CN110106202A - The preparation method and its cell of antitumor NK cell and application - Google Patents
The preparation method and its cell of antitumor NK cell and application Download PDFInfo
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- CN110106202A CN110106202A CN201910376948.XA CN201910376948A CN110106202A CN 110106202 A CN110106202 A CN 110106202A CN 201910376948 A CN201910376948 A CN 201910376948A CN 110106202 A CN110106202 A CN 110106202A
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Classifications
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- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- 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
- C12N2510/00—Genetically modified cells
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Abstract
The present invention discloses a kind of preparation method of transgenosis NK cell, step: chemokine receptors gene C CR7 gene and CXCR4 gene (a) being connected to slow virus carrier PHS-BVC-LW238, form recombination engineered vector；(b) recombination engineered vector is transfected into the NK92 cell, obtains transgenosis NK cell.Transgenosis NK cell can inhibit the growth of tumour well.
Description
Technical field
The invention belongs to medical domains, are related to new and effective antitumor NK cell and its development of knurl chemotactic.
Background technique
In recent years, application of the immunization therapy in therapeutic field of tumor is more and more wider, is commented within continuous 2 years 2014,2015
For year most important scientific breakthrough field, achievement has surmounted classic chemotherapy, radiotherapy and operation, has become the new hand for the treatment of of cancer
Section.Immunotherapy of tumors research including DC, CAR-T, NK cell and immunologic test point monoclonal antibody etc. is more and more deep.
It is now recognized that cellular immunotherapy has three for the bottlenecks of the curative effect of entity tumor: tumour lacks ideal
Target spot, knurl tissue infiltration activating immune cell is few and tumor microenvironment immunosupress.NK cell has powerful natural resist
Tumour and anti-infectious immunity effect, anti-tumor activity is i.e. recognizable without tumor-cell antigen (target spot) and to attack tumour thin
Born of the same parents have avoided solid tumor cell and have lacked ideal this obstacle of target spot.
NK cell has the function of antitumor, anti-infective and adjusting immune response etc., and NK cell does not need specific antigen
Stimulation, have preferable prospect in immunotherapy field.Early utilization NK cell and Tumor-infiltrating lymphocytes (LAK)
Carry out immunization therapy.
Tumour cell has evolved number of mechanisms to escape NK cell recognition, including MHCI class molecule and NK inhibition
The expression of receptors ligand.For the deliquescent resistance for overcoming tumour cell cell-mediated to NK, improve to tumour recognition capability,
Chimerical receptor is widely studied.Its principle: introducing Chimeric antigen receptor greatly will avoid inhibition signal being transmitted to NK cell
It is interior, to enhance NK cell anti-tumor ability.Currently, the method for common gene modification makes NK cell express tumor associated antigen
(TAA) specific Chimerical receptor, once contact antigen, Chimerical receptor signal can activate NK cell.Chimerical receptor must draw
NK cell function could effectively be enhanced by entering signal transduction structural domain, and the release including cytotoxicity and cell factor is the most frequently used
Be the signal domain from TCR-CD3 compound.
In recent years, NK cell because its unique advantage and mechanism of action as the immunization therapy for tumour increasingly by weight
Depending on [1], and become the new effective for the treatment of cancer.The relatively short service life of NK cell in the circulating cycle avoids many bases
The common dangerous cytokine storm [2] in the immunotherapy of T cell, thus adopt be transfused NK cell miss the target toxicity very
It is small.The particular advantages of NK cell are that they can kill tumour cell in the case where no specific antigen identifies, more
Certain effect, such as neoplastic hematologic disorder and melanoma [3-5] are obtained in kind oncotherapy.And NK cell has adopted infusion
It is successfully applied to leukaemia and other patients with hematological tumor [6-8].However, it is in other solid tumors including colon cancer
Therapeutic effect it is unsatisfactory [9,10].The key that multinomial research report influences the success or failure of NK adoptive cellular immunotherapy is to migrate
To the NK cell quantity [9,2,1,6] of tumor locus.The research for the killing of NK cell tumour, which is concentrated mainly on, at present promotes it
Existence, homeostatic proliferation etc. in vivo；And promotes NK cell chemotaxis, gathers tumor locus and be very important one
Link has not yet to see research report.This research is the emerging important directions [2,11] of clinical NK cellular immunotherapy.
It has recently been demonstrated that the NK cell of cytokine activation and amplification in vitro lack or express low-level chemotactic because
Sub- receptor, it means that the expression quantity of chemokine receptors may extremely close NK cell to this process of knurl chemotactic on NK cell
It is important.
Chemotactic factor (CF) and chemokine receptors play an important role [12] into tumor migration in lymphocyte.By NK cell
Effectively transporting tumor locus is the successful committed step of cancer immunotherapy.By carrying out clinic I to NK cellular immunotherapy
Phase test, Childs RW etc. have found that a kind of important restriction factor of influence patients with solid tumor NK cell infusion clinical effectiveness is NK
Cell fails enough migrate to tumor locus [13].Immunocyte can pass through the chemokine receptors and tumour point of cell surface
The chemotactic factor (CF) interaction secreted is directed into tumour [12].These features obtain more next in immunotherapy of tumors kind in recent years
More concerns.Previously have and give exogenous cytokines, or chemotatic factor expressing level is improved by transgenosis, to promote
Improve the report [13] of NK cell quantity in knurl into NK cell Proliferation, the shortcomings that this scheme is exogenous cytokines
Systemic side effects are big, it is difficult to control cytokine-expressing amount, the concentration for directly acting on NK cell is not also high.In addition, cell because
Sub- transgenosis modification mode by the restrictive control of ajor histocompatibility complex gene MHC molecule so that its application also by
To limitation.And pass through the chemokine receptors of gene modification NK cell surface, it both can be to avoid transgenosis modified cytokines not
Foot, and immunocyte can be made more to infiltrate to tumor microenvironment, enhance tumor-inhibiting action.
NK cell plays a significant role in body is antitumor, obtains high quantity, high purity N K using amplification in vitro method
Cell is the hot spot of research in recent years NK adoptive cellular immunotherapy.With amplification method progress, NK cell activity, purity are improved, and are
NK cell becomes adoptive immunotherapy and provides Important Platform.NK adoptive cellular immunotherapy is divided into two classes at present: self NK
Cell therapy and allosome NK cell therapy.
The application IL-2 induction such as Rosenberg patient PBMC obtains LAK cell, cooperates large dosage IL-2 to kidney, black
The patients such as plain tumor treat, and obtain 20-30% Tumor response rate, but large dosage IL-2 brings very big adverse reaction.With expansion
Increasing technology improves, and IL-2 dosage is reduced and NK cell activity, purity improve.It extracts and trains from Multiple Myeloma
It supports and amplifies NK cell, then with its Cytotoxicity in vitro autologous patient myeloma cell, fragmentation effect is obvious, shows that autologous NK cells have
Hoping becomes treatment myeloma new tool.Yang Mengyin etc. extracts Patients with gliomas peripheral blood mononuclear cells, through amplification in vitro culture
NK cell out treats recurrent malignant gliomas, significant in efficacy.Although multinomial research discovery autologous NK cells treatment tumour obtains
It must alleviate, still some patient can not benefit, and following reason may influence autologous NK cells treatment curative effect: 1. NK cell can only
The HLA molecular weight tumor cell that killing lacks and distribution type is inconsistent does not have lethal effect to the tumour cell of expression HLA molecule.Such as
Fruit patient tumors cell expresses HLA molecule.So the Inhibitory receptor of NK cell surface will play main function, and autologous patient swashs
NK cell living cannot play lethal effect.2. the tumour of research discovery recently may cause the reduction of NK cell quantity, i.e. patient
The effector cell's negligible amounts for being converted into NK cell, while the function of tumor patient NK cell and normal person can be stimulated in peripheral blood
Compared to there is different degrees of damage, the autologous NK cells hypofunction amplified may cause.
Based on autologous NK cells immunization therapy there are the shortcomings that and technical difficulty, many scholars attempt establish application
Allosome NK cell is used for tumor biotherapy.NK cell line NK3.3 and pernicious change cell origin including normal NK cells source
NK cell line YT, NK-92, NKL, HANK-1, NK-YS, KHYG1 and NKG.In above-mentioned NK cell line, NK-92 is only
One enters the cell line of clinical research, all shows efficiently to separate sources tumor cell line, such as leukaemia, breast cancer
Killing activity.The a large amount of activated receptors of its surface expression, such as NKp30, NKp46, NKG2D, NKG2E, CD28；Inhibit the expression of receptor
Seldom, only NKGA/B and low-level K1112DIA and ILT-2. lacks the KIRs of most of normal NK cells clonal expressions,
But retain the cytotoxicity that perforin/granzyme mediates.The researchs such as Cheng in recent years discovery NKG cell line be
CD56brightNK cell, height expression adhesion factor, Activating receptor and the relevant receptor of cell dissolution and molecule, kill tumour
The ability of cell is better than NK-92, NKL and YT, may become the NK cell line of tumor patient adoptive immunotherapy.Barkholt
Phase I clinical trial Deng the allosome NK cell therapy malignant tumour in application amplification in vitro is studies have shown that allosome NK cell therapy
Malignant tumour has no toxic side effect and graft-versus-host reaction, and NK cell is prompted to be likely to become the new road for the treatment of malignant tumor
Diameter.But allosome NK cell therapy tumour is there is also a series of problems, such as donor NK cell time-to-live length in recipient's body,
It can or can not be eliminated by the acquired immunity cell of patient itself, furthermore which the adverse reaction of allosome NK cell infusion has, it is clinical
Using whether safety etc., these still need to further study.
Foreign countries, which have, at present reaches grinding for the T cell quantity for increasing knurl part by reinforcing T lymphocyte chemotactic process
Study carefully report；Many researchs have shown that, chemotactic factor (CF) in conjunction with NK cell receptor after not only generate chemotactic process, can also activate NK itself
The killing activity to tumour cell.
It is the antitumor NK of novel gene engineering risen that NK cell (CAR-NK cell), which can be improved, in Chimerical receptor expression
Cell.Principle: introduce Chimeric antigen receptor will avoid inhibition signal being transmitted to NK significantly it is intracellular, so that it is thin to enhance NK
The antitumor and antiviral ability of born of the same parents.Currently, the method for mostly using gene modification makes NK cell express tumor associated antigen (TAA)
The Chimerical receptor of specificity, once contact antigen, Chimerical receptor signal can activate NK cell.Chimerical receptor must be introduced into signal
Transduction structural domain could effectively enhance NK cell function, and the release including cytotoxicity and cell factor, the most commonly used is come
From the signal domain of TCR-CD3 compound.The sustainable proliferation of CAR-NK cell, but knurl Chemotaxis Function is not enhanced,
And because solid tumor lacks promising target, the actual effect of CAR-NK is unsatisfactory.
Bibliography
1.Kremer V,Ligtenberg MA,Zendehdel R,Seitz C,Duivenvoorden A,
Wennerberg E et al.Genetic engineering of human NK cells to express
CXCR2improves migration to renal cell carcinoma.J Immunother Cancer.2017；5
(1):73.doi:10.1186/s40425-017-0275-9.
2.Childs RW,Berg M.Bringing natural killer cells to the clinic:ex
vivo manipulation.Hematology Am Soc Hematol Educ Program.2013；2013:234-
46.doi:10.1182/asheducation-2013.1.234.
3.Louis CU,Savoldo B,Dotti G,Pule M,Yvon E,Myers GD et al.Antitumor
activity and long-term fate ofchimeric antigen receptor-positive T cells in
patients with neuroblastoma.Blood.2011；118(23):6050-6.doi:10.1182/blood-2011-
05-354449.
4.Pule MA,Savoldo B,Myers GD,Rossig C,Russell HV,Dotti G et al.Virus-
specific T cells engineered to coexpress tumor-specific receptors:persistence
and antitumor activity in individuals with neuroblastoma.Nat Med.2008；14(11):
1264-70.doi:10.1038/nm.1882.
5.Simon B,Wiesinger M,Marz J,Wistuba-Hamprecht K,Weide B,Schuler-
Thurner B et al.The Generation of CAR-Transfected Natural Killer T Cells for
the Immunotherapy of Melanoma.Int J Mol Sci.2018；19(8).doi:10.3390/
ijms19082365.
6.Ruggeri L,Capanni M,Urbani E,Perruccio K,Shlomchik WD,Tosti A et
al.Effectiveness of donor natural killer cell alloreactivity in mismatched
hematopoietic transplants.Science.2002；295(5562):2097-100.doi:10.1126/
science.1068440.
7.Benson DM,Jr.,Cohen AD,Jagannath S,Munshi NC,Spitzer G,Hofmeister
CC et al.A Phase I Trial ofthe Anti-KIRAntibody IPH2101 and Lenalidomide in
Patients with Relapsed/Refractory Multiple Myeloma.Clin Cancer Res.2015；21
(18):4055-61.doi:10.1158/1078-0432.CCR-15-0304.
8.Miller JS,Soignier Y,Panoskaltsis-Mortari A,McNearney SA,Yun GH,
Fautsch SK et al.Successful adoptive transfer and in vivo expansion of human
haploidentical NK cells in patients with cancer.Blood.2005；105(8):3051-7.doi:
10.1182/blood-2004-07-2974.
9.Zhang Q,Zhang H,Ding J,Liu H,Li H,Li H et al.Combination Therapy
with EpCAM-CAR-NK-92 Cells and Regorafenib against Human Colorectal Cancer
Models.J Immunol Res.2018；2018:4263520.doi:10.1155/2018/4263520.
10.Carlsten M,Childs RW.Genetic Manipulation of NK Cells for Cancer
Immunotherapy:Techniques and Clinical Implications.Front Immunol.2015；6:
266.doi:10.3389/fimmu.2015.00266.
11.Childs RW,Carlsten M.Therapeutic approaches to enhance natural
killer cell cytotoxicity against cancer:the force awakens.Nat Rev Drug
Discov.2015；14(7):487-98.doi:10.1038/nrd4506.
12.Franciszkiewicz K,Boissonnas A,Boutet M,Combadiere C,Mami-Chouaib
F.Role ofchemokines and chemokine receptors in shaping the effector phase
ofthe antitumor immune response.Cancer Res.2012；72(24):6325-32.doi:10.1158/
0008-5472.CAN-12-2027.
13.Andreas Lundqvist MB,Aleah Smith,and Richard W.Childs.Bortezomib
Treatment to Potentiate the Anti-tumor Immunity of Ex-vivo Expanded
Adoptively InfusedAutologous Natural Killer Cells.Journal ofCancer.
[14]Gelsomino F,Barbolini M,Spallanzani A,et al.The evolving role of
microsatellite instability in colorectal cancer:a review[J].Cancer Treat Rev,
2016,51:19-26.
[15].Chatterjee A,Gogolak P,Blottiere HM,Rajnavolgyi E.The impact
ofATRA on shaping human myeloid cell responses to epithelial cell-derived
stimuli and on T-lymphocyte polarization.Mediators of inflammation.2015；2015:
579830.doi:10.1155/2015/579830.
[16] Zhang Chaofeng, Chen great Wei, cold person of outstanding talent, Zhu Songming, Fei Zhewei medium selection are suffered from colorectal cancer
Expression and its meaning China clinician's magazine (electronic edition) .2013 in person's peripheral blood；7(15):7231-3.
[17] expression and effect The 2nd Army Medical College journal .2014 of the Chemokines CC CL19 in colorectal cancer；35
(7):727-33.
[18] Zhang Lei, Sun Xuezhu, the expression and its meaning of Sun Jingzhou .SDF-1/CXCR4 and VEGF in Colon and rectum gland cancer
Adopted China Immunology Journal .2015 (5): 674-7.
Summary of the invention
More in summary, first aspect present invention provides a kind of preparation method of transgenosis NK cell, the preparation
Method are as follows:
Chemokine receptors gene is gone in NK cell, the institute that can be overexpressed the chemokine receptors gene is obtained
State transgenosis NK cell.
In some embodiments, the chemokine receptors gene is the chemokine receptors gene of NK cell；
In some embodiments, the chemokine receptors gene are as follows: people CCR7 gene and/or people's CXCR4 gene；
In some embodiments, No. GenBank of the people CCR7 gene are as follows: NM_001301714.1 (GeneID:
1236)；No. GenBank of the people CXCR4 gene are as follows: NM_001008540.2 (GeneID:7852)；
In some embodiments, the NK cell is allosome NK cell；
In some embodiments, the NK cell is NK92 cell；
In some embodiments, the chemokine receptors gene is stablized in the antitumor NK cell and is overexpressed.
In some embodiments, the preparation method includes the following steps:
(a) the chemokine receptors gene is connected on engineering carrier, forms recombination engineered vector；
(b) the recombination engineered vector is converted or transfects the NK cell, obtain the transgenosis NK cell；
In some embodiments, the engineering carrier is slow virus carrier, in some embodiments, the base
Because engineered vector is slow virus carrier PHS-BVC-LW238.
Second aspect of the present invention provides a kind of transgenosis NK cell, and the transgenosis NK cell is such as first party of the present invention
The transgenosis NK cell that preparation method described in face is prepared.
Third aspect present invention provides a kind of transgenosis NK cell, and the transgenosis NK cell can express chemotactic factor (CF)
Acceptor gene；
In some embodiments, the chemokine receptors gene is the chemokine receptors gene of NK cell；
In some embodiments, the chemokine receptors gene are as follows: people CCR7 gene and/or people's CXCR4 gene；
In some embodiments, No. GenBank of the CCR7 gene are as follows: NM_001301714.1 (GeneID:
1236)；No. GenBank of the CXCR4 gene are as follows: NM_001008540.2 (GeneID:7852).
Fourth aspect present invention provides a kind of recombination engineering expression vector, the recombination engineering expression vector
Middle recombination has chemokine receptors gene, and the gene engineering expression carrier can be used in conversion or transfected NK cells；
In some embodiments, the chemokine receptors gene is the chemokine receptors gene of NK cell；
In some embodiments, the chemokine receptors gene of the NK cell is people CCR7 gene and/or people CXCR4
Gene；
In some embodiments, No. GenBank of the people CCR7 gene are as follows: NM_001301714.1 (GeneID:
1236)；No. GenBank of the CXCR4 gene are as follows: NM_001008540.2 (GeneID:7852)；
In some embodiments, the recombination engineering expression vector comes from slow virus carrier, in some embodiment party
In formula, the recombination engineering expression vector comes from PHS-BVC-LW238.
Fifth aspect present invention provides a kind of kit, contains second aspect of the present invention or the present invention in the kit
Recombination engineering expression vector described in transgenosis NK cell or fourth aspect present invention described in the third aspect.
Sixth aspect present invention provide preparation method as described in the first aspect of the invention prepare anti-tumor agent or
Purposes in tumour chemotactic preparation.
It is thin that seventh aspect present invention provides the transgenosis NK as described in second aspect of the present invention or third aspect present invention
Born of the same parents or recombination engineering expression vector as described in the fourth aspect of the present invention or or fifth aspect present invention as described in reagent
Purposes of the box in killing tumor cell or tumour chemotactic.
It is an object of the present invention to provide a kind of new and effective reinforcings to the antitumor NK cell of knurl chemotactic and its development side
Method.By being overexpressed the chemokine receptors of NK cell, increases NK cell in conjunction with chemotactic factor (CF), strengthen the chemotactic mistake of NK cell
Journey and tumor activity is killed, and reaches the NK cell quantity for increasing knurl part and enhance activity.
A kind of antitumor NK cell of the novel and high-efficiency of knurl chemotactic, on established NK in vitro culture platform base,
NK-92 cytotostatic is set to be overexpressed chemokine receptors CCR7 and CXCR4 using gene modification technology, it is corresponding to knurl secretion
Chemotactic factor (CF) combine, hence it is evident that strengthen its chemotactic to tumour knurl, to solve the problems, such as NK lazy weight in knurl；Chemotactic because
The killing activity to tumour of NK cell can be significantly improved after sub- bind receptor again, activation knurl local immunity microenvironment is to mention
High curative effect；The new and effective antitumor NK cell of knurl chemotactic is succeeded in developing by these technological innovations.
People CCR7 and people's CXCR4 genetic fragment are connected to the slow virus carrier PHS-BVC- of linearisation by ligase
Carrier is transfected 293T cell after being sequenced successfully by LW238, is packed adult lentiviral particle, is then transfected NK92 cell, make NK92
Cell is overexpressed cross-film chemokine receptors CCR7 and CXCR4.
Chemokine receptors CCR7 and CXCR4 on NK92 cell membrane, CCL12, CCL19 with the secretion of tumour knurl,
The chemotactic factor (CF)s such as CXCL12 combine, according to NK cell chemotaxis principle, NK92 cell by along the concentration gradient of these chemotactic factor (CF)s,
Assemble to knurl local motion.
On established NK in vitro culture platform base, by PCR amplification include two chemokine receptors: people CCR7 and
The full length gene coded sequence of CXCR4 is carried with I-sceI enzyme and Xbal enzyme difference digestion overall length target gene and lentivirus transfer
CCR7 and CXCR4 genetic fragment, the PHS-BVC- of linearisation is connected to by ligase by body PHS-BVC-LW238 plasmid
LW238 carrier converts connection product into DH5 α, and converted product is applied in the solid medium of the benzyl containing ammonia, picking positive bacteria
It falls, with the PHS-AVC-LW750 of PCR method identification positive colony, transfects carrier by LIPO2000 after being sequenced successfully
293T cell packs adult lentiviral particle, then transfects NK92 cell, NK92 cell is made to be overexpressed chemokine receptors
CXCR7 and CXCR4.
For the major obstacle of the cellular immunotherapy curative effect of entity tumor (including but not limited to colon cancer): infiltration is arrived swollen
The activating immune cell lazy weight of tumor tissue and the immunosupress of knurl microenvironment, this method make NK using gene modification technology
Cell long-period is stablized (and can be transferred to filial generation NK cell) and is overexpressed: two cross-film chemokine receptors such as CCR7 and CXCR4, with
The chemotactic factor (CF) of colon cancer secretion: after CCL12, CCL19 and CXC12 are combined, strengthen the NK cell to colon cancer knurl tissue
Chemotactic process increases NK quantity in knurl；It can be improved again after receptor on Chemokines CC CL12, CCL19 combination NK cell membrane
Killing activity of the NK cell itself to colon cancer cell, and activated tumor local immunity microenvironment.
The anticancer usage of the antitumor NK cell of aforementioned knurl chemotactic: because of the curative effect of cellular immunotherapy entity tumor
Main bottlenecks have three: tumour lacks ideal target spot, activating immune cell is few in knurl and the immune suppression of tumor microenvironment
System, and NK cell has powerful natural antitumor immunization, anti-tumor activity is not necessarily to tumor-cell antigen (target spot) i.e.
It can recognize and attack tumour cell, avoided solid tumor cell and lacked this obstacle of promising target.For other two restriction
The NK92 cell of the principal element of its curative effect, the technology of the present invention preparation has obviously strengthened the chemotactic process to knurl of itself,
Increase NK cell quantity in knurl, increased activity preferably solves the problems, such as that the activity of NK cell in knurl is low few with quantity.
The existing technical solution for enhancing NK cell activity is: whole body is using cell factors such as IL-2, IL-12, or turns
Gene technology makes certain cell height of part express these cell factors, so that NK cell activity be made to enhance.But whole body uses cell
The toxicity of this scheme of the factor is big, and the concentration for acting on NK cell is not high；Transgenic technology makes certain cell of part, such as swollen
Oncocyte height expresses these cell factors, this scheme the disadvantage is that Cytokine Expression Level be difficult to control, directly act on NK
The concentration of cell is not also high.In addition, cytokine transgene modifies mode by ajor histocompatibility complex gene MHC molecule
Restrictive control, so that its application is also restricted.
NK cell is set to be overexpressed cross-film chemokine receptors by gene modification technology, by chemotactic factor (CF) mechanism of action,
Strengthen its Chemotaxis to tumour knurl, solves the problems, such as NK lazy weight in knurl；It again can after chemotactic factor (CF) bind receptor
Promote itself killing activity of NK cell, and activates local immunity microenvironment；Pass through the solution and technology of these key technical problems
The efficient antitumor NK cell of knurl chemotactic is succeeded in developing in innovation, to form the technical program.
It is now recognized that the bottlenecks of the curative effect of cellular immunotherapy entity tumor have three: the ideal target spot of tumour shortage,
The activating immune cell of knurl tissue infiltration lacks and tumor microenvironment immunosupress.It is swollen in the exhausted entity several greatly for accounting for malignant tumour
In the CAR-T cell therapy of tumor, fail to solve the problems, such as these three very well and the unsatisfactory curative effect that seems.NK cell has powerful natural
The effect of antitumor and anti-infectious immunity；Its anti-tumor activity is i.e. recognizable without tumor-cell antigen (target spot) and to attack tumour thin
Born of the same parents have avoided solid tumor cell and have lacked this obstacle of promising target.Therefore, before NK cell has good antitumor application thereof
Scape.
Exogenous cytokines are awarded, or Cytokine Expression Level is improved by transgenosis, so that NK cell be promoted to increase
It grows and improves NK cell quantity in knurl, the shortcomings that this scheme is that the systemic side effects of exogenous cytokines are big, transgenosis
Overexpression hold time it is short, be difficult to control cytokine-expressing amount, the concentration for directly acting on NK cell is also high.In addition, thin
Intracellular cytokine transgenosis modifies mode by the restrictive control of ajor histocompatibility complex gene MHC molecule, so that it is applied
Also it is restricted.Genetic modification technology can solve some limitations, such as CAR-NK cell.The sustainable proliferation of CAR-NK cell, but
Knurl Chemotaxis Function is not enhanced, and because solid tumor lacks promising target, the actual effect of CAR-NK is unsatisfactory.
Tumour may cause the reduction of NK cell quantity, i.e., the effector cell for being converted into NK cell can be stimulated in peripheral blood in patients
Negligible amounts, while the function of tumor patient NK cell is compared with normal people different degrees of damage, may cause and amplifies
Autologous NK cells hypofunction.The NK92 cell of the technology of the present invention preparation belongs to allosome NK cell, can obviously strengthen knurl and become
Change, increased activity, preferably solves the activity problems and count issue of autologous NK cells.
NK cell, which needs to enter tumour knurl tissue through capillary by chemotaxis, plays killing activity.This hair
It is bright so that NK is overexpressed chemokine receptors by gene modification technology, it is combined by the chemotactic factor (CF) discharged with tumour cell,
Make the autoactivation of NK cell and Chemotaxis is to knurl tissue, solves the problems, such as NK lazy weight in knurl；Activate local immunity
Microenvironment；By these technological innovations, the antitumor NK cell of new and effective knurl chemotactic, this immunization therapy have been succeeded in developing
New technology.
The technical solution is prepared with apparent knurl chemotactic, the NK cell of increased activity.
The technology of the present invention makes NK cytotostatic be overexpressed chemokine receptors by gene modification method, by strengthening NK
The chemotactic process of cell, hence it is evident that increase chemotactic to the NK cell number of knurl, and greatly improve the killing activity of NK cell, obtain
Inside and outside experiment confirms.This is key problem in technology point of the invention.The present invention is overexpressed chemokine receptors by stablizing, and strong
Change the knurl chemotactic of NK cell.
In the present invention, two kinds of chemokine receptors CCR7, CXCR4 of our first passage up-regulation NK92 cell surfaces make
It stablizes expression, and by CXCL12/CXCR4 and CCL19, CCL21/CCR7 can form a suitable ligand/receptor pair, uses
In NK cell-targeting tumour (ligand sources) migration of chemotactic expression CXCR4, CCR7 receptor.The application of this novel NK cell is obvious
NK cell itself is promoted to human colon cancer cell or the chemotactic and accumulation process of colon cancer tissue (chemotactic factor (CF) source), is increased
Knurl part NK cell quantity.Preferably solve the problems, such as that NK cell quantity is insufficient in tumor tissues, enhances the anticancer of NK
Effect.
Detailed description of the invention
Fig. 1 is Technology Roadmap of the invention.
Fig. 2 is the slow virus carrier schematic diagram that building is overexpressed chemokine receptors CCR7 and CXCR4.
A: the template plasmid (PHS-AVC-LW633) containing coding PROTEIN C CR7 and CXCR4；
B: the building schematic diagram of control group slow virus carrier, LW238 carrier include GFP fluorogene and puromycin resistance to
Medicine gene；
C: slow virus carrier PHS-AVC-LW749 building schematic diagram；
B-c: using I-sceI enzyme digestion CCR7 gene, is connected to PHS-BVC-LW238 plasmid, generates slow virus carrier
PHS-AVC-LW749, gene trunk structure are as follows:
PLA-hefla-CCR7-3Xflag-IRES-EGFP-P2A-PURO。
D: experimental group slow virus carrier can be overexpressed chemokine receptors CCR7 and CXCR4, and have expression GFP and resist
The function of puro；
The trunk coded sequence of e: slow virus carrier PHS-BVC-LW238, PHS-AVC-LW749 and PHS-AVC-LW750
Table.
Fig. 3 is that slow virus package carrier constructs schematic diagram (a:PSPAX2b:PMD2G).
Fig. 4 is to observe control vector (PHS-BVC-LW238) and experimental vehicle (PHS-AVC- using fluorescence inverted microscope
LW750 the photo of the transfection efficiency of 293T cell) is transfected.
In Fig. 5, a is the efficiency of infection photo with concentration experimental group virus liquid and control group virus liquid infection NK92 cell.
B-e uses RT-PCR and Western Blot technology validating experiment group on mRNA and protein level to infect respectively
NK92 cell has successfully been overexpressed chemokine receptors CCR7 and CXCR4, improves 5-8 times.
B: compared with the NK92 cell (LW238-NK92) of control viral solution infection, the NK92 of experimental group virus infection is thin
Born of the same parents (LW750-NK92) high expression CCR7 (p=0.03) in mRNA level in-site.
C: compared with LW238-NK92 cell, LW750-NK92 cell in mRNA level in-site high expression CXCR4 (p <
0.001)。
D: compared with LW238-NK92 cell, LW750-NK92 cell high expression CCR7 on protein level.
E: compared with LW238-NK92 cell, LW750-NK92 cell high expression CXCR4 on protein level.
In Fig. 6, the sequence of sequence respective figure abscissa from left to right of icon from top to bottom in this figure, aMTT test
In, when no chemotactic factor (CF), LW750NK92 cell, LW238NK92 cell compares nothing with the killing activity of 92 cell of wild type NK
Marked difference, in the presence of Chemokines CC CL12 and CCL21, LW750NK92 cell killing activity is significantly higher than wild type NK
92 cells improve 98%.
LW750NK92 cell in b-c Transwell test, LW238NK92 cell and 92 cell of untreated NK difference
Cell quantity (*: P < 0.05 of the chemotactic to knurl；**:P<0.001).
B: compared with 92 cell of LW238NK92 cell and untreated NK, LW750NK92 cell is to containing chemotactic factor (CF)
The chemotactic ability of the SW480 cell of CXCL12/SDF-1 α, HT29 cell, CACO2 cell significantly improves.
C: compared with 92 cell of LW238NK92 cell and untreated NK, LW750NK92 cell is to containing chemotactic factor (CF)
The chemotactic ability of the SW480 cell of CCL21, DLD-1 cell, COLO205 cell significantly improves.
In Fig. 7, a difference group SCID mice injects tumor formation situation after HT29 cell, and different colours indicate that HT29 cell is taken
The intensity of the red fluorescent protein of band, red are most strong.
B LW750NK92 cell (Fig. 7 b from left to right first and second SCID mouse), LW238NK92 cell (Fig. 7 b from a left side to
Third and fourth right SCID mouse) and 92 cell of untreated NK (Fig. 7 b from left to right the five, the six SCID mouse) in Mice Body
Quantity is infiltrated, different colours indicate that the intensity of EGFP albumen entrained by NK92 cell, red are most strong.
The fluorescence intensity comparative diagram (* * *: p < 0.0001) of c control group and experimental mice knurl region.
In Fig. 8, the variation of a experimental group, control group, untreated fish group mouse gross tumor volume after injecting different NK92 cells
(*:p<0.05).Figure abscissa indicates that the number of days after mouse inoculation tumour cell, ordinate indicate the subcutaneous tumor body of mice with tumor
Product size (unit: CM3)。
It is completely shelled after putting to death within each 4 mouse of b experimental group, control group, untreated fish group the 30th day after being inoculated with colon cancer cell
Except the comparison of gross tumor volume, scheme worm vernier caliper to open distance to be 1cm.
C-d experimental mice is the same as control group, untreated fish group survival time of mice (c:P=0.02 compared with survival rate；d:P
=0.021).
Specific embodiment
Technical solution in order to preferably explain the present invention, is discussed in detail the embodiment of the present invention with reference to the accompanying drawing.With
Lower embodiment should not be construed as further illustrating the present invention to fixation or limitation of the invention.Unless otherwise specified, real
Applying technical characteristic used in example and could alternatively be has equivalent or identity function or effect under the premise of without departing substantially from inventive concept
Other techniques known in the art features.
Core of the invention technology path
Referring specifically to Fig. 1.
Implement the major programme of technical solution of the present invention
A, for the major technical barrier for restricting solid tumor cell immunization therapy curative effect: the activation of tumor tissues is arrived in infiltration
The immunosupress of immunocyte quantity deficiency and knurl microenvironment, the technical program on NK cell injuring model platform base,
NK cell is set to be overexpressed chemokine receptors CCR7, CXCR4 using transgenic technology；
B, colon cancer Transplanted tumor model, human colon cancer cell strain limiting dilution assay to unicellular routine culture, harvest are established
Logarithmic growth phase cell is injected to SCID mice, observes mice with tumor life cycle and tumor size, and detection turns CCR7, CXCR4 gene
NK cell evaluates the suppression of transgenosis NK cell to the chemotactic of tumour knurl, Infiltrating and to the fragmentation effect of tumour cell
Tumor effect and toxicity；
C, the new and effective antitumor NK cell successfully to tumour knurl chemotactic is developed；The NK developed simultaneously is thin
Born of the same parents, which also retain, has natural anti-infectious immunity activity.
Materials and methods
Cell line and culture medium:
NK-92 cell is obtained from Hangzhou Pedagogic University (HZNU, Hangzhou, China).Cell culture is containing 20% tire ox
Serum (Gibco), 1% mycillin, in the α-MEM culture medium (Gibco) of 100U IL-2.It is placed in 5%CO2, 37 DEG C of culture
Case.
DLD-1 cell, HT29 cell, K562 cell, SW480 cell, COLO205 cell, CACO2 cell are purchased from upper marine
Qiao Xinzhou Biotechnology Co., Ltd (Shanghai, China).Cell culture is in (Gibco) containing 10% fetal calf serum, 1% green, strepto-
In 1640 culture medium of RPMI (Gibco) of element.It is placed in 5%CO2, 37 DEG C of incubator.
293T cell is presented by affiliated hospital, Hangzhou Pedagogic University translational medicine platform teacher Chen Yidan；
Carrier
(PLV indicates slow virus plasmid to PLV-hef1a-MCS-3xflag-IRES-EGFP-P2A-Puro plasmid；Hef1a is
Wide spectrum strong promoter starts protein expression；MCS is multiple cloning sites；3xflag is label, is detected for Western Blot；
IRES is albumen connecting element, and front and back connects 2 protein gene, protein gene can be made to separate expression；EGFP is green fluorescence
Albumen, observable efficiency of infection；P2A is albumen connecting element, and front and back connects 2 protein gene, protein gene can be made to separate
Expression；Puro is puromycin-resistant, is used for eukaryocyte resistance screening) it is synthesized by Beijing symphysis Gene Tech. Company Limited
(abbreviation PHS-BVC-LW238 plasmid)；Packaging plasmid PASPAX2, packaging plasmid PUMD2G are by affiliated hospital, Hangzhou Pedagogic University
Translational medicine platform teacher Chen Yidan present.
Embodiment 1: the building of slow virus carrier plasmid
Template plasmid (title: PHS-AVC-LW633) containing encoding human CCR7 and CXCR4 albumen is in advance in the application
The laboratory of people constructs, the coded sequence of CCR7 containing someone and people CXCR4 in PHS-AVC-LW633.The GenBank of people CCR7 is stepped on
Record number are as follows: NM_001301714.1 (GeneID:1236)；The GenBank accession number of people CXCR4 are as follows: NM_001008540.2
(GeneID:7852).In order to construct while being overexpressed the slow virus carrier plasmid of CCR7 gene and CXCR4 gene, design first
Primer
5 '-CAAAGTTTTTTTCTTCCATTTCAGGTAGGGATAAGCCACCATGTACTCCATCATTT GTT-3 ' and
5’-GTCTTTGTAGTCCATGATTACCCTGTTATCTGGGGAGAAGGTGGTGGTGGTCTCGGCCTCCACAC
TCAT-3 ' is connected to PHS- using I-sceI enzyme digestion CCR7 gene by the complete encoding sequence of PCR amplification CCR7 gene
BVC-LW238 plasmid (symphysis Gene Tech. Company Limited synthesizes by Beijing) generates slow virus carrier, is named as
PLA-hefla-CCR7-3Xflag-IRES-EGFP-P2A-PURO(PHS-AVC-LW749).Then design primer
5 '-GCATGACCCGCAAGCCCGGTGCCTGATAATAGTAATCAATTACGGGGTCATTAGTT C-3 ' and
5’-GCTTGATATCGAATTGCCGGAATTTTTAGCTGGAGTGAAAACTBTGAAGACGCTTGATATCGAAT
TGCCGGAATTTTTAGCTGGAGTGAAAACTBTGAAGACTCAGAC-3 ', the CXCR4 by PCR amplification containing separate promoters
Gene order, the CXCR4 gene using the digestion of Xbal enzyme containing separate promoters are connected to PHS-AVC-LW749 plasmid, and building is same
When the slow virus carrier plasmid PLA-hefla-CCR7-3Xflag-IRES-EGFP-P2A-PURO- comprising CCR7 and CXCR4
CMV-CXCR4(PHS-AVC-LW750).Serving the sequencing of Hai Shenggong biotech firm confirms to construct successfully, wraps in PHS-AVC-LW750
The coded sequence of gene containing CCR7 and CXCR4 gene.Respective carrier overview is referring to fig. 2.
Embodiment 2: slow virus plasmid packaging
Using 2000 reagent of Lipofectamine by recombined lentivirus vector PHS-AVC-LW750, control vector PHS-
BVC-LW238 is transiently transfected respectively with packaging plasmid PASPAX2, PUMD2G to package cell line 293T cell.Slow virus packaging
Vector construction can be found in Fig. 3.2 methods: (1) in the previous day of transfection by 293T cell inoculation in two holes of six orifice plates, every hole
Middle cell number about 6 × 105.It is cultivated in the culture medium of DMEM containing serum, when cell fusion degree reaches 70%-90%, discards and contain
The DMEM culture medium of serum-free is added in serum DMEM culture medium (containing 1% mycillin).(2) 1 holes are by 4 μ g slow virus carriers
PHS-AVC-LW750,3 μ g packaging plasmid PASPAX2 and 1 μ g packaging plasmid PMD2G are transiently transfected to 293T cell.It is used instead after 8h
The culture medium of DMEM containing serum.Fluorescence microscope transfection efficiency is used after 48h.(3) 4 μ g control vector (PHS- of another hole
BVC-LW238) and 3 μ g PASPAX2 and 1 μ g PMD2G packaging virus liquid, packing method is the same, control vector (PHS-BVC-
LW238 the photo of the transfection efficiency of 293T cell) and after experimental vehicle (PHS-AVC-LW750) packaging is transfected referring to fig. 4.Thus
It can be proved that the present embodiment slow virus plasmid is packed successfully, it can be used in transformed cells.Collect slow virus liquid, 4 DEG C, 3000g from
The heart 20 minutes, take supernatant.
Embodiment 3: infection NK92 cell
(α-MEM culture medium, 20%FBS, 0.2mM inositol, 0.1mM β-sulfydryl are used with T25 culture bottle culture NK92 cell
Ethyl alcohol, 0.02mM folic acid, 100U IL-2,5%CO2, 37 DEG C).Electricity microscopic observation cellular morphology daily, cell changes liquid every other day.It is logical
It is passed on when being often in logarithmic growth phase by 6-7 days NK92 cells by 1:2.On the day before infection, cell inoculation to 96 orifice plates, carefully
Born of the same parents' number about 5 × 104.When cell fusion degree reaches 70%-90%, serum-containing media is discarded, the MEM training of serum-free is added
It supports base (IL-2 containing 100U and 1% mycillin).100 μ l vial supernatants and 3 μ g/ are added in every hole when infection
mlpolybrene.It sucks supernatant afterwards for 24 hours, growth medium is added.It is carried with control vector (PHS-BVC-LW238) and experiment
It is independently inoculated with NK92 cell after body (PHS-AVC-LW750) packaging, uses fluorescence microscope cell infection after 48-72h
Efficiency.As a result referring to a in Fig. 5.Infected NK92 cell carries out screening in 1 week with the puromycin of 3 μ g/ml.It is collected after 1 week
Cell is simultaneously resuspended in the fresh MEM culture medium containing 100UIL-2 and 1% mycillin but without puromycin.
Embodiment 4:RT-PCR confirms that NK92 cell is overexpressed chemokine receptors CCR7, CXCR4
PHS-BVC-LW238 and PHS-AVC-LW750 is cracked using Trizol Reagent kit (Invitrogen)
The NK-92 cell transfected after packaging extracts mRNA.It is anti-that reverse transcription is carried out using reverse transcription cDNA synthetic agent box (TAKARA)
It answers.It is polluted to exclude gDNA, specific primer is used in PCR:
CXCR4PF-5'CCACGCCACCAACAGTCAGA3'；
CXCR4PR-5'GGCAAAGATGAAGTCGGGAATA3'；
CCR7PF-5'CATGCTCCTACTTCTTTGCATC3'；With
CCR7PR-5’CACTGTGGCTAGTATCCAGATG3’。
All primers are synthesized by Shanghai Sheng Gong biotech firm.
PCR product expands rear electrophoresis map referring to the b and c of Fig. 5.
Embodiment 5:Western Blot confirms that NK92 cell is overexpressed chemokine receptors CCR7, CXCR4
The NK92 cell of infection is cracked into (lysate ingredient are as follows: 10mM Tris-HCLpH 8.0 in lysis buffer；
140mM NaCl；1%Triton-X-100).Lysate is centrifuged, SDS-PAGE is carried out in reduction-state, is transferred on pvdf membrane
(Whatman GmbH,Dassel,Germany).Born respectively on one film rabbit source anti-CXCR4 monoclonal antibody (1:1000,
Abcam) and and HRP label goat anti-rabbit igg (1:1000, Abcam).It is mono- that the anti-CCR7 in rabbit source is born on another film respectively
Clonal antibody (1:10000, Abcam) and HRP label goat anti-rabbit igg (1:1000, Abcam), utilize biodiversity resources
System (BIO-RAD, USA) is visualized and is recorded to film.Using β-actin albumen as reference, for PHS-BVC-LW238
Parallel processing is distinguished with the NK92 cell transfected after PHS-AVC-LW750 packaging, as a result referring to d in Fig. 5 and e.
Embodiment 6:Transwell testing inspection NK92 cell migration ability
NK92 cell, LW238NK92 cell, the LW750NK92 cell of logarithmic growth phase respectively, it is 48 hours hungry, and
It is resuspended in no FBS α-MEM, makes in 100 μ l culture mediums containing 5 × 105A cell is added in the upper hole of the cell transwell, often
100 μ l of hole, every kind of cell inoculation are separately added into corresponding 4 cell lower opening without fetal calf serum in 4 upper holes
1640 culture medium of RPMI (as negative control group), SW480, HT29, the culture of CACO2 cell are free of the supernatant of FBS,
The human chemokine CXCL12 of 100ng/ml is added dropwise in three kinds of colon cancer cell culture supernatants afterwards, 3 multiple holes of every group of setting are put
After setting cell incubator 8 hours；In addition distinguish logarithmic growth phase NK92 cell, LW238NK92 cell, LW750NK92 cell,
It is 48 hours hungry, and be resuspended in no FBS α-MEM, make in 100 μ l culture mediums containing 5 × 105It is small that transwell is added in a cell
In the upper hole of room, every 100 μ l of hole, every kind of cell inoculation is separately added into not in corresponding 4 cell lower opening in 4 upper holes
1640 culture medium of RPMI (as negative control group) containing fetal calf serum, SW480, DLD-1, the culture supernatant of COLO205 cell
The human chemokine CCL21 of 50ng/ml is added dropwise in liquid in rear three kinds of human colon cancer cell culture supernatants, and every group setting 3 multiple
The NK92 cell for migrating across perforated membrane after placing cell incubator 8 hours, is assessed by counting the living cells in lower opening in hole
Quantity.The cell of non-through hole migration is removed using cotton swab.Cell on film lower surface is fixed with 4% paraformaldehyde,
At room temperature with 0.05% violet staining 15 minutes, and use microscopic counting.3 mirror images are randomly choosed under 40 power microscopes
Cell is counted, experiment is in triplicate.
Transwell experimental result shows that LW750NK92 is thin compared with LW238NK92 cell and wild type NK92 cell
Born of the same parents migrate to SW480 cell, HT29 cell, and cell quantity significantly increases in the supernatant of CACO2 cell α containing CXCL12/SDF-1
(see Fig. 6 b), (most persons increase to 280 from 169, and increase is nearly 70%), and difference is statistically significant (P < 0.05).Count NK92
Cell, LW238NK92 cell, LW750NK92 cell are migrated in 8h to the knot of three kinds of chemotactic factor (CF)s of CCL21 containing people (CCR7 ligand)
The NK cell quantity of the cell lower opening of colon-cancer cell supernatant, as the result is shown with LW238NK92 cell and wild type NK92 cell
Compare, LW750NK92 cell migration to SW480 cell, DLD-1 cell, COLO205 cell the CCL21 containing people supernatant in
Cell quantity significantly increase (see Fig. 6 c), (most persons increase to 214 from 142, increase it is nearly 50%), difference is statistically significant
(P < 0.05).Confirm that being overexpressed Chemokine receptor CXCR4 and CCR7 is remarkably improved NK92 cell in vitro to human colon carcinoma
The chemotactic ability of cell.
Embodiment 7:MTT method detects NK92 cell killing activity
NK92 cell, LW238-NK92 cell, LW750-NK92 cell respectively as effector cell (E, 2 × 1010/L),
K562 cell as target cell (T, 5 × 108/L).In 96 orifice plates, E (50 μ are added in each experimental group (E+T, E:T=40:1)
L), T (50 μ L).Three group controls: effector cell's control group E group, target cell control group T group and blank control group are set up simultaneously
(natural death group), every group sets three multiple holes.After being incubated for 8h, absorbance OD value is measured at wavelength 490nm.92 cell of NK kills
Hurt activity (%)=(1- (ODE+T-ODE)/ODT) × 100%, as a result as shown in a in Fig. 6, when no chemotactic factor (CF), LW750NK92
The cell killing activity of cell and 92 cell of LW238NK92 cell and untreated NK is without significant difference (P > 0.05), it was demonstrated that mistake
Expression Chemokine receptor CXCR4 and CCR7 do not influence the killing activity of NK92 cell.There are Chemokines CC CL12 and CCL21
In the presence of, LW750NK92 cell killing activity is significantly higher than 92 cell of wild type NK and LW238-NK92 cell, improves 98%.
This illustrates the LW750NK92 for being overexpressed Chemokine receptor CXCR4 and CCR7 in response to Chemokines CC CL12 and CCL21.
Embodiment 8: the building of microsatellite instability colon cancer Transplanted tumor model in Mice Body
Referring to the bibliography [14] of Gelsomino F et al., take 4 week old SCID mices (Harlan,
Indianapolis, IN), it is divided into experimental group, control group and untreated fish group, is raised in the pathogen-free conditions of standard, is guaranteed
The supply of food and water.The search procedure of related to animal all meets the ethical standard of research institution.Method: (1) tumor formation: packet
Containing 2 × 106PBS (pH7.4) 200ul of HT29 cell is subcutaneously injected to oxter on the right side of female mice.Red fluorescence is used in advance
Protein labeling kit (Shanghai jackshaft Xin Zhou Biotechnology Co., Ltd, article No. A1001-7), express that HT29 cell can red
Color fluorescin.(2) knurl measures: knurl uses two-dimensional measurement, weekly twice with digital calipers measurement.Once knurl is any
One vertical line is more than that 18mm or mouse show angor, i.e. execution mouse.Gross tumor volume calculation formula are as follows: V=0.52 × L
×W 2, V is gross tumor volume, and L is the longest diameter of tumour, and W is the diameter length perpendicular to longest diameter.(3) injection NK is thin
Born of the same parents: when knurl reaches about 5mm2When, experimental group includes 4 × 10 by tail vein injection6100 μ of PBS of LW750NK92 cell
L, control group injection include the 100 μ l of PBS of LW238NK92 cell, and untreated fish group injection includes the PBS of untreated NK92 cell
100 μ l, every 48h injection is primary, and for about 40 days, LW238NK92 and LW750NK92 cell was sick slowly by carrying GFP gene
The infection of toxin grain obtains, can be with constant expressing green fluorescent protein, using small animal living body imaging system in Mice Body
Fluorescence detection is carried out, NK92 cellular infiltration situation is compared.Utilize whole body optical imaging system (Xenogen, Caliper Life
Sciences, Hopkinton, MA) record mouse tumor image.(4) mouse is put to death, tumour is completely stripped.Experiment has carried out two
Secondary, the result of acquisition is similar.As a result as shown in Figure 7 and Figure 8, it was demonstrated that transgenosis NK92 cell is in vivo to human colon cancer cell
Chemotactic ability also significantly increases, and mouse tumor load can be made to mitigate, life span extension.
NK cell has powerful natural antitumor immunization；It can recognize and attack without tumor-cell antigen (target spot)
Tumour cell is hit, thus NK cell has unique antitumor advantage.But the system of the curative effect of NK cellular immunotherapy entity tumor
About bottleneck first is that the NK cell of knurl tissue infiltration is few, killing activity is low.It is controlled in the currently used NK cell of entity tumor
In treatment, this problem not can solve and aobvious unsatisfactory curative effect.
Meter (is calculated, imitate target than normally about 1:25) according to higher proportion by the killing activity 60% of NK cells against tumor cells
Calculate: 2500 NK cells besiege 100 tumour cells, and only 60 tumour cells are killed.Actually in human tumor knurl
Middle tumor cell number considerably beyond NK number (not calculating the part that tumour is constantly proliferated temporarily) here.Therefore, in in-vivo tumour tissue
NK cell number and killing activity are all much to be not enough to kill most of tumour cell, even if venoclysis NK cell, quantity
Also limited, and NK cell cannot enter knurl through capillary, this is also that currently used NK cell therapeutic approach treatment is real
The main reason for body tumor curative effect is undesirable.NK cell, which needs to enter knurl tissue through capillary by chemotaxis, to be played
Killing activity, to make up the deficiency on NK cell and tumour cell ratio, but also NK cell being capable of part after killing tumour
It is recycled, improves the utilization efficiency of NK cell.
Human colon cancer cell has been demonstrated that CXCL12/SDF-1 α and CCL19, CCL21 [15] can be secreted.Fei Zhewei etc.
The expression of CXCL12 in colorectal cancer patients blood is proved by 53 serum in patients with colorectal CXCL12 expressions of detection
It is significantly higher than normal person [16], Zhang Lei is equal to pass through CCL21 expression in 90 serum in patients with colorectal of detection, it was demonstrated that knot is straight
CCL21 level is in high expression [17] in patients with bowel cancer serum.Binding result: the chemotactic factor (CF)s such as CXL12 are in human colon adenocarcinoma
MRNA and protein expression level in tissue are much higher than Tumor-surrounding tissue [18], it was demonstrated that can in colon cancer body determined by us
It is poor to form CXCL12, CCL21 chemotactic factor (CF) concentration gradient: colon cancer tissue > patients serum > normal human serum；Pass through
CXCL12/CXCR4 and CCL19, CCL21/CCR7 can form a suitable ligand/receptor pair, express for chemotactic
The NK cell-targeting tumour (ligand sources) of CXCR4, CCR7 receptor migrates.Accordingly, the technology of the present invention is modified by transgenic technology
NK cell makes NK cytotostatic be overexpressed Chemokine receptor CXCR4, CCR7, by strengthening chemotactic process, responds these external sources
Property chemotactic factor (CF) and tumour endogenous generate chemotactic factor (CF) effect.This study demonstrates the chemotactics for being overexpressed CXCR4, CCR7
The NK92 cell of factor acceptor is redirected really and reinforcing is migrated to CXCL12/SDF-1 α and the source CCL19, CCL21, and point
(such as Fig. 6 b- is not confirmed in the transwell experiment of the human colon carcinoma supernatant containing CXCL12/SDF-1 α and CCL21
c)；NK92 cells in vitro chemotaxis after chemokine receptors up-regulation is remarkably reinforced really.MTT experiment confirms this novel simultaneously
NK cell (LW750NK92) is compared with wild type NK92 cell, its killing activity simultaneously unaffected (33%VS34%), difference
Not statistically significant (see Fig. 6 a), and under the conditions of having existing for chemotactic factor (CF) 100ng/mlCXCL12 and 50ng/mlCCL21,
Killing activity substantially enhances (34.5%VS 62%) compared with compareing NK cell (LW238NK92), and difference has obvious statistics to anticipate
Adopted (see Fig. 6 a).On this basis, we enter lotus knurl SCID mouse body by tracking tail vein injection with green fluorescent protein
The distribution of interior NK92 cell, it was demonstrated that by the NK92 cell (LW750NK92 cell) of our gene modifications, successfully navigated to
In the subcutaneous colon cancer knurl of mouse, observed in the internal fluoroscopic imaging systems result of toy, with lack chemotactic factor (CF)
The SCID mice with tumor of the LW238NK92 cell processing of receptor CCR7, CXCR4 is compared, and tumor area green fluorescence distributed areas are increased
And intensity obviously increases (as described in Fig. 7 a-b), it is found that NK cell (LW750NK92 cell) fluorescence intensity of chemotactic to knurl is bright
Aobvious to increase nearly 100%, difference is statistically significant (as shown in Figure 7 c), and the NK92 after illustrating chemokine receptors up-regulation is thin
Chemotaxis in colon cancer body is remarkably reinforced in born of the same parents.The effect and colon cancer tissue secrete CXCL12, CCL21, with cancer beside organism and
Serum formation concentration gradient is poor, thus the NK92 cell migration of chemotactic CCR7, CXCR4 expression quantity up-regulation, aggregation to tumor tissues
In it is directly related.
By recording and analyzing PBS group, LW238NK92 group, LW750NK92 group lotus knurl SCID mouse gross tumor volume, life cycle feelings
Condition shows tumor volume on the 23rd to PBS group 2.4cm3vs LW2382.1cm3Vs LW750 group 1.0cm3；(such as Fig. 8 a-b), 40 days
Existence: PBS group 0%vs LW238 group 0%vs LW750 group 63% (such as Fig. 8 c-d), it was demonstrated that up-regulation NK92 cell CXCR4, CCR7
Expression quantity can be enhanced NK92 cell inhibitor against colon carcinoma cells effect, extend mice with tumor life cycle.
It was found that the NK cell number of chemotactic to knurl obviously increases nearly 100% (as shown in Figure 7), and greatly improves its killing and live
Property (such as Fig. 6 shows NK killing activity: 30%vs 60%), these obtained inside and outside experiment confirm.
Above each embodiment is only intended to further illustrate the present invention, is not for limiting protection model of the invention
It encloses, it is all obviously to change based on equivalents made by design of the invention and to each technical solution of the invention
Into each falling within protection scope of the present invention.
Claims (9)
1. a kind of preparation method of transgenosis NK cell, the preparation method is that:
Chemokine receptors gene is gone in NK cell, obtains described turn that can be overexpressed the chemokine receptors gene
Gene NK cell.
2. preparation method as described in claim 1, it is characterised in that:
The chemokine receptors gene is the chemokine receptors gene of NK cell；
It is preferred that the chemokine receptors gene are as follows: people CCR7 gene and/or people's CXCR4 gene；
It is preferred that No. GenBank of the people CCR7 gene are as follows: NM_001301714.1；The GenBank of the people CXCR4 gene
Number are as follows: NM_001008540.2；
It is preferred that the NK cell is allosome NK cell；
It is preferred that the NK cell is NK92 cell；
It is overexpressed it is preferred that the chemokine receptors gene is stablized in the antitumor NK cell.
3. preparation method as described in claim 1, which is characterized in that the preparation method includes the following steps:
(a) the chemokine receptors gene is connected on engineering carrier, forms recombination engineered vector；
(b) the recombination engineered vector is converted or transfects the NK cell, obtain the transgenosis NK cell；
It is preferred that the engineering carrier is slow virus carrier, it is further preferred that the engineering carrier is slow virus carrier PHS-
BVC-LW238。
4. a kind of transgenosis NK cell, the transgenosis NK cell is preparation method as claimed in any one of claims 1-3
The transgenosis NK cell being prepared.
5. a kind of transgenosis NK cell, the transgenosis NK cell can express chemokine receptors gene；
It is preferred that the chemokine receptors gene is the chemokine receptors gene of NK cell；
It is preferred that the chemokine receptors gene are as follows: people CCR7 gene and/or people's CXCR4 gene；
It is preferred that No. GenBank of the CCR7 gene are as follows: NM_001301714.1；No. GenBank of the CXCR4 gene are as follows:
NM_001008540.2。
6. a kind of recombination engineering expression vector, recombination has chemokine receptors base in the recombination engineering expression vector
Cause, the gene engineering expression carrier can be used in conversion or transfected NK cells；
It is preferred that the chemokine receptors gene is the chemokine receptors gene of NK cell；
It is preferred that the chemokine receptors gene of the NK cell is people CCR7 gene and/or people's CXCR4 gene；
It is preferred that No. GenBank of the people CCR7 gene are as follows: NM_001301714.1；No. GenBank of the CXCR4 gene
Are as follows: NM_001008540.2；
It is preferred that the recombination engineering expression vector comes from slow virus carrier, it is further preferred that recombination engineering expression carries
Body comes from PHS-BVC-LW238.
7. a kind of kit, containing transgenosis NK cell as described in claim 4 or 5 or such as claim in the kit
Recombination engineering expression vector described in 6.
8. preparation method as claimed in any one of claims 1-3 is preparing the use in anti-tumor agent or tumour chemotactic preparation
On the way.
9. transgenosis NK cell as described in claim 4 or 5 or recombination engineering expression vector as claimed in claim 6
Or purposes of the kit in killing tumor cell or tumour chemotactic as claimed in claim 7.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111321169A (en) * | 2020-03-09 | 2020-06-23 | 杭州师范大学 | Genetically modified NK cell and preparation method and application thereof |
| CN112301059A (en) * | 2020-09-23 | 2021-02-02 | 杭州美中疾病基因研究院有限公司 | CAR-NK transgenic vector based on replication-defective recombinant lentivirus and construction method and application thereof |
| CN114807237A (en) * | 2022-05-12 | 2022-07-29 | 广东普罗凯融生物医药科技有限公司 | Preparation method and application of NK (natural killer) cells for over-expressing CD16a |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1629285A (en) * | 2003-12-19 | 2005-06-22 | 龚小迪 | Natural killer cell for transgenic expression cell chemotactic factors |
| WO2016085946A1 (en) * | 2014-11-24 | 2016-06-02 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Active cxcr4+ immune cells and methods for their production and use |
| WO2018022646A1 (en) * | 2016-07-25 | 2018-02-01 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of producing modified natural killer cells and methods of use |
| WO2018053270A1 (en) * | 2016-09-16 | 2018-03-22 | The General Hospital Corporation | Modified natural killer cells for the treatment of cancer |
-
2019
- 2019-05-07 CN CN201910376948.XA patent/CN110106202A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1629285A (en) * | 2003-12-19 | 2005-06-22 | 龚小迪 | Natural killer cell for transgenic expression cell chemotactic factors |
| WO2016085946A1 (en) * | 2014-11-24 | 2016-06-02 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Active cxcr4+ immune cells and methods for their production and use |
| WO2018022646A1 (en) * | 2016-07-25 | 2018-02-01 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methods of producing modified natural killer cells and methods of use |
| WO2018053270A1 (en) * | 2016-09-16 | 2018-03-22 | The General Hospital Corporation | Modified natural killer cells for the treatment of cancer |
Non-Patent Citations (6)
| Title |
|---|
| FRANZISKA ECKERT等: "《Potential Role of CXCR4 Targeting in the Context of Radiotherapy and Immunotherapy of Cancer》", 《FRONTIERS IN IMMUNOLOGY》 * |
| MIN CHENG等: "《Establishment, characterization, and successful adaptive therapy against human tumors of NKG cell, a new human NK cell line》", 《CELL TRANSPLANTATION》 * |
| NADJA MÜLLER等: "《Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma》", 《JOURNAL OF IMMUNOTHERAPY》 * |
| SRINIVAS S SOMANCHI等: "《Engineering lymph node homing of ex vivo-expanded human natural killer cells via trogocytosis of the chemokine receptor CCR7》", 《BLOOD》 * |
| TIZIANO INGEGNERE等: "《Human CAR NK Cells: A New Non-viral Method Allowing High Efficient Transfection and Strong Tumor Cell Killing》", 《FRONTIERS IN IMMUNOLOGY》 * |
| 吴霞等: "《趋化性细胞因子对NK细胞生物学功能的调控作用》", 《细胞与分子免疫学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321169A (en) * | 2020-03-09 | 2020-06-23 | 杭州师范大学 | Genetically modified NK cell and preparation method and application thereof |
| CN112301059A (en) * | 2020-09-23 | 2021-02-02 | 杭州美中疾病基因研究院有限公司 | CAR-NK transgenic vector based on replication-defective recombinant lentivirus and construction method and application thereof |
| CN114807237A (en) * | 2022-05-12 | 2022-07-29 | 广东普罗凯融生物医药科技有限公司 | Preparation method and application of NK (natural killer) cells for over-expressing CD16a |
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