CN106544321B - Universal CAR-T cell and its preparation method and application - Google Patents
Universal CAR-T cell and its preparation method and application Download PDFInfo
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Abstract
Universal CAR-T cell and its preparation method and application.The present invention provides a kind of universal CAR-T cell, expression specificity CAR and TCR is not expressed.The present invention also provides a kind of methods for preparing universal CAR-T cell, and described method includes following steps: 1) sgRNA molecule and Cas9 molecule are introduced in T cell；2) CAR molecule is introduced in the T cell；Wherein, step 2) can carry out before, after or at the same time in step 1), and the sgRNA molecule includes and a chain or the complementary targeting structural domain of β chain constant code area's (i.e. TRAC or TRBC) target region of gene from TCR.There is GVHD and the interference of potential TCR receptor signal in the T cell that universal CAR-T cell of the invention avoids transduction.
Description
Technical field
The present invention relates to immunologys and molecular biology field, especially a kind of logical more particularly to tumour immunotherapy
With type CAR-T cell and its preparation method and application.
Background technique
Tumour immunotherapy is received extensive attention and is applied in recent years, especially CAR-T (chimeric antigen
Receptor-engineered T cells) technology appearance, so that the mankind has been obtained landmark hair to the control of tumour
Exhibition.Application of the technology in oncotherapy starts from 1989.Its clinical applications result allows people to see the dawn for curing cancer:
Can not only work miracle in acute or chronic lymphocytic blood disease, some other solid tumor such as lymthoma,
Also there is infusive therapeutic effect in neuroblastoma.T cell applied in CAR-T technology is mainly derived from present
Patient itself imports and expands from the T cell of patient itself derived from peripheral blood by activation, CAR, then feeds back to patient's body
It is interior.But in many cases, since the state of an illness of patient is dangerous or the condition of patient itself does not allow again outside its in vivo extraction
All blood carries out the treatment of CAR-T cell；Even if even patient can provide peripheral blood, but the T sorted out from blood is thin
Cytoactive is not enough to carry out CAR transformation, this just limits the application range of CAR-T technology from a certain range.In order to solve this
A problem, allosome CAR-T are increasingly becoming a good selection.Meanwhile the source that allosome CAR-T technology solves T cell is asked
Topic will also lay the foundation for the commercialization of CAR-T popularization.
In body, it is a complicated process that T cell, which captures and eliminates heterologous antigen, needs ajor histocompatibility multiple
The common participation of object (MHC) and T cell receptor (TCR) are closed, wherein TCR plays key in the Process of Antigen that identification MHC is combined
Effect.There are two types of types, commonly referred to as MHC I class and MHC II class for major histocompatibility complex (MHC), anti-in addition to having
Former Presentation is also related to the rejection during organ transplant.Killer T cell is then the class having by its surface
Molecule, that is, T cell receptor (TCR) like antibody identifies and removes the antigen through MHC I class molecule submission.In allogeneic environment
In, the TCR signal transduction effect on CAR-T may generate potential influence to clinical response, and mutual between TCR and CAR
There has been no in-depth studies for effect.Meanwhile it feeding back allosome CAR-T cell and being possible to the wind for graft versus host disease(GVH disease) (GVHD) occur
Danger.
It is therefore desirable to have reducing the donorcells rejection of receptor and avoiding the occurrence of the improvement CAR-T curative effect of GVHD
Method.
Summary of the invention
The present invention in view of the foregoing drawbacks, using CRISPR/Cas9 technology to come from healthy volunteer (donor) T cell
It is modified, selectivity knocks out a chain and/or the β chain constant code area (TRAC and/or TRBC) of endogenous T cells receptor (TCR)
Gene, while Chimeric antigen receptor (CAR) is stablized into expression in T cell, to obtain expression specificity CAR while not table
Up to the CAR-T cell of TCR.
The first aspect of the present invention provides a kind of universal CAR-T cell, expression specificity CAR and does not express
TCR。
The second aspect of the present invention provides a kind of method for preparing universal CAR-T cell, and the method includes as follows
Step:
1) sgRNA molecule and Cas9 molecule are introduced in T cell；
2) CAR molecule is introduced in the T cell；
Wherein, step 2) can carry out before, after or at the same time in step 1), and the sgRNA molecule includes and comes from
The targeting structural domain of the target region complementation of a chain or β chain constant code area (i.e. TRAC or TRBC) gene of TCR.
The third aspect of the present invention provides the composition of the universal CAR-T cell comprising first aspect present invention.
The universal CAR-T cell that the fourth aspect of the present invention provides first aspect present invention is used to prepare treatment tumour
Drug purposes.
Universal CAR-T cell of the invention avoids the T cell for being infused into patient's body and GVHD and potential occurs
The interference of TCR receptor signal.The application will pave the way for the use of universal CAR-T cell, have great clinical meaning.
Detailed description of the invention
According to the detailed description carried out referring to the drawings, above and other aspects, features and advantages of the invention can become
It obtains clearer.
Fig. 1 shows sgRNA expression plasmid and Cas9 expression plasmid, and in different transfections, (liposome transfection, electricity turn and slow disease
Poison transfection) under the conditions of transfection efficiency comparison result.
Fig. 2 a and Fig. 2 b show the comparison results that different sgRNA cause the efficiency of genome mutation.
Fig. 3 shows the result that the region TRAC and TRBC knocked out through TCR is sequenced.
Fig. 4 shows the result that the site of missing the target on the potential human genome of the TRAC-sg3 of prediction is sequenced.
Fig. 5 shows the selection result of the T cell of negative, CD4 and the CD8 positive to TCR.
Fig. 6 show T cell, WT CAR-T andTCR-Comparison result of the CAR-T cell to the fragmentation effect of target cell.
Fig. 7 show T cell, WT CAR-T andTCR-CAR-T cell inhibits and kills the comparison of the effect of tumour cell
As a result.
Fig. 8 shows injection mouse T cell, people WT CAR-T and peopleTCR-After CAR-T cell, the change of average mice body weight
The comparison result of change.
Specific embodiment
As described above, the first aspect of the present invention provides a kind of universal CAR-T cell, expression specificity CAR is simultaneously
And do not express TCR.
In one embodiment, the cell origin is in healthy volunteer (or donor).
In one embodiment, the tcr gene of universal CAR-T cell of the invention is knocked.
In a specific embodiment, a chain of the TCR and/or β chain constant code area (i.e. TRAC and/or
TRBC) gene is knocked.TRAC and TRBC gene can be knocked out all, can also partially be knocked out；Can both struck
It removes, alternative one can also be knocked out；As long as cell cannot express active TCR after knocking out.
In a specific embodiment, a chain constant code area (i.e. TRAC) gene of the TCR is knocked.For example,
In a particular embodiment, a chain constant code area gene of TCR of the present invention be introduced into the cell TRAC-sg1-7 molecule it
One (being shown in Table 1) and 9 molecular moiety of Cas all knock out；It is preferred that a chain constant code area gene of the TCR is introduced into carefully
The TRAC-sg3 molecule and Cas9 molecule of born of the same parents knocks out.
In another specific embodiment, β chain constant code area (i.e. TRBC) gene of the TCR is knocked.Example
Such as, in a particular embodiment, the β chain constant code area gene of TCR of the present invention is introduced into TRBC-sg1-7 points of the cell
One of son (being shown in Table 1) and 9 molecular moiety of Cas all knock out；It is preferred that β chain constant code area's gene of the TCR is drawn
The TRBC-sg1 molecule and Cas9 molecule for entering cell knock out.
In one embodiment, CAR expressed by universal CAR-T cell of the invention can be known in the art
Any CAR, as long as it can make T cell identify cell surface antigen in a manner of human leucocyte antigen (HLA)-dependent/non-dependent, performance is killed
Wound effect.It is, for example, possible to use CAR disclosed in Chinese invention patent application 201510324558.X, tool of the invention
CAR used in body embodiment can refer to the application for a patent for invention disclosure.Specifically, CAR-T cell of the invention
The CAR of middle expression includes signal peptide, extracellular combined area, optional hinge area, transmembrane region and the intracellular signal area being linked in sequence.
Terms used herein " signal peptide " refer to short (the length 5- for guiding newly synthesized protein to shift to secretion access
30 amino acid) peptide chain.In the present invention, it can be used the signal peptide of the intracorporal various protein of people, such as body is endocrine
The signal peptide of Cytokine protein, leukocyte differentiation antigen (CD molecule).
In a specific embodiment, the signal peptide is GMCSF signal peptide, nucleotide sequence such as patent of invention
Apply shown in the SEQ ID No:1 in the sequence table of 201510324558.X.
In a specific embodiment, the signal peptide is CD8 signal peptide, nucleotide sequence such as patent of invention Shen
It please be shown in the SEQ ID No:2 in the sequence table of 201510324558.X.
In the present invention, the hinge area of various different antibodies or antigen receptor, especially CD can be used in the hinge area
The hinge area of molecule.In a specific embodiment, the hinge area can be selected from the hinge of the albumen such as CD8 or CD28
Area.The CD8 or CD28 is the native marker object on T cell surface.
In a specific embodiment, the hinge area is CD8 hinge area (CD8-hinge), nucleotide sequence
As application for a patent for invention 201510324558.X sequence table in SEQ ID No:6 shown in.
In a specific embodiment, the hinge area is CD28 hinge area (CD28-hinge), nucleotides sequence
It arranges shown in the SEQ ID No:7 in the sequence table such as application for a patent for invention 201510324558.X.
In the present invention, can be used the transmembrane region of various people's vivo proteins, especially various different antigen receptors across
Film area.It is preferable to use transmembrane region be CD molecule transmembrane region.In one embodiment, the transmembrane region can be selected from CD8
Or the transmembrane region of the albumen such as CD28.
In a specific embodiment, the transmembrane region is CD8 transmembrane region (CD8-TM), and nucleotide sequence is as sent out
Shown in SEQ ID No:8 in the sequence table of bright patent application 201510324558.X.
In a specific embodiment, the transmembrane region is CD28 transmembrane region (CD28-TM), and nucleotide sequence is such as
Shown in SEQ ID No:9 in the sequence table of application for a patent for invention 201510324558.X.
" the extracellular combined area " includes the scFv of specific recognition tumor cell surface antigen.
Terms used herein " scFv " refer to such antibody fragment --- it is comprising being connected by connector (linker)
The heavy chain variable region (variable region of heavy chain, VH) and light chain variable region (variable region connect
Of light chain, VL) recombinant protein, connector makes the two structural domains associated, to ultimately form antigen binding position
Point.Be typically of size of a complete antibody 1/6 of scFv.ScFv is preferably the amino acid sequence encoded by a nucleotide chain
Column.The scFv that the present invention uses can be by being used alone or in combination routine techniques known in the art, such as amino acid deletions, inserts
Enter, replace, increase, and/or recombinate and/or other method of modifying make further modification.According to a kind of amino acid sequence of antibody
Be listed in the method that this modification is introduced in its DNA sequence dna be for a person skilled in the art it is well known (see, for example,
Sambrook molecular cloning: laboratory manual, Cold Spring Harbor Laboratory (1989) N.Y.).The modification is excellent
It is selected in nucleic acid level and carries out.Above-mentioned scFv can also include its derivative.
Terms used herein " specific recognition " mean antigen recognizing district of the invention not with or substantially not with target
Any polypeptide cross reaction other than antigen.Degree of its specificity can be judged by immunological technique, including but unlimited
In immunoblotting, immunoaffinity chromatography, flow cytometry etc..
In one embodiment, the extracellular combined area includes specific recognition CD19, CEA, EGFR, GD2 or CD138
Deng scFv.
In a specific embodiment, the extracellular combined area includes the scFv (anti-of specific recognition CD19
CD19scFv)。
In a specific embodiment, the extracellular combined area includes through humanization modified specific recognition CD19
ScFv.
In a preferred embodiment, the extracellular combined area is through humanization modified specific recognition CD19's
ScFv-S1 (its nucleic acid sequence as application for a patent for invention 201510324558.X sequence table in SEQ ID No:4 shown in) or
ScFv-S2 (its nucleic acid sequence as application for a patent for invention 201510324558.X sequence table in SEQ ID No:5 shown in).
In the present invention, the intracellular signal area of various people's vivo proteins can be used, especially various different antigen receptors
Intracellular signal area.It is preferable to use intracellular signal area be CD molecule intracellular signal area.In one embodiment, the born of the same parents
Interior signaling zone can be selected from the intracellular signal area of CD3 ζ, Fc ε RI γ, CD28, CD137, CD134 albumen, and combinations thereof.CD3 points
Son is made of five subunits, and wherein 3 ITAM motifs are contained in CD3 ζ subunit (also known as CD3zeta, abbreviation Z), which is
Important signal zone of transformation in TCR-CD3 complex.CD3 δ Z is the CD3 ζ sequence without ITAM motif of mutation, in this hair
Generally as the building component of negative control in bright embodiment.Fc ε RI γ is mainly distributed on mast cell and basophil
Cellular surface contains an ITAM motif, similar with CD3 ζ in structure, distribution and function.Further, as described above, CD28,
CD137, CD134 are costimulatory signal molecules, are acted in the costimulation generated with its intracellular signal section after respective ligand binding
Cause the continuous proliferation of T cell, and can be improved the level of the T cell secretion cell factors such as IL-2 and IFN-γ, improves simultaneously
The time to live and antitumous effect of CAR-T cell in vivo.
There are many combinations in the present invention specifically used intracellular signal area comprising signaling zone chosen from the followings or combinations thereof:
CD28 signaling zone (CD28-signal), the sequence of nucleotide sequence such as application for a patent for invention 201510324558.X
Shown in SEQ ID No:10 in list；
CD137 signaling zone (CD137-signal), nucleotide sequence such as application for a patent for invention 201510324558.X's
Shown in SEQ ID No:11 in sequence table；With
CD3 ζ signaling zone (CD3 ζ-signal), the sequence of nucleotide sequence such as application for a patent for invention 201510324558.X
Shown in SEQ ID No:12 in list.
In a specific embodiment, CAR packet chosen from the followings expressed by universal CAR-T cell of the invention
The Chimeric antigen receptor egg of signal peptide, extracellular combined area, optional hinge area, transmembrane region and intracellular signal area containing sequential connection
It is white:
GMCSF-scFv-S1-CD8-hinge-CD8-TM-CD137-signal-CD3 ζ-signal (CAR1, nucleic acid sequence
It arranges shown in the SEQ ID No:13 in the sequence table such as application for a patent for invention 201510324558.X)；
GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD137-signal-CD3 ζ-signal (CAR2, nucleic acid sequence
It arranges shown in the SEQ ID No:14 in the sequence table such as application for a patent for invention 201510324558.X)；
(CAR3, nucleic acid sequence are as special in invented by GMCSF-scFv-S1-CD28-TM-CD28-signal-CD3 ζ-signal
Shown in SEQ ID No:15 in the sequence table of benefit application 201510324558.X)；
(CAR4, nucleic acid sequence are as special in invented by GMCSF-scFv-S2-CD28-TM-CD28-signal-CD3 ζ-signal
Shown in SEQ ID No:16 in the sequence table of benefit application 201510324558.X)；
GMCSF–scFv-S1–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal
(CAR5, nucleic acid sequence as application for a patent for invention 201510324558.X sequence table in as shown in SEQ ID No:17)；
GMCSF–scFv-S2–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal
(CAR6, nucleic acid sequence as application for a patent for invention 201510324558.X sequence table in SEQ ID No:18 shown in).
As described above, the second aspect of the present invention provides a kind of method for preparing universal CAR-T cell, the method
Include the following steps:
1) sgRNA molecule and Cas9 molecule are introduced in T cell；
2) CAR molecule is introduced in the T cell；
Wherein, step 2) can carry out before, after or at the same time in step 1), and the sgRNA molecule includes and comes from
The targeting structural domain of the target region complementation of a chain and/or β chain constant code area (i.e. TRAC and/or TRBC) gene of TCR.
In the method for the invention, the sgRNA molecule refers to that one section includes complementary with the target region of gene to be knocked out
Targeting structural domain nucleic acid sequence, can identify target DNA sequence dna and guide Cas9 molecule cut target site.
In the method for the invention, the Cas9 molecule refers to a kind of double-stranded DNA nuclease, being capable of drawing in sg RNA
It leads and lower target site is cut.
In one embodiment, the sgRNA molecule includes and a chain constant code area (i.e. TRAC) base from TCR
Because of the targeting structural domain of target region complementation.
In a specific embodiment, the sequence such as SEQ ID for the targeting structural domain that the sgRNA molecule is included
Shown in one of NOs:1-7.
In a preferred embodiment, the sequence of the targeting structural domain is as shown in SEQ ID NO:3.
In one embodiment, the sgRNA molecule includes and β chain constant code area (i.e. TRBC) base from TCR
Because of the targeting structural domain of target region complementation.
In a specific embodiment, the sequence such as SEQ ID for the targeting structural domain that the sgRNA molecule is included
Shown in one of NOs:8-14.
In a preferred embodiment, the sequence of the targeting structural domain is as shown in SEQ ID NO:8.
In one embodiment, by carrier construction by the sgRNA molecule and Cas9 molecule through liposome transfection, electricity
Turn or slow-virus transfection technology is introduced into the T cell.
In a preferred embodiment, the sgRNA molecule and Cas9 molecule are turned through electricity by skill by carrier construction
Art is introduced into the T cell.
In a further preferred embodiment, technology is turned for the sgRNA molecule by electricity and encodes Cas9 molecule
MRNA is introduced into the T cell.
In one embodiment, the CAR molecule is introduced into the T cell by slow-virus transfection technology.
In a specific embodiment, method of the invention further includes that separation and/or activation come before step 1)
The step of from the T cell of healthy volunteer's (or donor)；Preferably, the method further includes to universal after step 2)
The step of CAR-T cell is sorted；It is highly preferred that carrying out validity to resulting universal CAR-T cell again after sorting
Verifying.
As described above, the third aspect of the present invention provides the universal CAR-T cell comprising first aspect present invention
Composition.
In one embodiment, the composition also includes pharmaceutical diluent, excipient or carrier etc..
As described above, the fourth aspect of the present invention provides the universal CAR-T cell of first aspect present invention for making
The purposes of the drug of standby treatment tumour.
Illustrate the contents of the present invention below by way of specific embodiment.It should be understood that the specific embodiment is only to illustrate mesh
, it is not meant to that the contents of the present invention are only limitted to specific embodiment.
Embodiment 1: the preparation of universal CAR-T cell
1. the separation and activation of health donors T cell.
1) acquisition of health donors peripheral blood: 4 DEG C of refrigerators of peripheral blood of acquisition are temporary, interior for 24 hours, set via equipped with constant temperature
Standby transfer car(buggy) is transported to the laboratory GMP and is separated and cultivated.
2) preparation of peripheral blood mononuclear cells (PBMC): DPBS (Dulbecco's Phosphate is drawn with pipette
Buffered Saline) or physiological saline be added to step (1) acquisition peripheral blood in (1:1), dilution, haemocyte is dilute
Release liquid, be slowly added in the centrifuge tube equipped with lymphocyte separation medium (Ficoll or Histopaque-1077), with 800g from
After heart 20min, the tunica albuginea confluent monolayer cells above lymphocyte separation medium are drawn, are transferred in a new centrifuge tube, Lonza is added
Supernatant is abandoned after 15 culture medium of x-vivo (article No.: 04-418Q) centrifugation, retains the cell precipitation of centrifugation bottom of the tube to get periphery is arrived
Blood mononuclear cell.
3) separation and activation of T cell: obtained peripheral blood mononuclear cells is counted, is added according to the ratio of 1:1
The beads for entering to be coupled CD3/CD28 antibody, gently shakes 20min, and using the suction-operated of magnetic frame, the T for obtaining the CD3 positive is thin
Born of the same parents, T cell at this time are active, and are added complete medium (Lonza x-vivo 15+IL-2), are trained to T cell
Support amplification.
2. knocking out the tcr gene in the T cell obtained through step 1 using CRISPR/Cas9 system, concrete operation step is such as
Shown in lower:
1) for the sgRNA of a chain of TCR and β chain constant code area (TRAC, TRBC) gene design and plasmid construction.
For selecting 5 '-GN in first three exon sequence of the gene coding region TRAC and TRBC19-22The sequence of NGG-3 '
Column, and with Cas-OFFinder ensure its 3 ' end 8-12 base will not mispairing to genome other positions (http: //
www.rgenome.net/cas-offinder/).Selected sequence is shown in Table 1.
Table 1:sgRNA sequence
Synthetic primer, 5 '-ACCGN of positive-sense strand19-22- 3 ', 5 '-AAACN of antisense strand19-22- 3 ' (in just antisense strand
N19-22Sequence reverse complemental).The sequence fragment of synthesis is respectively taken into 22.5 μ L, with 5 μ 10 × TransTaq of L HiFi
Buffer II (full formula gold Transgene, Beijing) mixing, is slowly cooled to room temperature after 95 DEG C of heating 3min.Products therefrom is used
T4PNK (T4Polynucleotide Kinase/T4 polynueleotide kinase) carries out phosphatizing treatment, 37 DEG C of incubation 30min.
Product (Insert sgRNA) after phosphorylation is passed through into Golden Gate reaction forming to psgRNA carrier (Addgene respectively
Plasmid#53121 in).
Reaction system is as follows:
| 10X Buffer Tango | 1μL |
| DTT(50mM) | 0.2μL |
| ATP(10mM) | 1μL |
| Esp3I | 0.75μL |
| T4DNA ligase | 0.25μL |
| psgRNA vector(20ng/μL) | 2μL |
| Insert sgRNA | 0.2μL |
| ddH2O | 4.6μL |
After connection product use feeling state stbl3 conversion, (Life is sequenced using universal primer U6 in selected clone
Technology, Shanghai), the clone for being correctly inserted into sgRNA sequence is shaken into bacterium and extracts plasmid.
2) sgRNA expression plasmid and Cas9 expression plasmid are transferred to the Efficiency testing of T cell system and primary T cells
As follows compare sgRNA expression plasmid and Cas9 expression plasmid it is different transfection (liposome transfection, electricity turn and
Slow-virus transfection) under the conditions of transfection efficiency.
Liposome transfection: in the hole middle berth 1 × 10 of six orifice plates61 μ is added in the OPTI-MEM of 200 μ L in Jurkat cell
G pCas9 (Addgene Plasmid#53118), 1 μ g TRAC-sg3 expression plasmid are uniformly mixed, and add 4 μ L X-
TremeGENE HP DNA transfection reagent or 6 μ L 100nM polyethyleneimines (Polyethylenimine, PEI) or 6 μ L
Lipo2000 reagent, is stored at room temperature 15min after mixing, in inlet hole.Cell is gained into fresh culture culture after 6-8h.
It changes after liquid 72h and mCherry positive cell ratio is analyzed by fluidic cell sorting (FACS).
Electricity turns: taking 1 × 106Jurkat cell turns that 1 μ g pCas9,1 μ g TRAC- is added in Buffer in the electricity of 100 μ L
Sg3 expression plasmid is uniformly mixed, and is turned with LONZA electroporation electricity.The cell turned continues to cultivate.It changes thin by streaming after liquid 72h
Born of the same parents sort (FACS) and analyze mCherry positive cell ratio.
Slow-virus infection: 1 × 10 is taken6Jurkat cell is packaged with Cas9 and TRAC- according to the ratio addition of MOI=4
The slow virus of sg3, changes liquid afterwards for 24 hours, continues to cultivate 48h, analyzes mCherry positive cell ratio by fluidic cell sorting (FACS)
Example.
The comparison result of different transfection conditions efficiency is shown in Fig. 1.Comparison result shows, liposome transfection method transfection efficiency compared with
Low, electric shifting method cell transfecting efficiency is higher, and the two Cell viability is similar.
3) sgRNA causes the Efficiency testing of genome mutation
With 1 μ g pCas9,1 μ g pTRAC-sgRNA and/or 1 μ g pTRBC-sgRNA transfection 2 × 105HeLa cell, 72h
The genome for extracting transfection cell afterwards is template, and with Primer-Blast design primer, specifically amplification includes sgRNA sequence
One section of 300-1000bp size genomic fragment.The PCR product and 10 × NEB of 300-500ng are taken in 50 μ L systems
Buffer2 (NEB) is mixed, and is slowly cooled to room temperature after 95 DEG C of heating 3min.0.5 μ L T7E1 (NEB) is added 37 in products therefrom
DEG C incubate 15min, carry out agarose gel electrophoresis, electrophoretogram with Image J image analysis software analyze band cut efficiency,
Indicate that sgRNA generates the efficiency (such as Fig. 2 a and 2b) of InDel (insertion-deletion).
The results show that the TRAC-sg3 and TRBC-sg1 editorial efficiency with higher to target gene site, subsequent selection
The plasmid of expression TRAC-sg3 and TRBC-sg1 continues to test.
4) tcr gene of T cell system (Jurkat, SupT1 etc.) and primary T cell are knocked out
Turn technology using electricity, sgRNA and Cas9 are imported into T cell system (Jurkat, SupT1 etc.) and primary T is thin
In born of the same parents, the negative T cell of CD4 or the CD8 positive simultaneously of TCR is filtered out by flow sorting techniques or immunomagnetic bead technique.
The negative T cell of CD4 or the CD8 positive simultaneously of the TCR filtered out is extracted into genome (blood/cell/tissue base
Because of a group DNA extraction kit), with specific primer (TRAC:Forward:AGTCTGTCTGCCTATTCACCGA, Reverse:
CCTGGTGCATTCATGTGCCG；TRBC:Forward:GGATAGATGATCAGACAAGCCT, Reverse:
TGGTAGCTGGTCTCACCTAAT) PCR amplification TRAC and TRBC include the genome area for corresponding to sgRNA, PCR product respectively
It carries out TA clone and is sequenced to verify the knockout of TCR from molecular level, it as a result fig. 3, it is shown that can be successfully right
TRAC the and TRBC gene of TCR is edited, including insertion mutation and deletion mutation, both causes frameshift mutation, thus
The expression of TCR is inhibited from the level of gene.
It is predicted simultaneously for the site of missing the target on the potential human genome of TRAC-sg3 and TRBC-sg1, and to prediction
Possibility influence other gene expressions site areas of missing the target carry out amplification and T7E1 analysis, in order to it is true from molecular level
That recognizes TCR knocks out the knockout for not introducing the non-specific gene of off-target, as a result as shown in Figure 4, it can be seen that TRAC
Any mutation does not occur with the gene of TRBC, illustrates the system specificity meet demand.
The above results show that being transferred to TRAC-sg3 and TRBC-sg1 and tcr gene in the T cell screened is complete
It knocks out, meanwhile, do not find the gene mutation at potential site of missing the target.
The design and vector construction of 3.CAR
With reference to Chinese invention patent application 201510324558.X disclosure, especially embodiment 1 and 2, design packet
The Chimeric antigen receptor of signal peptide, extracellular combined area, optional hinge area, transmembrane region and intracellular signal area containing sequential connection
(CAR), and vector construction is carried out.The nucleotide sequence of the extracellular combined area is the antigen in the Chimeric antigen receptor of anti-CD19
Combined area (is named as anti-CD19scFv-S0, referred to as scFv-S0 herein, mouse is derived from, referring to J
Immunother.2009September；32 (7): nucleotide sequence (such as application for a patent for invention 689-702.)
Shown in SEQ ID No:3 in the sequence table of 201510324558.X) on the basis of carry out it is humanization modified and obtain.Ying Li
Solution, the principle of antibody humanization's transformation are while guaranteeing affinity of antibody, to the maximum extent by skeleton area (framework
Region, FM) source of people sequence is changed into, to reduce the immunogenicity of antibody.In this embodiment, by above-mentioned SEQ ID No:3
In antigen recognizing district remain unchanged, remaining sequence is changed accordingly, has carried out the humanization design more than 40 kinds,
Synthesize to obtain these sequences by the method for gene chemical synthesis again, the other parts of CAR molecule be all using round pcr, respectively from
Clone obtains in human cDNA library, then carries out bridging connection, the nucleotide sequence of CAR molecule is finally prepared.By these
CAR molecule is transferred to T cell, by comprising the T cell containing these CAR molecules through humanization modified sequence with comprising containing
The T cell of the CAR molecule of scFv-S0 is compared the killing ability of target cell, finally screens to obtain two kinds and changes through humanization
The extracellular combination region sequence made, being referred to as anti-CD19scFv-S1, (referred to as scFv-S1, nucleotide sequence is as sent out
Shown in SEQ ID No:4 in the sequence table of bright patent application 201510324558.X) and anti-CD19scFv-S2 is (referred to as
ScFv-S2, nucleotide sequence as application for a patent for invention 201510324558.X sequence table in SEQ ID No:5 shown in).
Below by taking CAR5 and CAR6 as an example, the preparation step of the nucleotide sequence of CAR molecule is illustrated.
Design of primers is carried out first, and primer sequence used in the present embodiment is as follows:
1-1:5 '-atgcttctcctggtgacaag-3 '
1-2:5 '-tgggatcaggaggaatgctg-3 '
2-1:5 '-TACATCTGGGCGCCCTTGGCCGG-3 '
2-2:5 '-GGAGCGATAGGCTGCGAAGTCGCG-3 '
3-1:5 '-AGAGTGAAGTTCAGCAGGAGCG-3 '
3-2:5 '-TTAGCGAGGGGGCAGGGCCT-3 '
4-1:5 '-atgcttctcctggtgacaagcc-3 '
4-2:5 '-TGAGGAGACGGTGACTGAGGTTCCTTGG-3 '
5-1:5 '-gcggccgcaattgaagttatgta-3 '
5-2:5 '-TTAGCGAGGGGGCAGGGCCTGC-3 '
6-1:5 '-CTAGACTAGTatgcttctcctggtgacaagcc-3 '
6-2:5 '-CGACGCGTTTAGCGAGGGGGCAGGGCCTGC-3 '
Using the cDNA library of people as template, respectively using 1-1 and 1-2,2-1 and 2-2,3-1 and 3-2 as primer, pass through PCR grams
Grand corresponding CAR molecular moiety, respectively GMCSF, CD28-TM+CD28-signal (this two parts is connected to) and CD3 out
ζ-signal.GMCSF+scFv segment is obtained by bridging primer 4-1 and 4-2 again, is obtained by bridging primer 5-1 and 5-2
CD28-TM+CD28-signal+CD3 ζ-signal segment then obtains complete CAR molecule by bridging primer 6-1 and 6-2
Nucleotide sequence, restriction enzyme site be SpeI and MluI.
By the nucleotide sequence of CAR molecule produced above through SpeI (Fermentas) and MluI (Fermentas) double enzymes
It cuts, connect the site SpeI-MluI for being inserted into slow virus pLenti-CMV-eGFP carrier, conversion through T4 ligase (Fermentas)
To competence E.coli (DH5 α), after being sequenced correctly, plasmid purification kit is used to extract (Qiagen) and plasmid purification, used
In subsequent experimental.
4. the building and amplification of universal CAR-T cell
As described in step 1, after the T cell in PBMC is sorted and activated using the beads of coupling CD3/CD28 antibody, use
Cell density is adjusted to 2 × 10 by 15 culture medium of Lonza x-vivo6cell/mL.It is packaged with according to the ratio addition of MOI=2-4
The slow virus (slow virus method, specific steps can be found in 3 part of embodiment of application for a patent for invention 201510324558.X) of CAR,
It changes liquid afterwards for 24 hours, the state of cell is observed after 48h, collects cell suspension, 5min is centrifuged with 400g, supernatant is abandoned, with x-vivo 15
Cell density is adjusted to 1 × 10 by culture medium7cell/mL.Use mMessage mMachine T7Ultra kit (Life
Technologies) kit prepares Cas9mRNA and TRAC-sg3mRNA, stand-by after purifying and elution.Cell and
MRNA mixing reaches its ultimate density in every 100 μ L containing 1 × 106A cell and 500ng mRNA (Cas9mRNA and TRAC-
Each 250ng of sg3mRNA).MRNA is imported in cell using electroporation (NEPA21).The upgrowth situation of observation cell daily, often
It carries out changing liquid every two days.After cell culture 11-12d, quality testing is carried out to resulting T cell.And in 12-14d to T cell
It is purified, specific method is shown in step 5, (that is, universal CAR-T cell, referred to herein simply as by finished product finallyTCR-
CAR-T cell) it is dispensed and is frozen.
The screening of the T cell of 5.TCR feminine gender, CD4 and the CD8 positive: flow sorting techniques or immunomagnetic bead technique are utilized
Filter out the negative T cell of CD4 or the CD8 positive simultaneously of TCR.
1) first step carries out negative sorting first with the immunomagnetic beads of coupling CD3 antibody, removes and still express CD3 in T cell
(TCR) cell (usually carrying out 2 negative sortings)；
2) second step takes the T cell of a small amount of first steps, carries out flow cytometer detection, while dyeing CD3, CD4 and CD8, if
CD3 positive rate < 0.01%, while CD4+CD8 positive rate > 90%, can carry out further work.In the present embodiment, CD3
Positive rate is 0%, while CD4+CD8 positive rate is 93.3% (Fig. 5).
Embodiment 2: universal CAR-T (TCR-CAR-T cell) validation verification
The universal CAR-T cell (that is, effector cell) that observation embodiment 1 obtains is white to B cell type acute lymphoblastic
The therapeutic effect of blood disease.
Cell biology verifying:
Steps are as follows for specific experiment:
Step 1: using Calcein-AM label target cell (that is, coming from B cell type Patients With Acute Lymphoblastic Leukemia
Cell)
1) Calcein-AM (Life Technology, Shanghai) is diluted to 1mg/mL with DMSO；
2) the full culture medium of target cell is resuspended at 1 × 106The density of a/mL；
3) 15 μM of Calcein-AM, 37 DEG C, 5%CO are added230min is cultivated, every 10min is mixed gently；
4) with 1500rpm centrifugation 5 minutes, supernatant is removed, is resuspended with full culture medium, twice of repetitive operation；
Step 2: being killed using effector cell to target cell
1) target cell marked is resuspended according to the density of 5000-50000/mL, 100 μ L is taken to be added to 96 orifice plates
In；
2) 100 μ L effector cells are added than (5:1) according to ET appropriate (effect target ratio), while with T cell and general periphery
Blood CAR-T (that is, WT CAR-T cell) is used as control cell, and every group 3 parallel, detects the fluorescence intensity (test of supernatant
release)；Individual A group (6 parallel), only target cell are designed, the fluorescence intensity of its spontaneous apoptosis cracking is detected
(spontaneous release)；Design individual B group (6 parallel), only target cell+2%Triton X-100, detection
The fluorescence intensity (maximum release) of its maximum cracking；
3) 37 DEG C, 5%CO2After cultivating 4h, centrifugation takes 75 μ L supernatants, is transferred on a new culture plate；
4) sample utilizes spectramax Gemini dual-scanning microplate
Spectrofluorimeter detects (excitation filter:485 ± 9nm；band-pass filter:530±9nm)；
The percentage of cell cracking: [(test release-spontaneous release)/(maximum is calculated as follows
release–spontaneous release)]*100。
Acquired results are as shown in Figure 6, it can be seen that universal CAR-T killing ability and WT CAR-T killing ability are basic
Unanimously.
Zoopery verifying:
One, the building of lymphoma mouse model:
1. cell line: human lymphoma cell system Daudi；
Daudi cell is human lymphoma cell system, and the human lymphoma mould of mouse can be constructed by hypodermic mode
Type.Its CD19 is expressed as the positive, can be used as the target cell of CAR-T cell.
2.Daudi cell culture
Daudi cell line is suspension cell line, can fast fast-growing in 1640 culture mediums (Gibco) containing 20%FBS
It is long.Cell density is 2-3 × 106It needs to pass on when/mL.It takes cell suspension in centrifuge tube when passage, is centrifuged 5 points with 500g
Clock abandons supernatant.Cell density is adjusted to 0.3-0.5 × 106/ mL continues to cultivate.In the case of normal growth, Daudi cell line
To pass on every other day, cell density maintains 0.3-3 × 106Between/mL.
3. cell line is inoculated with
Daudi cell is resuspended with physiological saline, adjusting its viable cell concentrations is 3 × 108A/mL, on ice by its with
Matrigel (BD, China) is mixed well according to the volume ratio of 2:1.It is inoculated with by hypodermic mode.
Successfully to grow 100mm3Tumour as the successful criterion of mouse lymph lymphoma model construction.Wherein tumour body
Product calculation formula are as follows: gross tumor volume (mm3)=tumour major diameter (mm) × tumour minor axis2(mm2)×0.5；
4. mouse lymph lymphoma model is administered.The record administration same day is D0.It is defeated that cell is carried out by way of tail vein injection
(200 μ L of PBS, 200 μ L of human T-cell (amount to 1 × 10 to note6/ only), 200 μ L of people WT CAR-T cell (amount to 1 × 106/ only),
PeopleTCR-200 μ L of CAR-T cell (amounts to 1 × 106/ only)), all equal single-doses of mouse.As a result as shown in Figure 7, it can be seen that
It is provided by the inventionTCR-CAR-T cell has the effect of inhibiting and killing tumour cell well.
Embodiment 3: universal CAR-T (TCR-CAR-T cell) security verification
Mouse GVHD model
The C57 male mice 30 (2.4Gy full-body exposure was given before 24 hours) of 6-8 week old, single tail vein injection is small
Mouse T cell, people's WT CAR-T cell and peopleTCR-CAR-T cell each 1 × 107(every group is randomly assigned 10 to a/mL, negative control
Group injection same volume PBS), starting Detailed clinical observation after injection cell 4 hours, (death rate, clinical symptoms, behavior, weight become
Change), observation is primary daily later, is observed continuously 60 days, whether generation to assess GVHD and its seriousness.
It can be seen from Table 2 that serious GVHD reaction all occurs for the mouse injected in the group of normal xenogenesis CAR-T cell,
And there is 70% dead mouse.And the CAR-T cell after TCR is knocked out hardly happens GVHD to body, illustrates CAR- after TCR knockout
T cell safety is higher.
Fig. 8 shows the variation of average mice body weight, inject mouse weight in normal xenogenesis CAR-T groups of cells it is obvious under
Drop, at most has dropped 30%, and does not inject the mouse weight in the group of CAR-T cell and the group of injection TCR knockout CAR-T cell
It is not decreased obviously, there is a little rise phenomenon on the contrary.Comprehensive all data show the safety of the CAR-T cell after TCR is knocked out
It is high.
Table 2: mouse anomaly statistics table
Claims (6)
1. a kind of method for preparing universal CAR-T cell, described method includes following steps:
1) sgRNA molecule and Cas9 molecule are introduced in T cell；
2) CAR molecule is introduced in the T cell；
Wherein, step 2) can carry out before, after or at the same time in step 1), and the sgRNA molecule include with from TCR's
The targeting structural domain of the target region complementation of a chain and/or β chain constant code area (i.e. TRAC and/or TRBC) gene；
And wherein it is described targeting structural domain sequence as shown in SEQ ID NO:3 or SEQ ID NO:8,
Wherein turn technology by electricity the mRNA of the sgRNA molecule and coding Cas9 molecule is introduced into the T cell.
2. method of claim 1, the method further includes separation and/or activation healthy volunteer or donor before step 1)
T cell the step of.
3. method for claim 2, wherein the method further includes sorting to universal CAR-T cell after step 2)
The step of.
4. method for claim 3, wherein carrying out validation verification to resulting universal CAR-T cell again after sorting.
5. the method for claims 1 or 2, wherein the CAR molecule is introduced into the T cell by slow-virus transfection technology.
6. using the universal CAR-T cell of described in any one of claim 1 to 55 described in any item method preparations, wherein described universal
CAR-T cell expression specificity Chimeric antigen receptor (CAR) and T cell receptor (TCR) is not expressed, wherein the CAR includes
The signal peptide of sequential connection, extracellular combined area, optional hinge area, transmembrane region and intracellular signal area；The nucleosides of the signal peptide
Acid sequence as application for a patent for invention 201510324558.X sequence table in shown in SEQ ID No:1 or SEQ ID No:2；It is described
SEQ ID No:4 or SEQ in the sequence table of the nucleotide sequence such as application for a patent for invention 201510324558.X of extracellular combined area
Shown in ID No:5；SEQ in the sequence table of the nucleotide sequence such as application for a patent for invention 201510324558.X of the hinge area
Shown in ID No:6 or SEQ ID No:7；The nucleotide sequence of the transmembrane region such as application for a patent for invention 201510324558.X's
In sequence table shown in SEQ ID No:8 or SEQ ID No:9；The nucleotide sequence such as application for a patent for invention in the intracellular signal area
In the sequence table of 201510324558.X shown in SEQ ID No:10, SEQ ID No:11 or SEQ ID No:12.
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| CN109456942A (en) * | 2017-09-06 | 2019-03-12 | 亘喜生物科技（上海）有限公司 | Universal Chimeric antigen receptor T cell technology of preparing |
| WO2019047932A1 (en) * | 2017-09-08 | 2019-03-14 | 科济生物医药（上海）有限公司 | Genetically engineered t cell and application thereof |
| TW201920661A (en) * | 2017-09-18 | 2019-06-01 | 大陸商博雅輯因（北京）生物科技有限公司 | Gene editing T cell and use thereof |
| CN107723275B (en) * | 2017-10-20 | 2020-09-04 | 重庆精准生物技术有限公司 | Universal CAR-T cell and preparation method and application thereof |
| CN107557342A (en) * | 2017-11-02 | 2018-01-09 | 广东万海细胞生物科技有限公司 | A kind of preparation method and applications of CAR Treg cells |
| CN109750035B (en) * | 2017-11-02 | 2020-06-05 | 上海邦耀生物科技有限公司 | sgRNA for targeting and guiding Cas9 protein to efficiently cleave TCR and B2M gene locus |
| CN110616187B (en) * | 2018-06-20 | 2021-12-03 | 西安桑尼赛尔生物医药有限公司 | Method for efficiently knocking chimeric antigen receptor gene into T cell specific genome site by CRISPR-Cas9 and application |
| CN110616188B (en) * | 2018-06-20 | 2023-05-09 | 上海隆耀生物科技有限公司 | Universal CAR-T cell and preparation method and application thereof |
| CN110616186A (en) * | 2018-06-20 | 2019-12-27 | 西安桑尼赛尔生物医药有限公司 | Method for increasing allogeneic T cell transplantation compatibility and application thereof |
| CN108913693B (en) * | 2018-07-23 | 2021-02-02 | 合肥一兮生物科技有限公司 | siRNA for knocking down human TRAC/TRBC gene, lentiviral vector, CAR-T cell construction method and application |
| CN109207501A (en) * | 2018-09-25 | 2019-01-15 | 深圳市菲鹏生物治疗股份有限公司 | Express the construction method of the sequence of universal Chimeric antigen receptor |
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| CN111676195A (en) * | 2019-03-11 | 2020-09-18 | 北京卡替医疗技术有限公司 | UCAR immune cell for treating T cell tumor |
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Address after: No. 316, floor 3, building 1, block B, No. 80, xingshikou Road, Haidian District, Beijing 100093 Patentee after: Beijing Yimiao Shenzhou Pharmaceutical Technology Co., Ltd Address before: 102206 Beijing City, Changping District small town life Zhongguancun Life Science Park Road No. 8 Building 1, Beijing University Medical Industrial Park 218 Patentee before: BEIJING IMMUNO SHENZHOU MEDICAL TECHNOLOGY Co.,Ltd. |