CN106350488A - Preparation method of CIK (cytokine-induced killer) blocked by PD-1 for cancer therapy - Google Patents
Preparation method of CIK (cytokine-induced killer) blocked by PD-1 for cancer therapy Download PDFInfo
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Abstract
The invention discloses a preparation method of CIK (cytokine-induced killer) blocked by PD-1 for cancer therapy and belongs to the field of medical treatment. The preparation method includes the coating step, the activating step, the cleaning step, the blocking step and the filtering step. In the coating step, an anti-human CD3 monoclonal antibody is coated in a cell culture flask; in the activating step, the CD3 monoclonal antibody, IFN gamma and IL-2 are utilized to activate and culture a lymphocyte; in the cleaning step, the lymphocyte which is activated and cultured by three times is cleaned; in the blocking step, the cleaned lymphocyte is suspended by normal saline to obtain suspended cell solution, the PD-1 monoclonal antibody is added into the suspended cell solution, and then suspending incubation is carried out; in the filtering step, the cell suspension obtained by incubation of the PD-1 monoclonal antibody is filtered and then is refilled with normal saline to obtain feedback cell suspension. The CIK blocked by PD-1 has the advantages of low consumption of the PD-1 monoclonal antibody, good blocking effect of PD-1 molecules, high killing activity and the like, and also has the advantages of operational simplicity, low preparation cost and convenience in clinical application.
Description
Technical field
The present invention relates to medical field, especially for the preparation method of the pd-1 closing cik for the treatment of tumor.
Background technology
Programmed death receptor 1 (pd-1) is a kind of protein molecule being expressed in t cell surface, when pd-1 and its part
The transcription of downstream nf- κ b gene can be suppressed, the secretion of suppression interferon-δ, thus suppress t cell when pd-l1 or pd-l2 combines
Killing activity.Pd-l1 or pd-l2 is expressed in kinds of tumor cells, such as pulmonary carcinoma, breast carcinoma, gastric cancer, the esophageal carcinoma, hepatocarcinoma, evil
Property melanoma, ovarian cancer, cancer of pancreas, the tumor tissues such as renal cell carcinoma and bladder transitional cell carcinoma high expression pd-l1.T cell table
It is that tumor cell is escaped that the pd-1 in face is combined caused t cell cytotoxic activity suppression with the pd-l1 being expressed on tumor cell
Keep away one of important mechanisms of immune surveillance and killing.Using pd-1 antibody or pd-l1 antibody blocking pd-1 and pd-l1, pd-l2
In conjunction with the tumor activity that kills of t cell being made to be not inhibited, thus improving the effect that t cell kills tumor.Research confirms to adopt pd-1 Dan Ke
The tumors such as grand antibody venoclysises treatment malignant melanoma, lung squamous cancer, colon cancer and renal carcinoma receive good therapeutic effect.So
And, venoclysises enter internal pd-1 antibody by by the hemodilution of human body 4000ml-5000ml, therefore in order to remain effective
Drug level needs the amount being transfused pd-1 antibody larger.Blood constituent, lymphoid tissue microenvironment structure and composition are complicated, no simultaneously
Only can cause pd-1 antibody loss also influence whether pd-1 antibody and t cell surface pd-1 combination.
Cik is the killing cell of cytokine profiles induction, and existing cik activation cultural method is easily obtained substantial amounts of effect
Cell.But these effector lymphocytes have higher pd-1 expression rate, this have impact on the tumor-killing effect of cik to a certain extent
And clinical efficacy.Applying for the stage patented technology cn201511018960.1, cn201610278810.2,
Cn201410573897.7 and cn201410573311.7, adds pd-1 during autologous peripheral blood culture in vitro of lymphocytes
Antibody, washes when collecting cell and abandons unconjugated pd-1 antibody.Treatment is generally existed with training base unit weight in lymphocyte incubation
1500ml about, the cumulative volume of training base is larger, and the consumption of pd-1 antibody is still larger, unconjugated pd-1 antibody after simultaneously cultivating
Discard with Pei Ji when washing cell, cause certain waste.In addition, in incubation the secretions of cell and metabolite,
The pd-1 antibody that can affect to a certain extent and the combination of pd-1, thus affect the sealing effect of pd-1.
Treatment is expensive with pd-1 antibody, adds pd-1 antibody, pd-1 antibody in venoclysises or cik incubation
The drawback such as consumption is big, high cost and pd-1 sealing effect susceptible, greatly have impact on the clinical practice of pd-1 antibody.
Content of the invention
It is an object of the invention to overcoming above-mentioned the deficiencies in the prior art, it is desirable to provide a kind of cell proliferation multiple is big, kill
Wound activity height and the preparation method of the low Tumor cytotoxicity cell of preparation cost.The technical scheme is that for treating
The pd-1 of tumor closes the preparation method of cik, comprises the steps of: anti-human cd3 monoclonal antibody coated cell culture bottle；Activation
Step, using described cd3 monoclonal antibody, ifn γ and il-2 activation culture lymphocyte；Cleaning step, cleaning swashs for three times
The described lymphocyte lived after cultivating；Closing step, with the lymphocyte after the described cleaning of 20-50ml normal saline suspension, obtains
To suspension cell solution, add pd-1 monoclonal antibody in described suspension cell solution, the 18-25 DEG C of incubation that is suspended obtains for 30 minutes
To cell suspension.
Preferably, the preparation method of the described pd-1 closing cik for treating tumor comprises step further: filters step
Suddenly, add cell-dispersing agent 200 mesh filter screen to add normal saline after filtering in described cell suspension to be fed back to 300ml
Liquid.
Preferably, described anti-human cd3 monoclonal antibody coated cell bottle comprises the steps of: using 0.01m ph9.6's
Pbs solution dilution first class purification anti-human cd3 monoclonal antibody is to concentration 50-70ng/ml；Described first class purification after dilution is resisted
People's cd3 monoclonal antibody is added in culture bottle, places more than 24 hours for 4 DEG C.
Preferably, described activation step comprise the steps of: by heparin anti-coagulating inject centrifuge tube a in, at a temperature of 4 DEG C with
3000rpm is centrifuged 5min；Take upper plasma to move into centrifuge tube b, 56 DEG C of water-bath 30min, be centrifuged with 3000rpm at a temperature of 4 DEG C
15min, collects supernatant, 4 DEG C save backup；Lower floor's hemocyte stays standby in centrifuge tube a；The normal saline described centrifuge tube of suspension
Described hemocyte in a, the density that the blood cell suspension being obtained is slowly added into equipped with 4ml is 1.077 ± 0.001
In the centrifuge tube of ficoll-hypaque, at a temperature of 4 DEG C, 20min is centrifuged with 3000rpm；Collect mononuclearcell confluent monolayer cells simultaneously
Using the centrifuging described cell of cleaning three times；Add anteserum-less substrate resuspended, prepared anteserum-less substrate suspension cell；Will be described
It is added in anteserum-less substrate suspension cell containing in the coated culture bottle of described anti-human cd3 monoclonal antibody, add inactivation blood
Starch and add into anteserum-less substrate, 37 DEG C, 5%co2Under the conditions of cultivate 24 hours；Ifn γ is added in culture bottle, 37 DEG C, 5%
co2Under the conditions of cultivate 4-6 days；Take the cell after culture move into cell culture bags in, add anteserum-less substrate, inactivation blood plasma and
Il-2,37 DEG C, 5%co2Under the conditions of cultivate 4-5 days；Add anteserum-less substrate and il-2,37 DEG C, 5%co2Under the conditions of cultivate 4-5
My god.
Preferably, also comprise before described cleaning step: testing sequence, detect cell quantity after carrying out cell culture, and
Using the mycoplasma in pcr technology for detection culture supernatant.
The invention has the benefit that the consumption of pd-1 monoclonal antibody is few, pd-1 molecule sealing effect is good, and pd-1 closes
Cik anti-tumor activity high.Have the characteristics that simple to operate, preparation cost is low and is easy to clinical practice simultaneously.
Brief description
Fig. 1 is the expression rate of the pd-1 molecule of cik cell；
Fig. 2 closes the recall rate of cik cell pd-1 molecule for pd-1 monoclonal antibody；
Fig. 3 is that the tumor cell destruction that cik closes cik with pd-1 compares.
Specific embodiment
Below in conjunction with the accompanying drawings, the invention will be further described to pass through specific embodiment.Following examples are descriptive
, it is not determinate it is impossible to protection scope of the present invention is limited with this.
Experiment equipment
Low temperature normal speed centrifuge (U.S., beckman), co2Cell culture incubator (Japan, sanyo), pipettor (U.S.,
Thermo), measuring pipette (U.S., kimble), 75cm2Culture bottle (U.S., corning), 15ml centrifuge tube (U.S.,
Corning), 50ml centrifuge tube (U.S., corning), cell scraper (U.S., corning), cellular filter (U.S.,
Corning), inverted microscope (Japan, nikon), cryopreservation tube (U.S., corning), 96 porocyte culture plates (U.S.,
Corning), microplate reader (U.S., biotek), flow cytometer (U.S., bd).
Experiment reagent
Ficoll-hypaque cell separation liquid (Tianjin Hao ocean biological product science and technology limited Company)), sodium chloride note
Penetrate liquid (Shijiazhuang four medicine company), heparin sodium (Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd), ifn- γ (Beijing Fourth Ring bio-pharmaceuticals
Company limited), il-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.), first class purification anti-human cd3 monoclonal antibody (U.S.,
Ebioscience), anteserum-less substrate (U.S., thermo), human serum albumin's (Shanxi health treasured limited public affairs of biological product share
Department), pembrolizumab (U.S., msd), fitc labelling Mus anti-human cd3 monoclonal antibody (U.S., bd), pe labelling rabbit anti-human
Pd-1 monoclonal antibody (U.S., ebioscience), hank ' s liquid (compound method: 1. solution A: nacl 160g, kcl8g,
mgso4·7h2o 2g、mgcl2·6h2O 2g is added to 800ml tri-distilled water, dissolving.cacl22.8g is dissolved in 100ml tri-distilled water, molten
It is added to after solution in above-mentioned 800ml solution, supply tri-distilled water to 1000ml.2. second liquid: na2hpo4·12h2o 3.04g、kh2po4
1.2g, glucose 20g add 800ml distilled water, dissolving.Add 0.4% phenol red solution 100ml, above-mentioned two liquid mixing, plus three and steam
Water is to 1000ml.1 part of solution A, 1 part of second liquid, 18 parts of filtrations of tri-distilled water, 8 pounds of 20min minute sterilizings, 4 DEG C of preservations are pressed during use),
Nacl (examination of traditional Chinese medicines Lu), kcl (examination of traditional Chinese medicines Lu), mgso4·7h2O (Tianjin Zhi Yuan Chemical Co., Ltd.), mgcl2·6h2O (my god
Jin Zhiyuan Chemical Co., Ltd.), cacl2(Tianjin Bo Di Chemical Co., Ltd.), na2hpo4·12h2O (Tianjin recovery science and technology
Development Co., Ltd), kh2po4(Tianjin Zhi Yuan Chemical Co., Ltd.), glucose (examination of traditional Chinese medicines Lu), phenol red (the upper graceful biology of Hypon
Science and Technology Ltd.), scg7901 stomach cancer cell (Shanghai cyropreservation center).
Cd3 monoclonal antibody is coated: the pbs solution using 0.01m ph9.6 dilutes first class purification anti-human cd3 monoclonal anti
Body, to concentration 50-70ng/ml, this anti-human cd3 monoclonal antibody is added to 75cm2In culture bottle, every bottle of 30-50ml, put for 4 DEG C
Put 1 to 7 day, using front exhaustion pbs solution.Or cd3 monoclonal antibody coated cell training after freeze-drying removes pbs
Foster bottle can 4 DEG C of preservations of longer-term.20ml training base cleaning bottle wall is added, keeping flat culture bottle makes the covering of training base be coated side before cell culture
Bottle wall, stands 10 minutes, inhales and abandons Pei Ji, standby.
Pd-1 close cik preparation: by 50ml heparin anti-coagulating inject 50ml centrifuge tube a in, at a temperature of 4 DEG C with
3000rpm is centrifuged 5min.Upper plasma is moved into 50ml centrifuge tube b, 56 DEG C of water-bath 30min, with 3000rpm at a temperature of 4 DEG C
Centrifugation 15min, collects supernatant, 4 DEG C save backup.
Add hank ' s liquid or normal saline to 48ml in described centrifuge tube a, mix.Blood cell suspension is slowly added to
To the ficoll-hypaque (density: in 15ml centrifuge tube 1.077 ± 0.001), 8ml/ manages, at a temperature of 4 DEG C equipped with 4ml
20min is centrifuged with 3000rpm.Collect mononuclearcell (pbmc) simultaneously to move it in 1 50ml centrifuge tube, plus hank ' s liquid or
Normal saline, to 50ml, is centrifuged 5min with 3000rpm at a temperature of 4 DEG C, abandons supernatant, be repeated 3 times.
Add the 10ml anteserum-less substrate above-mentioned sedimentation cell of suspension, cell suspension is added to anti-human cd3 monoclonal antibody
Coated 75cm2In culture bottle, add and state b pipe blood plasma 8ml, preferred blood plasma is patient's autologous plasma, can prevent anaphylaxiss,
And add anteserum-less substrate to 80ml, 37 DEG C, 5%co2Under the conditions of cultivate 24 hours.Ifn γ, ifn γ is added in culture bottle
Final concentration of 1000u/ml, 37 DEG C, 5%co2Under the conditions of cultivate 4-6 days about.Basis of microscopic observation cell covers with culture bottle
When, using Pei Ji in measuring pipette and bottle, cell is swept away from bottle wall and cell suspension is moved into 1500-2000ml cell
In culture bag, addition 500ml anteserum-less substrate, 10ml above-mentioned b pipe blood plasma and il-2 (final concentration of 300-500u/ml), 37 DEG C,
5%co2Under the conditions of cultivate 4-5 days.Add anteserum-less substrate 1000ml and il-2 (3 × 105-5×105U), 37 DEG C, 5%co2Bar
Cultivate 2 days under part.
Culture fluid 20ml is taken to carry out antibacterial culturing and utilize pcr technology for detection mycoplasma.Other cells are (thin in culture bag
Born of the same parents) continue culture 2 days, collect cell, at 4 DEG C, 5min is centrifuged with 3000rpm, abandons supernatant.Normal saline adds to 50ml, 4
At DEG C, 5min is centrifuged with 3000rpm, abandons supernatant, be repeated 3 times.
Plus 20-40ml normal saline, mix, add the anti-human pd-1 monoclonal antibody of 200 μ g (final concentration of 50-250 μ g/
Ml), mix.(18 DEG C -25 DEG C) incubation 30min of room temperature.Plus concentration is the human serum albumin 4-10ml of 200mg/ml, plus physiology
To 50ml, 200 mesh filter screens filter saline, and cell suspension is moved in transfusion bag, plus normal saline, to 300ml, heat sealing, glues
Label, for venous re-transfusion (total cellular score: 1 × 108-1×109).
Measure of merit
Take the cik of cik and pd-1 closing, carry out the double dyeing of cd3, pd-1 immunofluorescence and cellulotoxic experiment respectively.cd3、
The double Coloration experiment of pd-1 immunofluorescence: take each 0.5ml of cik cell suspension of cik and pd-1 closing to be added separately in 2 test tubes,
800rpm is centrifuged 5min, inhales and abandons supernatant, gently shakes, and mixes cell, it is mono- that each test tube is simultaneously introduced the anti-human cd3 of fitc labelling Mus
Clonal antibody and pe labelling rabbit anti-human pd-1 monoclonal antibody 100 μ l, mix, incubated at room 30 minutes.Add 5ml
0.01mph7.4pbs, is centrifuged 5min with 1000rpm at 4 DEG C, abandons supernatant, be repeated 3 times.1ml 0.01mph7.4pbs is added to hang
Floating cell, flow cytomery, before result display closing, the pd-1 positive cell rate of cik is 38.02% ± 16.43%, envelope
It is down to 3.12% ± 0.52%, after closing, pd-1 recall rate substantially reduces (p < 0.05), shows that the inventive method has good after closing
The effect of good closing pd-1 molecule, result is as shown in Figure 1 and Figure 2.
Cellulotoxic experiment: with the full culture medium of rpmi1640 (containing 10% calf serum, 100u/ml penicillin, 100u/ml
Streptomycin) cik of cik, pd-1 closing is tuned into 1 × 105C/ml, scg7901 tumor cell is tuned into 1 × 104c/ml.Respectively
Above-mentioned cell is added in 96 porocyte culture plates.A hole adds cik cell or the cik of pd-1 closing, 100 μ l/ holes；B hole adds
Scg7901 tumor cell, 100 μ l/ holes；After the every hole in c hole adds the cik of 100 μ l cik or pd-1 closings, add scg7901 and swell
The every hole of oncocyte 100 μ l.37 DEG C, 5%co2Under the conditions of cultivate 72 hours, in each hole add 5mg/ml mtt, 20 μ l/ holes, continue
Culture 4 hours.1000rpm is centrifuged 10min, inhales and abandons supernatant 180 μ l, adds dmso, 100 μ l/ holes, mixes, microplate reader 450nm is surveyed
Determine od value.Killing rate computational methods are killing rate=[1- (c-a)/b] × 100%.Cyto toxic experiment showed shows cik, pd-1
The tumor cell destruction of the cik of closing is respectively as follows: 47.82% ± 12.24% and 73.3% ± 15.62%, pd-1 closing
The tumor cell destruction of cik apparently higher than cik tumor cell destruction (p < 0.05) it was demonstrated that pd-1 close cik tumor
Killing activity significantly improves, and result is as shown in Figure 3.
Claims (5)
1. the pd-1 being used for treating tumor closes the preparation method of cik it is characterised in that comprising the steps of:
It is coated step, anti-human cd3 monoclonal antibody coated cell culture bottle；
Activation step, using described cd3 monoclonal antibody, ifn γ and il-2 activation culture lymphocyte；
Cleaning step, the described lymphocyte after three activation cultures of cleaning；
Closing step, with the lymphocyte after the described cleaning of 20-50ml normal saline suspension, obtains suspension cell solution, to institute
State addition pd-1 monoclonal antibody in suspension cell solution, the 18-25 DEG C of incubation that is suspended obtains cell suspension in 30 minutes.
2. the pd-1 for treating tumor according to claim 1 closes the preparation method of cik it is characterised in that further
Comprise step: filtration step, add cell-dispersing agent 200 mesh filter screen to add normal saline extremely after filtering in described cell suspension
300ml obtains feeding back liquid.
3. the pd-1 for treating tumor according to claim 1 closes the preparation method of cik it is characterised in that described
Anti-human cd3 monoclonal antibody coated cell culture bottle comprises the steps of: the pbs solution using 0.01m ph9.6 dilutes first class
Purification anti-human cd3 monoclonal antibody is to concentration 50-70ng/ml；By the anti-human cd3 monoclonal antibody of described first class purification after dilution
It is added in culture bottle, place more than 24 hours for 4 DEG C.
4. the pd-1 for treating tumor according to claim 1 closes the preparation method of cik it is characterised in that described swash
Step of living comprises the steps of:
Heparin anti-coagulating is injected in centrifuge tube a, at a temperature of 4 DEG C, 5min is centrifuged with 3000rpm；Upper plasma is taken to move into centrifuge tube
B, 56 DEG C of water-bath 30min, are centrifuged 15min with 3000rpm at a temperature of 4 DEG C, collect supernatant, 4 DEG C save backup；Lower floor's hemocyte
Stay standby in centrifuge tube a；
The described hemocyte that normal saline suspends in described centrifuge tube a, the blood cell suspension being obtained is slowly added into equipped with 4ml
Density be 1.077 ± 0.001 the centrifuge tube of ficoll-hypaque in, at a temperature of 4 DEG C with 3000rpm be centrifuged 20min；
Collect mononuclearcell confluent monolayer cells and adopt centrifuging to clean described cell three times；Add anteserum-less substrate resuspended, prepared depletion of blood
Clear training base suspension cell；
Described anteserum-less substrate suspension cell is added to containing in the coated culture bottle of described anti-human cd3 monoclonal antibody,
Add inactivation blood plasma and add into anteserum-less substrate, 37 DEG C, 5%co2Under the conditions of cultivate 24 hours；Ifn is added in culture bottle
γ, 37 DEG C, 5%co2Under the conditions of cultivate 4-6 days；The cell after culture is taken to move in cell culture bags, addition anteserum-less substrate,
Inactivation blood plasma and il-2,37 DEG C, 5%co2Under the conditions of cultivate 4-5 days；Add anteserum-less substrate and il-2,37 DEG C, 5%co2Condition
Lower culture 4-5 days.
5. the pd-1 for treating tumor according to claim 1 closes the preparation method of cik it is characterised in that in institute
Also comprise before stating cleaning step: testing sequence, detect cell quantity after carrying out cell culture, and using the culture of pcr technology for detection
Mycoplasma in supernatant.
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| CN108048397A (en) * | 2017-12-15 | 2018-05-18 | 海南博鳌银丰康养国际医院有限公司 | It is a kind of to utilize the method for targeting PD-1 antibody silence immunocyte negative regulation receptors |
| CN109913413A (en) * | 2019-03-05 | 2019-06-21 | 广州筑康生物技术有限公司 | A kind of T cell extracorporeal culturing method, its cell preparation and its application loading PD-1 antibody |
| CN110656084A (en) * | 2019-10-31 | 2020-01-07 | 秦森邦 | BAK cell and preparation kit and preparation method thereof |
| CN110747167A (en) * | 2019-10-31 | 2020-02-04 | 秦森邦 | Preparation method and application of hemizygous BAK cell |
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| CN109913413A (en) * | 2019-03-05 | 2019-06-21 | 广州筑康生物技术有限公司 | A kind of T cell extracorporeal culturing method, its cell preparation and its application loading PD-1 antibody |
| CN110656084A (en) * | 2019-10-31 | 2020-01-07 | 秦森邦 | BAK cell and preparation kit and preparation method thereof |
| CN110747167A (en) * | 2019-10-31 | 2020-02-04 | 秦森邦 | Preparation method and application of hemizygous BAK cell |
| CN110747167B (en) * | 2019-10-31 | 2021-11-02 | 秦森邦 | Preparation method and application of hemizygous BAK cell |
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