CN108864307A - The Chimeric antigen receptor of signal peptide optimization targeting CD19, the T cell and preparation method and application for expressing the Chimeric antigen receptor - Google Patents
The Chimeric antigen receptor of signal peptide optimization targeting CD19, the T cell and preparation method and application for expressing the Chimeric antigen receptor Download PDFInfo
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Abstract
The invention discloses the Chimeric antigen receptors that a kind of signal peptide optimizes targeting CD19,Express the T cell and preparation method and application of the Chimeric antigen receptor,The Chimeric antigen receptor of the signal peptide optimization targeting CD19 includes the transmembrane protein signal peptide of sequential series,The single-stranded variable region of anti-CD 19 antibodies,The hinge area of CD8 protein molecular,Transmembrane region,4-1BB costimulation structural domain and CD3 ζ intracellular signal transduction structural domain,Wherein,The amino acid sequence of the transmembrane protein signal peptide is as shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3,The amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID No.11 or SEQ ID No.12 or SEQ ID No.13,The Chimeric antigen receptor of targeting CD19 is more efficient in T cell,Stablize expression,Preferably to play the lethal effect to target cell.
Description
Technical field
The present invention relates to immunologys and technical field of molecular biology, optimize especially with regard to a kind of signal peptide and target
The Chimeric antigen receptor of CD19, the T cell and preparation method and application for expressing the Chimeric antigen receptor.
Background technique
Adoptive immunity cell therapy (adoptive cell therapy, ACT) refers to living from tumor patient separating immune
Property cell, after carrying out amplification in vitro with Function Identification, then feed back again to patient, thus direct killing tumour or excitation body
Immune response killing tumor cell.
Immunization therapy (Immunotherapy) is the 4th kind of tumor therapeuticing method after operation, radiotherapy, chemotherapy.It is embedding
Antigen receptor (chimeric antigen receptor, CAR) the T cell technology of conjunction is that a kind of cell rapidly developed recently is exempted from
Epidemic disease treatment technology, CAR are an artificial synthesized fusion receptors, include extracellular antigen binding domain in structure, transmembrane region, intracellular
Signal transduction area and costimulatory signal area.Extracellular region is the variable region of mab sequence for identifying tumor associated antigen
(single chain variable fragment, ScFV), transmembrane region connect extracellular region and intracellular region, and common cross-film is distinguished
Son has CD3, CD4, CD8 and CD28 etc., and intracellular region has immunoreceptor tyrosine activating motif (ITAM), and it is thin that the most commonly used is T
Born of the same parents' receptor TCR/CD3 ζ chain and immunoglobulin FC receptor FC ε RI γ, are mainly responsible for signal transduction functionality；People pass through molecule gram
Grand method recombinates said elements in vitro, forms recombinant plasmid, will recombinate matter by means such as viral vectors, electroporations
Grain is transduceed into T cell, followed by culture amplification in vitro, finally feeds back to the purpose for playing treatment tumour in patient body, this warp
The T cell for crossing gene modification and transformation is known as CAR-T cell.CAR-T cell identifies and combines tumour antigen by ScFV sections,
T cell is activated by CD3 ζ chain and costimulatory signal area again, the CAR-T cell recognition being transformed in this way, killing tumor cell just have
There are two major features：1. targeting；2. it is restricted to overcome MHC, that is, MHC is eliminated the reliance on, therefore breaches tumour cell and pass through
It lowers the expression of MHC and this problem of immunologic escape occurs.
In view of the advantage of CAR-T technology, technology development in recent years is swift and violent.On August 31st, 2017, Novartis (Novartis)
The breakthrough CAR-T cell therapy Kymriah (tisagenlecleucel, former name CTL019) of company obtains FDA approval listing,
This is first CAR-T cell therapy in human history, and indication is 25 years old or less of B-lineage Acute Lymphocyte Leukemia (B-ALL)
Patient.On October 18th, 2017, U.S. FDA have approved the CAR-T therapy Yescarta (axicabtagene of Kite Pharma
Ciloleucel it) lists, indication is certain types of non-Hodgkin lymphoma.
Signal peptide refers to a bit of polypeptide for guiding newly synthesized peptide chain to cross over cell membrane, generally by 15-30 amino acid
Residue composition, positioned at the N-terminal of new synthesis peptide chain.It is not only able to guide secretory protein or memebrane protein goes out born of the same parents, moreover it is possible to pilot protein matter
Different zones are properly positioned in the cell.Signal peptide plays an important role in the expression of foreign protein, selects suitable letter
Number peptide can promote the expression of recombinant protein.
Chimeric antigen receptor is transmembrane protein, anti-dependent on extracellular ScFV section identification target after it is expressed in T cell
It is former.There is document report signal peptide that can influence the expression of CAR, Seogkyoung etc. is during constructing IL13R CAR-T
It was found that the expression quantity of CAR is 78.8%, using hIL3 signal peptide as CAR using the signal peptide of VHCAMP as the signal peptide of CAR
Signal peptide, the expression quantity of CAR is 14.9%, illustrates that the selection of signal peptide is most important to the building of CAR.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The purpose of the present invention is to provide a kind of signal peptides to optimize the Chimeric antigen receptor of targeting CD19, expresses the inosculating antibody
The T cell and preparation method and application of original receptor enable to the Chimeric antigen receptor of targeting CD19 more to increase in T cell
Effect stablizes expression, preferably to play the lethal effect to target cell.
To achieve the above object, described the present invention provides the Chimeric antigen receptor that a kind of signal peptide optimizes targeting CD19
Chimeric antigen receptor includes the hinge of the single-stranded variable region of transmembrane protein signal peptide, anti-CD 19 antibodies of sequential series, CD8 protein molecular
Sequence, transmembrane region, 4-1BB costimulation structural domain and CD3 ζ intracellular signal transduction structural domain, wherein the Chimeric antigen receptor
The end N- is connected with transmembrane protein signal peptide with the heavy chain variable region of the single-stranded variable region of anti-CD 19 antibodies, the transmembrane protein signal peptide
Amino acid sequence as shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3, the amino of the Chimeric antigen receptor
Acid sequence is as shown in SEQ ID No.11 or SEQ ID No.12 or SEQ ID No.13, wherein separately includes transmembrane protein letter
The amino acid sequence of the Chimeric antigen receptor of number peptide amino acid sequence SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3
It is corresponding in turn in SEQ ID No.11, SEQ ID No.12 and SEQ ID No.13, the i.e. amino acid in transmembrane protein signal peptide
When sequence is as shown in SEQ ID No.1, the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID No.11；In cross-film
When the amino acid sequence of protein signal peptide is as shown in SEQ ID No.2, the amino acid sequence of the Chimeric antigen receptor such as SEQ
Shown in ID No.12；When the amino acid sequence of transmembrane protein signal peptide is as shown in SEQ ID No.3, the Chimeric antigen receptor
Amino acid sequence as shown in SEQ ID No.13.
The present invention has chosen above-mentioned three kinds of transmembrane protein signal peptides (CD8 signal peptide (SEQ ID No.1), GM-CSFR signal
Peptide (SEQ ID No.2) and light chain immunoglobulin К signal peptide (SEQ ID No.3)), to the antigen receptor sequence of targeting CD19
Column are optimized, so that CD19 CAR (that is, antigen receptor of targeting CD19) in T cell more express by stability and high efficiency, into
And preferably play the lethal effect to target cell.
In a preferred embodiment, the amino acid sequence of the light chain variable region of the single-stranded variable region of the anti-CD 19 antibodies
As shown in SEQ ID No.4, bonding pad of the amino acid sequence of heavy chain variable region as shown in SEQ ID No.5, between light and weight chain
Amino acid sequence as shown in SEQ ID No.6；The amino acid sequence of the hinge area of the CD8 protein molecular such as SEQ ID
Shown in No.7.
In a preferred embodiment, the amino acid sequence of the transmembrane region of the Chimeric antigen receptor such as SEQ ID
Shown in No.8, the amino acid sequence of the 4-1BB costimulation structural domain is as shown in SEQ ID No.9, the CD3 ζ intracellular signal
The amino acid sequence in conducting structure domain is as shown in SEQ ID No.10.
The present invention also provides the T cells for expressing above-mentioned Chimeric antigen receptor.
The present invention also provides the preparation methods of above-mentioned T cell, include the following steps：(1) chimeric antigen of CD19 is targeted
The acquisition of receptor slow virus carrier；(2) preparation and titration of the Chimeric antigen receptor slow virus of CD19 are targeted；(3) in turn
With the Chimeric antigen receptor slow-virus infection T cell for the targeting CD19 that step (2) prepares, the inosculating antibody of CD19 is targeted with completion
The preparation of original receptor T cell.
In a preferred embodiment, it is described targeting CD19 Chimeric antigen receptor slow virus carrier acquisition include with
Lower step：(1) gene order of the Chimeric antigen receptor of synthesis targeting CD19：Synthesize transmembrane protein signal peptide, anti-CD 19 antibodies
Single-stranded variable region, the hinge area of CD8 protein molecular, transmembrane region, 4-1BB costimulation structural domain and CD3 ζ intracellular signal transduction structure
The gene order in domain；(2) slow virus carrier of building expression Chimeric antigen receptor.
In a preferred embodiment, the gene order of Chimeric antigen receptor described in above-mentioned steps (1) such as SEQ ID
Shown in No.22 or SEQ ID No.23 or SEQ ID No.24.
In a preferred embodiment, the Chimeric antigen receptor slow virus preparation of the targeting CD19 and titration packet
Include following steps：(1) the 0th day bed board：0.25% pancreatin digests 293T cell, is layered on containing 10ml DMEM (containing 10%FBS)
10cm culture dish in, in 37 DEG C, 5%CO2Constant incubator culture, cell confluency degree reaches 90-95% after guaranteeing for 24 hours；(2)
293T cell transfecting：Transfection first 2 hours, 293T cell is changed into liquid, by the Chimeric antigen receptor slow virus carrier for targeting CD19 with
And assistant carrier is mixed with PEI, piping and druming uniformly, is stored at room temperature 13-17min, obtains DNA/PEI mixture, by what is prepared
DNA/PEI mixture is added dropwise in 293T cell, is continued culture 4-6 hours, is changed liquid；(3) target CD19 chimeric antigen by
The preparation of body slow virus：The cell conditioned medium of above-mentioned 293T cell transfecting 24 hours with 48 hours, 0.45 μM of filter filtering are collected respectively
Afterwards, 20,000rpm, 4 DEG C, ultracentrifugation is precipitated with PBS lytic virus, and -80 DEG C freeze viral pellet, for use；(4) slow virus
Titration：293T cell is laid on 24 orifice plates, 1 × 105/ hole, respectively by 0.25 μ L, 0.5 μ L, 1 μ L, 3 μ L, 10 μ L slow virus
Suspension infects 293T cell, after 48 hours, collects cell, streaming immunofluorescence dyeing, according to formula 105× [GFP positive cell
Number/viral volume (ml) × 1/100], calculate CAR-T19 lentivirus titers.
In a preferred embodiment, the Chimeric antigen receptor slow-virus infection T cell of CD19 will be targeted, to complete target
Include the following steps to the preparation of the Chimeric antigen receptor T cell of CD19：(1) blood is gone into centrifuge tube, with isometric 1640
Basal medium dilutes 2 times, is uniformly mixed；(2) 4ml Ficoll lymphocyte separation medium is transferred to new centrifuge tube, by 2
Times good blood of the above-mentioned dilution of volume is gently added to above Ficoll lymphocyte separation medium；(3) room temperature, horizontal centrifugal 15-
25min；(4) suction pipe gentle aspiration tunica albuginea layer, i.e. buffy coat are used, after being washed three times with PBS, is transferred to Tissue Culture Dish,
IL-2, the CD3 antibody of 1 μ g/ml and the CD28 antibody of 1 μ g/ml of 300U/ml is added, activates 24-48h；(5) CD19 will be targeted
The slow virus of Chimeric antigen receptor be added in cell, liquid is changed after 6-10h, continues culture 2-4 days；(6) streaming immunofluorescence contaminates
Color detects the expression of CAR 19, with clear efficiency of infection, if efficiency of infection illustrates CAR-T cell transduction 20% or more
Success continues culture 7-10 days, obtains the CAR-T cell quantity of enough feedbacks, successfully completes targeting CD19 Chimeric antigen receptor T
Cell preparation.
Above-mentioned Chimeric antigen receptor expresses the T cell of the Chimeric antigen receptor in preparation treatment hematologic malignancies or leaching
The drug of bar tumor or the application in preparation.
Compared with prior art, the present invention has the advantages that：
T cell is a kind of cell of more difficult transduction, although the appearance of lentiviruses transduction technology overcomes to a certain extent
This problem, but the T cell due to tumour patient and normal human T cells' state are different, so thin in gene editing modification T
In the immunotherapy of tumors of born of the same parents, however it remains because individual difference difference, the low situation of certain patient's T cell transduction efficiencies.And
One of the key of Chimeric antigen receptor T cell treatment seeks to a Chimeric antigen receptor T cell Successful transductions and enters T cell, and
It is required that it is stable, lasting in T cell surface expression, so that the antigen binding with target cell surface, plays killing target cell effect.
So optimizing from different perspectives, realization Chimeric antigen receptor is an extremely important problem in the expression of stablizing of T cell.
In the present invention, CD19 Chimeric antigen receptor sequence is after signal peptide optimizes, expressed in T cell it is more stable, efficient, such as
Shown in Fig. 1 streaming immunofluorescence dyeing, after optimizing signal peptide, the expression efficiency of CAR19 has obtained largely improving, maximum
1.37 times can be improved, so as to play more good therapeutic effect to CD19 positive B-cells leukaemia and lymthoma.
Detailed description of the invention
Fig. 1 is expression of the CD19 CAR in T cell according to an embodiment of the present invention；
Fig. 2 is lethal effect of the CD19 CAR according to an embodiment of the present invention to target cell.
Specific embodiment
With reference to the accompanying drawing, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention
Shield range is not limited by the specific implementation.
Unless otherwise explicitly stated, otherwise in entire disclosure and claims, term " includes " or its change
Changing such as "comprising" or " including " etc. will be understood to comprise stated element or component, and not exclude other members
Part or other component parts.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, it is unless otherwise specified, commercially commercially available.
Embodiment 1 targets the acquisition of the Chimeric antigen receptor slow virus carrier of CD19
(1) gene order of the Chimeric antigen receptor of synthesis targeting CD19：Synthesize transmembrane protein signal peptide, anti-CD 19 antibodies
Single-stranded variable region, the hinge area of CD8 protein molecular, transmembrane region, 4-1BB costimulation structural domain and CD3 ζ intracellular signal transduction structure
The gene order in domain, and add EcoRI restriction enzyme site at the end 5', add SalI restriction enzyme site at the end 3'；Wherein transmembrane protein signal peptide
Nucleotide sequence as shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, the anti-CD 19 antibodies are single-stranded can
Become the nucleotide sequence in area as shown in SEQ ID NO.17, the nucleotide sequence such as SEQ of the hinge area of the CD8 protein molecular
Shown in ID NO.18, the nucleotide sequence of the transmembrane region such as SEQ ID NO.19 shows, the core of the 4-1BB costimulation structural domain
Nucleotide sequence is as shown in SEQ ID NO.20, the nucleotide sequence such as SEQ ID of the CD3 ζ intracellular signal transduction structural domain
Shown in NO.21, the nucleotide sequence of the Chimeric antigen receptor of the anti-CD19 finally synthesized such as SEQ ID NO.22, SEQ ID
Shown in NO.23, SEQ ID NO.24；
(2) enter slow virus carrier with EcoRI and two restriction enzyme site digestion rear clones of SalI, sequencing is tested in sequencing, identification
Correct slow virus carrier transformed competence colibacillus cell Stbl3 is demonstrate,proved, carries out a large amount of of plasmid with the big extraction reagent kit of endotoxin-free plasmid
Extracting carries out plasmid using spectrophotometer and quantifies, then dispenses, -20 DEG C freeze.
Embodiment 2 targets the preparation and titration of the Chimeric antigen receptor slow virus of CD19
Material：293T cell is cultivated in the DMEM culture medium containing 10%FBS, consumptive material include DMEM (Hyclone,
SH30022.01), X-VIVO 15 (Lonza, 04-418Q), FBS (Gibcol, P30-3302), pancreatin (Gibco, 25200-
056), PBS (Hyclone, SH30256.01), PEI (polyethyleneimine) (24765-2), the 19 staining kit (Shanghai CAR-T
Offshore Biotechnology Co., Ltd), detailed process is as follows：
(1) the 0th day bed board：0.25% pancreatin digests 293T cell, is layered on containing 10ml DMEM's (containing 10%FBS)
In 10cm culture dish, 37 degree, 5%CO2Constant incubator culture, cell confluency degree reaches 90-95% after guaranteeing 24.
(2) 293T cell transfecting：First 2 hours of transfection, 293T cell changes liquid.CD19 Chimeric antigen receptor slow virus will be targeted
Carrier and assistant carrier are mixed with PEI, and piping and druming uniformly, is stored at room temperature 15min, obtains DNA/PEI mixture.By what is prepared
DNA/PEI mixture is added dropwise in 293 cells, is continued culture 4-6 hours, is changed liquid.
(3) the Chimeric antigen receptor slow virus preparation of CD19 is targeted：Collect respectively above-mentioned 293T cell transfecting 24 hours with
48 hours cell conditioned mediums, after 0.45 μM of filter filtering, 20,000rpm, 4 degree, ultracentrifugation is precipitated with PBS lytic virus ,-
80 DEG C freeze viral pellet, for use.
(4) lentivirus titers measure：293T cell is laid on 24 orifice plates, 1 × 105/ hole, respectively by 0.25 μ L, 0.5 μ L, 1
μ L, 3 μ L, 10 μ L slow virus suspensions infect 293T cell, after 48 hours, collect cell, streaming immunofluorescence dyeing, according to formula
105× [GFP positive cell number/virus volume (ml) × 1/100] calculates CAR-T19 lentivirus titers, sick slowly to CAR-T19
Toxic effect valence reaches 8 × 108/ ml can carry out next step test.
Embodiment 3 targets the Chimeric antigen receptor T cell preparation of CD19
15 milliliters of centrifuge tubes are added in 4 milliliters of room temperature Ficoll lymphocyte separation mediums, with 10 milliliters of pipettes by 8 milliliters
Diluted peripheral blood PBMC is added to above lymphocyte separation medium 1640 culture mediums in equal volume, 2000rpm, horizontal, room temperature, centrifugation
20 minutes, with 3 milliliters of suction pipe gentle aspiration tunica albuginea cellular layers, it is transferred to 15 milliliters of new centrifuge tubes, with 10 times of volumes
PBS is washed 3 times, and cell is resuspended with X-VIVO15 culture medium, carries out cell count, and adjustment cell concentration is 1 × 106/ milliliter is drawn
2 milliliters of lymphocyte suspensions, are transferred to 6 orifice plates, and the CD28 of the IL-2 of 300U/ml, the CD3 antibody of 1 μ g/ml and 1 μ g/ml is added
Antibody, activating T cell 24-48h, after T cell activation, the slow virus prepared infects T cell, infection with the ratio of Moi1.25
After 72 hours, streaming immunofluorescence dyeing is carried out using 19 staining kit of CAR-T, analyzes the efficiency of infection of CAR-T 19, point
It is most strong, most stable to analyse the CAR expression containing which kind of signal peptide, if efficiency of infection illustrates CAR-T cell transduction 20% or more
Success continues culture amplification 7-10 days, obtains the CAR-T cell quantity of enough feedbacks, successfully completes the inosculating antibody of targeting CD19
The preparation of original receptor T cell.As a result as shown in Figure 1, the CD19 of CD8 signal peptide, immunoglobulin signal peptide and GM-CSFR signal peptide
Chimeric antigen receptor is transduceed after T cell, and positive rate is respectively 22.1%, 27.9%, 20.3%, this result illustrates the excellent of signal peptide
CD19 Chimeric antigen receptor can be improved in the expression of T cell in change.
CD19 Chimeric antigen receptor T cell cytotoxicity functional verification.Using eFluor670 to CD19 positive target cell
NALM6 carries out viable cell labelling, by the CAR-T cell prepared and target cell with 0.5:1,1:1,3:1,6:1 effect target is than altogether
Culture after 4 hours, collects cell, and 7-AAD is dyed, 7-AAD positive cell in flow cytometer showed eFluor670 positive cell group
Ratio judges CAR-T to the killing-efficiency of target cell, as a result as shown in Fig. 2, CD19 Chimeric antigen receptor T cell and target cell
It is common to be incubated for 5 hours, in effect target than 0.5:Under conditions of 1, target cell apoptosis rate is 15% or so, imitates target than 3:1 and 6：1 item
Under part, target cell apoptosis rate is respectively 31% and 29%, it can be seen that with the raising of effect target ratio, target cell apoptosis rate liter
Height illustrates that killing rate is improving.CD19 Chimeric antigen receptor T cell and target cell are incubated for 15 hours jointly, imitate target than 0.5:1,
3:1,6:1 corresponding target cell apoptosis rate is respectively 58%, 63% and 59%, this illustrates chimeric antigen prepared by the present invention
Recipient T cells just have dose dependent and time dependence to the killing of target cell in a certain range, also reflect from side
Its specificity.
Present invention optimizes CD19 Chimeric antigen receptor T cell sequence, the CAR-T cell prepared using this sequence, CD19
The expression of CAR is higher, more stable, can preferably kill CD19 positive target cell, has extensive prospect in oncotherapy.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Sequence table
<110>Beijing multi-win Time Technology Co., Ltd
<120>The Chimeric antigen receptor of signal peptide optimization targeting CD19, the T cell for expressing the Chimeric antigen receptor and preparation side
Method and application
<130> P181069DD1F
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213>CD8 signal peptide (Homo sapiens)
<400> 1
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 2
<211> 22
<212> PRT
<213>GM-CSFR signal peptide (Homo sapiens)
<400> 2
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Pro Leu Leu Ile Pro
20
<210> 3
<211> 20
<212> PRT
<213>Light chain immunoglobulin К signal peptide (Homo sapiens)
<400> 3
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
20
<210> 4
<211> 107
<212> PRT
<213>CAR19 ScFV light chain variable region (Homo sapiens)
<400> 4
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 5
<211> 120
<212> PRT
<213>CAR ScFV heavy chain variable region (Homo sapiens)
<400> 5
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 6
<211> 15
<212> PRT
<213> Gly-Ser linker（Homo sapiens）
<400> 6
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 7
<211> 45
<212> PRT
<213>CD8 protein molecular hinge area (Homo sapiens)
<400> 7
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> 8
<211> 24
<212> PRT
<213>Transmembrane region (Homo sapiens)
<400> 8
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 9
<211> 42
<212> PRT
<213>4-1BB costimulatory molecules structural domain (Homo sapiens)
<400> 9
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 10
<211> 112
<212> PRT
<213>CD3 ζ intracellular signal transduction structural domain (Homo sapiens)
<400> 10
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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> 11
<211> 486
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 11
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
20 25 30
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
50 55 60
Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro
65 70 75 80
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
85 90 95
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
145 150 155 160
Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly
165 170 175
Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly
180 185 190
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser
195 200 205
Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
210 215 220
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys
225 230 235 240
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
245 250 255
Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
340 345 350
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
Gln Ala Leu Pro Pro Arg
485
<210> 12
<211> 487
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 12
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Pro Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
145 150 155 160
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
165 170 175
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
180 185 190
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
195 200 205
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
210 215 220
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
225 230 235 240
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
245 250 255
Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
420 425 430
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
450 455 460
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
465 470 475 480
Met Gln Ala Leu Pro Pro Arg
485
<210> 13
<211> 485
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 13
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser
20 25 30
Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp
35 40 45
Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val
50 55 60
Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser
85 90 95
Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn
100 105 110
Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
130 135 140
Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu
145 150 155 160
Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val
165 170 175
Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val
180 185 190
Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg
195 200 205
Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met
210 215 220
Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His
225 230 235 240
Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
245 250 255
Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
260 265 270
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
275 280 285
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
290 295 300
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
305 310 315 320
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
325 330 335
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
340 345 350
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
355 360 365
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
370 375 380
Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
385 390 395 400
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
405 410 415
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
420 425 430
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
435 440 445
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
450 455 460
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
465 470 475 480
Ala Leu Pro Pro Arg
485
<210> 14
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60
ccc 63
<210> 15
<211> 66
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ccccctgctg 60
atcccc 66
<210> 16
<211> 60
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60
<210> 17
<211> 726
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720
tcctca 726
<210> 18
<211> 135
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 19
<211> 72
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 20
<211> 126
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 21
<211> 336
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 22
<211> 1458
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccagg 60
cccgacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120
accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180
ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240
tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300
caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360
ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420
ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480
ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540
cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600
tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660
gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720
cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780
gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960
gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140
agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440
caggccctgc cccctcgc 1458
<210> 23
<211> 1461
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ccccctgctg 60
atccccgaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120
gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180
aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240
ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300
gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360
ggagggggga ccaagctgga gatcacaggt ggcggtggct cgggcggtgg tgggtcgggt 420
ggcggcggat ctgaggtgaa actgcaggag tcaggacctg gcctggtggc gccctcacag 480
agcctgtccg tcacatgcac tgtctcaggg gtctcattac ccgactatgg tgtaagctgg 540
attcgccagc ctccacgaaa gggtctggag tggctgggag taatatgggg tagtgaaacc 600
acatactata attcagctct caaatccaga ctgaccatca tcaaggacaa ctccaagagc 660
caagttttct taaaaatgaa cagtctgcaa actgatgaca cagccattta ctactgtgcc 720
aaacattatt actacggtgg tagctatgct atggactact ggggccaagg aacctcagtc 780
accgtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960
ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1020
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1080
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1140
aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1200
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440
atgcaggccc tgccccctcg c 1461
<210> 24
<211> 1455
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 120
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 180
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 240
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 300
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 360
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 420
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 480
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 540
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 600
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 660
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 720
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 780
tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960
cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1020
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1080
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1140
gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1200
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440
gccctgcccc ctcgc 1455
Claims (10)
1. a kind of Chimeric antigen receptor of signal peptide optimization targeting CD19, which is characterized in that the Chimeric antigen receptor includes suitable
The concatenated transmembrane protein signal peptide of sequence, the single-stranded variable region of anti-CD 19 antibodies, the hinge area of CD8 protein molecular, transmembrane region, 4-1BB
Costimulation structural domain and CD3 ζ intracellular signal transduction structural domain, wherein believed with transmembrane protein at the end N- of the Chimeric antigen receptor
Number peptide is connected with the heavy chain variable region of the single-stranded variable region of anti-CD 19 antibodies, and the amino acid sequence of the transmembrane protein signal peptide is such as
Shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3, the amino acid sequence of the Chimeric antigen receptor such as SEQ ID
Shown in No.11 or SEQ ID No.12 or SEQ ID No.13, wherein separately include transmembrane protein signal peptide amino acid sequence
The amino acid sequence of the Chimeric antigen receptor of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 is corresponding in turn in SEQ
ID No.11, SEQ ID No.12 and SEQ ID No.13.
2. Chimeric antigen receptor as described in claim 1, which is characterized in that the light chain of the single-stranded variable region of anti-CD 19 antibodies
The amino acid sequence of variable region as shown in SEQ ID No.4, the amino acid sequence of heavy chain variable region as shown in SEQ ID No.5,
The amino acid sequence of bonding pad between light and weight chain is as shown in SEQ ID No.6；The amino of the hinge area of the CD8 protein molecular
Acid sequence is as shown in SEQ ID No.7.
3. Chimeric antigen receptor as described in claim 1, which is characterized in that the amino of the transmembrane region of the Chimeric antigen receptor
Acid sequence is as shown in SEQ ID No.8, and the amino acid sequence of the 4-1BB costimulation structural domain is as shown in SEQ ID No.9, institute
The amino acid sequence of CD3 ζ intracellular signal transduction structural domain is stated as shown in SEQ ID No.10.
4. expressing the T cell of the described in any item Chimeric antigen receptors of claim 1-3.
5. the preparation method of T cell as claimed in claim 4, which is characterized in that include the following steps：
(1) acquisition of the Chimeric antigen receptor slow virus carrier of CD19 is targeted；
(2) preparation of the Chimeric antigen receptor slow virus of CD19 is targeted；
(3) the Chimeric antigen receptor slow-virus infection T cell of the targeting CD19 prepared with step (2), to complete targeting CD19
Chimeric antigen receptor T cell preparation.
6. the preparation method of T cell as claimed in claim 5, which is characterized in that the Chimeric antigen receptor of the targeting CD19
Slow virus carrier is included the following steps：
(1) gene order of the Chimeric antigen receptor of synthesis targeting CD19：It is single-stranded to synthesize transmembrane protein signal peptide, anti-CD 19 antibodies
Variable region, the hinge area of CD8 protein molecular, transmembrane region, 4-1BB costimulation structural domain and CD3 ζ intracellular signal transduction structural domain
Gene order；
(2) slow virus carrier of building expression Chimeric antigen receptor.
7. the preparation method of T cell as claimed in claim 6, which is characterized in that Chimeric antigen receptor described in step (1)
Gene order is as shown in SEQ ID No.22 or SEQ ID No.23 or SEQ ID No.24.
8. the preparation method of T cell as claimed in claim 6, which is characterized in that the Chimeric antigen receptor of the targeting CD19
Slow virus preparation includes the following steps：
(1) bed board：0.25% pancreatin digests 293T cell, is layered in the culture dish containing DMEM, in 37 DEG C, 5%CO2Constant temperature training
Case culture is supported, cell confluency degree reaches 90-95% after guaranteeing for 24 hours；
(2) 293T cell transfecting：First 2 hours of transfection, changes liquid for 293T cell, will target the Chimeric antigen receptor slow virus of CD19
Carrier and assistant carrier are mixed with PEI, and piping and druming uniformly, is stored at room temperature 13-17min, obtains DNA/PEI mixture, will prepare
Good DNA/PEI mixture is added dropwise in 293T cell, is continued culture 4-6 hours, is changed liquid；
(3) the Chimeric antigen receptor slow virus preparation of CD19 is targeted：Above-mentioned 293T cell transfecting 24 hours and 48 small is collected respectively
When cell conditioned medium, after being filtered with filter, ultracentrifugation, with PBS lytic virus precipitate, -80 DEG C freeze viral pellet, for use.
9. the preparation method of T cell as claimed in claim 8, which is characterized in that the Chimeric antigen receptor for targeting CD19 is slow
Virus infection T cell is included the following steps with completing the Chimeric antigen receptor T cell preparation of targeting CD19：
(1) blood is gone into centrifuge tube, is diluted with 1640 isometric basal mediums, is uniformly mixed；
(2) lymphocyte separation medium is transferred to new centrifuge tube, 2 times of good blood of the above-mentioned dilution of volume are gently added to lymph
Above cell separating liquid；
(3) room temperature, horizontal centrifugal 15-25min；
(4) suction pipe gentle aspiration buffy coat is used, after being washed three times with PBS, Tissue Culture Dish is transferred to, adds human IL-2, CD3
Antibody and CD28 antibody activate 24-48h；
(5) slow virus for targeting the Chimeric antigen receptor of CD19 is added in cell, is incubated for 6-10h, changes liquid, continue to cultivate 2-4
It；
(6) expression of streaming immunofluorescence dyeing detection CAR 19, with clear efficiency of infection, if efficiency of infection 20% with
On, continue culture 7-10 days, then successfully completes the Chimeric antigen receptor T cell preparation of targeting CD19.
10. the described in any item Chimeric antigen receptors of claim 1-3 or expression Chimeric antigen receptor as claimed in claim 4
T cell preparation treatment hematologic malignancies or lymthoma drug or preparation in application.
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