CN106544321A - Universal CAR T cells and its production and use - Google Patents
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Abstract
Universal CAR T cells and its production and use.The invention provides a kind of universal CAR T cells, its expression specificity CAR and TCR is not expressed.Present invention also offers a kind of method for preparing universal CAR T cells, methods described comprises the steps：1) introducing sgRNA molecules and Cas9 molecules in T cell；2) the introducing CAR molecules in the T cell；Wherein, step 2) can be in step 1) before, afterwards or while carry out, and the sgRNA molecules are comprising the complementary targeting domain of the target region with a chains from TCR or β chain constant codes area (i.e. TRAC or TRBC) gene.There is GVHD and the interference of potential TCR receptor signals in the T cell that the universal CAR T cells of the present invention avoid transduction.
Description
Technical field
The present invention relates to immunology and biology field, more particularly to tumour immunotherapy, especially a kind of logical
With type CAR-T cell and its production and use.
Background technology
Tumour immunotherapy is widely paid close attention in recent years and is applied, especially CAR-T (chimeric antigen
Receptor-engineered T cells) technology appearance, make control of the mankind to tumour obtain landmark
Exhibition.Application of the technology in oncotherapy starts from 1989.Its clinical practice result allows people to see the dawn for curing cancer：
Not only can 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 technologies is mainly derived from present
Patient itself, imports and expands through activation, CAR from the T cell of patient itself derived from peripheral blood, then feed back to patient's body
It is interior.But in many cases, as the state of an illness of patient is dangerous or patient itself condition does not allow to extract outer in vivo out from which again
All blood carries out the treatment of CAR-T cells；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 transformations, and this just limits the range of application of CAR-T technologies from certain limit.In order to solve this
Individual problem, allosome CAR-T are increasingly becoming a good selection.Meanwhile, allosome CAR-T technologies solve the source of T cell and ask
Commercialization for CAR-T is also promoted and is laid the foundation by topic.
In body, it is a complicated process that T cell is captured and eliminates heterologous antigen, needs ajor histocompatibility multiple
The common participation of compound (MHC) and φt cell receptor (TCR), wherein TCR play key in the Process of Antigen that identification MHC is combined
Effect.MHC (MHC) has two types, commonly referred to MHC I classes and MHC II classes, except with anti-
Former Presentation is also related to the rejection during organ transplant.Killer T cell is then the class having by its surface
It is antigen that φt cell receptor (TCR) recognized and removed Jing MHC I quasi-molecule submissions like the molecule of antibody.In allogeneic environment
In, the TCR signal transductions effect on CAR-T may produce potential impact to clinical response, and mutual between TCR and CAR
Effect not yet has in-depth study.Meanwhile, feed back the wind that allosome CAR-T cells are possible to graft versus host disease(GVH disease) (GVHD) occur
Danger.
It is therefore desirable to have reduce acceptor donorcells rejection and avoid the occurrence of GVHD improve CAR-T curative effects
Method.
The content of the invention
The present invention is directed to drawbacks described above, using CRISPR/Cas9 technologies to the T cell from healthy volunteer (donor)
Modified, selectively knocked out a chains and/or the β chain constant codes area (TRAC and/or TRBC) of endogenous T cells acceptor (TCR)
Gene, while Chimeric antigen receptor (CAR) is stably expressed in T cell, so as to obtain expression specificity CAR while not table
Up to the CAR-T cells of TCR.
A first aspect of the present invention provides a kind of universal CAR-T cells, its expression specificity CAR and does not express
TCR。
A second aspect of the present invention provides a kind of method for preparing universal CAR-T cells, and methods described includes as follows
Step：
1) introducing sgRNA molecules and Cas9 molecules in T cell；
2) the introducing CAR molecules in the T cell；
Wherein, step 2) can be in step 1) before, afterwards or while carry out, and the sgRNA molecules comprising with from
The complementary targeting domain of the target region of a chains or β chain constant codes area (i.e. TRAC or TRBC) gene of TCR.
A third aspect of the present invention provides the composition of the universal CAR-T cells comprising first aspect present invention.
A fourth aspect of the present invention provides the universal CAR-T cells of first aspect present invention to be used to prepare treatment tumour
Medicine purposes.
The universal CAR-T cells of the present invention avoid the T cell being infused in patient's body and GVHD and potential occur
TCR receptor signals are disturbed.The application will be that the use of universal CAR-T cells is paved the way, with great clinical meaning.
Description of the drawings
According to the detailed description for carrying out referring to the drawings, the above and other aspects, features and advantages of the present invention can become
Obtain clearer.
Fig. 1 shows that (liposome transfection, electricity turn and slow disease in different transfections for sgRNA expression plasmids and Cas9 expression plasmids
Poison transfection) under the conditions of transfection efficiency comparative result.
Fig. 2 a and Fig. 2 b show that different sgRNA cause the comparative result of the efficiency of genome mutation.
Fig. 3 shows the result are sequenced by the TRAC and TRBC regions knocked out to Jing TCR.
Fig. 4 shows the result is sequenced by the site of missing the target on the potential human genomes of TRAC-sg3 to prediction.
Fig. 5 shows the selection result to the T cell that TCR is negative, CD4 and CD8 is positive.
Fig. 6 show T cell, WT CAR-T andTCR-Comparative result of the CAR-T cells to the fragmentation effect of target cell.
Fig. 7 show T cell, WT CAR-T andTCR-The comparison of the effect of CAR-T Carbazole alkaloids and kill tumour cell
As a result.
Fig. 8 shows injection mouse T cell, people WT CAR-T and peopleTCR-After CAR-T cells, the change of average mice body weight
The comparative result of change.
Specific embodiment
As described above, a first aspect of the present invention provides a kind of universal CAR-T cells, its expression specificity CAR is simultaneously
And do not express TCR.
In one embodiment, the cell derived is in healthy volunteer (or donor).
In one embodiment, the tcr gene of universal CAR-T cells of the invention is knocked.
In a specific embodiment, a chains of the TCR and/or β chain constant codes area (i.e. TRAC and/or
TRBC) gene is knocked.TRAC and TRBC genes all can be knocked out, it is also possible to partly be knocked out；Can be struck with the two
Remove, it is also possible to knock out alternative one；As long as cell can not express activated TCR after knocking out.
In a specific embodiment, a chain constant codes area (i.e. TRAC) gene of the TCR is knocked.For example,
In a particular embodiment, a chain constant codes area gene of TCR of the present invention be introduced into the cell TRAC-sg1-7 molecules it
One (being shown in Table 1) and 9 molecular moieties of Cas are all knocked out；It is preferred that, a chain constant codes area gene of the TCR is introduced into carefully
The TRAC-sg3 molecules of born of the same parents and Cas9 molecules are knocked out.
In another specific embodiment, β chain constant codes area (i.e. TRBC) gene of the TCR is knocked.Example
Such as, in a particular embodiment, the β chain constant codes area gene of TCR of the present invention is introduced into TRBC-sg1-7 point of the cell
One of son (being shown in Table 1) and 9 molecular moieties of Cas are all knocked out；It is preferred that, β chain constant code area's genes of the TCR are drawn
The TRBC-sg1 molecules and Cas9 molecules for entering cell is knocked out.
In one embodiment, the CAR expressed by universal CAR-T cells of the invention can be known in the art
Any CAR, as long as which can make T cell that cell surface antigen is recognized in the way of human leucocyte antigen (HLA)-dependent/non-dependent, performance is killed
Wound is acted on.It is, for example possible to use the CAR disclosed in Chinese invention patent application 201510324558.X, the tool of the present invention
CAR used in body embodiment refers to the application for a patent for invention disclosure.Specifically, CAR-T cells of the invention
The CAR of middle expression includes signal peptide, extracellular land, optional hinge area, transmembrane region and the intracellular signal area being linked in sequence.
Terms used herein " signal peptide " refers to short (length 5- of the protein of the new synthesis of guiding to secretion path transfer
30 amino acid) peptide chain.In the present invention, it is possible to use the signal peptide of the various protein in human body, such as body are endocrine
The signal peptide of Cytokine protein, leukocyte differentiation antigen (CD molecules).
In a specific embodiment, the signal peptide be GMCSF signal peptides, its nucleotide sequence such as patent of invention
SEQ ID No in the sequence table of application 201510324558.X:Shown in 1.
In a specific embodiment, the signal peptide be CD8 signal peptides, its nucleotide sequence such as patent of invention Shen
SEQ ID No that please be in the sequence table of 201510324558.X:Shown in 2.
In the present invention, the hinge area can use the hinge area of various different antibodies or antigen receptor, particularly CD
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 are the native marker things on T cell surface.
In a specific embodiment, the hinge area be CD8 hinge areas (CD8-hinge), its nucleotide sequence
Such as the SEQ ID No in the sequence table of application for a patent for invention 201510324558.X:Shown in 6.
In a specific embodiment, the hinge area be CD28 hinge areas (CD28-hinge), its nucleotides sequence
SEQ ID No in the sequence table of row such as application for a patent for invention 201510324558.X:Shown in 7.
In the present invention, it is possible to use the transmembrane region of various people's vivo proteins, particularly various different antigen receptors across
Film area.The transmembrane region for preferably using is the transmembrane region of CD molecules.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 regions (CD8-TM), and its nucleotide sequence is as sent out
SEQ ID No in the sequence table of bright patent application 201510324558.X:Shown in 8.
In a specific embodiment, the transmembrane region is CD28 transmembrane regions (CD28-TM), and its nucleotide sequence is such as
SEQ ID No in the sequence table of application for a patent for invention 201510324558.X:Shown in 9.
The scFv of " extracellular land " comprising specific recognition TCSA.
Terms used herein " scFv " refers to such antibody fragment --- which is to include by joint (linker) even
The weight chain variable district (variable region of heavy chain, VH) for connecing and light chain variable district (variable region
Of light chain, VL) recombinant protein, joint cause the two domains be associated, to ultimately form antigen binding position
Point.ScFv's is typically of size of the 1/6 of a complete antibody.ScFv is preferably by the amino acid sequence of a nucleotide chain coding
Row.The scFv that the present invention is used such as amino acid deletions, can be inserted by being used alone or in combination routine techniques known in the art
Enter, replace, increase, and/or recombinate and/or other method of modifying are further modified.According to a kind of amino acid sequence of antibody
The method that this modification is introduced in being listed in its DNA sequence dna is known (see, for example, for a person skilled in the art
Sambrook molecular clonings：Laboratory manual, Cold Spring Harbor Laboratory (1989) N.Y.).The modification is excellent
Being selected in nucleic acid level is carried out.Above-mentioned scFv can also include its derivative.
Terms used herein " specific recognition " mean the present invention antigen recognizing district not with or substantially not with target
Any polypeptide cross reaction beyond antigen.Its specific degree can be judged by immunological technique, including but do not limit
In Western blotting, immunoaffinity chromatography, flow cytometry etc..
In one embodiment, the extracellular land includes specific recognition CD19, CEA, EGFR, GD2 or CD138
Deng scFv.
In a specific embodiment, scFv (anti-of the extracellular land comprising specific recognition CD19
CD19scFv)。
In a specific embodiment, the extracellular land includes Jing humanization modified specific recognition CD19
ScFv.
In a preferred embodiment, the extracellular land is humanization modified specific recognitions CD19 of Jing
ScFv-S1 (the SEQ ID No in the sequence table of its nucleotide sequence such as application for a patent for invention 201510324558.X:Shown in 4) or
ScFv-S2 (the SEQ ID No in the sequence table of its nucleotide sequence such as application for a patent for invention 201510324558.X:Shown in 5).
In the present invention, it is possible to use the intracellular signal area of various people's vivo proteins, particularly various different antigen receptors
Intracellular signal area.The intracellular signal area for preferably using is the intracellular signal area of CD molecules.In one embodiment, the born of the same parents
Interior signaling zone can be selected from CD3 ζ, the intracellular signal area of Fc ε RI γ, CD28, CD137, CD134 albumen, and combinations thereof.CD3 point
Son is made up of five subunits, and wherein containing 3 ITAM motifs, the motif is for CD3 ζ subunits (also known as CD3zeta, abbreviation Z)
Signal zone of transformation important in TCR-CD3 complexs.CD3 δ Z are the not CD3 ζ sequences with ITAM motifs of mutation, at this
In bright embodiment generally as negative control structure component.Fc ε RI γ are mainly distributed on mast cell and basophil
Cellular surface, which contains an ITAM motif, in structure, is distributed and functionally similar with CD3 ζ.In addition as it was previously stated, CD28,
CD137, CD134 are costimulatory signal molecules, the costimulation effect that its intracellular signal section is produced after with respective ligand binding
Cause the continuous proliferation of T cell, and the level that T cell secretes the cell factor such as IL-2 and IFN-γ can be improved, while improving
CAR-T cells time to live in vivo and antitumous effect.
There is multiple combination in the specifically used intracellular signal area of the present invention, and which is included selected from following signaling zone or its combination：
CD28 signaling zones (CD28-signal), the sequence of its nucleotide sequence such as application for a patent for invention 201510324558.X
SEQ ID No in list:Shown in 10；
CD137 signaling zones (CD137-signal), its nucleotide sequence such as application for a patent for invention 201510324558.X
SEQ ID No in sequence table:Shown in 11；With
CD3 ζ signaling zones (CD3 ζ-signal), the sequence of its nucleotide sequence such as application for a patent for invention 201510324558.X
SEQ ID No in list:Shown in 12.
In a specific embodiment, the CAR expressed by the universal CAR-T cells of the present invention is selected from following bag
Chimeric antigen receptor egg containing the signal peptide being linked in sequence, extracellular land, optional hinge area, transmembrane region and intracellular signal area
In vain：
GMCSF-scFv-S1-CD8-hinge-CD8-TM-CD137-signal-CD3 ζ-signal (CAR1, its nucleic acid sequence
SEQ ID No in the sequence table of row such as application for a patent for invention 201510324558.X:Shown in 13)；
GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD137-signal-CD3 ζ-signal (CAR2, its nucleic acid sequence
SEQ ID No in the sequence table of row such as application for a patent for invention 201510324558.X:Shown in 14)；
(CAR3, its nucleotide sequence are as special in invented for GMCSF-scFv-S1-CD28-TM-CD28-signal-CD3 ζ-signal
SEQ ID No in the sequence table of profit application 201510324558.X:Shown in 15)；
(CAR4, its nucleotide sequence are as special in invented for GMCSF-scFv-S2-CD28-TM-CD28-signal-CD3 ζ-signal
SEQ ID No in the sequence table of profit application 201510324558.X:Shown in 16)；
GMCSF–scFv-S1–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal
(CAR5, its nucleotide sequence as application for a patent for invention 201510324558.X sequence table in such as SEQ ID No:Shown in 17)；
GMCSF–scFv-S2–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal
(CAR6, the SEQ ID No in the sequence table of its nucleotide sequence such as application for a patent for invention 201510324558.X:Shown in 18).
As described above, a second aspect of the present invention provides a kind of method for preparing universal CAR-T cells, methods described
Comprise the steps：
1) introducing sgRNA molecules and Cas9 molecules in T cell；
2) the introducing CAR molecules in the T cell；
Wherein, step 2) can be in step 1) before, afterwards or while carry out, and the sgRNA molecules comprising with from
The complementary targeting domain of the target region of a chains and/or β chain constant codes area (i.e. TRAC and/or TRBC) gene of TCR.
In the method for the invention, the sgRNA molecules refer to that includes the target region complementation with gene to be knocked out
Targeting domain nucleotide sequence, its can recognize target DNA sequence dna and guide Cas9 molecules cutting target site.
In the method for the invention, the Cas9 molecules refer to a kind of double-stranded DNA nuclease, and which being capable of drawing in sg RNA
Lead and lower target site is cut.
In one embodiment, the sgRNA molecules comprising with a chain constant codes area (i.e. TRAC) base from TCR
Because of the complementary targeting domain of target region.
In a specific embodiment, the sequence such as SEQ ID of the targeting domain included by the sgRNA molecules
NOs:Shown in one of 1-7.
In a preferred embodiment, the sequence such as SEQ ID NO of the targeting domain:Shown in 3.
In one embodiment, the sgRNA molecules comprising with β chain constant codes area (i.e. TRBC) base from TCR
Because of the complementary targeting domain of target region.
In a specific embodiment, the sequence such as SEQ ID of the targeting domain included by the sgRNA molecules
NOs:Shown in one of 8-14.
In a preferred embodiment, the sequence such as SEQ ID NO of the targeting domain:Shown in 8.
In one embodiment, by carrier construction by the sgRNA molecules and Cas9 molecule Jing liposome transfections, electricity
Turn or slow-virus transfection technology is introduced in the T cell.
In a preferred embodiment, the sgRNA molecules and Cas9 molecule Jing electricity are turned by skill by carrier construction
Art is introduced in the T cell.
In a further preferred embodiment, technology is turned by the sgRNA molecules by electricity and Cas9 molecules are encoded
MRNA is introduced in the T cell.
In one embodiment, the CAR molecules are introduced in the T cell by slow-virus transfection technology.
In a specific embodiment, the method for the present invention is in step 1) also include before separating and/or activate
From the T cell of healthy volunteer's (or donor) the step of；Preferably, methods described is in step 2) after also include to universal
The step of CAR-T cells are sorted；It is highly preferred that carrying out validity to the universal CAR-T cells of gained again after sorting
Checking.
As described above, a third aspect of the present invention provides the universal CAR-T cells comprising first aspect present invention
Composition.
In one embodiment, the composition is also comprising pharmaceutically useful diluent, excipient or carrier etc..
As described above, a fourth aspect of the present invention provides the universal CAR-T cells of first aspect present invention to be used to make
The purposes of the medicine of standby treatment tumour.
Present disclosure is illustrated below by way of specific embodiment.It should be understood that the specific embodiment only illustrates mesh
, it is not meant to that present disclosure is only limitted to specific embodiment.
Embodiment 1：The preparation of universal CAR-T cells
1. the separation of health donors T cell and activation.
1) collection of health donors peripheral blood：4 DEG C of refrigerators of peripheral blood of collection are kept in, in 24h, via setting equipped with constant temperature
Standby transfer car(buggy) is transported to GMP laboratories and is separated and cultivated.
2) preparation of PMNC (PBMC)：With pipette, extract DPBS (Dulbecco's Phosphate
Buffered Saline) or physiological saline be added to (1 in the peripheral blood that step (1) is gathered：1), dilute, 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, is proceeded in a new centrifuge tube, add Lonza
15 culture medium (article No.s of x-vivo：Supernatant is abandoned after 04-418Q) being centrifuged, is retained the cell precipitation of centrifugation bottom of the tube, that is, is obtained periphery
Blood mononuclear cell.
3) separation and activation of T cell：The PMNC for obtaining is counted, according to 1:1 ratio adds
Enter to be coupled the beads of CD3/CD28 antibody, gently shake 20min, using the suction-operated of magnetic frame, obtain the positive T of CD3 thin
Born of the same parents, T cell now are active, and add complete medium (Lonza x-vivo 15+IL-2), T cell is trained
Support amplification.
2. the tcr gene in the T cell that Jing steps 1 are obtained is knocked out using CRISPR/Cas9 systems, concrete operation step is such as
Shown in lower：
1) the sgRNA designs of a chains and β chain constant codes area (TRAC, TRBC) gene for TCR and plasmid construction.
For selecting 5 '-GN in first three exon sequence of TRAC and TRBC gene coding regions19-22The sequence of NGG-3 '
Row, and with Cas-OFFinder guarantee its 3 ' 8-12 base held will not mispairing to genome other positions (http://
www.rgenome.net/cas-offinder/).Selected sequence is shown in Table 1.
Table 1：SgRNA sequences
Synthetic primer, positive-sense strand are 5 '-ACCGN19-22- 3 ', antisense strand is 5 '-AAACN19-22- 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 L 10 × TransTaq HiFi
Buffer II (full formula gold Transgene, Beijing) mix, and are slowly cooled to room temperature after heating 3min in 95 DEG C.Products therefrom is used
T4PNK (T4Polynucleotide Kinase/T4 polynueleotide kinases) carries out phosphatizing treatment, 37 DEG C of incubation 30min.
By the product (Insert sgRNA) after phosphorylation respectively by Golden Gate reaction formings to psgRNA carrier (Addgene
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 the conversion of connection product use feeling state stbl3, selected clone is sequenced (Life using universal primer U6
Technology, Shanghai), the clone for being correctly inserted into sgRNA sequences is shaken into bacterium and extracts plasmid.
2) sgRNA expression plasmids and Cas9 expression plasmids proceed to the Efficiency testing of T cell system and primary T cells
It is as follows compare sgRNA expression plasmids and Cas9 expression plasmids different transfections (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 plates6Jurkat cell, adds 1 μ in the OPTI-MEM of 200 μ L
G pCas9 (Addgene Plasmid#53118), 1 μ g TRAC-sg3 expression plasmids are well mixed, and add 4 μ L X-
TremeGENE HP DNA transfection reagents or 6 μ L 100nM polyethyleneimines (Polyethylenimine, PEI), or 6 μ L
Lipo2000 reagents, are stored at room temperature 15min, in inlet hole after being well mixed.Cell is gained into fresh culture culture after 6-8h.
MCherry positive cell ratios are analyzed by fluidic cell sorting (FACS) after changing liquid 72h.
Electricity turns：Take 1 × 106Jurkat cell, adds 1 μ g pCas9,1 μ g TRAC- in the electricity of 100 μ L turns Buffer
Sg3 expression plasmids, are well mixed, and are turned with LONZA electroporations electricity.The cell for having turned continues culture.Pass through streaming after changing liquid 72h thin
Born of the same parents' sorting (FACS) analysis mCherry positive cell ratios.
Slow-virus infection：Take 1 × 106Jurkat cell, adds according to the ratio of MOI=4 and is packaged with Cas9 and TRAC-
The slow virus of sg3, changes liquid after 24h, continue culture 48h, analyze mCherry positive cell ratios by fluidic cell sorting (FACS)
Example.
The comparative result of different transfection conditions efficiency is shown in Fig. 1.Comparative result shows, liposome transfection method transfection efficiency compared with
Low, electric shifting method cell transfecting efficiency is higher, and both Cell viabilities are similar to.
3) sgRNA causes the Efficiency testing of genome mutation
2 × 10 are transfected with 1 μ g pCas9,1 μ g pTRAC-sgRNA and/or 1 μ g pTRBC-sgRNA5HeLa cells, 72h
The genome for extracting transfectional cell afterwards is template, designs primer with Primer-Blast, and specifically amplification includes sgRNA sequences
One section of 300-1000bp size genomic fragment.The PCR primer and 10 × NEB of 300-500ng are taken in 50 μ L systems
Buffer2 (NEB) mixes, and is slowly cooled to room temperature after 95 DEG C of heating 3min.Products therefrom adds 0.5 μ L T7E1 (NEB) 37
DEG C 15min is incubated, enters row agarose gel electrophoresis, electrophoretogram analyzes the efficiency of band cutting with Image J image analysis softwares,
Indicate that sgRNA produces the efficiency (such as Fig. 2 a and 2b) of InDel (insertion-deletion).
As a result show, TRAC-sg3 and TRBC-sg1 has the higher editorial efficiency to target gene site, follow-up to choose
The plasmid of expression TRAC-sg3 and TRBC-sg1 continues experiment.
4) knock out the tcr gene of T cell system (Jurkat, SupT1 etc.) and primary T cells
Turn technology using electricity, sgRNA and Cas9 are imported to into T cell system (Jurkat, SupT1 etc.) and primary T are thin
In born of the same parents, TCR is filtered out by flow sorting techniques or immunomagnetic bead technique negative while the T cell of the CD4 or CD8 positives.
By the TCR for filtering out negative CD4 or CD8 simultaneously positive T cell extraction 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 amplifications TRAC and TRBC includes the genome area of correspondence sgRNA, PCR primer respectively
Carry out the knockout of TA clones and sequencing TCR is verified from molecular level, as a result fig. 3, it is shown that can be successfully right
TRAC the and TRBC genes of TCR enter edlin, including insertion mutation and deletion mutation, both cause frameshift mutation, so as to
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 genomes of TRAC-sg3 and TRBC-sg1, and to prediction
Possibility affect the site areas of missing the target of other gene expressions carry out amplification and T7E1 analyze, in order to it is true from molecular level
The knockout for recognizing TCR does not have the knockout of the non-specific gene for introducing off-target, as a result as shown in Figure 4, it can be seen that TRAC
There is no any mutation with the gene of TRBC, illustrate the special sexual satisfaction demand of the system.
Result above shows, proceeds to TRAC-sg3 and TRBC-sg1 and the tcr gene in the T cell screened is complete
Knock out, meanwhile, do not find the gene mutation of potential site of missing the target.
The design of 3.CAR and vector construction
With reference to Chinese invention patent application 201510324558.X disclosure, particularly embodiment 1 and 2, design bag
Chimeric antigen receptor containing the signal peptide being linked in sequence, extracellular land, optional hinge area, transmembrane region and intracellular signal area
(CAR), and carry out vector construction.The nucleotide sequence of the extracellular land is the antigen of the Chimeric antigen receptor in anti-CD19
(here is named as anti-CD19scFv-S0, referred to as scFv-S0, and which derives from mouse, referring to J for land
Immunother.2009September；32(7):689-702.) nucleotide sequence is (such as application for a patent for invention
201510324558.X sequence table in SEQ ID No:Shown in 3) on the basis of carry out it is humanization modified and obtain.Ying Li
Solution, the principle of antibody humanization's transformation is while affinity of antibody is ensured, to greatest extent by skeleton area (framework
Region, FM) change as people's source sequence, to reduce the immunogenicity of antibody.In this embodiment, by above-mentioned SEQ ID No:3
In antigen recognizing district keep it is constant, remaining sequence is changed accordingly, the humanization design more than 40 kinds has been carried out,
These sequences are obtained by the synthesis of the method for gene chemical synthesis again, the other parts of CAR molecules are all to utilize round pcr, respectively from
In human cDNA library, clone obtains, and then carries out bridging connection, finally prepares the nucleotide sequence of CAR molecules.By these
CAR molecules proceed to T cell, by the T cell comprising the CAR molecules containing the humanization modified sequences of these Jing with contain
The T cell of the CAR molecules of scFv-S0 is compared to the killing ability of target cell, and final screening obtains two kinds of Jing humanizations and changes
The extracellular combination region sequence made, which is referred to as anti-CD19scFv-S1, and (referred to as scFv-S1, its nucleotide sequence is as sent out
SEQ ID No in the sequence table of bright patent application 201510324558.X:Shown in 4) and anti-CD19scFv-S2 is (referred to as
ScFv-S2, the SEQ ID No in the sequence table of its nucleotide sequence such as application for a patent for invention 201510324558.X:Shown in 5).
Below by taking CAR5 and CAR6 as an example, the preparation process of the nucleotide sequence of CAR molecules is illustrated.
Design of primers is carried out first, and the 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’
CDNA library with people as template, respectively with 1-1 and 1-2,2-1 and 2-2,3-1 and 3-2 as primer, by PCR gram
It is grand go out corresponding CAR molecular moieties, respectively GMCSF, CD28-TM+CD28-signal (this two parts is connected to) and CD3
ζ-signal.GMCSF+scFv fragments are 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 fragments, subsequently obtain complete CAR molecules by bridging primer 6-1 and 6-2
Nucleotide sequence, restriction enzyme site be SpeI and MluI.
By nucleotide sequence Jing SpeI (Fermentas) of CAR molecules produced above and the double enzymes of MluI (Fermentas)
Cut, Jing T4 ligases (Fermentas) connects the SpeI-MluI sites for inserting slow virus pLenti-CMV-eGFP carriers, conversion
To competence E.coli (DH5 α), Jing after sequencing is correct, (Qiagen) plasmid purification are extracted using plasmid purification kit, used
In subsequent experimental.
4. the structure of universal CAR-T cells and amplification
As described in step 1, after the T cell in PBMC being sorted and activated using the beads for being coupled CD3/CD28 antibody, use
Cell density is adjusted to 2 × 10 by 15 culture mediums of Lonza x-vivo6cell/mL.Ratio according to MOI=2-4 is added and is packaged with
The slow virus (slow virus method, concrete steps can be found in 3 part of embodiment of application for a patent for invention 201510324558.X) of CAR,
Liquid is changed after 24h, the state of observation of cell after 48h collects cell suspension, and 5min is centrifuged with 400g, supernatant is abandoned, with x-vivo 15
Cell density is adjusted to 1 × 10 by culture medium7cell/mL.Using mMessage mMachine T7Ultra kit (Life
Technologies) kit prepares Cas9mRNA and TRAC-sg3mRNA, stand-by after purifying and wash-out.Cell and
MRNA mixes so as to which ultimate density contains 1 × 10 in reaching every 100 μ L6Individual cell and 500ng mRNA (Cas9mRNA and TRAC-
The each 250ng of sg3mRNA).MRNA is imported in cell using electroporation (NEPA21).The upgrowth situation of daily observation of cell, often
Carry out every two days changing liquid.After cell culture 11-12d, quality testing is carried out to the T cell of gained.And in 12-14d to T cell
Purified, concrete grammar is shown in step 5, finally by finished product (that is, universal CAR-T cells, referred to herein simply asTCR-
CAR-T cells) carry out dispensing and frozen.
5.TCR is negative, the screening of the T cell that CD4 and CD8 is positive：Using flow sorting techniques or immunomagnetic bead technique
Filter out TCR negative while the T cell of the CD4 or CD8 positives.
1) first step, carries out negative sorting first with the immunomagnetic beads for being coupled CD3 antibody, still expresses CD3 in removing T cell
(TCR) cell (generally carrying out 2 negative sortings)；
2) second step, takes the T cell of a small amount of first step, carries out flow cytometer detection, while CD3, CD4 and CD8 are dyeed, if
CD3 positive rates ＜ 0.01%, while CD4+CD8 positive rates ＞ 90%, you can carry out further work.In the present embodiment, CD3
Positive rate is 0%, while CD4+CD8 positive rates are 93.3% (Fig. 5).
Embodiment 2：Universal CAR-T (TCR-CAR-T cells) validation verification
The universal CAR-T cells (that is, effector cell) that observation embodiment 1 is obtained are white to B cell type acute lymphoblastic
The therapeutic action of blood disease.
Cell biology is verified：
Specific experiment step is as follows：
The first step：Target cell is marked (that is, from B cell type Patients With Acute Lymphoblastic Leukemia using Calcein-AM
Cell)
1) Calcein-AM (Life Technology, Shanghai) is diluted to into 1mg/mL with DMSO；
2) it is target cell is resuspended into 1 × 10 with full culture medium6The density of individual/mL；
3) 15 μM of Calcein-AM, 37 DEG C, 5%CO are added2Culture 30min, gently mixes per 10min；
4) it is centrifuged 5 minutes with 1500rpm, removes supernatant, it is resuspended with full culture medium, repeat twice；
Second step：Target cell is killed using effector cell
1) it is the target cell for having marked is resuspended according to the density of 5000-50000/mL, take 100 μ L and be added to 96 orifice plates
In；
2) according to appropriate ET (effect target ratio) than (5:1) 100 μ L effector cells are added, while with T cell and general periphery
, used as compared with control cells, 3 per group parallel for blood CAR-T (that is, WT CAR-T cells), detects the fluorescence intensity (test of supernatant
release)；Design single A groups (6 parallel), only target cell, detect the fluorescence intensity of its spontaneous apoptosis cracking
(spontaneous release)；Design single B groups (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 culture 4h, centrifugation takes 75 μ L of supernatant, 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 lysis is calculated as follows：[(test release-spontaneous release)/(maximum
release–spontaneous release)]*100。
Acquired results are as shown in Figure 6, it can be seen that it is basic with WT CAR-T killings ability that universal CAR-T kills ability
Unanimously.
Zoopery is verified：
First, the structure of lymphoma mouse model：
1. clone：Human lymphoma cell system Daudi；
Daudi cells are human lymphoma cell systems, and the human lymphoma mould of mouse can be built by hypodermic mode
Type.Its CD19 is expressed as the positive, can be used as the target cell of CAR-T cells.
2.Daudi cell culture
Daudi clones are suspension cell line, can fast-growing soon in 1640 culture mediums (Gibco) containing 20%FBS
It is long.Cell density is 2-3 × 106Need to pass on during/mL.When passing on, obtained cell suspension is centrifuged 5 points with 500g in centrifuge tube
Clock, abandons supernatant.Cell density is adjusted to into 0.3-0.5 × 106/ mL, continues culture.In the case of normal growth, Daudi clones
To pass on every other day, cell density maintains 0.3-3 × 106Between/mL.
3. clone inoculation
With the resuspended Daudi cells of physiological saline, its viable cell concentrations is adjusted for 3 × 108Individual/mL, on ice by its with
Matrigel (BD, China) is according to 2：1 volume ratio is fully mixed.It is inoculated with by hypodermic mode.
Successfully to grow 100mm3Tumour as the successful criterion of mouse lymph lymphoma model construction.Wherein tumour body
Accumulating computing formula is：Gross tumor volume (mm3)=tumour major diameter (mm) × tumour minor axis2(mm2)×0.5；
4. mouse lymph lymphoma model administration.The record administration same day is D0.Cell is carried out by way of tail vein injection defeated
(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 cells (amount to 1 × 106/ only),
PeopleTCR-200 μ L of CAR-T cells (amount to 1 × 106/ only)), the equal single-dose of all mouse.It is as a result as shown in Figure 7, it can be seen that
What the present invention was providedTCR-CAR-T cells have the effect for suppressing and killing tumour cell well.
Embodiment 3：Universal CAR-T (TCR-CAR-T cells) security verification
Mouse GVHD models
The C57 male mices 30 (2.4Gy full-body exposures were given before 24 hours) of 6-8 week old, single tail vein injection is little
Mouse T cell, people's WT CAR-T cells and peopleTCR-CAR-T cells each 1 × 107Individual/mL (is randomly assigned 10, negative control per group
Group injection same volume PBS), injection cell starts Detailed clinical observation after 4 hours (death rate, clinical symptoms, behavior, body weight become
Change), daily observation later once, Continuous Observation 60 days, whether to assess the generation of GVHD and its seriousness.
It can be seen from Table 2 that all there is serious GVHD reactions in the mouse in the group of the normal xenogenesis CAR-T cells of injection,
And have 70% dead mouse.And the CAR-T cells after TCR is knocked out hardly occur GVHD to body, CAR- after TCR knockouts is illustrated
T cell security is higher.
Fig. 8 shows the change of average mice body weight, under the Mouse Weight in the normal xenogenesis CAR-T groups of cells of injection is obvious
Drop, at most have dropped 30%, and the group and injection TCR of not injecting CAR-T cells knocks out the Mouse Weight in the group of CAR-T cells
It is not decreased obviously, has a little rise phenomenon on the contrary.Comprehensive all data, show the security of the CAR-T cells after TCR knockouts
It is high.
Table 2：Mouse anomaly statistics table
Claims (10)
1. a kind of universal CAR-T cells, its expression specificity Chimeric antigen receptor (CAR) and do not express φt cell receptor
(TCR)。
2. universal CAR-T cells of claim 1, wherein the cell derived is in healthy volunteer (or donor).
3. universal CAR-T cells of claim 1 or 2, wherein the encoding gene of the TCR is knocked；Preferably, it is described
The a chains and/or β chain constant codes area (i.e. TRAC and/or TRBC) gene of TCR is knocked；It is highly preferred that a chains of the TCR
And/or β chain constant codes area gene be introduced into one of TRAC-sg1-7 molecules of the cell and/or TRBC-sg1-7 molecules it
One and Cas 9 molecule is knocked out；Most preferably, a chains of the TCR and/or β chain constant codes area gene are introduced into cell
TRAC-sg3 molecules and/or TRBC-sg1 molecules and Cas9 molecules are knocked out.
4. universal CAR-T cells of any one of claim 1-3, wherein the CAR includes the signal peptide, born of the same parents being linked in sequence
Outer land, optional hinge area, transmembrane region and intracellular signal area；Preferably, the nucleotide sequence of the signal peptide is as invented
SEQ ID No in the sequence table of patent application 201510324558.X:1 or SEQ ID No:Shown in 2；Preferably, it is described extracellular
SEQ ID No in the sequence table of the nucleotide sequence of land such as application for a patent for invention 201510324558.X:4 or SEQ ID
No:Shown in 5；Preferably, in the sequence table of the nucleotide sequence of the hinge area such as application for a patent for invention 201510324558.X
SEQ ID No:6 or SEQ ID No:Shown in 7；Preferably, the nucleotide sequence of the transmembrane region such as application for a patent for invention
201510324558.X sequence table in SEQ ID No:8 or SEQ ID No:Shown in 9；Preferably, the intracellular signal area
SEQ ID No in the sequence table of nucleotide sequence such as application for a patent for invention 201510324558.X:10、SEQ ID No:11 or
SEQ ID No:Shown in 12.
5. universal CAR-T cells of claim 4, wherein the such as application for a patent for invention of the nucleotide sequence of the CAR
201510324558.X sequence table in SEQ ID No:13、SEQ ID No:14、SEQ ID No:15、SEQ ID No:
16、SEQ ID No:17 or SEQ ID No:Shown in 18.
6. a kind of method for preparing universal CAR-T cells, methods described comprise the steps：
1) introducing sgRNA molecules and Cas9 molecules in T cell；
2) the introducing CAR molecules in the T cell；
Wherein, step 2) can be in step 1) before, afterwards or while carry out, and the sgRNA molecules comprising with from TCR's
The complementary targeting domain of the target region of a chains and/or β chain constant codes area (i.e. TRAC and/or TRBC) gene.
7. the method for claim 6, methods described is in step 1) before also include separating and/or activate healthy volunteer and (or supply
Person) T cell the step of；Preferably, methods described is in step 2) after also include what universal CAR-T cells were sorted
Step；It is highly preferred that carrying out validation verification to the universal CAR-T cells of gained again after sorting.
8. the method for claim 6 or 7, wherein the sequence such as SEQ ID NOs of the targeting domain:Shown in one of 1-14；
Preferably, the sequence such as SEQ ID NO of the targeting domain:3 or SEQ ID NO:Shown in 8；And wherein by building load
Body is by the sgRNA molecules and Cas9 molecule Jing liposome transfections, electricity turn or slow-virus transfection technology is introduced in the T cell；
Preferably, the sgRNA molecules and Cas9 molecule Jing electricity are turned technology and is introduced in the T cell by carrier construction；More preferably
Ground, turns technology by electricity and the mRNA of the sgRNA molecules and coding Cas9 molecules is introduced in the T cell；Preferably, it is described
CAR molecules are introduced in the T cell by slow-virus transfection technology.
9. a kind of composition, which includes the universal CAR-T cells of any one of claim 1-5；Preferably, the composition
Pharmaceutically useful diluent, excipient or carrier are included also.
10. the universal CAR-T cells of any one of claim 1-5 are used for the purposes of the medicine for preparing treatment tumour.
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