CN110079504A - A kind of CAR-T cell containing unstable structure domain and preparation method thereof and adjust CAR-T cell function method - Google Patents
A kind of CAR-T cell containing unstable structure domain and preparation method thereof and adjust CAR-T cell function method Download PDFInfo
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Abstract
A kind of CAR-T cell containing unstable structure domain and preparation method thereof and adjust CAR-T cell function method, encoding gene containing unstable structure domain in CAR-T cell, and the encoding gene for removing the 3 ' ends and unstable structure domain of the CAR gene of terminator codon is directly connected to, and constitutes fusion；The fusion protein of above-mentioned fusion coding is expressed in CAR-T cell, which is CAR molecule, and C-terminal is unstable structure domain, is positioned at cell membrane.The preparation method of CAR-T cell containing unstable structure domain of the invention, process is few, and step is easily operated, is easy to implement industrialization；The method disclosed by the invention for adjusting the CAR-T cell function containing unstable structure domain, the quantity of the i.e. adjustable CAR molecule of the corresponding small molecule compound in unstable structure domain need to only be added, and the presence in unstable structure domain will not influence the function of CAR molecule itself.
Description
Technical field
The present invention relates to immunologys and technical field of cell biology, the CAR-T specifically containing unstable structure domain
Cell and preparation method thereof and adjusting CAR-T cell function method.
Background technique
In cellular immunotherapy field, CAR-T therapy has become research hotspot, and cardinal principle is through Chimeric antigen receptor
The T cell of modification, can specificity identification tumor associated antigen, make the targeting of T effector cell, killing activity and persistently
Property be above the immunocyte routinely applied, and tumor by local immunosupress microenvironment can be overcome and break host immune tolerance shape
State.Currently, going deep into research, people increasingly consider the safety issue in CAR-T application, i.e., how accurately to control
The activity of antigen caused by CAR-T cell (such as cytokine storm), and other side effects that may cause are reduced, to CAR-T
The activity of cell realizes precise controlling etc..
The Dimerized technology CID of chemical induction when small-molecule drug rimiducid is added, can cause separated two
A monomer combines, and when being connected when activating or inhibiting original part close to formation dimer on each monomer, can rise and activate or inhibit work
With final realization controls activity switch with drug.CID technology combination CAR-T technology, that is, develop suicide switch CIDeCAR with
Activity switch GoCAR-T, to provide the method more safer and more effective than existing cellular immunotherapy.Wherein, commit suiside switch CIDeCAR
Technology is that the suicide PROTEIN C aspase9 of Apoptosis will be caused to be connected on drug binding domains monomer, when oral chemical drugs
After object, cause Caspase9 Dimerized, thus the active cell apoptosis program that is activated, and then cause CAR-T Apoptosis, from
CAR-T, stopped reaction are removed in vivo.And activity switch GoCAR-T technology is that the co-activation of CAR is signally attached to drug combination
In domain monomer, if not after Oral Chemical drug, CAR-T cell only cannot be started by the contact of tumour cell, by oral
Chemicals, it is possible to provide co-activation signal, starting and activation CAR-T reaction.
It developed a kind of protein regulation system dependent on intracellular breakdown machine in recent years, can pass through using cell membrane
Small molecule compound ligand and specific domain realize the adjusting to proteins of interest, so-called specific domain refers to egg
White corresponding structural domain encoding gene is after mutation, and in the case where small molecule compound ligand is not present, which becomes
It is unstable, easily degraded by intracellular protease body, and combine smaller ligand after, which tends towards stability, such knot
Structure domain is known as unstable structure domain (Destabilizing domains；DD).Unstable structure domain is general at present and immunological regulation
The effect of factor connection, these factors may enhance adoptive cellular therapy, but need accurate control to optimize its therapeutic effect, mesh
Preceding technology can not the time of accuracy controlling cells play therapeutic effect or the expression of cell factor, this makes many potential
Using being difficult to safely and effectively dispose, lack adjustability but also the potent albumen of transient expression be difficult to it is safely expressed.
Summary of the invention
To solve the above problems, the object of the present invention is to provide a kind of CAR-T cell containing unstable structure domain and its
Preparation method and adjusting CAR-T cell function method.
The present invention to achieve the above object, is achieved through the following technical solutions:
A kind of CAR-T cell containing unstable structure domain, the coding base in CAR-T cell containing unstable structure domain
Cause, and the encoding gene for removing the 3 ' ends and unstable structure domain of the CAR gene of terminator codon is directly connected to, and constitutes fusion
Gene；The fusion protein of above-mentioned fusion coding is expressed in CAR-T cell, which is CAR molecule, and C-terminal is
Unstable structure domain, is positioned at cell membrane.
In CAR-T cell, due to the characteristic in unstable structure domain, the fusion protein of above-mentioned expression is easily dropped by proteasome
Solution, causes CAR molecule on cell membrane that cannot accumulate；When unstable structure domain, corresponding small molecule compound ligand is present in CAR-
When in T cell, above-mentioned fusion protein is not degraded by proteasome, and CAR molecule is accumulated on cell membrane.It is unstable in fusion protein
The presence of constant domain will not influence the function of CAR molecule itself.
The present invention, without limiting, can derive from document report or operation to the type in unstable structure domain and source
Person screens to obtain.
A kind of preferred CAR-T cell containing unstable structure domain, the unstable structure domain be dihydrofoilic acid also
The unstable structure domain of protoenzyme DHFR, the unstable structure domain of FKBP12 albumen are based on estrogen receptor ligands binding structural domain
The unstable structure domain ERLBP of development.
The invention also includes the preparation methods of the CAR-T cell containing unstable structure domain, comprising the following steps:
1. the encoding gene in unstable structure domain is connect with CAR gene end, gene fusion construct；Specifically, will not
The encoding gene in rock-steady structure domain is placed in 3 ' ends of CAR gene, connects and composes fusion；
2. by step, 1. gained fusion gene cloning is inserted into slow virus plasmid；
3. packaging virus, preparation carries the slow virus of fusion；
4. with step, 3. gained and is expanded slow-virus infection T cell.
The invention also discloses the methods for adjusting the CAR-T cell function containing unstable structure domain, and unstable knot is added
The corresponding small molecule compound in structure domain.
The preferred method for adjusting the CAR-T cell function containing unstable structure domain, when the unstable structure domain is
The unstable structure domain of dihyrofolate reductase DHFR, corresponding small molecule compound are trimethoprim TMP；When the unstable knot
Structure domain is the unstable structure domain of FKBP12 albumen, and corresponding small molecule compound is Shied-1；When the unstable structure domain is
Based on the unstable structure domain ERLBP of estrogen receptor ligands binding structural domain development, corresponding small molecule compound is 4- hydroxyl
Tamoxifen.
The present invention has the advantage that compared with prior art
In the CAR-T cell of the application meaning, the encoding gene at 3 ' ends and DD of the CAR gene of terminator codon is removed
It is directly connected to, constitutes fusion；The fusion protein of fusion coding, N-terminal are CAR molecule, and C-terminal is unstable structure
Domain；The unstable characteristic of DD is directly passed to CAR molecule, comprising the entire fusion protein including CAR molecule by intracellular protein
The degradation of enzyme body, causes CAR molecule on cell membrane that cannot accumulate；In the presence of DD smaller ligand, exist comprising CAR molecule
Interior entire fusion protein is not degraded by proteasome, and CAR molecule is accumulated on cell membrane.Unstable structure domain in fusion protein
Presence, will not influence the normal function of CAR molecule itself.By way of artificially adding this simplicity of small molecule, cell is adjusted
The quantity of CAR molecule on film, and then change the function of CAR-T cell.Herein described method is directly on protein level to CAR
Molecular amounts are adjusted, and adjust quick.
The preparation method of CAR-T cell containing unstable structure domain of the invention, process is few, and step is easily operated, just
In realization industrialization；The method disclosed by the invention for adjusting the CAR-T cell function containing unstable structure domain, need to only be added not
The quantity of the i.e. adjustable CAR molecule of the corresponding small molecule compound in rock-steady structure domain, and the presence in unstable structure domain, will not
Influence the function of CAR molecule itself.
Detailed description of the invention
The schematic illustration of CAR-T cell function of Fig. 1 small molecule compound adjusting containing unstable structure domain；
The sequential structure of Fig. 2 DD-CAR (DD is unstable structure domain, and ScFv is single-chain antibody)；
The CAR molecule amount of Fig. 3 small molecule TMP adjusting DHFR-CD19CAR-T cell surface；
Fig. 4 small molecule TMP adjusts anti-tumor effect in DHFR-CD19CAR-T cell body；
Fig. 5 small molecule Shield adjusts FKBP-CD19CAR-T cell to the lethal effect of Raji cell.
Specific embodiment
The object of the present invention is to provide a kind of CAR-T cell containing unstable structure domain and preparation method thereof and adjusting
CAR-T cell function method, is achieved through the following technical solutions:
A kind of CAR-T cell containing unstable structure domain, the coding base in CAR-T cell containing unstable structure domain
Cause, and the encoding gene for removing the 3 ' ends and unstable structure domain of the CAR gene of terminator codon is directly connected to, and constitutes fusion
Gene；The fusion protein of above-mentioned fusion coding is expressed in CAR-T cell, which is CAR molecule, and C-terminal is
Unstable structure domain, is positioned at cell membrane.
In CAR-T cell, due to the characteristic in unstable structure domain, the fusion protein of above-mentioned expression is easily dropped by proteasome
Solution, causes CAR molecule on cell membrane that cannot accumulate；When unstable structure domain, corresponding small molecule compound ligand is present in CAR-
When in T cell, above-mentioned fusion protein is not degraded by proteasome, and CAR molecule is accumulated on cell membrane.It is unstable in fusion protein
The presence of constant domain will not influence the function of CAR molecule itself.
A kind of preferred CAR-T cell containing unstable structure domain, the unstable structure domain be dihydrofoilic acid also
The unstable structure domain of protoenzyme DHFR, the unstable structure domain of FKBP12 albumen are based on estrogen receptor ligands binding structural domain
The unstable structure domain ERLBP of development.
The invention also includes the preparation methods of the CAR-T cell containing unstable structure domain, comprising the following steps:
1. the encoding gene in unstable structure domain is connect with CAR gene end, gene fusion construct；Specifically, will not
The encoding gene in rock-steady structure domain is placed in 3 ' ends of CAR gene, connects and composes fusion；
2. by step, 1. gained fusion gene cloning is inserted into slow virus plasmid；
3. packaging virus, preparation carries the slow virus of fusion；
4. with step, 3. gained and is expanded slow-virus infection T cell.
The invention also discloses the methods for adjusting the CAR-T cell function containing unstable structure domain, and unstable knot is added
The corresponding small molecule compound in structure domain；The additive amount of small molecule compound is 0-1000 μm of ol/L.As shown in Figure 1, three in Fig. 1
Angular is small molecule compound, as its stabilization is made behind ligand binding unstable structure domain, so that cell surface CAR molecular number
It is accumulated.
The preferred method for adjusting the CAR-T cell function containing unstable structure domain, when the unstable structure domain is
The unstable structure domain of dihyrofolate reductase DHFR, corresponding small molecule compound are trimethoprim TMP；When the unstable knot
Structure domain is the unstable structure domain of FKBP12 albumen, and corresponding small molecule compound is Shied-1；When the unstable structure domain is
Based on the unstable structure domain ERLBP of estrogen receptor ligands binding structural domain development, corresponding small molecule compound is 4- hydroxyl
Tamoxifen.
The source of partial sequence or plasmid etc. in the embodiment of the present invention:
The sequence source of DHFR is document Iwamoto M,T,Lundberg C,Kirik D,Wandless
TJ.A general chemical method to regulate protein stability in the mammalian
central nervous system.Chem Biol.2010Sep 24；17(9):981-8.
Single-chain antibody (ScFV) sequence of CD19CAR is obtained according to following documents: Nicholson IC, Lenton KA,
Little DJ,Decorso T,Lee FT,et al.(1997)Construction and characterisation ofa
functional CD19specific single chain Fv fragment for immunotherapy of B
lineage leukaemia and lymphoma.Mol Immunol 34:1157–1165.
FKBP amino acid sequence is obtained by following documents: Banaszynski LA, Chen LC, Maynard-Smith
LA,Ooi AG,Wandless TJ.A rapid,reversible,and tunable method to regulate
protein function in living cells using synthetic small molecules.Cell.2006Sep
8；126(5):995-1004.
Based on the unstable structure domain (ERLBD) of estrogen receptor ligands binding structural domain development, small molecule is corresponded to
Conjunction object is 4-hydroxytamoxifen, and the acquisition methods of ERLBD have related introduction in the following documents: Miyazaki Y, Imoto
H,Chen LC,Wandless TJ.Destabilizing domains derived from the human estrogen
receptor.J Am Chem Soc.2012Mar 7；134(9):3942-5.
PLVX-EF1 α-IRES-mCherry plasmid is common slow virus plasmid, can be bought from Takara company, Cat.#
631987.CD3 sorts magnetic bead purchase in Mei Tian Ni company, people CD3microbeads, orderNO.130-050-101.
The CAR-T cell function containing unstable structure domain is adjusted using the corresponding small molecule compound in unstable structure domain
When, the additive amount of small molecule compound is generally in 0-1000 μm of ol/L.
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1 prepares DHFR-CD19CAR-T cell
One, the fusion of full genome synthesis DHFR and CD19CAR
Unstable structure domain DD selects the unstable structure of dihyrofolate reductase (Dihydrofolate reductase)
Domain, abbreviation DHFR；Its small molecule compound ligand is trimethoprim (TMP)
The sequence of DHFR is the transmembrane region of SED ID NO:1, CD19CAR, costimulatory signal area, CD3 ζ sequence nucleotide sequence are
ScFV sequence is connected by SED ID NO:2 according to the positional relationship of Fig. 2 with transmembrane region, costimulatory signal area, CD3 ζ sequence, structure
CD19CAR is built, sequence is SEQ ID NO:3；
Full genome synthesizes the fusion of DHFR and CD19CAR, makes fusion upstream restriction enzyme site XbaI, downstream enzyme
Enzyme site is BamHI；Fusion (DHFR-CD19CAR) sequence is SEQ ID NO:4；In this fusion, DHFR coding
The connection of the CD3- ζ chain end of gene and CD19CAR, in this way in track fusion resulting fusion protein, DHFR is located at fusion
PROTEIN C end.
Two, building carries the slow virus plasmid of DHFR-CD19CAR fusion
With XbaI and BamHI double digestion said gene segment and pLVX-EF1 α-IRES-mCherry plasmid；Glue is cut to return
It receives；The connection of conventional molecular biological method；Connection product is converted into Stbl3 competent cell, is coated with LB culture medium flat plate (ammonia benzyl
Penicillin resistance)；Picking monoclonal to LB culture medium is stayed overnight；Small upgrading grain, XbaI and BamHI double digestion attached gel electrophoresis are tested
Demonstrate,prove it；Utilize the further plasmid order-checking proving correctness of universal primer；Plasmid is stored in -20 DEG C, is named as pLVX-DHFR-
CD19CAR。
The present embodiment universal primer is bought in Sangon Biotech (Shanghai) Co., Ltd..
Three, preparation carries the slow virus of DHFR-CD19CAR fusion protein
1, plasmid mentions greatly
By pLVX-DHFR-CD19CAR plasmid routine transformation stbl3 competence, picking monoclonal is (interior to 500ml conical flask
Culture medium containing 200mlLB), it is incubated overnight, extracts plasmid using the big extraction reagent kit of Tiangeng plasmid, ddH2O elutes plasmid,
Nanadrop measures plasmid concentration greater than 1.2 μ g/ μ l.
2, virus packaging
(1) it prepares A liquid: 1.5mlEP being taken to manage, opti-MEM 1.2ml is added, it is as follows to be added each plasmid, vortex oscillation 5~
10s is thoroughly to mix；
Plasmid mass ratio:
psPAX2 7.5μg
pMD2.G 2.5μg
pLVX-DHFR-CD19CAR 20μg
(2) it prepares B liquid: taking 15ml centrifuge tube, take opti-MEM 1.5ml, lipo200060 μ l to be added in centrifuge tube, whirlpool
Rotation oscillation 5-10s；
(3) A liquid is added in B liquid, vortex oscillation 5-10s, room temperature 20min；
(4) 293T prepares: 293T cells spreads 10cm plate, until 293T cells degrees of fusion reaches 80%；
(5) A, B mixed liquor are uniformly dripped in above-mentioned 10cmplate, stands 3-5min, gently shakes up, 37 DEG C of incubators
Cultivate about 60h；
(6) 2000rpm is centrifuged 10min, removes cell fragment, 0.45 μm of membrane filtration；
(7) supernatant is collected, packing is placed in -80 DEG C of preservations.
Four, the slow-virus infection T cell of DHFR-CD19CAR fusion is carried
1, CD3 positive T cell screens
(1) PBMC cell is collected, tally counts.
(2) the CD3 sorting magnetic bead of doses is added in cell suspension, (every 10720ul is added in cell
Microbeads it) hangs on；
(3) sorting column is put on sorting frame, is rinsed and is divided with 0.5ml buffer (PBS+0.5%BSA+0.5mM EDTA)
Select column；
(4) cell suspension crosses column, abandons column liquid.
(5) will sorting column remove, the T cell of screening is poured in culture bottle/ware with Buffer, be added complete medium and
IL-2 culture；
2, T cell activation
(1) T cell counts, and 24 orifice plates are added in 5E+5/w T cell；
(2) anti-CD3300ng/ml, IL-2100IU/ml is added in every hole；
(3) 37 DEG C are incubated overnight；
3, RetroNectin is coated with culture plate
(1) PBS dilutes RetroNectin 4ug/ml, is coated with culture plate 3ug/cm2；
(2) culture plate is added in the RetroNectin of appropriate volume, room temperature 2 hours or 4 DEG C overnight；
24 porocyte culture plates: the hole 0.5ml/；
6 porocyte culture plates: the hole 2ml/；
(3) RetroNectin is discarded, adds the PBS of the 2%BSA of appropriate volume to terminate, room temperature 30min；
24 porocyte culture plates: the hole 0.5ml/；
6 porocyte culture plates: the hole 2ml/；
(4) BSA solution is discarded, it is primary with PBS board-washing, discard supernatant liquid.
Note: after RetroNectin can be closed with sealed membrane, 4 DEG C are saved one week.
4, slow-virus infection T cell
(1) by virus according to MOI=50 value be added in coated 24 orifice plate of RetroNectin, 32 DEG C of 3000rpm from
Heart 2h；
(2) 24 orifice plates are added in T cell, cell number is no more than the hole 5E+5/, 32 DEG C of centrifugation 2h of 3000rpm；
(3) appropriate culture medium (or not supplementing, no more than 1ml) is supplemented, IL-2 100IU/ml, polybrene is added
8ug/ml；
(4) 37 DEG C of cultures, 5%CO2Culture.
Five, DHFR-CD19CAR-T cell expands
1, DHFR-CD19CAR-T cell expands
37 DEG C of cultures, 5%CO2Culture.It is added appropriate 15 serum free medium of X-vivo, IL-2 100IU/ml, every other day
Liquid or liquid feeding are changed, keeps cell density in 1*10^6/ml or so.
2, infection rate measures
Take 1*10^5 cell, the ratio of stream measuring mCherry luminescent cell number, through detecting, infection rate 45%.
Embodiment 2
DHFR-CD19CAR-T cell membrane surface CAR molecular number prepared by embodiment 1 is adjusted using TMP
The DHFR-CD19CAR-T cell that Example 1 constructs, is placed in 24 orifice plates, 5*10^5, every hole CAR-T cell,
It is respectively 0nM, 10nM, 100nM, 1000nM, 10000nM that DHFR ligand-trimethoprim to final concentration, which is added,；Every group 3 parallel；
Culture is for 24 hours；
Take CD19 albumen (Biotinylated Human CD19, the Fc Tag, ultra of biotin labeling
Sensitivity (primary amine labeling), ACROBiosystems, Cat.No.CD9-H8259), FITC label
Streptavidin (FITC-SA, Biolegend, Cat.No.405201), according to corresponding instructions method carry out flow cytometer detection,
The average fluorescent strength of CAR positive cell group is measured, film surface CAR molecular number is more, and fluorescence intensity is stronger.
As shown in figure 3, average fluorescent strength increases with the increase of TMP concentration, dose dependent is presented, which show
The CAR molecular number of the adjustable DHFR-CD19CAR-T cell membrane surface of TMP.
Embodiment 3
Anti-tumor effect in DHFR-CD19CAR-T body prepared by embodiment 1 is adjusted using TMP
1, male NSG mouse (8 weeks) are selected, NSG mouse is in SPF environment and feeds；It is divided into A, B, C group, each 6；
2, conventional method culture Raji cell；Raji cell is collected, cell is resuspended in PBS；Every NSG mouse tail vein injection
2*10^6 cell is designated as Day 0 (the 0th day)；
3, (the 3rd day) Day 3, collects DHFR-CD19CAR-T cell, and cell is resuspended in PBS；For B, C group NSG mouse, tail
It is injected intravenously 2*10^6 DHFR-CD19CAR-T cell/mouse；The PBS of A group NSG mouse injection same volume；C group mouse drink
TMP (0.25mg/ml) is added in water.
4, observation mouse survival state, record mouse diing time draw survivorship curve, as shown in figure 4, TMP can daily
To extend survival time of mice, show that TMP can adjust the anti-tumor effect of DHFR-CD19CAR-T in body.
Embodiment 4
Prepare FKBP-CD19CAR-T cell
One, the fusion of full genome synthesis FKBP and CD19CAR
1, DD selects the unstable structure domain of FKBP12 albumen, abbreviation FKBP, and correspondence small molecule compound is Shield-
1；
2, FKBP amino acid sequence is shown in SEQ ID NO:5；
3, the fusion of full genome synthesis FKBP and CD19CAR, makes fusion upstream restriction enzyme site XbaI, downstream
Restriction enzyme site is BamHI；Fusion (FKBP-CD19CAR)；In this fusion, FKBP encoding gene and CD19CAR's
The connection of CD3- ζ chain end, in this way in track fusion resulting fusion protein, FKBP is located at fusion protein C-terminal.
Two, building carries the slow virus plasmid of FKBP-CD19CAR fusion
1, with XbaI and BamHI double digestion said gene segment and pLVX-EF1 α-IRES-mCherry plasmid；Glue is cut to return
It receives；The connection of conventional molecular biological method；
2, connection product is converted into Stbl3 competent cell；It is coated with LB culture medium flat plate (amicillin resistance)；
3, picking monoclonal is to LB culture medium overnight incubation；
4, small upgrading grain, XbaI and BamHI double digestion attached gel electrophoresis verify it；Utilize the further plasmid of universal primer
Sequence verification correctness；The universal primer is bought in Sangon Biotech (Shanghai) Co., Ltd.；
5, plasmid is stored in -20 DEG C, is named as pLVX-FKBP-CD19CAR.
Three, preparation carries the slow virus of FKBP-CD19CAR fusion
1, plasmid mentions greatly
By pLVX-FKBP-CD19CAR plasmid routine transformation stbl3 competence, picking monoclonal is (interior to 500ml conical flask
Culture medium containing 200mlLB), it is incubated overnight, extracts plasmid, ddH using the big extraction reagent kit of Tiangeng plasmid2O elutes plasmid,
Nanadrop measures plasmid concentration greater than 1.2 μ g/ μ l.
2, virus packaging
(1) it prepares A liquid: 1.5ml EP being taken to manage, opti-MEM 1.2ml is added, it is as follows to be added each plasmid, vortex oscillation 5-
10s is thoroughly to mix.
Plasmid mass ratio:
psPAX2 7.5μg；
pMD2.G 2.5μg；
pLVX-FKBP-CD19CAR 20μg；
(2) it prepares B liquid: taking 15ml centrifuge tube, opti-MEM 1.5ml is added, opti-MEM is added in 60 μ l of lipo2000
In, vortex oscillation 5-10s；
(3) A liquid is added in B liquid, vortex oscillation 5-10s, room temperature 20min；
(4) 293T prepares: 293T cells spreads 10cm plate, until 293T cells degrees of fusion reaches 80%；
(5) A, B mixed liquor are uniformly dripped in above-mentioned 10cm plate, stands 3-5min, gently shakes up, 37 DEG C of incubators
Cultivate about 60h；
(6) 2000rpm is centrifuged 10min, removes cell fragment.0.45 μm of membrane filtration；
(7) supernatant is collected, packing is placed in -80 DEG C of preservations.
Four, the slow-virus infection T cell of the slow virus of FKBP-CD19CAR fusion is carried
1, CD3 positive T cell screens
(1) PBMC cell is collected, tally counts；
(2) the CD3 sorting magnetic bead of doses is added in cell suspension, (every 10720ul is added in cell
Microbeads), hang on；
(3) sorting column is put on sorting frame, is rinsed and is divided with 0.5ml buffer (PBS+0.5%BSA+0.5mM EDTA)
Select column；
(4) cell suspension crosses column, abandons column liquid；
(5) will sorting column remove, the T cell of screening is poured in culture bottle/ware with Buffer, be added complete medium and
IL-2 culture.
2, T cell activation
(1) T cell counts, and 24 orifice plates are added in 5E+5/w T cell；
(2) anti-CD3 300ng/ml, IL-2 100IU/ml is added in every hole；
(3) 37 DEG C of culture 6h are incubated overnight.
3, RetroNectin is coated with culture plate
(1) PBS dilutes RetroNectin 4ug/ml, is coated with culture plate 3ug/cm2；
(2) culture plate is added in the RetroNectin of appropriate volume, room temperature 2 hours or 4 DEG C overnight；
24 porocyte culture plates: the hole 0.5ml/；
6 porocyte culture plates: the hole 2ml/；
(3) RetroNectin is discarded, adds the PBS of the 2%BSA of appropriate volume to terminate, room temperature 30min；
24 porocyte culture plates: the hole 0.5ml/；
6 porocyte culture plates: the hole 2ml/；
(4) BSA solution is discarded, it is primary with PBS board-washing, discard supernatant liquid；
Note: after RetroNectin can be closed with sealed membrane, 4 DEG C are saved one week.
4, slow-virus infection T cell
(1) by virus according to MOI=50 value be added in coated 24 orifice plate of RetroNectin, 32 DEG C of 3000rpm from
Heart 2h；
(2) 24 orifice plates are added in T cell, cell number is no more than the hole 5E+5/, 32 DEG C of centrifugation 2h of 3000rpm；
(3) appropriate culture medium (or not supplementing, no more than 1ml) is supplemented, IL-2100IU/ml, polybrene is added
8ug/ml；
(4) 37 DEG C of cultures, 5%CO2Culture.
Five, FKBP-CD19CAR-T cell expands
1, CAR-T cell expands
37 DEG C, 5%CO2Culture, is added appropriate 15 serum free medium of X-vivo, IL-2 100IU/ml changes liquid every other day
Or liquid feeding, keep cell density in 1*10^6/ml or so.
2, infection rate measures
Take 1*10^5 cell, the ratio of stream measuring mCherry luminescent cell number, through detecting, infection rate 35%.
Embodiment 5
Shild-1 adjusts FKBP-CD19CAR-T cell killing function
The FKBP-CD19CAR-T cell constructed is taken, is placed in 24 orifice plates, 5*10^5, every hole CAR-T cell is added
Fkbp ligand body-Shild-1 to final concentration be respectively 0nM, 1000nM；Every group 3 parallel；Culture is for 24 hours.
Divide and partly collect above-mentioned cell, centrifugation removal supernatant replaces fresh culture.Using Raji as target cell, according to effect
Target ratio 5:1 building killing system, flow cytometer detection lethal effect, as shown in Figure 5, the results showed that 1000nM Shild-1 killing is added
Rate is 63.9%, and when Shild-1 not being added, lethal effect 28.5%.
Sequence table
<110>Shandong University The Second Hospital
<120>a kind of CAR-T cell containing unstable structure domain and preparation method thereof and the adjusting cell function side CAR-T
Method
<130> 20190506A-1
<141> 2019-05-06
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 477
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atctctctga tcgccgccct ggcagtggac cacgtgatcg gcatggagac cgtgatgcca 60
tggaacctgc ccgccgatct ggcctggttc aagcggaaca ccctgaataa gccagtgatc 120
atgggcagac acacatggga gtccatcggc aggcccctgc ctggacgcaa gaatatcatc 180
ctgagctccc agcccagcac cgacgatagg gtgacatggg tgaagtccgt ggacgaggca 240
atcgcagcat gcggcgatgt gcccgagatc atggtcatcg gcggcggcag agtgtacgag 300
cagttcctgc ctaaggccca gaagctgtat ctgacccaca tcgacgccga ggtggagggc 360
gacacacact ttcctgatta cgagccagac gattgggagt ccgtgttctc tgagtttcac 420
gacgccgatg cccagaactc tcacagctat tgttttgaga tcctggagcg gagataa 477
<210> 2
<211> 660
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60
catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120
tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 180
tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240
atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300
gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 360
cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 420
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 480
aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 540
gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 600
ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 660
<210> 3
<211> 1503
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcgac 60
atccagatga cacagactac atcctccctg tctgcctctc tgggagacag agtcaccatc 120
agttgcaggg caagtcagga cattagtaaa tatttaaatt ggtatcagca gaaaccagat 180
ggaactgtta aactcctgat ctaccataca tcaagattac actcaggagt cccatcaagg 240
ttcagtggca gtgggtctgg aacagattat tctctcacca ttagcaacct ggagcaagaa 300
gatattgcca cttacttttg ccaacagggt aatacgcttc cgtacacgtt cggagggggg 360
actaagttgg aaataacacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 420
agtaatggct ccacctccgg ctccggcaag cccggctccg gcgagggctc caccaagggc 480
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 540
acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 600
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 660
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 720
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 780
tacggtggta gctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 840
attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 900
catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 960
tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 1020
tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1080
atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 1140
gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1200
cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1260
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 1320
aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1380
gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1440
ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1500
taa 1503
<210> 4
<211> 1989
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tctagaatgc ttctcctggt gacaagcctt ctgctctgtg agttaccaca cccagcattc 60
ctcgacatcc 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
ggggggacta agttggaaat aacacgggct gatgctgcac caactgtatc catcttccca 420
ccatccagta atggctccac ctccggctcc ggcaagcccg gctccggcga gggctccacc 480
aagggcgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 540
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 600
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 660
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 720
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 780
tattactacg gtggtagcta tgctatggac tactggggtc aaggaacctc agtcaccgtc 840
tcctcaattg aagttatgta tcctcctcct tacctagaca atgagaagag caatggaacc 900
attatccatg tgaaagggaa acacctttgt ccaagtcccc tatttcccgg accttctaag 960
cccttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 1020
gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 1080
atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 1140
ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 1200
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1260
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgcagaga 1320
aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1380
tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1440
cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1500
cctcgcatct ctctgatcgc cgccctggca gtggaccacg tgatcggcat ggagaccgtg 1560
atgccatgga acctgcccgc cgatctggcc tggttcaagc ggaacaccct gaataagcca 1620
gtgatcatgg gcagacacac atgggagtcc atcggcaggc ccctgcctgg acgcaagaat 1680
atcatcctga gctcccagcc cagcaccgac gatagggtga catgggtgaa gtccgtggac 1740
gaggcaatcg cagcatgcgg cgatgtgccc gagatcatgg tcatcggcgg cggcagagtg 1800
tacgagcagt tcctgcctaa ggcccagaag ctgtatctga cccacatcga cgccgaggtg 1860
gagggcgaca cacactttcc tgattacgag ccagacgatt gggagtccgt gttctctgag 1920
tttcacgacg ccgatgccca gaactctcac agctattgtt ttgagatcct ggagcggaga 1980
taaggatcc 1989
<210> 5
<211> 107
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro
1 5 10 15
Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp
20 25 30
Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe
35 40 45
Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala
50 55 60
Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr
65 70 75 80
Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr
85 90 95
Leu Val Phe Asp Val Glu Leu Leu Lys Pro Glu
100 105
Claims (5)
1. a kind of CAR-T cell containing unstable structure domain, it is characterised in that: contain unstable structure in CAR-T cell
The encoding gene in domain；And the encoding gene for removing the 3 ' ends and unstable structure domain of the CAR gene of terminator codon directly connects
Composition fusion is connect, and expresses the fusion protein of above-mentioned fusion coding in CAR-T cell, which is located at thin
On after birth.
2. a kind of CAR-T cell containing unstable structure domain according to claim 1, it is characterised in that: it is described not
Rock-steady structure domain is the unstable structure domain of dihyrofolate reductase DHFR, the unstable structure domain of FKBP12 albumen or based on female
The unstable structure domain ERLBP of androgen receptor ligand binding structural domain development.
3. the preparation method of the CAR-T cell described in claim 1 containing unstable structure domain, it is characterised in that: including with
Lower step:
1. the encoding gene in unstable structure domain is connect with CAR gene end, gene fusion construct；Specifically, by unstable
The encoding gene of structural domain is placed in 3 ' ends of CAR gene, connects and composes fusion；
2. by step, 1. gained fusion gene cloning is inserted into slow virus plasmid；
3. packaging virus, preparation carries the slow virus of fusion；
4. with step, 3. gained and is expanded slow-virus infection T cell.
4. the method for adjusting the CAR-T cell function described in claim 1 containing unstable structure domain, it is characterised in that: add
Enter the corresponding small molecule compound in unstable structure domain.
5. the method according to claim 4 for adjusting the CAR-T cell function containing unstable structure domain, feature exist
In: when the unstable structure domain that the unstable structure domain is dihyrofolate reductase DHFR, corresponding small molecule compound is first
Oxygen benzyl pyridine TMP；When the unstable structure domain that the unstable structure domain is FKBP12 albumen, corresponding small molecule compound is
Shied-1；When the unstable structure domain is the unstable structure domain developed based on estrogen receptor ligands binding structural domain
ERLBP, corresponding small molecule compound are 4-hydroxytamoxifen.
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| WO2022026577A1 (en) * | 2020-07-31 | 2022-02-03 | Nantbio, Inc. | Chimeric t cell receptors, nucleic acids, and methods of making and using the same |
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