CN105602903A - Antitumor stem cell antigen OCT4 (octamer-binding transcription factor 4) specific CTL (cytotoxic T lymphocyte) and preparation method thereof - Google Patents
Antitumor stem cell antigen OCT4 (octamer-binding transcription factor 4) specific CTL (cytotoxic T lymphocyte) and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an antitumor stem cell antigen OCT4 (octamer-binding transcription factor 4) specific CTL (cytotoxic T lymphocyte) and a preparation method thereof. A directionally-targeted nano-liposome-carried tumor stem cell antigen is adopted to prepare the antigen-specific T lymphocyte. According to the tumor stem cell antigen-specific T lymphocyte preparation technique, the novel nano-liposome-carried tumor stem cell antigen is adopted, and the tumor stem cell is used as the therapeutical target, thereby inducing the more efficient targeted killing on the specific CTL of the tumor stem cell, and directly aiming at the tumor generation and relapse roots; and thus, the tumor therapy can implement a permanent cure. The 72-hour quick-maturated dendritic cell is adopted to greatly shorten the optimal therapeutical time window for the tumor patient. The engineering cell is adopted to amplify the antigen-specific T lymphocyte, thereby generating a high proportion of Tcm with higher killing capacity. The preparation method of the tumor stem cell antigen OCT4 specific T lymphocyte is simple and easy to implement; the immune target spot aims at the tumor stem cell; and thus, the antitumor stem cell antigen OCT4 specific CTL has high antigenicity and favorable stability.
Description
Technical field
The present invention relates to Life Sci-Tech field, relate in particular to a kind of resisting tumour stem cells antigen OCT4 spyOpposite sex CTL and preparation method thereof.
Background technology
In China, malignant tumour has become one of important illness of harm humans health, and every annual morbidity is235.23/10 ten thousand people, the death rate is 148.81/10 ten thousand. In recent years along with science and technology, medical diagnosis and treatmentThe development of level and raising, but the overall mortality rate of tumour and survival rate be still not improved, and recurrence and transfer are complied withIt is so the main cause that causes cancer prognosis not good. Along with the further investigation to oncobiology characteristic, recognize at presentFor some malignant entity tumors (glioma, lung cancer, breast cancer and colorectal cancer etc.) are a kind of Diseases of Hematopoietic Stem Cells,Be that tumour derives from tumor stem cell, tumor stem cell is called again tumour initiator cell. This class cell only accounts for wholeTumour cell sub-fraction, but self-renewal capacity and multi-lineage potential there is, be to form different differentiation journeysThe tumour of degree and tumour be the root of growth constantly, may be the root of oncotherapy failure. Further research is found,Tumor stem cell not only has more metastatic potential compared with other tumour cells, and chemotherapy, radiation are had more to resistance. CauseHow this, kill stem cell in whole tumour to obtain stable and permanent the shrinking back of tumour, and even curing is orderPre-neoplastic is learned the focus and emphasis of area research. There is in the recent period multinomial research report Oct4 in Several Kinds of Malignancy groupKnit or clone in unconventionality expression, prompting Oct4 except being active in the growth course of stem cell, also very likelyParticipate in into the generating process of body cell and tumour, this correlation research for Oct4 and malignant tumour provides thinkingAnd direction.
Oct4 is one of member of Pit-Oct-Unc (POU) transcription factor family, is produced by POU5F1 gene code, being positioned on No. 6 chromosomes (6p21.31), length is 16.40kb, belongs toAlbuminoid. Oct4 geneBeing exactly one of them key gene, being also referred to as Oct3, POU5F1, OTF3 or OTF4, is POU transcription factorA member in family. Oct4 can maintain the undifferentiated state of embryonic stem cell and promote it constantly to breed, and itIn generation, propagation and the atomization of tumour, play certain effect. Research is found, the overexpression of Oct-4Can increase stem cell and change malignant cell potential into, suppress its expression and can suppress sending out of malignant tumourRaw, development, and the height of Oct-4 expression and the grade malignancy of genital system have significant positiveGuan Xing. OCT4 only exists in tumor stem cell, and tumor stem cell be the minute quantity that exists there is oneselfThe original neoblast of renewal, high tumorigenesis power, invasiveness and drug resistance. To maintaining tumor stem cell oneself moreNewly, the characteristic such as tumorigenesis power, invasiveness and drug resistance plays an important role, the treatment of target OCT4 will be to eliminateLung cancer stem cell provides new approaches. For the novel therapies of tumor stem cell will with existing operation, chemotherapy, radiotherapyCombine etc. conventional method, bring new hope and Gospel to cancer patient. Therefore, the pin taking OCT4 as target spotThe targeted therapy of tumor stem cell will be had to important clinical meaning. On the one hand, may be as providing clinicallyJudge the molecular indexes of tumor prognosis; On the other hand, can utilize it to carry out antitumor controlling as molecule target geneTreat, overcome the repellence of tumor chemoradiotherapy, improve the treatment curative effect of tumour.
In human cytotoxic history, there were three large sharp weapon with cancer fight: operation, chemotherapy, controls with radiationTreat. So far, immune scientist Si Tanman utilizes the BMDC (Dendriticcell, DC) of previously having found,Excite the immunity of self to remove tumour, utilize the achievement in research of oneself, life was extended to by the several months of expecting4 years. Because he obtained Nobel Prize in Physiology or Medicine to the contribution in immune field in 2011, at tumor area alsoStart a new world---tumour immunity cell therapy. His achievement in research is widely used in nowClinical, become the 4th kind for the treatment of of cancer mode after operation, Radiotherapy chemotherapy. Due to tumour immunity cellTreatment can combined surgery, chemotherapy, radiotherapy are precisely removed tumors remaining cell, prevents recurrence and shifts, and strengthensImmunosurveillance, promotes life in patients and time, and there is no the toxic and side effect of chemicotherapy and operationHuge injury. In recent years, being subject to paying close attention to more and more widely in clinical practice, is the focus of studying both at home and abroad,Be acknowledged as the tumor therapeuticing method of tool prospect.
Adoptive immunity cell therapy refers to by inputting self or allochthonous tumor-killing cell is treatedThe method of tumour. It not only can correct the low of cellular immune function, promotes the function of Host Anti-tumor Immunity,Directly performance antineoplastic action. Wherein the treatment of adopting of cytotoxic T cell has special excellentMore property. The treatment of adopting of cytotoxic T cell is entity tumor, and especially tumor associated antigen is clearer and more definiteThe study hotspot of malignant mela noma, colorectal cancer, liver cancer, prostate cancer, lung cancer etc. Specific CTL s is (thinCellular toxicity T cell) generation be by by the DC of tumour antigen load maturation (BMDC) stimulate TThereby cell obtains the specific CTL clone of antigen. This CTL can identify and kill and wound that to carry corresponding tumour anti-Former malignant cell, just can be applied to clinical treatment through a large amount of amplifications. This methods for the treatment of is compared otherThe features such as methods for the treatment of has easy acquisition, workable, and targeting is good, security height.
But the maturity that the transfer of adopting property T cell also faces a lot of problems: DC is low, immune tolerance,Lazy weight of specific, activated T cell etc. Because DC amplification has very large difficulty, so this alsoIndirectly limit the quantity of specific, activated T cell. Tumour immunity taking dendritic cell vaccine as representative is controlledTreatment becomes the 4th kind of antitumor therapy after operation, chemotherapy and radiotherapy, mainly utilizes the antigen of tumour cellMaterial incentive body produces specific tumor cell immunologic cytotoxicity, thereby reaches the object of tumors destroyed. With traditionAntineoplaston compare, the immunization therapy of DC vaccine mediation has the plurality of advantages such as security is good, specificity is high,Lung cancer, colon cancer, prostate cancer, breast cancer, melanoma etc. are widely used at present treating all kinds of perniciousTumour, but its clinical efficacy still needs further to be improved. BMDC (DC) is the strongest as function in bodyAntigen presenting cell, be that excitating organism produces the primary core link of anti-tumor immune response. It can absorb anti-Former, and antigenic information is offered to the lymphocyte to CD8+T by MHC-I quasi-molecule, can inducing specificCytotoxic T lymphocyte (cytotoxicTlymphocyte, CTL) generates, and then kills tumour cell,Be applicable to the immunotherapy of kinds of tumors. Therefore, improving DC is to strengthen it to the intake of antigen and the ability of offeringThe Critical policies of clinical efficacy.
Nanometer grade liposome is the spherical entity being formed by phospholipid bilayer shell parcel aqueous core, and its structure is similarBiomembrane is a kind of good biocompatibility and nontoxic nano material. It can seal water-soluble and fat-soluble medicine,There is drug dose, slowly-releasing and the targeting NO release medicine etc. of minimizing advantage, thereby it is anti-to be widely used in nanoscaleThe exploitation of tumour medicine. In addition, nanometer grade liposome is also a kind of good antigen vectors, not only can wrap upThe antigen of series of physicochemical different in kind and immunologic adjuvant, protected protein polypeptide antigen is not degraded, and can also urgeEnter antigen presenting cell engulfing and presenting, the specific immune response of raising body antigen. Based on above thisA little advantages, nanometer grade liposome is as a kind of novel vaccine carrier, just gradually for bacterial vaccine, viral epidemic diseaseThe developments such as seedling, anti parasitic vaccine and anti-tumor vaccine. Positive charge is carried on neutral fats plastid and surfaceCation nanometer level liposome is the most frequently used nano vaccine carrier. Wherein it is worth noting especially that cation receivesMeter level liposome, it is good protein/polypeptide antigen vectors still not, or a kind of novel immunologic adjuvant,Can direct activation antigen presenting cell, strengthen vaccine-induced immune response.
The cellular immunity result for the treatment of of tumour is still undesirable at present, and main cause comprises following two aspects: 1) tumourSuffer from and have immunosuppressant microenvironment, on the one hand, the T lymphocyte of tumor tissues produces immune tolerance, anotherAspect, tumour cell can be secreted panimmunity inhibiting factor, and induction produces regulatory T lymphocyte or inhibitionDCs function. 2) antigen of applying in immunotherapy of tumors is expressed on the tumour cell having broken up mostly, and swellsKnurl stem cell is not expressed these antigens, and the immunologic cytotoxicity bringing out reacts not for tumor stem cell. AddThe tumour antigen that current conventional DC adds patient tumors lysis to obtain, makes DC load tumour antigen, although thisKind method can make DC absorb patient's tumour antigen, but due to the antigenicity of tumour antigen a little less than, DC is to swollenTumor antigen intake is less, and its submission antigenic capacity is also just lower, makes the killing activity of the CTL cell of its generationDeficiency, and it is limited to utilize simple cell factor to amplify amplification CTL cell quantity, and the Tcm ratio of generation is little,And then affect antineoplaston effect.
Summary of the invention
The present invention is directed to the problems such as existing tumour immunity cell antitumor curative effect is not good, propose to adopt target dendronOn shape cell, the cationic-liposome of C-type agglutinin receptor, as antigen vectors, wraps up tumor stem cell antigenOct4, by greatly improving ingestion efficiency and the antigen presentation ability of DC cell to tumour antigen, strengthens it and resistsFunction of tumor, a large amount of expansion of antigen specific T-cells of incorporation engineering cell and cell factor and Tcm, therebyStrengthen antitumor immune function. In addition the antigen-specific that, the present invention is prepared with tumor stem cell antigen OCT4Property CTL innovation be: the target taking tumor stem cell as treatment, can induce more effective targetThe specificity cell toxicity T lymphocyte of killing tumor cells stem cell, the root that directly occurs and recur for tumourSource, makes oncotherapy have more " effecting a permanent cure " property.
To achieve these goals, the technical measures that the present invention takes are:
A preparation method for resisting tumour stem cells antigen OCT4 specific CTL, comprises the following steps:
Step 1, preparation is enclosed with the target BMDC C-type agglutinin of tumor stem cell antigen OCT4The cationic-liposome carrier of acceptor;
Step 2, utilizes cationic-liposome carrier that described step 1 obtains by tumor stem cell antigen OCT4Be carried on ripe DC cell;
Step 3, utilizes the ripe DC cell induction that described step 2 obtains to obtain the pouring of OCT4 specificity TBar cell and central Memorability T lymphocyte.
In order further to optimize technique scheme, the technical measures that the present invention takes also comprise:
Further, in described step 1, specifically comprise the following steps:
Step 1, provides polyglycol derivatization sphingomylin, and the sugar of C-type agglutinin type is connected to poly-Ethylene glycol derivatization sphingomylin, obtains the sugar-modified polyglycol derivatization second of C-type agglutinin typeHydramine phosphatide;
Step 2, by the sugar-modified poly-second two of the C-type agglutinin type obtaining in cationic lipid and step 1Alcohol derivatization sphingomylin is dissolved in respectively in the mixed solvent of chloroform and methyl alcohol, obtains mixed liquor;
Step 3, by the mixed liquor rotation evaporate to dryness obtaining in step 2, makes it to form one deck equal with Rotary EvaporatorsEven film, adds the PBS buffer solution or the pure water that contain tumor stem cell antigen OCT4 to put after vacuum dryingPut 4 DEG C of ultrasonic aquations, pushed that to obtain being enclosed with the target dendron shape of tumor stem cell antigen OCT4 after film thinThe cationic-liposome carrier of born of the same parents C-type agglutinin receptor.
Preferably, the antigen of tumor stem cell described in step 1 OCT4 is selected from the restricted Tumor Stem of HLA-A0201Cellular antigens OCT4 forgives epitope peptide 15 peptides, as shown in SEQNO.1; Or artificial synthetic, transformation orThe OCT4 Antigenic Peptide of sudden change. Particularly, OCT4 protein sequence holds C end (360AA) to be from N:MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN
Wherein, forgiving epitope peptide 15 peptide sequences is: DVVRVWFCNRRQKGK
Preferably, the sugar of described C-type agglutinin type is mannose or mannoside.
Further, in above-mentioned steps two, specifically comprise the following steps:
Step 1, is suspended from basal medium by PMNC and adds DC cell induction culture medium,Plant in Tissue Culture Plate, at 37 DEG C, 5%CO2, under saturated humidity, cultivate and carry out DC cell induction;
Step 2, has tumor stem cell antigen to adding appropriate described step 1 to obtain load in DC cellThe cationic-liposome carrier of OCT4, hatches altogether and cultivates 6 hours; Then add DC to urge maturation medium,Continue to cultivate 24-48 hour, obtain the ripe DC cell of load tumor stem cell antigen OCT4.
Further, in above-mentioned steps three, specifically comprise the following steps:
Step 1, by the ripe DC cell induction T cell obtaining in described step 2, the T cell after inductionWith engineering cell Mixed culture, add cell factor and cultivate;
Step 2, adds the culture medium that contains cell factor every other day according to cell density, make cell maintain certain modelThe density growth of enclosing, until 2-3 week, harvesting, obtains OCT4 T lymphocyte specific and maincenterProperty Memorability T lymphocyte.
Preferably, above-mentioned cationic-liposome comprises phosphatidyl-ethanolamine bilayer, polyglycol derivatization phosphorusAcyl monoethanolamine and mannose. More preferably, described phosphatidyl-ethanolamine bilayer is by phosphatidyl-ethanolamineCationic lipid and the molecular composition of polyglycol derivatization phospholipid acyl monoethanolamine.
Preferably, above-mentioned polyglycol derivatization phospholipid acyl monoethanolamine and described mannose form mannose-modifiedPolyglycol derivatization phospholipid acyl sphingomylin. More preferably, above-mentioned cationic-liposome is at phospholipid moleculeOn polar group, connect the polyglycol derivatization phospholipid acyl sphingomylin of described mannose-modified.
Preferably, described cell factor is selected from people's recombinant cytokine IL-2, IL-12 and IL-18 cell factor.
Preferably, the K562 cell of the described engineering cell people 4-1BBL that has been artificial cell transfecting and IL-21Strain.
The present invention also provides the preparation-obtained specific CTL according to above-mentioned preparation method on the other hand; AndThe specific CTL preparing is in the application of preparing in antineoplastic.
The present invention adopts technique scheme, compared with prior art, has following technique effect:
The present invention adopts the nano liposomes load tumor stem cell antigen with directed target to prepare antigen-specificProperty T lymphocyte. Compared with conventional antigenspecific T lymphocyte technology of preparing, the present invention prepares tumourStem cell antigen T lymphocyte specific technology not only adopts novel nano liposome load tumor stem cell anti-Former, the target taking tumor stem cell as treatment, can induce the spy of more effective target killing tumor stem cellOpposite sex cytotoxic T lymphocyte, the root that directly occurs and recur for tumour, has more oncotherapyThe property of " effecting a permanent cure "; And adopt 72 hours fast-ripenin BMDCs, more ripe than conventional 7-8 days resultsDC can greatly shorten the time window of tumor patient optimal treatment; Adopt engineering cell to amplify amplification resists simultaneouslyFormer T lymphocyte specific, can produce the Tcm that a high proportion of kill capability is stronger. Tumor Stem of the present invention is thinPreparation method is simple and easy to do for extracellular antigen OCT4 T lymphocyte specific, and immune target spot is for tumor stem cell,Antigenicity is strong and have good stability, and convenient operation is adapted at clinical tumor and crosses in the immunization therapy of continuous property and applyPromote.
Brief description of the drawings
Fig. 1 is 72 hours fast-ripenin maturing dendritic cell phenotypic evaluation results in embodiment bis-.
Fig. 2 is that in embodiment tri-, tumor stem cell antigen OCT4CTL CF IFN-r detects knotReally.
Fig. 3 is the Tcm phenotype of the tumor stem cell antigen OCT4 specific CTL of amplification in embodiment tetra-Testing result.
Detailed description of the invention
The preparation method who the invention provides a kind of resisting tumour stem cells antigen OCT4 specific CTL, comprisesFollowing steps:
Step 1, preparation is enclosed with the target BMDC C-type agglutinin of tumor stem cell antigen OCT4The cationic-liposome carrier of acceptor;
Step 2, utilizes cationic-liposome carrier that described step 1 obtains by tumor stem cell antigen OCT4Be carried on ripe DC cell;
Step 3, utilizes the ripe DC cell induction that described step 2 obtains to obtain the pouring of OCT4 specificity TBar cell and central Memorability T lymphocyte.
The present invention adopts the sugar-modified cationic-liposome of C-type agglutinin type as antigen vectors, parcelTumor stem cell antigen, for the preparation of antigenspecific T lymphocyte. Described cationic-liposome, comprisesPhosphatidyl-ethanolamine bilayer, polyglycol derivatization phospholipid acyl monoethanolamine and mannose; Described C-The sugar of type agglutinin type is mannose or mannoside. Described phosphatide bimolecular ball by phosphatidyl-ethanolamine sun fromSub-fat and the molecular composition of described polyglycol derivatization phospholipid acyl monoethanolamine; Described mannose is at described polyethylene glycolDerivatization phospholipid acyl monoethanolamine one end forms the polyglycol derivatization phospholipid acyl sphingomylin of mannose-modified.This cationic-liposome spreads out by the polyethylene glycol that connects mannose-modified on the polar group of phospholipid moleculeBiochemical phosphatidyl-ethanolamine phosphatide. Described tumour antigen is the restricted tumor stem cell antigen of HLA-A0201OCT4 epitope peptide 9 peptides, forgive epitope peptide 15 peptides and forgive epitope peptide 30 peptides. Described engineering cell acrossThe artificial cell of the K562 cell line of film expression people 4-1BBL and IL-21.
Below by specific embodiment, the present invention is carried out to detailed and concrete introduction, so that better understand thisBright, but following embodiment does not limit the scope of the invention.
The preparation method of embodiment mono-resisting tumour stem cells antigen OCT4 specific CTL
Preparation method of the present invention mainly comprises three bulk steps, and the present embodiment is the general preparation side of the present inventionMethod.
The sugar-modified cationic-liposome antigen vectors of the first, C-type agglutinin type is made, concrete operations asUnder:
First prepare the polyglycol derivatization phospholipid acyl sphingomylin that mannose or mannoside are modified. By sweetReveal sugar or mannoside and be connected in polyglycol derivatization by the mode of aldehyde radical-amino or hydroxyl-amino condensationOn the amino of phosphatidyl-ethanolamine phosphatide, thereby obtain the polyglycol derivatization phospholipid of mannose or mannosideAcyl sphingomylin. Then by the polyglycol derivatization phosphorus of cationic lipid DOTAP and mannose or mannosideAcyl sphingomylin is dissolved in respectively chloroform-methanol (2:1), and according to also mixing by a certain percentage, the two is mixedAfter closing, obtaining mixed liquor is placed in round-bottomed flask. The rotation of described mixed liquor is steamed with stable Rotary EvaporatorsDry, make it to form the uniform film of one deck, with after vacuum drying removal in 12 hours remaining chloroform and methyl alcohol,Add the PBS buffer solution or the pure water that contain tumor stem cell antigen OCT4 and place 4 DEG C of ultrasonic aquation 3-5 of water-bathMinute, after extrusion film, be positioned over 4 DEG C of aquations 12 hours. By the ultrasonic 1-3 minute of water-bath, use syringeFilter polycarbonate membrane once, obtain the swollen of the upper C-type agglutinin receptor of described target BMDC (DC)Knurl stem cell antigen OCT4 cationic-liposome carrier, is stored in 4 DEG C.
The preparation of the BMDC of the second, 72 hour Fast Load tumor stem cell antigen, concrete operations are as follows:
By human peripheral blood single nucleus cell with 3.0-5.0*106Individual/ml is suspended from basal medium, adds DC cellInducing culture, plants in Tissue Culture Plate, at 37 DEG C, and 5%CO2, under saturated humidity, cultivate and carry out DC cell and lureLead.
Second day, in DC cell, add appropriate tumor stem cell antigen OCT4, hatch altogether and cultivate 6 hours.Then add DC to urge maturation medium, continue to cultivate 24-48 hour, obtain load tumor stem cell antigenThe ripe DC cell of OCT4.
The 3rd, the preparation method of rapid expansion of antigen specific T lymphocyte and Tcm, concrete operations are as follows:
T cell after the ripe DC induction that tumor stem cell antigen OCT4 is presented and engineering cell are according to 1:The ratio Mixed culture of 2-1:4, adds 1ug/mlanti-humanCD3 antibody, 1ug/mlanti-humanCD28 antibody, people's recombinant cytokine IL-2 of 300IU/ml and 10ng/mlhumanIL-12 cell because ofSon, is incubated in 37 DEG C, 5%CO altogether2, under saturated humidity, cultivate.
Add every other day the people's recombinant cytokine IL-2 and the 10ng/ml that contain 300IU/ml according to cell densityThe culture medium of humanIL-18 cell factor makes cell number maintain 1.3-2*106The density range of individual/ml,Continue to supplement fresh culture medium, until two or three week, harvesting.
The ripe phenotypic evaluation of embodiment bis-DC
In the present embodiment, from peripheral blood, gather and separate mononuclearcell, with 3.0-5.0*106Individual/ml'sDensity is suspended from AIM-V serum-free medium, then adds cell factor IL-4 and GM-CSF induction in nutrient solutionDC forms, and is then placed in 37 DEG C, 5%CO2In incubator, cultivate, second day, in DC cell culture fluid, addThe cationic-liposome of the tumor stem cell OCT4 antigen that enters to wrap, concentration is that 1-2.5ug/ml hatches 6 altogetherHour, in this backward DC cell, add DC to urge maturation medium, continue to cultivate 24-48 hour, bornCarry the DC cell of tumour tumor stem cell antigen. The ripe phenotypic evaluation result of DC is as accompanying drawing 1. A in figure, BThe mDC maturity that is two donor detects data, and CD83 is the maturity symbol of DC, CD80, and CD86 isCostimulatory molecules. The simultaneously two positive expression positives of the mDCCD83CD80 of two donor and CD83CD86Rate reaches more than 98%. Wherein in A, the two positive expressions of CD83CD80 are 98.6% and the two sun of CD83CD86Property is expressed as 98.8%; In B, the two positive expressions of CD83CD80 are 98.3% and the two positive expressions of CD83CD86Be 98.1%.
The secretion of the cytotoxic cytokines of the DC induction OCT4CTL of 3 72 hours fast-ripenins of embodimentDetect
In the DC cell of the load tumor stem cell antigen obtaining, add CTL inducible factor in embodiment bis-,Induction 4-7 days, middle every two and half amounts are added the culture medium that contains CTL inducible factor. Treat that CTL has inducedAfter, add the BFA of 10ng/ml concentration, after 4 hours, centrifugal collections CTL, with streaming antibody I FN-r withCD8 staining examine CF IFN-r secreting, expressing (as accompanying drawing 2). A in figure, B is two donorThe CF IFN-r of mDC induced tumor stem cell antigen Oct4CTL detects data. Loaded groupFor the liposome group of parcel OCT4 antigen, peptide group is independent OCT4 antigen group. Wherein in A figureThe positive secreting, expressing rate of peptide group IFN-r is the positive secreting, expressing rate of 2.89%, loaded group IFN-rBe 9.39%. In B figure, the positive secreting, expressing rate of peptide group IFN-r is 2.97%, loaded group IFN-rPositive secreting, expressing rate is 6.06%.
Tcm phenotype and ratio measuring in embodiment tetra-engineering cell amplification OCT4CTL
After centrifugal collection CTL, CTL and engineering cell Mixed culture, mixed proportion is 2-4:1. Engineering cellIn advance after gamma ray 100Gy radiation and CTL Mixed culture. Add 1ug/mlanti-humanCD3,1ug/mlAnti-humanCD28 antibody, people's recombinant cytokine of 10ng/mlhumanIL-12 and 300IU/mlIL-2 cell factor, is incubated in 37 DEG C, 5%CO altogether2, under saturated humidity, cultivate. Mend every other day according to cell densityAdd people's recombinant cytokine IL-2 of containing 300IU/ml and 10ng/mlhumanIL-18 cell factorCulture medium makes cell number maintain 1.3-2*106The density range of individual/ml, continues to supplement fresh culture medium,Until 7-10 days, harvesting; According to cell concentration can repetitive stimulation once, stimulate 7-10 days, tool at every turnGymnastics is done the same. CTL Flow cytometry (as accompanying drawing 3) after cultivation. A in figure, B is two donorUtilize engineering cell amplification mDC induced tumor stem cell antigen SOX-2CTLTcm to detect data. PeptideGroup is independent Oct4 antigen amplification group, and Background group is unloaded liposome amplification group, LoadedGroup is the liposome amplification group of parcel Oct4 antigen. Wherein peptide amplification group in A figure, CD62LCCR7Two positive expression rate are 2.72%; Background amplification group, the two positive expression rate of CD62LCCR7 are2.16%; The two positive expression rate of loaded amplification group CD62LCCR7 are 26.00%. Peptide amplification in figure BGroup, the two positive expression rate of CD62LCCR7 are 8.93%; Background amplification group, the two sun of CD62LCCR7Property expression rate is 4.43%; The two positive expression rate of loaded amplification group CD62LCCR7 are 26.60%.
The present invention adopts the nano liposomes load tumor stem cell antigen with directed target to prepare antigen-specificProperty T lymphocyte. Compared with conventional antigenspecific T lymphocyte technology of preparing, the present invention prepares tumourStem cell antigen T lymphocyte specific technology not only adopts novel nano liposome load tumor stem cell anti-Former, the target taking tumor stem cell as treatment, can induce the spy of more effective target killing tumor stem cellOpposite sex cytotoxic T lymphocyte, the root that directly occurs and recur for tumour, has more oncotherapyThe property of " effecting a permanent cure "; And adopt 72 hours fast-ripenin BMDCs, more ripe than conventional 7-8 days resultsDC can greatly shorten the time window of tumor patient optimal treatment; Adopt engineering cell to amplify amplification resists simultaneouslyFormer T lymphocyte specific, can produce the Tcm that a high proportion of kill capability is stronger. Tumor Stem of the present invention is thinPreparation method is simple and easy to do for extracellular antigen OCT4 T lymphocyte specific, and immune target spot is for tumor stem cell,Antigenicity is strong and have good stability, and is adapted at clinical tumor and crosses in the immunization therapy of continuous property and carry out application.
Above specific embodiments of the invention be have been described in detail, but it is just as example, the present invention alsoBe not restricted to specific embodiment described above. To those skilled in the art, any the present invention is carried outEquivalent modifications and substitute also all among category of the present invention. Therefore, do not departing from spirit of the present invention and modelEnclose lower done equalization conversion and amendment, all should contain within the scope of the invention.
Claims (12)
1. a preparation method for resisting tumour stem cells antigen OCT4 specific CTL, is characterized in that, comprise withLower step:
Step 1, preparation is enclosed with the target BMDC C-type agglutinin receptor of tumor stem cell antigen OCT4Cationic-liposome carrier;
Step 2, the cationic-liposome carrier that utilizes described step 1 to obtain is negative by tumor stem cell antigen OCT4Be loaded in ripe DC cell;
Step 3, utilizes ripe DC cell induction that described step 2 obtains to obtain OCT4 specificity T lymph thinBorn of the same parents and central Memorability T lymphocyte.
2. preparation method according to claim 1, is characterized in that, specifically comprises following in described step 1Step:
Step 1, provides polyglycol derivatization sphingomylin, and the sugar of C-type agglutinin type is connected to poly-second twoAlcohol derivatization sphingomylin, obtains the sugar-modified polyglycol derivatization monoethanolamine of C-type agglutinin typePhosphatide;
Step 2, spreads out the sugar-modified polyethylene glycol of the C-type agglutinin type obtaining in cationic lipid and step 1Biochemical alcohol amine phosphatide is dissolved in respectively in the mixed solvent of chloroform and methyl alcohol, obtains mixed liquor;
Step 3, by the mixed liquor rotation evaporate to dryness obtaining in step 2, makes it to form one deck uniform with Rotary EvaporatorsFilm, adds the PBS buffer solution or the pure water that contain tumor stem cell antigen OCT4 to place 4 DEG C after vacuum dryingUltrasonic aquation, pushed the target BMDC C-that obtains being enclosed with tumor stem cell antigen OCT4 after filmThe cationic-liposome carrier of type agglutinin receptor.
3. preparation method according to claim 1, is characterized in that, specifically comprises following in described step 2Step:
Step 1, is suspended from basal medium by PMNC and adds DC cell induction culture medium, plant inTissue Culture Plate, at 37 DEG C, 5%CO2, under saturated humidity, cultivate and carry out DC cell induction;
Step 2, has tumor stem cell antigen OCT4 to adding appropriate described step 1 to obtain load in DC cellCationic-liposome carrier, hatch altogether cultivate 6 hours; Then add DC to urge maturation medium, continue trainingSupport 24-48 hour, obtain the ripe DC cell of load tumor stem cell antigen OCT4.
4. preparation method according to claim 1, is characterized in that, specifically comprises following in described step 3Step:
Step 1, by the ripe DC cell induction T cell obtaining in described step 2, T cell and work after inductionJourney cell co-cultivation, adds cell factor and cultivates;
Step 2, add the culture medium that contains cell factor every other day according to cell density, make cell maintain certain limitDensity growth, until 2-3 week, harvesting, obtains OCT4 T lymphocyte specific and central noteThe property recalled T lymphocyte.
5. preparation method according to claim 1, is characterized in that, described cationic-liposome comprises phosphatideAcyl monoethanolamine bilayer, polyglycol derivatization phospholipid acyl monoethanolamine and mannose.
6. preparation method according to claim 5, is characterized in that, described phosphatidyl-ethanolamine bilayerBy phosphatidyl-ethanolamine cationic lipid and the molecular composition of polyglycol derivatization phospholipid acyl monoethanolamine.
7. preparation method according to claim 5, is characterized in that, described polyglycol derivatization phospholipid acylMonoethanolamine and described mannose form the polyglycol derivatization phospholipid acyl sphingomylin of mannose-modified.
8. preparation method according to claim 1, is characterized in that, tumor stem cell described in step 1 is anti-Former OCT4 is selected from the restricted tumor stem cell antigen of HLA-A0201 OCT4 epitope peptide 15 peptides, as SEQShown in NO.2; Or artificial synthetic, OCT4 Antigenic Peptide transformation or sudden change.
9. preparation method according to claim 4, is characterized in that, described engineering cell is that artificial cell turnsDye the K562 cell line of people 4-1BBL and IL-21.
10. preparation method according to claim 4, is characterized in that, described cell factor is selected from people's restructuringCell factor IL-2, IL-12 and IL-18 cell factor.
11. according to the preparation-obtained specific CTL of claim 1-10 any one preparation method.
12. specific CTLs as claimed in claim 11 are in the application of preparing in antineoplastic.
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