CN108822216B - Carry the Chimeric antigen receptor and its application of truncation or not truncated nature cell toxin receptor signal structure - Google Patents
Carry the Chimeric antigen receptor and its application of truncation or not truncated nature cell toxin receptor signal structure Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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Abstract
The present invention discloses a kind of Chimeric antigen receptor (CAR), comprising: antigen-binding domains (scfv) and signal transduction structural domain, wherein signal transduction structural domain includes the first conducting structure domain and the second conducting structure domain, antigen-binding domains of connecting between the first conducting structure domain and the second conducting structure domain.It is extremely low that cytokine levels are secreted when CAR structure disclosed by the invention is by antigenic stimulus, can guarantee that clinical application safety, i.e. clinical application safety are higher well；And exo-antigen positive tumor cell killing ability is stronger, anti-tumor activity is more preferable.
Description
Technical field
The present invention relates to immunotherapy of tumors technical fields, and in particular to a kind of carrying truncation or not truncated nature cell
The Chimeric antigen receptor and its application of toxin receptor signal structure.
Background technique
Chimeric antigen receptor (CAR) is the core component of CAR-T, and using ligand binding domains characteristic, CAR can be directed to institute
The immunocyte of selection redirects its specificity and reactivity, therefore assigns the non-dependent mode of T cell HLA and identify tumour antigen
Ability, this enable by CAR transformation T cell identifies wider mesh compared to nave T cell surface receptor TCR
Mark.It include tumor associated antigen (tumor-associated antigen, a TAA) combined area in the basic engineering of CAR
(the scFV section for being typically derived from monoclonal antibody antigen bond area), an extracellular hinge area, a transmembrane region and a born of the same parents
Interior signaling zone.
Current traditional CAR-T is significant in efficacy for neoplastic hematologic disorder, but entity tumor curative effect is not obvious, and limits it
In clinical application.For safety, cytokine release syndrome (CRS) is the common complication of CAR-T cell therapy,
It even can threat to life.CAR-T cell infusion in vivo after, since Chimeric antigen receptor and corresponding tumor associated antigen are special
Property combine, CAR-T cell is activated and starts to be proliferated, and causes cytokine cascade release, multiclass immune response is mediated, to draw
The clinical manifestations, i.e. CRS such as fever, low blood pressure, expiratory dyspnea, blood coagulation disorders, end-organ obstacle are played, traditional CAR-T is by anti-
Primary stimuli secrete cytokines level directly influences the severity of CRS generation.For validity, firstly, by solid tumor
The tumor stroma of relevant fibroblast (CAFs) composition provides a physical barriers, CAFs for the infiltration of CAR-T cell
Can also extracellular matrix secretion albumen T cell is separated from cancer cell；Secondly, the metabolism ring of solid tumor cancer microenvironment
Border is unfavorable for the persistence of traditional CAR-T cell, once because tumour formation is activated, tumour cell will stop passing through oxidation
Phosphorylation generates ATP and is converted to aerobic glycolysis, causes tumor environment to become acid, here it is so-called " Wa Shi effect " pH
Value is down to 6.5 from 7.4；Finally, tumor microenvironment causes anaerobic condition further to generate immunosupress, it is thin in low-oxygen environment tumour
Born of the same parents generate a kind of HIF1- ɑ molecule, it is thin by attracting control T cell (Tregs) to weaken traditional CAR-T to tumor locus
Born of the same parents' anti-tumor function, Tregs inhibit immune response, therefore limit therapeutic effect of the tradition CAR-T for entity tumor.
Summary of the invention
One of the objects of the present invention is to provide a kind of safety is more preferable, curative effect more significantly carries nature cell toxicity
The Chimeric antigen receptor (CAR) of receptor NKp44 signal structure.
The second object of the present invention is to provide the application of the Chimeric antigen receptor.
The third object of the present invention is to provide a kind of signal transduction structural domain of Chimeric antigen receptor.
The fourth object of the present invention is to provide the application of the signal transduction structural domain.
Details are as follows for the specific technical solution of above-mentioned purpose of the invention:
A kind of Chimeric antigen receptor (CAR) comprising: antigen-binding domains (scfv) and signal transduction structural domain,
Middle signal transduction structural domain includes the first conducting structure domain and the second conducting structure domain, and the first conducting structure domain and the second conduction are tied
It connects between structure domain antigen-binding domains.
Concatenated first conducting structure domain, antigen-binding domains and second pass in Chimeric antigen receptor of the present invention
Transduction domain becomes the multichain form that can transmit activation signal after antigen-binding domains molecule of the antigen binding, will swash
Signal living is transferred to the immunocyte for expressing it, realizes the effect of immunization therapy.
The Chimeric antigen receptor is multichained construction Chimeric antigen receptor, is conducted with the first conducting structure domain and second
Structural domain forms the signal transduction structural domain of CAR, and antigen-binding domains may specifically bind the ligand into target and lure
The activation of immunocyte is led, immune response is generated.
Second conducting structure of the present invention domain is DAP12, and the second conducting structure domain passes through T2A and antigen-binding domains
Series connection.
DAP12 of the present invention is a transmembrane domain, is widely present in from natural killer cells, granulocyte, list
Core/Macrophage Surface, for transmitting activation signals, nucleotide sequence is as shown in SEQ ID NO.1, and amino acid sequence is such as
Shown in SEQ ID NO.2.
T2A of the present invention is for the second conducting structure domain and the antigen-binding domains of connecting, and nucleotide sequence is such as
Shown in SEQ ID NO.3, amino acid sequence is as shown in SEQ ID NO.4.
First conducting structure of the present invention domain can for truncated or not truncated NKp44, NKp46, NKp30 or
NKG2D, wherein the nucleotide sequence of NKp44 full length gene is shown in that NCBI, GenBank:AJ225109.1, amino acid sequence are shown in
NCBI, GenBank:CAB39168.1；The nucleotide sequence of NKp46 full length gene is shown in NCBI, Accession:NM_
004829.6, amino acid sequence is shown in NCBI, Accession:NP_004820；The nucleotide sequence of NKp30 full length gene is shown in
NCBI, Accession:NM_147130.2, amino acid sequence are shown in NCBI, Accession:NP_667341.1；NKG2D gene
The nucleotide sequence of overall length is shown in that NCBI, Accession:AF461811, amino acid sequence are shown in NCBI, GenBank:
AAL65233.1.Described truncated NKp44, NKp46, NKp30 or NKG2D indicate the C-terminal interception of its full length amino acid sequence
Amino acid sequence.
In order to improve the safety and curative effect of Chimeric antigen receptor of the present invention, the present invention also provides a kind of preferred the
One conducting structure domain is selected from truncated NKp44 amino acid sequence, is named as NKp44cut, NKp44 of the present inventioncutFor
NKp44 full length amino acid sequence is close to the polypeptide of 100~160 amino acid of C-terminal, more preferably NKp44 full length amino acid sequence
Close to 120~160 amino acid of C-terminal polypeptide, further preferably NKp44 full length sequence amino acid sequence close to C-terminal 140~
The polypeptide of 160 amino acid, perhaps with foregoing polypeptides sequence have 80% or more identity nucleotide sequence also or with this
Sequence has the nucleotide sequence of 85% or more identity, also or with the sequence has the nucleotides sequence of 90% or more identity
Column also or with the sequence have the nucleotide sequence of 95% or more identity.
It is NKp44 full length amino acid sequence close to C-terminal 160 the present invention also provides a kind of preferred first conducting structure domain
The polypeptide of a amino acid, amino acid sequence is as shown in SEQ ID NO.8, and nucleotide sequence is as shown in SEQ ID NO.7.
Antigen-binding domains of the present invention can carry out this field conventional selection according to different tumor targets.
More specifically, Chimeric antigen receptor of the present invention is DAP12, T2A, antigen-binding domains and first conduct
Structural domain is connected sequentially through 2-10 arbitrary amino acid.The sequence and number of 2-10 arbitrary amino acid of the invention
Chimeric antigen receptor effect described herein is had not significant impact, can be arbitrary 2-10 amino acid sequences.
Such as retroviral vector can be used by encoding chimeric antigen receptor in Chimeric antigen receptor of the present invention
Nucleic acid be transferred in immunocyte such as T cell, when Chimeric antigen receptor combination target antigen, the first conducting structure domain and antigen
Binding structural domain, which separates, to be formed activation signal and is transferred to its immunocyte of expression.
The present invention also provides a kind of immunocytes with the Chimeric antigen receptor.
The present invention also provides the Chimeric antigen receptor or the immunocyte with the Chimeric antigen receptor is exempted from tumour
Application in epidemic disease treatment.
The present invention provides a kind of signal transduction structural domain, including the first conducting structure domain and the second conducting structure domain.
First conducting structure of the present invention domain and the second conducting structure domain are specifically bound in antigen-binding domains
Activation signal is transferred to its immunocyte of expression by the multichain form for becoming can to transmit activation signal after antigen, and realization is exempted from
The effect of epidemic disease treatment.
Second conducting structure of the present invention domain is DAP12.DAP12 of the present invention is a transmembrane domain, extensively
It is general to be present in from natural killer cells, granulocyte, Monocytes/Macrophages surface, for transmitting activation signals, nucleotide sequence
As shown in SEQ ID NO.1, amino acid sequence is as shown in SEQ ID NO.2.
First conducting structure of the present invention domain can for truncated or not truncated NKp44, NKp46, NKp30 or
NKG2D, wherein the nucleotide sequence of NKp44 full length gene is shown in that NCBI, GenBank:AJ225109.1, amino acid sequence are shown in
NCBI, GenBank:CAB39168.1；The nucleotide sequence of NKp46 full length gene is shown in NCBI, Accession:NM_
004829.6, amino acid sequence is shown in NCBI, Accession:NP_004820；The nucleotide sequence of NKp30 full length gene is shown in
NCBI, Accession:NM_147130.2, amino acid sequence are shown in NCBI, Accession:NP_667341.1；NKG2D gene
The nucleotide sequence of overall length is shown in that NCBI, Accession:AF461811, amino acid sequence are shown in NCBI, GenBank:
AAL65233.1.Described truncated NKp44, NKp46, NKp30 or NKG2D indicate to intercept from the C-terminal of its full length amino acid sequence
Amino acid sequence.
In order to improve the safety and curative effect that signal transduction structural domain of the present invention is formed by Chimeric antigen receptor, the present invention
A kind of preferred first conducting structure domain is also provided, truncated NKp44 amino acid sequence is selected from, is named as NKp44cut, this hair
The bright NKp44cutIt is NKp44 full length amino acid sequence close to the polypeptide of 100~160 amino acid of C-terminal, more preferably
NKp44 full length amino acid sequence is close to the polypeptide of 120~160 amino acid of C-terminal, further preferably NKp44 full length sequence ammonia
Base acid sequence or with foregoing polypeptides sequence has 80% or more identity close to the polypeptide of 140~160 amino acid of C-terminal
Nucleotide sequence also perhaps with the sequence there is the nucleotide sequence of 85% or more identity to have also or with the sequence
The nucleotide sequence of 90% or more identity also or with the sequence has the nucleotide sequence of 95% or more identity.
It is NKp44 full length amino acid sequence close to C-terminal 160 the present invention also provides a kind of preferred first conducting structure domain
When the polypeptide of a amino acid, amino acid sequence is as shown in SEQ ID NO.8, and nucleotide sequence is as shown in SEQ ID NO.7.
Signal transduction structural domain of the present invention combines the first conducting structure domain to form CAR with transmembrane receptor DAP12
Signal transduction structural domain, the ligand in target is specifically combined as CAR, the activation of immunocyte can be induced, generate
Immune response.
The present invention also provides application of the signal transduction structural domain in Chimeric antigen receptor or immunotherapy of tumors.
Close C-terminal of the present invention refers to the polypeptide intercepted since first amino acid of C section, such as close to C section 100~
The polypeptide of 160 amino acid is indicated since first amino acid of C section until any one amino acid in the 100th~160
Polypeptide.
Beneficial effects of the present invention:
(1) secretion cytokine levels are extremely low when CAR structure according to the present invention is by antigenic stimulus, can be well
Guarantee that clinical application safety, i.e. clinical application safety are higher；
(2) CAR structure according to the present invention confirms its significant curative effect to entity tumor by external functional experiment, because
This present invention can be applied not only to the treatment of neoplastic hematologic disorder, and can expand application of the CAR-T in treatment of solid tumors；
(3) CAR structure exo-antigen positive tumor cell killing ability according to the present invention is stronger, and anti-tumor activity is more
It is good.
Detailed description of the invention
Fig. 1 is the slow virus carrier containing unlike signal domain structure；
Fig. 2 is that MSLN4CAR-T cell infection slow virus is identified after 7 days by flow cytomery T cell surface
The positive expression rate of the CAR structure of MSLN antigen；
Fig. 3 is cell proliferative conditions after CAR-T cell infection slow virus；
Fig. 4 is the secretion situation of MSLN4CAR-T cell IL-2 under antigenic stimulus；
Fig. 5 is the secretion situation of MSLN4CAR-T cell IFN-γ under antigenic stimulus；
Fig. 6 is different groups of CAR-T IL-2, the secretion situation of IFN-γ under antigenic stimulus；
Fig. 7 is different groups of CAR-T to antigen positive tumor cell line lethal effect.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.It is not specified in embodiment specific
The experimental method of condition, usually according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker etc. write)
Described in condition or according to the normal condition proposed by manufacturer.Test material as used in the following examples, such as without special theory
It is bright, it is to be commercially available from routine biochemistry reagent shop.
Embodiment 1 contains DAP12-T2A-scFv-NKp44cutThe building of the CAR slow virus of this 4 elements
To prove to contain DAP12-NKp44cutThe CAR-T cell in intracellular signal domain is relatively in the past reported to contain 4-1BB-
The CAR-T cell of CD3 ζ, DAP12-KIRS2 and list DAP12 stimulus signal is more advantageous, it is therefore desirable to which building is not containing respectively
With the viral vectors of stimulus signal combination.It with the single-chain antibody for targeting people's mesothelin (MSLN) is unified extracellular in the present embodiment
The structure for identifying antigen, is respectively necessary for constructing following 5 Chimeric antigen receptors (Fig. 1):
MSLN(scfv)-CD8α-4-1BB-CD3ζ (MSLN1)
DAP12-T2A-MSLN(scfv) (MSLN2)
DAP12-T2A-MSLN(scfv)-KIRS2 (MSLN3)
DAP12-T2A-MSLN(scfv)-NKp44cut (MSLN4)
DAP12-T2A-MSLN(scfv)-NKp44wt (MSLN5)
1, the gene order of the Chimeric antigen receptor of targeting people mesothelin of the synthesis containing different stimulus signals intracellular
Synthesis successively contains the single-chain antibody scfv of natural kill activated receptor (abbreviation DAP12), T2A, anti-human mesothelin
(abbreviation MSLN (scfv)), nature cell toxin receptor (abbreviation NKp44wt), truncated nature cell toxin receptor is (referred to as
NKp44cut), structure is as shown in Figure 1.Wherein the nucleotide sequence of DAP12 is as shown in SEQ ID NO.1, and amino acid sequence is such as
Shown in SEQ ID NO.2, the nucleotide sequence of T2A as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4,
The nucleotide sequence of single-chain antibody (MSLN) scfv of anti-human mesothelin is as shown in SEQ ID NO.5, amino acid sequence such as SEQ
Shown in ID NO.6, the nucleotide sequence of NKp44 full length gene is shown in that NCBI, GenBank:AJ225109.1, amino acid sequence are shown in
NCBI, GenBank:CAB39168.1, NKp44cutNucleotide sequence as shown in SEQ ID NO.7, amino acid sequence such as SEQ
Shown in ID NO.8, the nucleotide and amino acid sequence of KIRS2 is shown in patent (for adoptive immunotherapy with Chimerical receptor
Target cytotoxic cell, publication number: CN107580628A), the nucleotide sequence and amino acid sequence of CD8 α -4-1BB-CD3 ζ
Column are shown in patent (Methods for treatment of cancer, US 20130309258A1).
2, the slow virus carrier of building expression Chimeric antigen receptor
PELNS Dap12-T2A-MSLN-KIRS2 is saved by Nanjing Ka Ti medical science and technology Co., Ltd, or according to document
(Enxiu Wang et al.Generation of Potent T-cell Immunotherapy for Cancer Using
DAP12-Based,Multichain,Chimeric Immunoreceptors.2015,Cancer Immunology
Research, 3 (7): method disclosed in 815) is constructed, and truncates NKp44cutGene chemical synthesis is by Nanjing Jin Sirui biotechnology
Company synthesizes and provides pUC19-NKp44cutPlasmid, by plasmid pELNS Dap12-T2A-MSLN-KIRS2 and pUC19-
NKp44cutBy NheI, SalI double digestion (being purchased from Takara company), endonuclease reaction by specification is carried out, and obtains pELNS
Dap12-T2A-MSLN segment about 8900bp, truncated NKp44cutThe DNA fragmentation of segment about 483bp, then uses QIAquick Gel Extraction Kit
(Takara company) carries out DNA fragmentation recycling, and specific method is shown in specification, recycle acquisition pELNS Dap12-T2A-MSLN and
NKp44cutGene, then by target fragment NKp44cutPass through T4 ligase with carrier segments pELNS Dap12-T2A-MSLN
(being purchased from Takara company) is attached, and obtains the slow virus carrier of expression Chimeric antigen receptor, is named as pELNS Dap12-
T2A-MSLN-NKp44cut(abbreviation MSLN4).5 μ L slow virus carrier MSLN4 are converted in Escherichia coli TOP10 competent cell
(be purchased from Nanjing An Jieyou Biotechnology Co., Ltd), picking monoclonal after 37 DEG C of culture 16h, the monoclonal of picking is in 37 DEG C of items
Plasmid extraction kit (being purchased from Takara company) extracting plasmid is used after cultivating 12h under part, specific method is shown in specification.
Construct pELNS MSLN-CD8 α -4-1BB-CD3 ζ (abbreviation MSLN1) respectively according to the method described above；pELNS
Dap12-T2A-MSLN (abbreviation MSLN2)；PELNS Dap12-T2A-MSLN-KIRS2 (abbreviation MSLN3)； pELNS Dap12-
T2A-MSLN-NKp44cut(abbreviation MSLN4)；pELNS Dap12-T2A-MSLN-NKp44wt(abbreviation MSLN5) slow virus carries
Body.
3, slow virus is packed
The present embodiment packs slow virus and uses calcium phosphate method, the specific steps are as follows:
(1) 293T cell passes on every other day
Each T150 cell bottle plantation 5 × 106A cell.After 48 hours, cell number should reach million/bottle of 20-25.
(2) 293T cell spreads bottle
A) by taking 1 T150 Tissue Culture Flask as an example, cell is gently washed twice with 1 × PBS of about 15ml
B) 3ml0.25% pancreatin -2.21mM EDTA is added
C) cell detachment is waited until, the DMEM culture medium that 12ml 10% (wt) FBS (being purchased from Gibico) is added (is purchased from
Corning) into the cell to have fallen off
D) it collects and cell is transferred to sterile centrifugation tube, 1000rpm is centrifuged 10 minutes
E) supernatant is sopped up, precipitating is resuspended in the DMEM culture solution of 10ml 10% (wt) FBS.
F) cell count calculates 12 × 10 according to cell concentration6Volume required for a cell
G) the DMEM culture solution of cell and 10% (wt) FBS of 25ml are merged, is put into T150 cell bottle, jog makes
It obtains cell and is evenly distributed to 37 DEG C of cell bottle bottom, overnight incubation in 5%CO2 incubator.
(3) cell transfecting
Cell is observed, cell density reaches about 80%-90%, can start to transfect at this time
A) 30-60 minutes before transfection, culture solution is softly sopped up.
B) it mixes Plasmid DNA and calcium chloride solution needs 28ug pRSV.rev (to be purchased from for one T150 bottles
Invitrogen company), 28ug pGAG-Pol (is purchased from Invitrogen company), and 11ug pVSVG (is purchased from Invitrogen
Company), 23ug recombinant slow virus expression plasmid pELNS Dap12-T2A-MSLN-NKp44cut, it is molten to be added to 1.5ml calcium chloride
In liquid, mix.
C) 1.5ml BBS solution is added in 15ml sterile centrifugation tube, DNA- calcium chloride solution is mixed with 1ml pipette tips
After be added drop-wise in BBS solution, rapidly mix 15-20 under, be incubated at room temperature 25-30 minutes.
D) with 5ml pipette DNA- calcium chloride-BBS mixture (being purchased from the green skies Bioisystech Co., Ltd in Shanghai)
Uniformly it is added dropwise in T150 bottles.It is cultivated in 37 DEG C of cell incubators containing 5% carbon dioxide, 6h changes liquid.
E) liquid is changed after 6h.Culture plate is shaked gently for several times with some calcium phosphate precipitations that sufficiently suspend, and it is heavy to suck phosphoric acid calcium
The culture solution in shallow lake is added the DMEM culture solution of 5% fresh (wt) FBS of 20ml, continues to cultivate.
(4) viral supernatants are collected for the first time
A) the 293T cells and supernatant that the previous day transfects is collected into centrifuge tube, 1000rpm is centrifuged 5 minutes, label, temporarily
It is stored in 4 DEG C of refrigerators.
B) the DMEM culture medium of 20ml 5% (wt) FBS preheated in advance is added in cell bottle, 37 DEG C of cell incubators
Continue overnight incubation.
(5) vial supernatant (48h/ the 4th day) is collected second.
(6) supernatant is filtered
The supernatant collected twice is concentrated in together, removes cell fragment with 0.45 μm of membrane filtration.
(7) viral concentration
4 DEG C, 12000-24000rpm centrifuged overnight
(8) virus storage
After centrifugation, whole supernatants are toppled over, the DMEM culture medium that fresh 5% (wt) FBS is added is resuspended, viral packing is carried out,
It is spare to deposit in -80 DEG C of refrigerators rapidly
(9) slow virus titer determination
A) virus infection 293T cell
293T cell is spread into 24 orifice plates before infection, takes and has purified 200 μ L of concentrating virus and be added in 293T cell, for 24 hours
Liquid is changed with the DMEM culture medium containing 10%FBS (wt) afterwards, is centrifuged 5min under the conditions of 1200r/min after infecting 72h to collect carefully
Born of the same parents extract genome.
B) genome is extracted
Genome extraction agent box is to operate purchased from Takara company by kit specification
C) qPCR measures virus titer
Reaction system is as follows: 12.5 μ L of Probe qPCR Mix (is purchased from Takara), and 0.5 μ L of upstream primer is (by Nanjing gold
Si Rui synthesis), 0.5 μ L of downstream primer (being synthesized by Nanjing Jin Sirui), 1 μ L of probe (being synthesized by Nanjing Jin Sirui), 2 μ L of template,
8.5 μ L of aqua sterilisa, reaction system are 25 μ L, and reaction condition is arranged to specifications, after reaction, with analysis software number
According to according to standard curve calculating virus titer.Calculated result shows that virus titer is 1 × 106TU/ml。
Embodiment 2, virus infection T cell
1, the separation activation and virus infection of T cell
(1) separation of human peripheral blood mononuclear cell
Peripheral blood about 10ml, room temperature (18-25 DEG C) natural subsidence about 30min are acquired with the heparin tube containing anti-coagulants, is received
Collect upper plasma, the upper plasma of collection is centrifuged 10min under the conditions of 5000r/min, 1:1 is added to lymphocyte by volume
In separating liquid (being purchased from the ocean Tianjin Hao biological products science and technology limited Company), gradient centrifugation, 3000r/min, centrifugation
30min, after centrifugation, centrifuge tube is by upper lower leaf: first layer is plasma layer；The second layer is lymphocyte tunica albuginea layer；Third layer
For transparent separation liquid layer；4th layer of red blood cell layer.Draw lymphocyte tunica albuginea layer, and washed 2 times with PBS, be centrifuged twice with
1500r/min is centrifuged 10min, and cell is resuspended in PBS, and it is complete that 5% autologous plasma+300IU/ml recombinant human il-2+KBM581 is added
Culture medium culture human peripheral blood mononuclear cell.
(2) slow-virus infection T lymphocyte
With containing the 5% freshly prepd single core of autologous plasma+300IU/ml recombinant human il-2's+KBM581 complete medium culture
Cell PBMC, IL-2 are purchased from R&D Systems, and KBM581 is purchased from Corning, and addition CD3/CD28 Dynabeads exempts within the 0th day
The corresponding slow virus of 0.25MOI is added in epidemic disease magnetic bead (being purchased from invitrogen) activating T cell, progress slow-virus infection in first 3 days
Culture medium is changed to containing 5% autologous plasma+300IU/ after 48h as blank control by carrier, the T lymphocyte being uninfected by
Ml recombinant human il-2's+KBM581 complete medium continues culture 7-9 days.
2, in T cell CAR positive rate detection
By the T cell of the virus infection of culture to the 7th day, 1200r/min is centrifuged 5min, and it is thin to collect to abandon supernatant to the greatest extent
Born of the same parents are resuspended cell with the PBS solution containing volume fraction 1%FBS, and are 1 × 10 by cell adjustment density5Life is added in a/ml
Object element marks sheep anti mouse F (ab)2(Jackson ImmunoResearch company), adds Streptavidin-PE (BD
Biosciences company), 4 DEG C of incubation 15min, PBS solution is washed 2 times, and flow cytometer is detected, and is passed through as the result is shown
Culture in 7 days is crossed, the positive rate of CAR-T cell CAR: MSLN1 infection group and viral infection group positive rate 41%, MSLN2 infection group and viral infection group sun
Property rate 52%, MSLN3 infection group and viral infection group positive rate 59%, MSLN4 infection group and viral infection group positive rate 61% (Fig. 2), MSLN5 virus
Infected group positive rate 49%.
The influence of embodiment 3, virus infection CAR-T cell by cell proliferation
After the complete T cell of each group virus infection, by T cell 5% autologous plasma+300IU/ml recombined human containing volume fraction
IL-2+KBM581 complete medium counts primary for every 1-2 days.Then T lymphocyte growing state is observed, as a result such as Fig. 3 institute
Show.The result shows that cell is still capable of forming typical proliferating clones group, by cell after the virus of infection expression CAR
Counted, draw the visible infection MSLN4CAR-T cell Proliferation of cell Proliferation curve and MSLN1, MSLN2, MSLN3,
MSLN5CAR-T proliferation is similar, slightly weaker than the T cell of uninfecting virus (NTD in Fig. 3) proliferative capacity.
Embodiment 4, the cytokine secretion for detecting virus infection CAR-T cell
(1) method that cytokines measurement uses Elisa is carried out using R&D company kit.
(2) dilution of standard items: preparing 1ml centrifuge tube 7, and first standard is added in each centrifuge tube in number consecutively number
Then 500 μ L of product dilution takes 500 μ L of original content standard items to be added to 1 in the centrifuge tube for the number of finishing, mixes well, then
It takes 500 μ L to be added in second centrifuge tube in the centrifuge tube, mixes well；Take 500 μ L that third is added in the centrifuge tube again
In centrifuge tube, mix well；It takes 500 μ L to be added in the 4th centrifuge tube in the centrifuge tube again, mixes well；Again this from
It takes 500 μ L to be added in the 5th centrifuge tube in heart pipe, mixes well；Take 500 μ L that the 6th centrifugation is added in the centrifuge tube again
Guan Zhong is mixed well；It takes 500 μ L to be added in the 7th centrifuge tube in the centrifuge tube again, mixes well.
(3) it is marked with quasi- sample wells on enzyme mark coating plate, sequentially adds standard items 100 the μ L, each concentration 2- of various concentration
3 parallel holes.
(4) be loaded: blank well is respectively set, and (blank control wells are replaced with water, the antibody of enzyme marking reagent and biotin labeling
Operation is as before), sample to be tested hole, 100 μ L of product is first loaded in sample to be tested hole on enzyme mark coating plate, sample is added on enzyme by sample-adding
Target hole bottom, does not touch hole wall as far as possible, shakes gently mixing
(5) it is incubated for: being placed at room temperature for and be incubated for 2h
(6) it washs: discarding liquid, dry, every hole adds 200 μ L cleaning solutions, discards after static 30s, be so repeated 3 times, and claps
It is dry
(7) add antibody: 100 μ L detection antibody being added on enzyme mark coating plate
(8) it is incubated for: biconditional operation (5)
(9) it washs: biconditional operation (6)
(10) label: 100 μ L horseradish peroxidase-labeled Streptavidins are added in every hole
(11) it is incubated for: being protected from light and be placed at room temperature for incubation 20min
(12) it washs: biconditional operation (6)
(13) develop the color: 100 μ L of developing solution is added in every hole, and gently concussion mixes, and is protected from light and is placed at room temperature for incubation 20min
(14) terminate: 50 μ L of terminate liquid is added in every hole, terminates reaction
(15) measure: with blank value school zero, 450nm wavelength sequentially measures the absorbance (OD value) in each hole, measures Ying Jia
Enter and is carried out in 15min after terminate liquid.
Select that antigenic expression has discrepant target cell and MSLN4CAR-T is co-cultured, detection MSLN4CAR-T by
Response effect secretion IL-2 is generated to antigenic stimulus and IFN-γ is horizontal, and target cell selects OVCAR3 (MSLN high expression), SKOV3
(MSLN low expression), 293T (MSLN negative) show that MSLN4CAR-T institute when by MSLN antigenic stimulus is special with this
Property secret out of IL-2 and IFN-γ, as a result reflect that MSLN4CAR has discrepant target cell for antigenic expression and produces
Different response effects.The CAR-T of MSLN4 when MSLN high expression target cell OVCAR3 is co-cultured significant secretion of gamma-IFN and
IL-2 (Fig. 4 and Fig. 5) shows that MSLN4CAR has response effect for the tumour cell of antigen positive.
On the other hand, when comparing MSLN1, MSLN2, MSLN3, MSLN4, MSLN5CAR-T and OVCAR3 co-cultivation, each group
Secrete IL-2 and IFN-γ cytokine levels.As a result as shown in fig. 6, MSLN4CAR-T secretion level compared with MSLN2 and
MSLN5CAR-T group is obviously improved and (since MSLN2CAR-T antitumous effect is very weak, therefore does not have secrete cytokines), and compared with
MSLN1 and MSLN3 secretory volume has a degree of decline.Cell in vitro cytokine secretion the result shows that MSLN4CAR-T and
MSLN5CAR-T generates lower level cell factor, may improve clinical application safety.
Embodiment 5, the assessment of virus infection CAR-T cells in vitro fragmentation effect
(1) respectively cultivate target cell OVCAR3 cell (MSLN overexpression cell line), (MSLN low expression is thin for SKOV3 cell
Born of the same parents strain), 293T (strain of MSLN negative cells) and effector cell's MSLN1, MSLN2, MSLN3, MSLN4, MSLN5 group CAR-T it is thin
Born of the same parents.
(2) target cell and effector cell are collected, 1500rpm/min is centrifuged 5min, abandons supernatant
(3) target cell and effector cell is resuspended with 10%FBS+1640 complete medium
(4) real-time cell analysis system (RTCA) is utilized, in aerial addition 50 μ L, 1640 culture medium of E-Plate16
(5) baseline is detected using RTCA, determines that selected holes contact is normal
(6) setting effect target ratio is 0:1,1:1,5:1,10:1
(7) E-Plate16 is taken out, according to effect target ratio, uniformly mixed 100 μ L of target cell suspension is added in every hole, makes
Every hole kind cell number is 104cells/100μL。
(8) E-Plate16 is placed in incubator, with 37 DEG C, under the conditions of 5%CO2, is placed overnight
(9) second days, E-Plate16 is taken out, the 50 corresponding effector cells of μ L are added, calculated and effector cell 8h is added
Killing rate afterwards.
(10)
Testing result is as shown in Figure 7.The CAR-T of MSLN4 is substantially better than the effect of MSLN antigen positive tumor cytotoxicity
Remaining four groups, especially it is apparently higher than MSLN2 and MSLN5 group.Fig. 6 and Fig. 7 result synthesis display, killing experiments in vitro result table
Bright truncated nature cell toxin receptor, i.e. NKp44cutMake the first signal transduction structural domain on the basis for improving CAR safety
On have very strong anti-tumor activity, the CAR signaling zone design is conducive to clinical application.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Nanjing Ka Ti medical science and technology Co., Ltd
<120>it carries the Chimeric antigen receptor of truncation or not truncated nature cell toxin receptor signal structure and its answers
With
<160> 8
<170> SIPOSequenceListing 1.0
<210> 2
<211> 339
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60
ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120
ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180
gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240
aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300
gtctacagcg acctcaacac acagaggccg tattacaaa 339
<210> 2
<211> 113
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu
1 5 10 15
Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp
20 25 30
Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met
35 40 45
Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu
50 55 60
Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg
65 70 75 80
Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly
85 90 95
Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr
100 105 110
Lys
<210> 3
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccct 57
<210> 4
<211> 19
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 5
<211> 720
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
caggtacaac tgcagcagtc tgggcctgag ctggagaagc ctggcgcttc agtgaagata 60
tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaagcagagc 120
catggaaaga gccttgagtg gattggactt attactcctt acaatggtgc ttctagctac 180
aaccagaagt tcaggggcaa ggccacatta actgtagaca agtcatccag cacagcctac 240
atggacctcc tcagtctgac atctgaagac tctgcagtct atttctgtgc aagggggggt 300
tacgacggga ggggttttga ctactggggc caagggacca cggtcaccgt ctcctcaggt 360
ggaggcggtt caggcggcgg tggctctagc ggtggtggat cggacatcga gctcactcag 420
tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagtgccagc 480
tcaagtgtaa gttacatgca ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540
atttatgaca catccaaact ggcttctgga gtcccaggtc gcttcagtgg cagtgggtct 600
ggaaactctt actctctcac aatcagcagc gtggaggctg aagatgatgc aacttattac 660
tgccagcagt ggagtaagca ccctctcacg tacggtgctg ggacaaagtt ggaaatcaaa 720
<210> 6
<211> 240
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe
50 55 60
Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile
130 135 140
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser
145 150 155 160
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser
165 170 175
Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
180 185 190
Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile
195 200 205
Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp
210 215 220
Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu Ile Lys
225 230 235 240
<210> 7
<211> 483
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
aactctgtct ctaagtccgt cagattctat ctggtggtat ctccagcctc tgcctccaca 60
cagaccccct ggactccccg cgacctggtc tcttcacaga cccagaccca gagctgtgtg 120
cctcccactg caggagccag acaagcccct gagtctccat ctaccatccc tgtcccttca 180
cagccacaga actccacgct ccgccctggc cctgcagccc ccattgccct ggtgcctgtg 240
ttctgtggac tcctcgtagc caagagcctg gtgctgtcag ccctgctcgt ctggtggggg 300
gacatatggt ggaaaaccgt gatggagctc aggagcctgg atacccaaaa agccacctgc 360
caccttcaac aggtcacgga ccttccctgg acctcagttt cctcacctgt agagagagaa 420
atattatatc acactgttgc aaggactaag ataagcgatg atgatgatga acacactttg 480
tga 483
<210> 8
<211> 160
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Asn Ser Val Ser Lys Ser Val Arg Phe Tyr Leu Val Val Ser Pro Ala
1 5 10 15
Ser Ala Ser Thr Gln Thr Pro Trp Thr Pro Arg Asp Leu Val Ser Ser
20 25 30
Gln Thr Gln Thr Gln Ser Cys Val Pro Pro Thr Ala Gly Ala Arg Gln
35 40 45
Ala Pro Glu Ser Pro Ser Thr Ile Pro Val Pro Ser Gln Pro Gln Asn
50 55 60
Ser Thr Leu Arg Pro Gly Pro Ala Ala Pro Ile Ala Leu Val Pro Val
65 70 75 80
Phe Cys Gly Leu Leu Val Ala Lys Ser Leu Val Leu Ser Ala Leu Leu
85 90 95
Val Trp Trp Gly Asp Ile Trp Trp Lys Thr Val Met Glu Leu Arg Ser
100 105 110
Leu Asp Thr Gln Lys Ala Thr Cys His Leu Gln Gln Val Thr Asp Leu
115 120 125
Pro Trp Thr Ser Val Ser Ser Pro Val Glu Arg Glu Ile Leu Tyr His
130 135 140
Thr Val Ala Arg Thr Lys Ile Ser Asp Asp Asp Asp Glu His Thr Leu
145 150 155 160
Claims (7)
1. a kind of Chimeric antigen receptor, characterized by comprising: antigen-binding domains and signal transduction structural domain, wherein signal
Conducting structure domain includes the first conducting structure domain and the second conducting structure domain, the first conducting structure domain and the second conducting structure domain it
Between connect antigen-binding domains；Second conducting structure domain is DAP12, and the second conducting structure domain passes through T2A and antigen knot
Close structural domain series connection；The first conducting structure domain amino acid sequence is as shown in SEQ ID NO.8.
2. Chimeric antigen receptor according to claim 1, which is characterized in that concatenated in the Chimeric antigen receptor
One conducting structure domain, antigen-binding domains and the second conducting structure domain, after antigen-binding domains molecule of the antigen binding
Activation signal is transferred to its immunocyte of expression, realizes immunization therapy by the multichain form for becoming can to transmit activation signal
Effect.
3. Chimeric antigen receptor according to claim 1, which is characterized in that the DAP12 nucleotide sequence such as SEQ ID
Shown in NO.1, amino acid sequence is as shown in SEQ ID NO.2；The T2A nucleotide sequence is as shown in SEQ ID NO.3, amino
Acid sequence is as shown in SEQ ID NO.4.
4. Chimeric antigen receptor according to claim 1, which is characterized in that the first conducting structure domain nucleotides sequence
Column are as shown in SEQ ID NO.7.
5. Chimeric antigen receptor according to claim 1, which is characterized in that the Chimeric antigen receptor be DAP12,
T2A, antigen-binding domains and the first conducting structure domain are connected sequentially through 2-10 arbitrary amino acid.
6. having the immunocyte of Chimeric antigen receptor described in claim 1.
7. the described in any item Chimeric antigen receptors of Claims 1 to 5 or immunocyte as claimed in claim 6 are used in preparation
Application in immunotherapy of tumors drug.
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| CN109734814A (en) * | 2019-02-12 | 2019-05-10 | 南京卡提医学科技有限公司 | The purposes of engineering T cell treating cancer with immunity receptor |
| CN111850013B (en) * | 2019-06-25 | 2021-05-18 | 浙江康佰裕生物科技有限公司 | Chimeric antigen receptor with synergistic co-stimulation receptor and application thereof |
| WO2022083590A1 (en) * | 2020-10-19 | 2022-04-28 | 南京卡提医学科技有限公司 | Chimeric receptor containing dap 12 and co-stimulatory signal molecule signal domain, and method for using same |
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Non-Patent Citations (3)
| Title |
|---|
| "Co-expression of mFat-1 and pig IGF-1 genes by recombinant plasmids in modified chitosan nanoparticles and its synergistic effect on mouse immunity;Qi Xiong, et al.;《SCIENTIFIC REPORTS》;20171207;Recombinant plasmid construction |
| "Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors;Enxiu Wang, et al.;《Cancer Immunology Research》;20150731;第3卷(第7期);摘要，第818页图1A-1B,第815页右栏最后1段-第816页左栏第1、3段,第816页左栏最后1段-右栏第1 段,第817-824页Results |
| Accession No：AJ225109.1，Homo sapiens mRNA for NKp44 NK cell activating receptor;Cantoni,C.，et al.;《Genbank database》;20081007;FEATURES和ORIGIN |
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