weight, normal
Health status, gender, diet, administration time, route of administration, elimination factor, drug collocation and prevention or treatment specified disease
The many reasons such as degree change, although the dosage of aforementioned pharmaceutical compositions is the state, weight, disease by patient
Course of disease degree, drug form, route of administration and period are different, but those skilled in the art can suitably select, one day
Dosage is 0.0001~50mg/kg or 0.001~50mg/kg.It can be administered once with one day, can also divide and be administered several times.
Above-mentioned dosage does not limit the scope of the invention from the aspect of any.Pharmaceutical composition of the invention can be prepared into as follows
Dosage form, i.e. pill, sugar coated tablet, capsule, liquor, gel, syrup, slurries, suspending agent.
Invention effect
The natural killer cells being induced in the present invention is to cancer, especially biliary tract cancer cell, wherein especially to liver liner
Road cancer cell has excellent killing ability, therefore can effectively prevent or treat cancer of bile ducts in liver.
Detailed description of the invention
Fig. 1 shows the design signals for utilizing nude mice to design (in-vivo) experiment in vivo in one embodiment of this invention
Figure, Fig. 1 (a) be equivalent to for assess by the concentration dependent of SMT01 natural killer cells produced by the present invention safety and
The design of experiment of toxicity assessment.In addition, Fig. 1 (b) be equivalent to for assess it is thin by SMT01 natural kill produced by the present invention
The design of experiment of the concentration dependent efficiency of born of the same parents.
Fig. 2 indicates the flow cytometric analysis results of the SMT01 of (ex-vivo) external in one embodiment of the invention amplification
With the cytotoxicity analysis to biliary tract cancer cell line.Fig. 2 (a) indicates the phenotype of analysis SMT01 cell mass as a result, being
The phenotype of analysis NK cell has used anti-CD56/CD16 antibody, in order to analyze T cell group has used anti-CD 3 antibodies,
Anti-CD 40 antibody has been used in order to analyze DC and B cell.Fig. 2 (b) is for biliary tract cancer cell line and Extrahepatic Bile Duct Carcinoma in liver
HuCCT-1 (biliary tract cancer cell line in liver), SNU1196 (extrahepatic biliary passages cancer cell line) and SNU478 (the weary Te Shi pot of cell strain
Abdomen cancer cell line) SMT01 cytolytic ability analysis result.Its K562 cell strain belongs to positive control.
The tumor growth inhibitory effect of Fig. 3 expression SMT01.Fig. 3 (a), Fig. 3 (b) and Fig. 3 (c) indicate processing G1~G5's
The variation of nude mice and tumour picture, gross tumor volume and the tumor weight isolated by the nude mice.
Internal (in-vivo) experiment of nude mouse of Fig. 4 expression SMT01.Fig. 4 (a) indicates the variation of weight, and Fig. 4 (b) is indicated
The volume change of tumour.Herein, slide calliper rule (Califer, CD-15CX, Mitsutoyo K.K. (Mitutoyo), Japan) is utilized
Gross tumor volume is determined according to the following formula: gross tumor volume (mm3)=L (mm) × W2 (mm2)×1/2.Measure each group average swollen
Knurl weight is as follows: G1,2.904g (IR:0.0%)；G2,1.892g (IR:34.8%)；G3,2.292g (IR:21.1%)；G4,
(2.252g IR:22.4%)；G5,1.385g (IR:52.3%).Wherein, IR:IR (%)=(1- T/ is determined using following formula
C) ×× 100 (T: the average tumor weight of experiment group and positive controls, C: the average tumor weight of negative control group).
Fig. 5 indicates the H&E dyeing and IHC experimental result of nude mouse tumor.In each group the global shape of representative tumour and
CD56IHC result is observed at low power objective (12.5X), and the photo of amplification is shown in right side (100X).Known to G2~
CD56- positive NK cell is along the gap distribution (red arrow) between tumor nodule in G4 group.
Fig. 6 indicates the observation result in terms of the histology of the tumour cell of apoptotic cell and the natural killer cells of infiltration.
Fig. 6 (a) indicates that the TUNEL coloration result of all groups of tumor biopsy is further able in G2~G5 group compared with G1 group
Observe TUNEL positive cell.Fig. 6 (b) indicates IHC experimental result, and a and b are indicated along the loose group between tumor nodule
Knit the G2 group serial section of the NCR1- positive natural killer cells of display infiltration (red arrow), c and d indicate to show CD56 and
The photo of the serial section of the G3 group of NCR1- positive natural killer cells.E~h indicates the photo of the serial section of G3 group, with
The tumour cell of the IHC detection of aspartic acid proteolytic enzyme -3 (caspase-3) antibody using Anti-Human containing cysteine
, antiapoptotic signals mainly observe on the infiltrating sites of natural killer cells.I and j indicates the serial section of G4 group
Photo, in tumour aspartic acid proteolytic enzyme -3a positive cell (black arrow) of the people containing cysteine it is observed that
It is observed on the position (red arrow) of the natural killer cells of infiltration.K~l indicates the photo of the serial section of G4 group, containing half
Aspartic acid proteolytic enzyme -3a the positive cell (black arrow) of cystine is deposited along the crumbly texture in tumor nodule gap
It is the position (red arrow) of natural killer cells infiltration.M~p indicates display and natural killer cells in G1 and G5 group
The unrelated tumour of infiltration in most aspartic acid proteolytic enzyme -3a positive cell (black arrows containing cysteine for being distributed
Head) serial section photo.
Specific embodiment
As follows, the present invention is described in detail by following examples.But following examples only illustrate the present invention, this
The content of invention is not limited to these embodiments.
Embodiment
1. the preparation of natural killer cells
PBMC has been separated after obtaining blood by 2 healthy donors.Utilize MACSxpress (Milteyi
Biotec, Germany) cut off CD3+T cell.The PBMC washing 2 that T cell has been removed is taken second place using DPBS buffer
Afterwards, in NK amplification culture medium (ALyS505NK-IL2 1000IU/ml, cell science and technical research institute (Cell comprising 20ml
Science&Technology Inst)) T75 flask in cultivated.It is thin every 3 days natural kill in IL-2 activation
Additional fresh culture solution, culture move back T175 flask in 5~7 days in born of the same parents.The proliferation of natural killer cells is to achieve the goal
It is persistently additional until cell quantity to inject fresh culture solution, it has further cultivated 7~14 days.Use automatic cell counter
(automatic cell counter) is with the natural killer cells that trypan blue (trypan blue) algorithm is that measurement is proliferated
The survival rate and quantity of (SMT01 cell).
2. being designed with the experiment in vivo of nude mice
According to OECD excellent laboratories administrative standard (OECD Good Laboratory Practice regulations
(revision of ENV/MC/CHEM (98) 17 1997)) and the laboratory for non-clinical study excellent laboratories administrative standard
(Good Laboratory Practice) (21CFR Part 58, food and drug administration (Food and Drug
Administration), the U.S.'s (on April 1st, 2015) the, by (Charles River NCr nude mouse (BALB/cSlc-nu/nu)
Laboratory (Charles River Laboratories), Japanese SLC Co., Ltd. (Japan SLC, Inc))
Operation and raising in microisolator (trade (brand) name) cage.
In order to assess the safety and toxicity of dose-dependent, prepare the natural kill obtained above of 3 kinds of different amounts
Cell (Fig. 1 (a)).It is injected as low dosage, using 2 X 10 for being equivalent to people64 X 10 of cell/kg dosage4Cell/dynamic
Object is injected as intermediate dosage and high dosage, has been respectively adopted to 2 X 105Cell/animal and 1 X 106Cell/animal.It
Afterwards, (ex-vivo) by the natural killer cells comprising proliferation and repairs group to 50 heros and female mouse (6 weeks left sides in vitro
Each weight of the right female mouse of hero is 18.7~22.5g and 16.1~18.8g), totally 27 weeks every 3 weeks, vein note is carried out 2 times a week
It penetrates.The intravenous injection of 18 SMT01 cells has been carried out altogether.
The effect experiment (Fig. 1 (b)) of assessment SMT01 usage variance has been carried out for same nude mice.Specifically, right
Each group (G1~G5) comprising 10 nude mices is by HuCCT1 cell (5 X 106Cell/0.2mL) it is injected into subcutaneous, transplanting simultaneously
Survival.G1, physiological saline (negative control group)；G2-G4 injects SMT01；G5, gemcitabine (Gemsitabin)+cis-platinum
(cisplatin) (Gem+CDDP) (positive controls).It is 84~119mm that 8 gross tumor volumes are screened on each group3(weight
Range implements treatment for the mouse of 19.3~20.5g).
Be described in more detail, for the nude mice comprising HuCCT-1 tumour every 10 days totally 5 intravenous injection SMT01.It is positive right
According to group for by gemcitabine (Gem), cis- dichlorodiamine close platinum (Cisplatin Diamine Platinum Dichloride,
CDDP it) is administered respectively with the dosage of 120mg/kg and 3mg/kg.When injecting SMT01,3 mouse groups are according to following differences
Dosage is administered.The low dosage of G2-G4:G2 (4 X 104Cell/animal)；G3, intermediate dosage (2 X 105Cell/animal)；
G4, high dosage (1 X 106Cell/animal).G1 and G5 is respectively equivalent to negative control group (physiological saline) and positive controls
(CDDP+Gem).Disposable syringe (26G, 1ml) is used when cell infusion.Injected slurry volume is 0.2ml/, is finally injected
It is evaluated after so that the mouse with tumour is gone euthanasia after 14 days.(Fig. 1 (b))
3. human cell line
The people's biliary tract cancer cell line HuCCT-1 (biliary tract cancer cell line in liver) used in this experiment is purchased from health science
Resources for research library (Health Science Research Resources Bank) (Osaka, Japan), SNU1196 (liver outer bladder
Road cancer cell line) and SNU478 ((weary Te Shi ampulla cancer cell line) purchased from Korea Cell strain bank (Soul, South Korea) buy.?
Include 5%CO2Humidified ambient under, supplement by 10% fetal bovine serum (GIBCO), 100U/ml penicillin and 100mg/ml chain
Cell strain has been cultivated in the RPMI-1640 culture medium (GIBCO) of mycin.
4. phenotype flow cytometry (Phenotypicflow cytometric analysis)
In order to analyze the cell mass of SMT01, by cell with the PE Anti-Human CD56 being coupled and Anti-Human CD40 antibody and FITC
Anti-Human CD3 and Anti-Human the CD16 monoclonal antibody (BD Bo Aosaisi (Biosciences)) of coupling dye.In order to contaminate
Color, after phosphate buffer (phosphate buffer saline, PBS) washs 2 SMT01, in FACS dye solution
It suspends again in (PBS comprising 0.5% bovine serum albumin(BSA)).Then cell is dyed 30 at 4 DEG C using antibody appropriate
Minute.After carrying out 3 washings using PBS buffer solution, in FACS stream type cell analyzer (Beckton Dickinson
AriaIII cell) is analyzed.In order to analyze NK cell mass, natural killer cells is defined as CD56+/CD3-And CD56+/CD16+。
5. cytotoxicity analysis
Using Cell counting Kit (Cell Counting Kit-8, CCK-8), (more Ji polymoleculars count company
(Dojindo Mol.Tech), the U.S.) measurement natural killer cells cellular cytoxicity activity.In order to assess for people's cancer of bile ducts
The cellular cytoxicity activity of the natural killer cells of cell strain has used K562 cell as positive controls.In 96- orifice plate
1 X 10 is inoculated on each hole4After the SMT01 effector cell (effector cells) of cell, cultivate 24 hours.It uses
CCK8 kit determines target cell strain in 3 kinds of different effector-target (E:T) cell proportions (1:5,1:1 and 5:1)
Cells survival rate.Use the absorbance of microplate reader (microplate reader) measurement at 450 nm.Cytotoxicity result
It measures as follows.
Cytotoxicity (%)=100%- ((ODe+t-ODb)-(ODe-ODb))/(ODt-ODb) X100% (OD, extinction
Degree；E, effector；T, target；B, blank).
6. histology, immunohistochemistry (IHC) and TUNEL analysis
In order to which histologic analysis is fixed on paraffin after nude mice separation HuCCT-1 tumour.Prepare 4 μm of tissue
Slice is dyed as IHC experiment using hematoxylin-Yihong (H&E).It is Anti-Human that IHC, which tests used antibody,
CD56 (BD science (Sciences)), (the natural cytotoxicity triggering of nature cell toxin receptor 1
Receptor 1, NCR1) (BD science (Sciences)) and aspartic acid proteolytic enzyme of the Anti-Human containing cysteine
(Caspase) 3a antibody (Abcam).The original of the apoptotic cell using TUNEL dyeing is implemented using the glass slide of tumor biopsy
Position (in situ) analysis.TUNEL analysis is using TumorTACS in Apoptosis in-situ detection reagent box (R&D system
(Systems), Minneapolis, the U.S.).In order to which micro- sem observation has used Olympus (Olympus) MVX10, use
Olympus (Olympus) BX51 glass slide.
7. statistical analysis
Weight, tumour body are assessed using SAS (version 9.3, SAS software study institute (SASInstitute Inc), the U.S.)
Long-pending and tumor weight.Each numerical value is indicated with being averaged ± SD.In order to which homoscedasticity has used Bartlett inspection, in order to unite
Meter learns conspicuousness and Dunnett t- has been used to examine.Statistical significance is determined in P < 0.05.
8. by PBMC amplification SMT01, the natural killer cells of cytotoxicity
In order to produce SMT01, after having separated natural killer cells from human peripheral blood with MACSexpress, excision
CD3 positive T cell.Then, it will have been cultivated in the presence of IL-2 14 days by isolated natural killer cells.In order to determine SMT01
Cell mass uses the lymphocytes such as identification natural killer cells (CD56/CD3), T cell (CD3), dendritic cell and B cell
(CD40) antibody dyes the natural killer cells through amplification in vitro.In order to determine the hypotype of natural killer cells
CD56/CD16 antibody is used.It is thin that natural kill is shown as with the intracellular majority being amplified according to CD56 and CD3 phenotype
Born of the same parents.Confirm CD3+T cell group is distributed with 0.2% low-level.(Fig. 2 (a)) is confirmed in SMT01 cell mass
99.2% be CD56+/CD3-Natural killer cells.CD56 can be observed in natural killer cells simultaneouslydimAnd
CD56brightPhenotype includes more CD56 especially in SMT01dimNatural killer cells.
Have rated the cell lysis activity (Fig. 2 (b)) of SMT01.In order to evaluate the cytotoxicity of natural killer cells, make
The quantity of viable bacteria target cell is determined with Cell counting Kit -8 (CCK-8).In order to compare in people liver
(intrahepatic) and the existence of outer (extrahepatic) cancer of bile ducts (cholangiocarcinoma, the CC) cell strain of liver
Rate is used with K562 cell as positive control.The cell lysis activity increased is shown to K562 cell SMT01.For
For biliary tract cancer cell line, SMT01 has been confirmed compared with SNU1196 the and SNU478 cell strain of extrahepatic biliary passages cancer cell line,
More effectively biliary tract cancer cell line HuCCT1 in lethal liver.This demonstrate the natural killer cells pair being induced according to the present invention
In the cell dissolution effect highly significant of biliary tract cancer cell line in liver.
9. the in vitro study of the SMT01 of pair nude mice
In order to assess SMT01 dose-dependent safety, toxicity and effect, using 3 kinds of natural killer cells with
Amount implements internal (in-vivo) experiment (table 1,2).SMT01 has been injected intravenously in nude mice tail.At this point, SMT01
Low dosage is set as being equivalent to 2 X 10 of people64 X 10 of cell/kg4Cell (cells)/animal (animal), setting are intermediate
Dosage and high dosage are respectively set as 2 X 105Cell/animal and 1 X 106Cell/animal.In order to assess the peace of dose-dependent
Full property and toxicity, every 3 weeks, inject SMT01 2 times a week, total co-injection 18 times, every time using 3 different dosages
(table 1, Fig. 1).As a result, the SMT01 for having confirmed injection research on maximum utilized quantity also has safety without special lethal response
Property.Although not including on drawing, the result for observing the internal viscera of all SMT01 processing groups does not find and kills naturally
That hurts cell gives relevant any toxicity, while also not observing the lymphocytic any variation of T and B, shows of the invention
SMT01 does not have immune-related side effect (table 1).
To biliary tract cancer cell line, the internal cell lysis activity of SMT01 is had evaluated.In order to test, gallbladder is implemented to nude mice
The heterograft of road cancer cell.In more detail, to the subcutaneous with 5X10 of the back of every group of 10 nude mices6Cell/ml amount is moved
HuCCT-1 cell is planted.After biliary tract cancer cell transplantation 8 days, screening is transplanted successfully naked with similar gross tumor volume
After mouse, experiment in vivo has been carried out.In order to handle, to being screened in each processing group comprising negative control (G1, physiological saline)
8 mouse administration is implemented according to the method for the following table 2 and Fig. 1.To as the same of positive controls (G5, CDDP+Gem)
The mouse of quantity implements chemical administration.Divide 5 administration SMT01 (G2~G4) of dosage of 3 kinds of various concentrations.It is taken from nude mice
Gross tumor volume and weight (Fig. 3) are determined after tumour.
After G1, G2, G3, G4 and G5 group are administered 55 days respectively, the mean tumour volume observed is respectively 5324,
2769,3491,3193 and 2167mm3(Fig. 3 (b)).It observed in all processing groups other than negative control group (G1)
Tumor growth inhibits.It can be seen that the average weight of individually separated tumour out is widely different in processing group and negative control group.
(Fig. 3 (a)).It is obvious to the growth inhibitory effect of cancer of bile ducts that SMT01 of the invention can be observed in result in this way.Although
It observed maximum growth inhibitory effect in chemical administration group, but there is no statistical significance.All processing groups are determined
Changes of weight the result is that observed weight in chemical administration group is decreased obviously (Fig. 4 (a)), and has confirmed and drink
Eat unrelated (Fig. 4 (b)).But, weight maintenance can be observed to a certain degree in SMT01 processing group of the invention.Thus illustrate certainly
The treatment of Natural killer cell and quality of life are closely related, the weight to work in oncotherapy as important parameter
Maintain that there is effective effect.
Pass through In vivo study as above, it was demonstrated that SMT01 of the invention is excellent for the cell lysis activity of biliary tract cancer cell in liver
It is different, special side effect will not be caused in vivo.
[table 1]
[table 2]
10. the active Histological Effects evaluation of SMT01 in nude mouse tumor
The cell lysis activity (Fig. 5 and 6) of SMT01 is had rated using tumor tissue section.As shown in figure Fig. 3, in order to make
It makes histotomy the tumour separated from nude mice is fixed on paraffin.In order to confirm the HuCCT- for the nude mice for injecting SMT01
Whether natural killer cells is infiltrated in 1 tumour, in order to which IHC experiment has used Anti-Human's class CD56 antibody.For TUNEL analysis
Same histotomy is used.
IHC experimental result for the histotomy obtained from G1 (physiological saline group) and G5 (chemical administration group) be
Background level shows CD56 positive signal, and the opposite injection group (G2-G4) in SMT01, which is shown, is distributed mainly on tumour
The IHC signal of the CD45 cell in the tubercle gap (tumor nodules) (cleavage).That is, can confirm that SMT01 is infiltrated to naked
In the HuCCT-1 tumour formed in mouse (Fig. 5).In addition, infiltration of the IHC signal for pipe can be confirmed mainly in inside tumor group
It knits block and density is lesser partially observable (Fig. 5, G2-G4, arrow).
The infiltration for observing the CD56 positive NK cell for infiltrating into tumor tissues is become apparent under high-frequency microscope
(Fig. 6 (b)).Shown in such as figure Fig. 6 (b), being injected in the mouse group of G2~G4 of SMT01 can be observed that CD56 is positive to be killed naturally
Hurt cell, is concentrated mainly on the position infiltrated by the tumor tissues of vascular cell heterograft.It is thin for natural kill
The IHC of born of the same parents' activated receptor NCR1 experimentally can be seen similar as a result, confirming permeation cell is exactly natural killer cells (Fig. 6
(b), the column NCR1).Apoptosis letter has been detected by the IHC experiment of TUNEL dyeing and apoptosis markers caspase-3a
Number (Fig. 6 (a), (b)).As a result, withering for most tumors cell can be observed in the tumour for being filled with SMT01 of the invention
Die (Fig. 6 (a)).In addition, the cell that Apoptosis can be observed is very more in the aggregate site of natural killer cells, it is seen then that trip
The natural killer cells separated out shows that cell lysis activity, SMT01 of the invention are outstanding for the tumour cell near tumor vessel
It has significant cytotoxicity to biliary tract cancer cell line in liver.
Although the embodiment of the present invention is described in detail, the scope of the claims of the invention is without being limited thereto, not
In the range of technology main idea documented by claims, those skilled in the art can carry out a variety of modifications and deformation.
It can various modification and deformation in the technical scope of record.
Claims (8)
1. the abductive approach of natural killer cells characterized by comprising
CD3 is removed from peripheral blood mononuclear cells+The step of T cell；And
By the removal CD3+Training of the peripheral blood mononuclear cells of T cell in the interleukin 2 comprising 500~1500IU/ml
Support the step of cultivating 10~25 days in base.
2. the abductive approach of natural killer cells as described in claim 1, which is characterized in that the removal CD3+Outside T cell
All blood monocytes are cultivated in the culture medium of the interleukin 2 comprising 800~1200IU/ml.
3. the abductive approach of natural killer cells as described in claim 1, which is characterized in that by the removal CD3+T cell
Peripheral blood mononuclear cells behind the culture of T75 flask 5~7 days comprising 10~30ml culture medium, move on to T175 flask be further cultured for 7~
14 days.
4. the abductive approach of natural killer cells as described in claim 1, which is characterized in that the culture medium is to include 0.5
The sodium chloride (NaCl) of~0.6 weight %, the D-Glucose of 0.4~0.5 weight %, 0.2~0.3 weight % amino acid,
The NaHCO of 0.15~0.25 weight %3And 0.15~0.25 weight % HEPES (4- (2- ethoxy) -1- piperazine ethanesulfonic acid).
5. the abductive approach of natural killer cells as described in claim 1, which is characterized in that the natural killer cells includes
With CD56brightOr CD56dimPhenotype natural killer cells.
6. the prevention or treatment pharmaceutical composition of cancer of bile ducts, which is characterized in that as effective component comprising according to claim 1
Natural killer cells made from method described in any one of~5.
7. pharmaceutical composition as claimed in claim 6, which is characterized in that the cancer of bile ducts is cancer of bile ducts in liver.
8. pharmaceutical composition as claimed in claim 6, which is characterized in that the composition includes 1 X 104A~1 X 1010It is a
Natural killer cells.
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| KR1020180042103A KR20190118788A (en) | 2018-04-11 | 2018-04-11 | Natural killer cells for treating biliary tract cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090068141A1 (en) * | 2006-03-06 | 2009-03-12 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | Autologous natural killer cells and lymphodepleting chemotherapy for the treatment of cancer |
| CN107460168A (en) * | 2017-10-09 | 2017-12-12 | 天津长和生物技术有限公司 | The amplification cultivation method of NK culture matrix and NK |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090068141A1 (en) * | 2006-03-06 | 2009-03-12 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | Autologous natural killer cells and lymphodepleting chemotherapy for the treatment of cancer |
| CN107460168A (en) * | 2017-10-09 | 2017-12-12 | 天津长和生物技术有限公司 | The amplification cultivation method of NK culture matrix and NK |
Non-Patent Citations (1)
| Title |
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| SATORU SAITO等: "Ex Vivo Generation of Highly Purified and Activated Natural Killer Cells from Human Peripheral Blood", 《HUMAN GENE THERAPY METHODS》 * |
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