CN108949695A - A kind of non-virus carrier co-expresses CAR-T cell construction and its application of IL18 - Google Patents
A kind of non-virus carrier co-expresses CAR-T cell construction and its application of IL18 Download PDFInfo
- Publication number
- CN108949695A CN108949695A CN201810922418.6A CN201810922418A CN108949695A CN 108949695 A CN108949695 A CN 108949695A CN 201810922418 A CN201810922418 A CN 201810922418A CN 108949695 A CN108949695 A CN 108949695A
- Authority
- CN
- China
- Prior art keywords
- cell
- car
- ser
- gly
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- 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
Abstract
The invention discloses a kind of preparation methods of CD19 specific chimeric antigen receptor T cell for co-expressing IL18.IL2 the and anti-CD3/CD28 magnetic bead antibody of given dose is added by the way that sleeping beauty's plasmid transposon system containing CD19 CAR and IL18 to be transfected into T cell in the present invention, to obtain the CD19 CAR-T cell of coexpression IL18.Resulting T cell can specifically killing tumor cell, while the IL18 expressed can dramatically increase the fragmentation effect of T cell.
Description
Technical field
The present invention relates to immunocyte preparation fields, in particular it relates to which a kind of IL-18 coexpression is special in CD19
Sex-mosaicism antigen receptor T cell and application thereof.
Technical background
Chimeric antigen receptor T cell treats (Chimeric antigen receptor T cell therapy, CAR-T
It therapy is) a kind of emerging immunotherapy of tumors, which be transformed by the T cell to patient's body, thin in T
The Chimeric antigen receptor (Chimeric antigen receptors, CARs) that a kind of tumour-specific is expressed on after birth, makes it
It can be by the pathway activation of tumour-specific, non-MHC molecule dependence, to play antitumor action.CAR be it is a kind of across
Membrane receptor is mainly made of extracellular identification structural domain, transmembrane region and activation structure domain intracellular, and extracellular identification structural domain is usually
The single-chain antibody (single-chain variable fragment, scFv) of tumour cell characteristic surface molecule, it is intracellular to swash
Inactivation domain is the CD3 ζ of T cell receptor (T cell receptor, TCR), when CAR extracellular single-chain antibody and tumour cell
Identification combines, and can activate CD3 ζ C, and then directly activate T cell.
So far three generations CAR has been developed, wherein second generation CAR is most study, most application prospect, and second generation CAR molecule exists
A T cell costimulatory molecules, such as CD28,4-1BB are also coupled between transmembrane region and activation structure domain, in costimulatory molecules
Synergistic effect under, CAR-T cells show go out more longlasting proliferation can for and stronger anti-tumor function.
In recent years, transformation CAR-T cell makes its express certain cell factors, can function, existence and differentiation to CD8+T cell have
Facilitation, these factors include IL-12, IL-15, IL-7 and IL21 etc., certain cell factor is such as coupled on CAR molecule such as
IL-15 co-expresses cell factor and CAR in T cell, and it is thin also to significantly improve the internal of T cell, survival in vitro and killing target
The ability of born of the same parents.Compared with exogenous cytokines, the cell factor of CAR-T cell coexpression acts only on tumor by local micro-loop
Border, the influence of no hemodilution, and will not normal tissue bring side effect.
Cell factor IL18 is a kind of protein with anti-tumor activity, and IL-18 may act on the effects such as T, B and NK cell
The proliferation and differentiation of cell promote the expression of IFN-γ and the release of Ke Limei, to play its anti-tumor function.IL-18 and
IL12 also can be done directly on CD8+T cell and NK cell, it is induced to generate more IFN-γ, release granzyme B, expression
FasL, thus further killing tumor cell.Meanwhile IL18 because its relatively less toxic characteristic and with the collaboration of traditional remedies
It acts on noticeable.
Currently, including viral vectors and non-virus carrier two ways by the mode of cellular integration into T cell.Wherein viral vectors
Although host cell gene group can be integrated into higher copy number, the preparation tool of viral vectors acquires a certain degree of difficulty, especially
Large scale preparation；Virus of waiting a moment also has the potential risk of safety.Sleeping beauty transposon stand (Sleeping Beaut, SB) is used as table
Simple up to non-virus carrier structure, operation is easy, and can be inserted into large fragment nucleic acid sequence, and to compare slow virus very low for cost.Have
Studies have shown that the gene modification T cell that SB is mediated can expand 2,200 to 2,500 times in vitro, CAR expression rate is up to 84%, not
It was found that illustrating the clinical application potentiality of the great CAR-T cell of SB transposons/transposase system with the presence of hot spot is integrated.
Summary of the invention
The technical problem to be solved by the present invention is to by transfecting sleeping beauty's plasmid transposon system containing CD19 CAR and IL18
Enter in T cell, while IL2 the and anti-CD3/CD28 magnetic bead antibody of given dose is added, to obtain coexpression IL18's
CD19 CAR-T cell, and prove its potential application in treatment of solid tumors.Specifically, the present invention provides a kind of coexpression
The construction method of the CD19 CAR-T cell of IL18, and assign the ability that T cell killing has CD19 target tumor cell.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
The first purpose of this invention is the Chimeric antigen receptor molecule IL18 CD19 CAR of building coexpression IL18 and CD19,
It includes signal peptide, antigen binding domain, hinge area, transmembrane region, irritates signal transduction area, CD3 ζ signal transduction area, T2A small peptide altogether
Area is expressed with IL18.
In order to advanced optimize above-mentioned CD19 specific chimeric antigen receptor, the anti-CD19 scFv antibody sequence is from this
Oneself disclosed FMC63 of field, the monoclonal antibody amino acid sequence is as shown in SEQ ID No.3.Further, described compared with sequence
For the region CD8hinge, amino acid sequence is as shown in SEQ ID NO.4.Further, transmembrane region described in the transmembrane region is
CD28 transmembrane region, amino acid sequence is as shown in SEQ ID N0.5.
Further, the costimulatory signal conducting region intracellular is CD28 and 4-1BB, amino acid sequence such as SEQ ID NO.6
It is shown.
Further, the intracellular signal area is T cell signal transduction active region CD3 ζ, amino acid sequence such as SEQ ID
Shown in NO.7.
Further, the intracellular cytokine coexpression signal is IL18, and amino acid sequence is as shown in SEQ ID NO.8.
Further, the structure based on the above CD19 CAR molecule, the Chimeric antigen receptor of the present invention are CMV-
CSF2RA-CD19 scFv-CD8 α-CD28-4-1BB-CD3 ζ-T2A-IL18 (RZ-FMC63-28bbZ-IL18), structure is such as
Shown in Fig. 1, nucleotide and amino acid sequence are as shown in SEQ ID No.1 and SEQ ID NO.2.
A second object of the present invention is to provide transposons recombinant plasmid/swivel base enzyme systems of CD19 CAR for co-expressing IL18 a kind of
System.
Further, the recombinant expression carrier be original recombinant expression carrier be sleeping beauty's transposon vector pT/SVNeo (with
Under be abbreviated as pT/S), map is shown in Fig. 2.After restriction enzyme Bgl II/Not I double digestion, with contain Bgl II/
The nucleic acid fragment of the CD19 CAR-IL18 of Not I double enzyme site is configured to recombination Transposon plasmid by DNA T4 ligase
pT/SVNeo-CMV-CD19 CAR-IL18。
Further, recombination transposase plasmids SB11 and recombination Transposon plasmid pT/SVNeo-CMV-CD19 CAR-IL18 are pressed
According to the ratio mixing of mass ratio 1:3, to obtain transposons recombinant plasmid/swivel base enzyme system of the CD19 CAR of coexpression IL18
System.
Third object of the present invention is to provide a kind of nucleic acid containing coexpression IL18 and CD19 specific chimeric antigen receptor
The host cell of recombinant expression carrier.
Further, the host cell is T cell or the cell mass containing T cell.
Further, by pT-CMV-CD19 CAR-IL18 transposons and auxiliary transposase SB11 and PEIpro cationic polymer
According to mass ratio 1:4 transfecting T cells, the CD19 CAR-T cell of the CD19 CAR-IL18 positive can get.
The invention further relates to a kind of construction methods of above-mentioned host cell comprising the construction step of recombinant expression carrier and general
The step of recombinant expression carrier is transduceed to host cell.
Finally, the present invention also provides a kind of above-mentioned coexpression IL18 and CD19 specific chimeric antigen receptor, above-mentioned coding CD19
The nucleic acid of specific chimeric antigen receptor, above-mentioned recombinant expression carrier and host cell answering in mammal tumor treatment
With.
Further, mammalian solid tumors include but is not limited to blood tumor, oophoroma tumor, breast cancer, endometrium
Cancer, melanoma, cancer of pancreas, lung cancer, neuroblastoma etc..
More specifically, above-mentioned application is a kind of CAR-T cell for co-expressing IL18 and CD19, pass through SB transposons/swivel base
Enzyme system electrotransfection T cell to impart T cell expression IL18 and identify the ability of CD19 molecule, and targets the CD19 positive
Mammalian solid tumors.
Detailed description of the invention
Fig. 1 is to co-express CD19 specific chimeric antigen receptor (CAR) molecular structure of IL18 to illustrate schematic diagram:
Fig. 2 illustrates schematic diagram for transposon vector pT/SVNeo structure:
Fig. 3 is expression rate of the FCM analysis CAR molecule in T cell；
Fig. 4 is expression of the IL18 gene in T cell；
Fig. 5 is that the cell subsets after T cell, CD19 CAR-T cell and IL18 CD19 CAR-T cell and target cell effect becomes
Change situation；
Fig. 6 is the lethal effect of T cell, CD19 CAR-T cell and IL18 CD19 CAR-T cell and target cell Daudi.
Specific implementation
The present invention provides a kind of sleeping beauty's plasmid transposon system electricity containing CD19 CAR and IL18 and is transferred in T cell, simultaneously plus
Enter IL2 the and anti-CD3/CD28 magnetic bead antibody of given dose, so that the T cell of coexpression IL18 and CD19 CAR is obtained, and
Prove its potential application in treatment of solid tumors.
The present invention provides the Chimeric antigen receptor molecule CD19 of building building coexpression IL18 and CD19 in embodiment one
The method of scFv-IL18.Embodiment two provides the method that CAR molecule is transfected into T.Embodiment three provides building coexpression
The CD19 CAR-T cell of IL18 and the ability of CAR-T cell killing tumour target cell.
With reference to the accompanying drawings and examples, further description of the specific embodiments of the present invention.Following embodiment is only used for
Clearly illustrate technical solution of the present invention, and not intended to limit the protection scope of the present invention.
Embodiment one
The method for present embodiments providing the Chimeric antigen receptor molecule CD19 CAR-IL18 of building coexpression IL18 and CD19
The building of CD19 antibody scFv: by gene chemical synthesis CD19 antibody FMC63 genetic fragment (Nanjing Jin Sirui), exist respectively
BamH I and Xho I restriction enzyme site is added in FMC63 upstream and downstream, and segment is built into pUC57 carrier；
FMC63-28bbZ vector construction: by the restricted interior digestion of BamH I and Xho I to existing carrier pCDH-CMV-
CSF2RA-Trop2scFv-CD8 α-CD28-4-1BB-CD3 ζ-T2A-CopGFP and pUC57-FMC63 carrier carries out double digestion,
Target endonuclease bamhi progress T4 ligase is attached reaction respectively, finally obtains pCDH-CMV-CSF2RA-FMC63scFv-
CopGFP then replace with IL18 by CD8 α-CD28-4-1BB-CD3 ζ-T2A-CopGFP by In-Fusion PCR
To pCDH-CMV-CSF2RA-FMC63scFv-CD8 α-CD28-4-1BB-CD3 ζ-T2A-IL18, abbreviation pCDH-CMV-CD19
CAR-IL18.It expands to obtain the CD19 with Bag II and Not I by Bag II upstream primer and Not I downstream primer
The segment is then linked into the pT2/SVNeo carrier of Bag II and Not I double digestion, to obtain by CAR-IL18 segment
pT2/SVNeo-CMV-CD 19 CAR-IL18。
Embodiment two
This example is the preparation of CD19 scFv-IL18 CAR-T cell
Sleeping beauty transposon stand recombinant plasmid/transposase system: it is extracted by the big extraction agent box of QIAGEN endotoxin-free plasmid
The transposase plasmids SB11 of 19 CAR-IL18 of pT2/SVNeo-CMV-CD and sleeping beauty, will recombination transposase plasmids SB11 with
It recombinates Transposon plasmid pT-CMV-CD19 CAR-IL18 to mix according to the ratio of mass ratio 1:3, to obtain coexpression IL18
With transposons recombinant plasmid/transposase system of CD19 scFv.T cell transduction: according to EasySeq kit (STEMCELL)
Specification separates T cell respectively, then by T cell in 37 DEG C of culture 3h, with T cell culture medium (SuperCultureTML100)
Viral supernatants are diluted 2 times with IL2, T cell is incubated overnight in the supernatant, uses SuperCulture again laterTML100 (contains
Have IL2) culture 48h after it is spare.
Co-express the preparation of the CD19 CAR-T cell of IL18: pT-CMV-CD19 CAR-IL18 transposons and auxiliary transposase
SB11 and PEIpro cationic polymer are mixed in the T cell culture medium of serum-free according to mass ratio 1:4
(SuperCultureTML100), 4 DEG C of placement 15-30min are changed to normal serum-free T after mixture transfecting T cells 4-6h
Cell culture medium detects the transduction efficiency of CAR molecule after 72h with the expression of Flow cytometry GFP, as shown in figure 3,
The positive rate of IL18-CD19 CAR T cell is about 50%；It co-cultures 8-10 days, changes a not good liquor within during which every 3 days, can be obtained
The CD19 CAR-T cell of the CD19 scFv-IL18 positive.It is thin to collect T cell, CD19 CAR-T cell, IL18 CD19 CAR T
Born of the same parents extract total mRNAA respectively, the expression of its cell factor IL18 is detected through PCR, the results show that in IL18 CD19
High expression IL18 in CAR-T cell, and almost without expression in other two kinds of T cells, it is consistent with experimental design, see Fig. 4.
Embodiment three
The present embodiment is lethal effect of the CD19 CAR-T cells in vitro to tumour cell for expressing IL18.
The density of Daudi, Nalm-6 tumour cell is adjusted to 2 × 10 in 24 porocyte culture plates5A/ml, to 24 orifice plates
(quantity is 1 × 10 to the middle CD19 scFv-IL18 CAR-T cell that 500 μ L preparation is added5It is a)；Similarly, by effector cell:
Target ration (effect: target ratio) mixes effector cell and target cell according to the ratio of 5:1,2.5:1,1:1,0.5:1,0:1,1:0
It closes uniformly (being shown in Table 1), and supplements complete medium to 1ml, be uniformly mixed normal culture 6h.It collects thin after each group co-cultures
Born of the same parents, the anti-CD19 fluorescent antibody staining of employment, through flow cytometry CD19+ positive cell percentage, with the initial target cell of each group
Percentage compares, and each group target cell killing rate is calculated.It can be with from flow cytometry scatter plot from flow cytometry scatterplot
Find out, after killing 6h, can also be detected in CD19 CAR-T cell and significantly divide group, and co-expresses IL18 CD19 CAR-T
In the co-culture system of cell, the ratio of CD19+ cell mass significantly reduces (see Fig. 5), and calculated result shows control group (CD19
CAR-T cell) to target cell killing rate 50%, and IL18 CD19 CAR-T cell killing efficiency is co-expressed up to 90% (see figure
6).The result shows that the CD19 CAR-T cell of IL18 expression has the ability of very strong killing target cell, both had very strong anti-
Tumour function.
The mixed proportion table of table 1 different effect cell and target cell
Sequence table
<110>Hangzhou Rong Ze Biotechnology Co., Ltd
<120>the CAR-T cell construction of non-virus carrier coexpression IL18 a kind of and its application
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2241
<212> DNA
<213>artificial synthesized (artificial synthesis)
<400> 1
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcgga 60
tccgatattg tgctgaccca gagccccaag ttcatgagca ccagcgtggg cgatagagtg 120
agcgtgacct gcaaggcaag ccagaacgtg ggaacaaacg tggcctggta ccaacagaaa 180
cccggccaaa gccctaagcc cctgatttac agcgccacct acagaaatag cggcgtgccc 240
gacagattta caggaagcgg cagcggaacc gatttcacac tgaccatcac caacgtgcag 300
agcaaagacc tggccgacta cttctgccag cagtacaaca gataccccta caccagcgga 360
ggaggaacaa agctggagat caagagaggt ggtggtggtt ctggcggcgg cggctccggt 420
ggtggtggtt ctcaagtgca actgcaacag agcggagccg aactggtgag acccggaagc 480
agcgtgaaga tcagctgcaa ggcttccggc tacgccttta gcagctactg gatgaactgg 540
gtgaagcaga gacctggaca gggactggaa tggatcggcc agatttaccc tggagacggc 600
gacacaaact acaacggcaa gttcaagggc caagctacac tgaccgccga caaaagcagc 660
agcaccgcct atatgcagct gagcggactg accagcgaag atagcgctgt gtacttctgc 720
gccagaaaga ccatcagcag cgtggtggac ttctacttcg actactgggg acaaggcacc 780
accctgacag tgagcagcga attcaccacg acgccagcgc cgcgaccacc aacaccggcg 840
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900
ggcgcagtgc acacgagggg gctggacttc gcctgtgatt tttgggtgct ggtggtggtt 960
ggtggagtcc tggcttgcta tagcttgcta gtaacagtgg cctttattat tttctgggtg 1020
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1080
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1140
tccaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1200
caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1260
tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 1320
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1380
agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1440
ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1500
ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1560
aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgcgaggg cagaggaagt 1620
cttctaacat gcggtgacgt ggaggagaat cccggcccta tggctgctga accagtagaa 1680
gacaattgca tcaactttgt ggcaatgaaa tttattgaca atacgcttta ctttatagct 1740
gaagatgatg aaaacctgga atcagattac tttggcaagc ttgaatctaa attatcagtc 1800
ataagaaatt tgaatgacca agttctcttc attgaccaag gaaatcggcc tctatttgaa 1860
gatatgactg attctgactg tagagataat gcaccccgga ccatatttat tataagtatg 1920
tataaagata gccagcctag aggtatggct gtaactatct ctgtgaagtg tgagaaaatt 1980
tcaactctct cctgtgagaa caaaattatt tcctttaagg aaatgaatcc tcctgataac 2040
atcaaggata caaaaagtga catcatattc tttcagagaa gtgtcccagg acatgataat 2100
aagatgcaat ttgaatcttc atcatacgaa ggatactttc tagcttgtga aaaagagaga 2160
gaccttttta aactcatttt gaaaaaagag gatgaattgg gggatagatc tataatgttc 2220
actgttcaaa acgaagacta g 2241
<210> 2
<211> 746
<212> PRT
<213>artificial synthesized (artificial synthesis)
<400> 2
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Lys Phe Met
20 25 30
Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln
35 40 45
Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
50 55 60
Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro
65 70 75 80
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr
100 105 110
Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
145 150 155 160
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
165 170 175
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
180 185 190
Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
195 200 205
Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
210 215 220
Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
225 230 235 240
Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp
245 250 255
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Glu Phe Thr Thr Thr Pro
260 265 270
Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
275 280 285
Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His
290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val
305 310 315 320
Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile
325 330 335
Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr
340 345 350
Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln
355 360 365
Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly
370 375 380
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
385 390 395 400
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
405 410 415
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
420 425 430
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
435 440 445
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
450 455 460
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
465 470 475 480
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
485 490 495
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
500 505 510
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
515 520 525
Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser Leu Leu Thr Cys
530 535 540
Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Ala Glu Pro Val Glu
545 550 555 560
Asp Asn Cys Ile Asn Phe Val Ala Met Lys Phe Ile Asp Asn Thr Leu
565 570 575
Tyr Phe Ile Ala Glu Asp Asp Glu Asn Leu Glu Ser Asp Tyr Phe Gly
580 585 590
Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp Gln Val
595 600 605
Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met Thr Asp
610 615 620
Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile Ser Met
625 630 635 640
Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser Val Lys
645 650 655
Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile Ser Phe
660 665 670
Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp Ile
675 680 685
Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met Gln Phe
690 695 700
Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys Glu Arg
705 710 715 720
Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly Asp Arg
725 730 735
Ser Ile Met Phe Thr Val Gln Asn Glu Asp
740 745
<210> 3
<211> 110
<212> PRT
<213>artificial synthesized (artificial synthesis)
<400> 3
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30
Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asp Asp Asn Lys Arg Pro Ser Gly Val Leu Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu
85 90 95
Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 4
<211> 45
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 4
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 5
<211> 16
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 5
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
1 5 10 15
<210> 6
<211> 96
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 6
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Glu Gly Gly
1 5 10 15
Cys Glu Leu Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg
20 25 30
Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp
35 40 45
Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
50 55 60
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
65 70 75 80
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
85 90 95
<210> 7
<211> 112
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 7
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 8
<211> 193
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 8
Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met
1 5 10 15
Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn
20 25 30
Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile
35 40 45
Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro
50 55 60
Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg
65 70 75 80
Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met
85 90 95
Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys
100 105 110
Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile
115 120 125
Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly
130 135 140
His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe
145 150 155 160
Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys
165 170 175
Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu
180 185 190
Asp
Claims (3)
1. a kind of preparation method for the CD19 specific chimeric antigen receptor T cell for co-expressing IL18, by the way that CD19 will be contained
Sleeping beauty's plasmid transposon system electricity of CAR and IL18 is transferred in T cell, so that the CD19 CAR-T for obtaining coexpression IL18 is thin
Born of the same parents.
2. sleeping beauty's plasmid transposon system according to claim 1, which is characterized in that its system include CD19 CAR and
Sleeping beauty's plasmid pT/SVNeo and SB11 plasmid of IL, it is characterised in that:
(1) CMV-CD19 CAR-IL18 is built into pT/SVNeo plasmid using Bgl II and Not I restriction enzyme site and forms weight
Group carrier pT-CMV-CD19 CAR-IL18；
(2) PEIpro cationic polymer transfection reagent transfection pT/SVNeo-CMV-CD19 CAR-IL18 transposons and auxiliary is utilized
Transposase SB11 is helped, PEIpro and total plasmid mass ratio are 4:1, transposase SB11 and transposons pT-CMV-CD19 CAR-IL18
Mass ratio is 1:3；
(3) IL2 and magnetic cell ratio that addition concentration is 30ng/ml to 120ng/ml in 1 day to 3 days after electrotransfection are 3:1
Anti-CD3/CD28 antibody magnetic bead；
(4) then, by the cell culture of activation 7-10 days.
3. CD19 CAR molecule according to claim 1, which is characterized in that including the extracellular antigen recognizing district of CD19, hinge
Area, transmembrane region, intracellular signal area, the area T2A and IL18 express area, nucleotide and amino acid sequence such as sequence SEQ ID No.1
With shown in SEQ ID No.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810922418.6A CN108949695A (en) | 2018-08-14 | 2018-08-14 | A kind of non-virus carrier co-expresses CAR-T cell construction and its application of IL18 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810922418.6A CN108949695A (en) | 2018-08-14 | 2018-08-14 | A kind of non-virus carrier co-expresses CAR-T cell construction and its application of IL18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108949695A true CN108949695A (en) | 2018-12-07 |
Family
ID=64469027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810922418.6A Withdrawn CN108949695A (en) | 2018-08-14 | 2018-08-14 | A kind of non-virus carrier co-expresses CAR-T cell construction and its application of IL18 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108949695A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022220648A1 (en) * | 2021-04-15 | 2022-10-20 | 주식회사 유틸렉스 | Hla-dr-specific chimeric antigen receptor, and use thereof |
| US11547728B2 (en) | 2019-12-17 | 2023-01-10 | Juventas Cell Therapy Ltd. | CD19-targeted chimeric antigen receptor and use thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016014530A1 (en) * | 2014-07-21 | 2016-01-28 | Novartis Ag | Combinations of low, immune enhancing. doses of mtor inhibitors and cars |
| CN105408473A (en) * | 2013-05-14 | 2016-03-16 | 得克萨斯州大学系统董事会 | Human application of engineered chimeric antigen receptor (CAR) T-cells |
| CN105624107A (en) * | 2015-09-21 | 2016-06-01 | 深圳市科晖瑞生物医药有限公司 | Expansion method of various lymphocyte subpopulations and application of expansion method |
| KR20170093254A (en) * | 2014-12-29 | 2017-08-14 | 노파르티스 아게 | Methods for producing chimeric antigen receptor-expressing cells |
| CN107075482A (en) * | 2014-04-07 | 2017-08-18 | 诺华股份有限公司 | Use anti-CD19 Chimeric antigen receptors treating cancer |
| WO2018126317A1 (en) * | 2017-01-05 | 2018-07-12 | Helix Biopharma Corporation | Vegfr-2 car immune cells to treat cancers |
| CN108624607A (en) * | 2017-03-16 | 2018-10-09 | 上海恒润达生生物科技有限公司 | Target the Chimeric antigen receptor of mesothelin and method and purposes to its dual modification |
| CN109055430A (en) * | 2018-08-14 | 2018-12-21 | 杭州荣泽生物科技有限公司 | A kind of preparation method for co-expressing IL18 and CCL19 albumen and targeting MUC1 gene C AR-T cell |
-
2018
- 2018-08-14 CN CN201810922418.6A patent/CN108949695A/en not_active Withdrawn
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105408473A (en) * | 2013-05-14 | 2016-03-16 | 得克萨斯州大学系统董事会 | Human application of engineered chimeric antigen receptor (CAR) T-cells |
| CN107075482A (en) * | 2014-04-07 | 2017-08-18 | 诺华股份有限公司 | Use anti-CD19 Chimeric antigen receptors treating cancer |
| WO2016014530A1 (en) * | 2014-07-21 | 2016-01-28 | Novartis Ag | Combinations of low, immune enhancing. doses of mtor inhibitors and cars |
| KR20170093254A (en) * | 2014-12-29 | 2017-08-14 | 노파르티스 아게 | Methods for producing chimeric antigen receptor-expressing cells |
| CN105624107A (en) * | 2015-09-21 | 2016-06-01 | 深圳市科晖瑞生物医药有限公司 | Expansion method of various lymphocyte subpopulations and application of expansion method |
| WO2018126317A1 (en) * | 2017-01-05 | 2018-07-12 | Helix Biopharma Corporation | Vegfr-2 car immune cells to treat cancers |
| CA3049276A1 (en) * | 2017-01-05 | 2018-07-12 | Helix Biopharma Corporation | Vegfr-2 car immune cells to treat cancers |
| CN108624607A (en) * | 2017-03-16 | 2018-10-09 | 上海恒润达生生物科技有限公司 | Target the Chimeric antigen receptor of mesothelin and method and purposes to its dual modification |
| CN109055430A (en) * | 2018-08-14 | 2018-12-21 | 杭州荣泽生物科技有限公司 | A kind of preparation method for co-expressing IL18 and CCL19 albumen and targeting MUC1 gene C AR-T cell |
Non-Patent Citations (3)
| Title |
|---|
| BILIANG HU 等: "Augmentation of Antitumor Immunity by Human and Mouse CAR-T Cel", 《CELL REPORTS》 * |
| 夏涌: "靶向间皮素的嵌合抗原受体修饰T细胞治疗卵巢癌实验研究", 《万方学位论文》 * |
| 胡雪菲: "共表达细胞因子IL-18和IL-21的嵌合抗原受体T细胞（CAR-T）的构建及其功能研究", 《中国优秀硕士学位论文全文数据库(电子期刊)医药卫生科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11547728B2 (en) | 2019-12-17 | 2023-01-10 | Juventas Cell Therapy Ltd. | CD19-targeted chimeric antigen receptor and use thereof |
| JP2023502190A (en) * | 2019-12-17 | 2023-01-23 | 合源生物科技(天津)有限公司 | CD19-targeted chimeric antigen receptor and uses thereof |
| JP2023502191A (en) * | 2019-12-17 | 2023-01-23 | 合源生物科技(天津)有限公司 | Combination of plasmids and their application in the preparation of engineered immune cells |
| JP7430202B2 (en) | 2019-12-17 | 2024-02-09 | 合源生物科技(天津)有限公司 | Combination of plasmids and their application in the preparation of modified immune cells |
| JP7439128B2 (en) | 2019-12-17 | 2024-02-27 | 合源生物科技(天津)有限公司 | CD19 targeting chimeric antigen receptor and its use |
| WO2022220648A1 (en) * | 2021-04-15 | 2022-10-20 | 주식회사 유틸렉스 | Hla-dr-specific chimeric antigen receptor, and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105177031B (en) | T cell of Chimeric antigen receptor modification and application thereof | |
| Zhang et al. | CAR-NK cells for cancer immunotherapy: from bench to bedside | |
| CN110872577B (en) | Modified immune cells and uses thereof | |
| JP2021192623A (en) | Switch costimulatory receptors | |
| Morgan et al. | Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma | |
| Wargo et al. | Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression | |
| Sun et al. | MHC class II restricted neoantigen: a promising target in tumor immunotherapy | |
| JP2022025097A (en) | Transposase polypeptides and uses thereof | |
| CN109803983B (en) | Specific chimeric antigen receptor T cell targeting NKG2DL, preparation method and application thereof | |
| Redmond et al. | Defects in the acquisition of CD8 T cell effector function after priming with tumor or soluble antigen can be overcome by the addition of an OX40 agonist | |
| EP3692057B9 (en) | Modified epidermal growth factor receptor peptides for use in genetically-modified cells | |
| JP2021525073A (en) | Fc Epsilon CAR | |
| CN109280086B (en) | Hypoxia-inducible chimeric antigen receptor specifically activated by tumor microenvironment | |
| CN113481165B (en) | CAR-T secreting bispecific T cell adaptors and uses for treating solid tumors | |
| CN113286874A (en) | Chimeric Antigen Receptor (CARs) compositions and methods of use | |
| CN111892661B (en) | Chimeric antigen receptor and application thereof in preparation of products for treating tumors | |
| CN113416260B (en) | Claudin18.2-targeted specific chimeric antigen receptor cell and preparation method and application thereof | |
| JP2021506262A (en) | NKG2D DARIC receptor | |
| JP2024036482A (en) | DARIC interleukin receptor | |
| CN112522208A (en) | Transgenic tumor infiltrating lymphocyte and application thereof | |
| CN108949695A (en) | A kind of non-virus carrier co-expresses CAR-T cell construction and its application of IL18 | |
| WO2021239020A1 (en) | Immunotherapy method for combining chimeric antigen receptor and type i interferon and application thereof | |
| JP2022500038A (en) | MR1 restricted T cell receptor for cancer immunotherapy | |
| CN111875708B (en) | Chimeric antigen receptor T lymphocyte and application thereof in preparation of product for treating solid tumor | |
| CN111205361B (en) | Interleukin 21 protein (IL21) mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20181207 |
|
| WW01 | Invention patent application withdrawn after publication |