CN109097396A - A method of preparing the CAR-T cell of targeting mesothelin - Google Patents

Abstract

The present invention provides a kind of methods of CAR-T cell for preparing targeting mesothelin, include following steps: 1) nucleic acid of the CAR coded sequence containing targeting mesothelin antigen being imported PBMC, obtain initial cell；2) initial cell, culture described in mesothelin antigen and anti-CD28 antibody contact 1), wherein the state of the mesothelin antigen and the anti-CD28 antibody is non-immobilization.Method of the invention greatly simplifies operating process, reduces costs without first wrapping matrix that is fixed or suspending；And the CAR-T cell of the targeting mesothelin obtained is all increased significantly in the ratio of positive rate, the gentle Effector memory T cell of proliferation water, there is the tumor cell killing potential with CAR-T cell phase same level made from mesothelin antigen and CD28 antibody coating method simultaneously, but cytokine levels are decreased obviously, and reduce the potential risk of cytokines release syndrome (CRS) generation.

Description

A method of preparing the CAR-T cell of targeting mesothelin

Technical field

The present invention relates to Medical Immunology fields, more particularly to a kind of side of CAR-T cell for preparing targeting mesothelin Method.

Background technique

It is had become by the adoptive immunity cell therapy technology of representative of Chimeric antigen receptor (CAR) T cell treatment technology The hot fields of oncotherapy new technology development at present, the adoptive immunotherapy based on T lymphocyte take in Partial tumors Obtained certain effect.It goes through to list in the U.S. in the Kymriah of Novartis in 2017 and the Yescarta of Kate's pharmacy, but its Price is up to 47.5 ten thousand dollars and 37.3 ten thousand dollars respectively.Fancy price is main reason is that this current two CAR-T is thin Born of the same parents' drug requires separation PBMC cell of drawing blood from patient's body for new patient every time all using self-treating scheme It prepares again afterwards, complicated operating process causes cost high and uncontrollable.The preparation of mature products C AR-T cell at present Method generally includes to take a blood sample from patient's body, separation T cell, activation T cell, CAR gene modification T cell obtain CAR-T cell, CAR-T cell is expanded, then the CAR-T cell that amplification obtains is made into after preparation, (Clinical is fed back to patient manufacturing of CAR T cells:foundation of a promising therapy.Wang X,Rivière I Mol Ther Oncolytics.2016Jun 15；3) several steps.The low temperature that may also contain CAR-T cell in this is protected It deposits, recover and the processes such as cold chain transportation.Any one of process link is entirely made and used to require to be accompanied by strictly Quality control, which is let pass, to be examined.

The in vitro amplification of T cell needs activation lasting, appropriate.The activation of T cell needs the first signal of specificity (such as CD3) and costimulation second signal, as CD28,4-1BB or OX40 are carried out by TCR.Present most common T cell stimulation side Method is stimulation after being coated with porous plate with CD3 and anti-CD28 antibody, is stimulated or used mould using artificial antigen presenting cell (AAPC) The pearl of quasi- APC cell has been coated with magnetic bead or the nano-beads stimulation of CD3 and anti-CD28 antibody as clinical grade is other.Wherein The consistency superparamagnetism pearl of Dynabeads CD3/CD28 be covalent coupling CD3 antigen and anti-CD28 antibody.As The readily available clinical grade CD3+T cell selection of a generation and activation reagent, Dynabeads CD3/CD28 is extensively by different realities Room is tested applied in clinical test.More biotech companies have developed the other T based on pearl of readily available clinical grade Cell-stimulating reagent, the Miltenyi MACS GMP of the CTS Dynabeads CD3/28 including Invitrogen, U.S. day Ni The Juno Stage of ExpAct Treg pearl and Miltenyi MACS GMP TransAct CD3/28 pearl and Zhu Nuo Expamer technology.Miltenyi MACS GMP ExpAct Treg pearl be covalent coupling CD3- biotin, CD28 and antibiosis The paramagnetic bead of object element antibody.By adjusting magnetic bead-T cell ratio, Miltenyi MACS GMP ExpAct Treg pearl can be with For expanding modulating T cell and conventional T cells pedigree.It ends up in CAR-T cell production procedure and usually requires to remove magnetic bead, such as Magnetic bead is removed by using Dynal ClinExVivo MPC magnet.Miltenyi MACS GMP TransAct CD3/28 pearl It is the poly nanomatrix that coupling has CD3 or anti-CD28 antibody, with biodegradability, therefore in CAR-T cell formula Without removal before being formed.

Mesothelin is a kind of glycoprotein being anchored on cytoplasma membrane by phosphatidylinositols area (GPI), and height is expressed in more Mesothelin gene encodes the precursor protein of 69kDa a kind of, the processed embrane-associated protein for forming a 40kDa in kind tumor tissues It is referred to as falling off for megakaryocyte-potentiating factor (MPF) with a 31kDa and segment and releases extracellular, we is usually said Mesothelin refers to the segment being anchored on film, can be divided into Region I, tri- regions II, III according to its protein structure.It On the one hand the domain NF can be activated to swash by its GPI structural domain, MAPK and PI3K intracellular signaling pathway, promotes cell Proliferation, supports Anti-apoptotic；On the other hand lead to abnormal cell adherence with its receptor CA125/MUC16 interaction, cancer cell is promoted to turn It moves.Since mesothelin is overexpressed in Several Kinds of Malignancy (celiothelioma, oophoroma, cancer of pancreas, gastric cancer, cholangiocarcinoma etc.), It is a very promising tumor-specific therapies target spot.

The step of preparation flow of conventional CAR-T cell sequence usually first sorts T cell, is stimulated in vitro at present Amplification, then imports CAR gene again.And it is also above-mentioned CD3 that carry out stimulates the first signal and second signal used in vitro And CD28.The t cell activation reagent based on magnetic bead or nano-beads of existing clinical grade is used for CD3 and CD28 Design carry out covalent coupling.The problems of this way is that the indifference stimulation of CD3 and anti-CD28 antibody is easy Cause the exhaustion of T cell.If furthermore activation T cell method is using CD3 antigen and anti-CD28 antibody coating matrix stimulation, coating The operation of step will increase the complexity of preparation process, may bring more uncertain factors.If engaged in trade using above-mentioned The coupling of the clinical grade of product has the magnetic bead of CD3 and/or anti-CD28 antibody or nano-beads to be stimulated, then makes CAR-T cell The cost of preparation is difficult to decline, and influences the promotion and popularization in CAR-T cell clinical application.Therefore, also lack now including behaviour The preparation CAR-T cell for the step of making easy, cost controllably and capable of effectively activating T cell, as preparation targets mesothelin The method of CAR-T cell.

Summary of the invention

The technical problem to be solved by the present invention is in current CAR-T cell preparation process usually using CD3 and CD28 is likely to result in T cell respectively as the operation of the first signal and the second signal activation T cell and exhausts, while will be as thorn Swashing agent coating matrix stimulation T cell will increase the complexity of preparation process, and commodity in use clinical grade covalent coupling CD3 is anti- Former and/or anti-CD28 antibody magnetic bead or nano-beads reagent cause preparation cost high, uncontrollable, provide a kind of new preparation The method for targeting the CAR-T cell of mesothelin.The process employs with this field routinely taken first activation T cell, after lead Enter the opposite way of the operation of CAR gene, it first will be thin for the CAR channel genes T of mesothelin after sorting obtains T cell Born of the same parents, then reuse mesothelin antigen and anti-CD28 antibody cooperatively stimulates T cell, and the mesothelin antigen and anti- CD28 antibody is directly added into system in the form of on-fixed together and is stimulated the CAR-T cell of targeting mesothelin, nothing Matrix such as orifice plate need to be first coated with or use the magnetic bead or nano-beads material of coupling mesothelin antigen and/or anti-CD28 antibody, greatly Ground simplifies the process of mesothelin CAR-T cell preparation while reducing costs, and passes through the preparation containing the activation step Mesothelin CAR-T cell obtained by method has higher positive rate and the CAR-T cellular level phase with conventional method preparation When the lethality for target cell, and the expression of Some cytokines and CD3 and anti-CD28 antibody coating orifice plate stimulation CAR-T cell obtained reduces the wind of potential cytokines release syndrome (CRS) generation compared to significantly decreasing afterwards Danger.

Specifically, the present invention solves the technical problems to be solved by the invention by the following technical programs.

One aspect of the present invention provides a kind of method for preparing CAR-T cell comprising following steps: 1) will contain targeting The nucleic acid of Chimeric antigen receptor (Chimeric Antigen Receptor) coded sequence of mesothelin antigen imports peripheral blood list A nucleus (PBMC) obtains initial cell；2) initial thin described in the mesothelin antigen and anti-CD28 antibody contact 1) Born of the same parents, culture, wherein the state of the mesothelin antigen and the anti-CD28 antibody is non-immobilization.

In a preferred embodiment, the mesothelin antigen is overall length mesothelin.

In a preferred embodiment, the mesothelin antigen is mesothelin Domain III, amino acid sequence SEQ Shown in 21-131 of ID NO:2.

In a preferred embodiment, the mesothelin antigen preferably includes IgG Fc, and the Ig Fc is preferably IgG4Fc。

In a preferred embodiment, the mesothelin antigen is merged by hinge with IgG Fc, the preferably hinge For CD8 hinge.

In a preferred embodiment, the mesothelin antigen includes also signal peptide in N-terminal, and the signal peptide is preferably Light chain signal peptide.

In a preferred embodiment, the mesothelin antigen is mesothelin Domain III antigen, is believed in N-terminal and light chain The fusion of number peptide is merged by CD8 hinge with IgG4Fc in C-terminal, and amino acid sequence is shown in SEQ ID NO:2.

In a preferred embodiment, the nucleic acid is nucleic acid construct or mRNA, it is therefore preferable to nucleic acid construct.

In a preferred embodiment, the nucleic acid construct is expression vector, is optionally viral vectors, it is therefore preferable to Slow virus carrier derived from I type HIV, such as pWPT, pWPXL.

In a preferred embodiment, the nucleic acid construct is expression vector, is optionally Transposon System carrier, excellent Selection of land is Sleeping Beauty Transposon System carrier or PiggyBac Transposon System carrier transformation or not being transformed, PiggyBac Transposon System carrier being more preferably transformed or not being transformed, the PiggyBac swivel base being most preferably transformed Subsystem carrier, the pNB328 Transposon System carrier as disclosed in CN105154473A.

In a preferred embodiment, the nucleic acid is mRNA, and it is described embedding to contain 5 ' end cap minor structures, 5 ' UTR, coding Close open reading frame (ORF), 3 ' UTR the and polyA tails of antigen receptor.

In a preferred embodiment, the method for the importing be selected from infestation with virus particles, electricity turn, liposome transfection, Any one of calcium phosphate transfection and particle gun, it is therefore preferable to appointing in infestation with virus particles, electricity turn and liposome transfection One kind, more preferably infestation with virus particles or electricity turn, and most preferably electricity turns.

In a preferred embodiment, the anti-CD28 antibody be anti-CD28 monoclonal antibody or polyclonal antibody, preferably Ground is anti-CD28 monoclonal antibody, more preferably Humanized monoclonal antibodies, even more preferably for humanization source of mouse or The anti-CD28 monoclonal antibody in rabbit source, the most preferably anti-CD28 monoclonal antibody of humanization source of mouse.

In a preferred embodiment, the temperature of the culture is 37 DEG C；And/or the CO of the culture₂Concentration is 5%； And/or culture medium used in the culture is the AIM-V containing 2% fetal calf serum；And/or the time of the culture is 5-10 It.

In a preferred embodiment, also contain cell factor in culture medium used in the culture；Preferably, described Cell factor is IL-2, final concentration of 500IU/mL；And/or the cell factor is added when the culture carries out 0-6 hours The culture medium；Preferably, the culture medium is added when the culture carries out 6 hours in the cell factor.

In a preferred embodiment, the contact is to have the mesothelin antigen and the anti-CD28 antibody directly In the system containing the initial cell, direct interaction is generated with the surface of the initial cell.Preferably, between described Skin element antigen is mesothelin Domain III, and the anti-CD28 antibody is monoclonal antibody.

In a preferred embodiment, the state of the mesothelin antigen and the anti-CD28 antibody is solution form； Preferably, the final concentration of 0.1-3 μ g/mL of the mesothelin antigen, the final concentration of 0.1-3 μ g/mL of the anti-CD28 antibody.

In a preferred embodiment, the state of the mesothelin antigen and the anti-CD28 antibody is solution form； The final concentration of 0.1-1 μ g/mL of the mesothelin antigen, the final concentration of 0.1-1 μ g/mL of the anti-CD28 antibody.

In a preferred embodiment, the state of the mesothelin antigen and the anti-CD28 antibody is solution form； The final concentration of 0.5-1 μ g/mL of the mesothelin antigen, the final concentration of 0.5-1 μ g/mL of the anti-CD28 antibody.

In a preferred embodiment, the state of the mesothelin antigen and the anti-CD28 antibody is solution form； The final concentration of 0.1 μ g/mL of the mesothelin antigen, the final concentration of 0.1 μ g/mL of the anti-CD28 antibody.

In a preferred embodiment, the state of the mesothelin antigen and the anti-CD28 antibody is solution form； The final concentration of 0.5 μ g/mL of the mesothelin antigen, the final concentration of 0.5 μ g/mL of the anti-CD28 antibody.

In a preferred embodiment, the state of the mesothelin antigen and the anti-CD28 antibody is solution form； The final concentration of 1 μ g/mL of the mesothelin antigen, the final concentration of 1 μ g/mL of the anti-CD28 antibody.

In a preferred embodiment, the state of the mesothelin antigen and the anti-CD28 antibody is solution form； The final concentration of 3 μ g/mL of the mesothelin antigen, the final concentration of 3 μ g/mL of the anti-CD28 antibody.

Detailed description of the invention

Fig. 1: Meso3CAR with Muc1CAR gene structure display；

Fig. 2A: ConT-C329, cMeso3CAR-T-C329 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under conditions of fMeso3CAR-T-C329 cell obtained positive rate result；

Fig. 2 B:ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under the conditions of fMeso3CAR-T-D70 cell obtained positive rate result；

Fig. 2 C:ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under the conditions of fMeso3CAR-T-D71 cell obtained positive rate result；

Fig. 3 A:ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under the conditions of fMeso3CAR-T-D70 cell obtained growth curve；

Fig. 3 B:ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under the conditions of fMeso3CAR-T-D71 cell obtained increment curve；

Fig. 4 A: after being contacted with PANC-1 cell and SKOV-3 cell, cMeso3CAR-T-D70, cMeso3CAR-T-D71 and FMeso3CAR-T-D70 and fMeso3CAR- obtained under conditions of various concentration mesothelin III antigen+anti-CD28 antibody stimulation The level of T-D71 cell secretion of cytokines IL-2；

Fig. 4 B: after being contacted with PANC-1 cell and SKOV-3 cell, cMeso3CAR-T-D70, cMeso3CAR-T-D71 and FMeso3CAR-T-D70 and fMeso3CAR- obtained under conditions of various concentration mesothelin III antigen+anti-CD28 antibody stimulation The level of T-D71 cell secretion of cytokines IL-4；

Fig. 4 C: after being contacted with PANC-1 cell and SKOV-3 cell, cMeso3CAR-T-D70, cMeso3CAR-T-D71 and FMeso3CAR-T-D70 and fMeso3CAR- obtained under conditions of various concentration mesothelin III antigen+anti-CD28 antibody stimulation The level of T-D71 cell secretion of cytokines IL-10；

Fig. 4 D: after being contacted with PANC-1 cell and SKOV-3 cell, cMeso3CAR-T-D70, cMeso3CAR-T-D71 and FMeso3CAR-T-D70 and fMeso3CAR- obtained under conditions of various concentration mesothelin III antigen+anti-CD28 antibody stimulation The level of T-D71 cell secretion of cytokines IFN-γ；

Fig. 5: ConT-C329, cMeso3CAR-T-C329 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under conditions of fMeso3CAR-T-C329 cell T cell obtained exhaust factor PD-1 expression testing result；

Fig. 6 A:ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under the conditions of fMeso3CAR-T-D70 cell obtained effector T cell flow cytometer detection result；

Fig. 6 B:ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under the conditions of effector T cell flow cytometer detection result in fMeso3CAR-T-D71 cell obtained；

Effector T cell percentage statistical result histogram in C: Fig. 6 A of Fig. 6 and Fig. 6 B；

Fig. 7 A:ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under conditions of fMeso3CAR-T-D70 cell obtained respectively to the killing curve of pancreatic carcinoma PANC-1；

Fig. 7 B:ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under conditions of fMeso3CAR-T-D70 cell obtained respectively to the killing curve of ovarian cancer cell SKOV-3；

Fig. 7 C:ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under conditions of fMeso3CAR-T-D71 cell obtained respectively to pancreatic carcinoma PANC-1 and ovarian cancer cell SK-OV-3 Killing curve；

Fig. 7 D:ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under conditions of fMeso3CAR-T-D71 cell obtained respectively to pancreatic carcinoma PANC-1 and ovarian cancer cell SK-OV-3 Killing curve；

The positive rate result of Fig. 8 A:cMuc1CAR-T-D42 and fMuc1CAR-T-D42 cell；

The positive rate result of Fig. 8 B:cMuc1CAR-T-D43 and fMuc1CAR-T-D43 cell.

Specific embodiment

The step of method for preparing CAR-T cell of this field routine includes that separation obtains PBMC, with CD3 and anti-at present CD28 antibody activation amplification T cell turns to import transgenosis CAR in T cell with virion or electricity.It is obtained by aforesaid operations It, may respectively as the operation of the first signal and the second signal activation T cell using CD3 and CD28 during CAR-T cell It will cause T cell exhaustion, and the amplification of T cell carried out again by being coated with after matrix as the CD3 of stimulant and anti-CD28 antibody Stimulation increases the complexity of preparation process, and the clinical grade covalent coupling CD3 antigen of commodity in use and/or anti-CD28 are anti- The magnetic bead or nano-beads reagent of body cause preparation cost high.The present inventor uses by further investigation and repetition test, discovery The way opposite with the first activation T cell, the rear importing operation of CAR gene that the prior art is taken, i.e., obtain T cell in sorting Then cooperatively stimulate T thin with anti-CD28 antibody with mesothelin antigen again mesothelin CAR channel genes T cell first afterwards Born of the same parents, and the mesothelin antigen is directly added into system to targeting mesothelium together with anti-CD28 antibody is in the form of on-fixed The CAR-T cell of element is stimulated, and without first wrapping matrix that is fixed or suspending, greatly simplifies targeting mesothelin The operating process of CAR-T cell preparation reduces costs simultaneously.And the CAR-T for the targeting mesothelin that this method prepares is thin Born of the same parents are increased significantly in ratio several respects of positive rate, the gentle Effector memory T cell of proliferation water, while having anti-with mesothelin The tumor cell killing potential of CAR-T cell phase same level made from former and anti-CD28 antibody coating method, and the expression of cell factor Level significantly decreases compared with CAR-T cell obtained after mesothelin antigen and anti-CD28 antibody coating orifice plate stimulation, The potential risk that cytokines release syndrome (CRS) generation is reduced under the premise of not reducing killing tumour ability, thus complete At the present invention.

Part term of the present invention is defined below.

Unless otherwise noted, the application uses technical term according to common usage.Essential term in molecular biology Definition can be in Benjamin Lewin, Genes X, published by Jones&Bartlett Publishers, and 2009； With Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16volumes, 2008 with found in other similar bibliography.

In the present invention, term " antigen " defines expression when introducing internal in the text, is identified by immune system any Substance.Such as the segment or structural domain of albumen, albumen, such as the extracellular domain of cell transmembrane albumen.

Term " Chimeric antigen receptor " (Chimeric Antigen Receptor, CAR) is defined as artificial reconstructed in the text Receptor, can will identify the specific molecular (such as antibody) of cell surface antigen, be anchored on immunocyte (such as T cell) On, make immunocyte identification antigen and kills the cell of its identification antigen of expression.CAR usually successively includes optional signal Polypeptide such as single-chain antibody, hinge area, transmembrane region and the intracellular signal area of peptide, combination cell membranous antigen.In general, combination cell film is anti- Former polypeptide can be natural polypeptides or artificial synthetic polypeptide；Preferably, artificial synthetic polypeptide is single-chain antibody or Fab segment.

Term " coded sequence " is defined as directly determining the amino of its protein product (such as CAR) in nucleic acid sequence in the text The part of acid sequence.The boundary of coded sequence is usually the ribosome binding site by holding opening code-reading frame upstream close to mRNA 5 ' Point (for prokaryotic cell) and the transcription terminator determination that opening code-reading frame downstream is held close to mRNA 3 '.Coded sequence can be with Include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.

Term " peripheral blood mononuclear cells " (Peripheral Blood Mononuclear Cell, PBMC) is in the text Be defined as any cell with round individual cells core in peripheral blood, including but not limited to T cell, B cell, NK cell, Macrophage and monocyte；It does not include red blood cell, blood platelet, neutrophil leucocyte, eosinophil or basophilic granulocyte.

Term " nucleic acid construct " is defined as the artificial constructed nucleic acid that can be introduced into target cell or tissue in the text Section.DNA construct is typically comprised, refers to the nucleotide sequence for the coding target protein being subcloned into carrier DNA insertion.

What term " expression vector " was defined as recombinantly or synthetically generating in the text is used to express purpose nucleic acid in target cell Nucleic acid construct or carrier (such as exogenous nucleic acid or transgenosis).Typically, purpose nucleic acid express express target protein matter.

Term " antibody " is defined as the immunoglobulin molecules in conjunction with antigentic specificity in the text, can be for derived from nature Source or complete immunoglobulin derived from recombinant sources, and can be the immune response part of intact immunoglobulins.Antibody is logical It is often the tetramer of immunoglobulin molecules.Antibody in the present invention can exist in a variety of forms, and including but not limited to more grams Grand antibody, monoclonal antibody, Fv, Fab, F (ab) '₂, scFv, humanized antibody, heavy chain antibody and single domain antibody.

Term " polyclonal antibody " is defined as in the text containing there are many antibody generated by internal different B cell pedigree secretions Antibody mixture.The antibody of each of these kind of B cell pedigree secretion is all directed to identical specific antigen, but every kind of antibody institute For the specific antigen on epitope it is different.

Term " monoclonal antibody " is defined as showing the antibody of single binding specificity in the text, only in conjunction with single antigen Epitope.It includes the traditional antibody comprising the heavy chain-light chain tetramer generated by identical immunocyte, this is immune Cell origin is in the clone of a unique parental cell.Its also include can in conjunction with single epitope Fv, Fab, Fab ', F(ab’)₂, scFv, humanized antibody, heavy chain antibody and single domain antibody.

Term " humanized antibody " is defined as the antibody sequence phase of the sequence mankind natural with its after modification in the text The non-human source antibodies improved than similarity.Wherein at least one antibody combining site (complementary determining region, CDR) (such as CDR3, it is excellent Select all six CDR) replaced by the CDR from the human antibody with expectation specificity and/or its at least one area FR is resisted by people The area Ti FR replaces, and optionally, the constant region of non-source of people is replaced by the constant region of human antibody.

Term " on-fixed " is defined as proteantigen or antibody not with any matrix with covalently or non-covalently in the text Mode is connected.The matrix can be fixed matrix, such as orifice plate, culture dish, the slide of solid, be also possible to suspension Matrix, such as magnetic bead, nano particle.

Term " immobilization " is defined as antibody in the text, such as anti-CD28 antibody in a manner of covalently or non-covalently and discrete phase Connection, the matrix can be fixed matrix, such as orifice plate, culture dish, the slide of solid, be also possible to the base to suspend Matter, such as magnetic bead, nano particle.

Therefore the present invention provides a kind of method of CAR-T cell for preparing targeting mesothelin comprising 1) will contain targeting The nucleic acid of the Chimeric antigen receptor coded sequence of mesothelin antigen imports peripheral blood mononuclear cells (PBMC), obtains initial thin Born of the same parents；2) initial cell, culture described in the mesothelin antigen and anti-CD28 antibody contact 1), wherein the mesothelin antigen State with the anti-CD28 antibody is non-immobilization.

In the present invention, the mesothelin antigen is overall length mesothelin antigen or mesothelin Domain III antigen, it is preferable that For mesothelin Domain III antigen；Preferably, the mesothelin Domain III antigen includes IgG Fc.Preferably, the IgG Fc is IgG4Fc, and amino acid sequence is as shown in 144-250 of SEQ ID NO:2.The mesothelin Domain III antigen It can be merged by hinge with the IgG Fc, it is preferable that the hinge is CD8 hinge, amino acid sequence such as SEQ ID Shown in 132-143 of NO:3.

In the present invention, the mesothelin antigen can also include signal peptide in N-terminal, it is preferable that the signal peptide is light chain Signal peptide, amino acid sequence is shown in 2-20 in SEQ ID NO:2.

In the present invention, the mesothelin Domain III antigen can be merged in N-terminal with light chain signal peptide, be passed through in C-terminal CD8 hinge is merged with IgG4Fc, and amino acid sequence is shown in SEQ ID NO:2.

Chimeric antigen receptor in the method for the CAR-T cell of above-mentioned preparation targeting mesothelin provided by the invention can be The Chimeric antigen receptor of this field routine, comprising extracellular antigen binding domain, hinge area, transmembrane region, costimulatory signal area intracellular and Intracellular signal area.

In the present invention, the extracellular antigen binding domain can be the antigen binding molecules of this field routine, it is therefore preferable to anti- Body, such as single-chain antibody (scFv) or single domain antibody.

In certain embodiments, the extracellular antigen binding domain is the scFv for mesothelin, it is therefore preferable between being directed to The scFv of skin element Domain III.The DNA encoding sequence of the scFv for mesothelin Domain III is preferably SEQ ID Shown in 64-813 of NO:7.

In the present invention, the hinge area refers to the region between the functional areas heavy chain immunoglobulin CH1 and CH2.Suitable for this The hinge area of invention can be selected from the extracellular hinge of the extracellular hinge area of CD8a, IgG1Fc CH2CH3 hinge area, IgD hinge area, CD28 Any one or more of the extracellular hinge area of sequence, IgG4Fc CH2CH3 hinge area and CD4.In certain embodiments, originally Text uses CD8 hinge area or IgG4Fc CH2CH3 hinge area.

In the present invention, the transmembrane region can be CD28 transmembrane region, CD8 transmembrane region, CD3 ζ transmembrane region, CD134 transmembrane region, One of CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region；Preferably CD8 transmembrane region.

In the present invention, it is thin that the costimulatory signal area intracellular can be selected from CD28, CD134/OX40, CD137/4-1BB, lymph Born of the same parents' specific proteins tyrosine kinase (LCK), induced T lymphocyte costimulating factor (ICOS) and DNAX Activating protein-1 0 (DAP10) intracellular domain.In certain embodiments, the intracellular domain of the costimulatory signal molecule is the born of the same parents of CD28 Intracellular domain, sequence can be the sequence of the CD28 intracellular domain of this field routine.In certain embodiments, described total The intracellular domain of stimulus signal molecule is the intracellular domain of CD137/4-1BB, what sequence can be conventional for this field The sequence of CD137/4-1BB intracellular domain.

In certain embodiments, the Chimeric antigen receptor successively contains from N-terminal to C-terminal: targeting mesothelin antigen ScFv, CD8 hinge area, CD8 transmembrane region, the intracellular domain of CD137/4-1BB and CD3 ζ intracellular signal domain.In certain embodiment party In case, the Chimeric antigen receptor also contains signal peptide, it is preferable that the signal peptide is CD8 signal peptide, and coded sequence is Shown in SEQ ID NO:7 4-63.In certain embodiments, the Chimeric antigen receptor successively contains from N-terminal to C-terminal: Optional CD8 signal peptide, scFv, CD8 hinge area, CD8 transmembrane region, the intracellular domain of CD137/4-1BB and CD3 ζ letter intracellular Number domain.

In the present invention, the source of the peripheral blood mononuclear cells (PBMC) can be the source of this field routine, such as It is separated and is obtained from the blood of donor subject by Ficoll density-gradient centrifugation method, can also bought commercially available PBMC。

In the present invention, the nucleic acid of the Chimeric antigen receptor coded sequence containing targeting mesothelin antigen can be ability The nucleic acid of domain routine, including DNA and mRNA.It can be the inosculating antibody containing targeting mesothelin antigen when the nucleic acid is DNA The nucleic acid construct of the coded sequence of original receptor can be operated it includes the coded sequence of the CAR of targeting mesothelin antigen and with it The promoter of ground connection preferably also includes the enhancer sequence being operably connected with the promoter.The nucleic acid construct Body can be the nucleic acid construct of this field routine, it is therefore preferable to expression vector, more preferably virus system expression vector, such as Slow virus system expression carrier, adenovirus expression carrier or adeno-associated virus (AAV) expression vector or swivel base based on I type HIV Subsystem expression vector, as Sleeping Beauty Transposon System expression vector or the expression of PiggyBac Transposon System carry Body.When the nucleic acid is mRNA, it includes 5 ' end cap minor structures, 5 ' UTR, the open readings for encoding the Chimeric antigen receptor Frame (ORF), 3 ' UTR and polyA tails.

In certain embodiments, the nucleic acid of the Chimeric antigen receptor coded sequence containing targeting mesothelin antigen is PNB328 Transposon System carrier disclosed in CN105154473A, it includes the targets as shown in SEQ ID NO:7 64-813 To the coded sequence of the Chimeric antigen receptor of mesothelin Domain III.

In the present invention, the method for the importing can be the method for this field routine, including infestation with virus particles, electricity turn, Any one of liposome transfection, calcium phosphate transfection and biolistic transformation.Preferably, the method for the importing is that electricity turns, more excellent Selection of land, the condition that the electricity turns are that its number is selected to carry out for the condition of U014 using Lonza Nucleofactor 2b.

It in certain embodiments, will be comprising targeting mesothelin Domain III as shown in SEQ ID NO:7 64-813 The pNB328 Transposon System carrier of coded sequence of Chimeric antigen receptor transduceed by electricity into being isolated from donor subject's blood The PBMC of liquid obtains initial cell.

In the present invention, the contact is to be directly present in the mesothelin antigen and the anti-CD28 antibody containing described In the system of initial cell, direct interaction is generated with the surface of the initial cell.Preferably, the mesothelin antigen is Mesothelin Domain III, the anti-CD28 antibody are the anti-CD28 monoclonal antibody of source of mouse.

In the present invention, the on-fixed refers to the mesothelin antigen or the anti-CD28 antibody not with any matrix with altogether Valence or the state of non-covalent fashion connection.The matrix is the matrix of this field routine, including fixed matrix, such as orifice plate, culture Ware, slide etc. also include the matrix to suspend, such as magnetic bead, nano particle.

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 0.1-3 μ g/mL of the mesothelin Domain III antigen, it is preferable that be 0.1-1 μ g/mL, more preferably Ground is 0.5-1 μ g/mL；The final concentration of 0.1-3 μ g/mL of the source of mouse anti-CD28 antibody, it is preferable that be 0.1-1 μ g/mL, more It preferably, is 0.5-1 μ g/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III will be by that will contain its coded sequence PB transposon vector pNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, the final concentration of mesothelin The concentration formed after initial cell described in antigen and anti-CD28 antibody contact 1).

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 0.1 μ g/mL of the mesothelin Domain III antigen；The source of mouse anti-CD28 antibody it is final concentration of 0.1μg/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III, which passes through, carries the PB transposons containing its coded sequence Body pNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, described final concentration of with the mesothelin antigen and described The concentration formed after initial cell described in anti-CD28 antibody contact 1).

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 0.5 μ g/mL of the mesothelin Domain III antigen；The source of mouse anti-CD28 antibody it is final concentration of 0.5μg/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III, which passes through, carries the PB transposons containing its coded sequence Body pNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, described final concentration of with the mesothelin antigen and described The concentration formed after initial cell described in anti-CD28 antibody contact 1).

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 1 μ g/mL of the mesothelin Domain III antigen；Final concentration of 1 μ of the source of mouse anti-CD28 antibody g/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III passes through the PB transposon vector containing its coded sequence PNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, described final concentration of with the mesothelin antigen and described anti- The concentration formed after initial cell described in the contact 1) of CD28 antibody.

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 3 μ g/mL of the mesothelin Domain III antigen；Final concentration of 3 μ of the source of mouse anti-CD28 antibody g/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III passes through the PB transposon vector containing its coded sequence PNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, described final concentration of with the mesothelin antigen and described anti- The concentration formed after initial cell described in the contact 1) of CD28 antibody.

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 0.1 μ g/mL of the mesothelin Domain III antigen；The source of mouse anti-CD28 antibody it is final concentration of 0.5μg/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III, which passes through, carries the PB transposons containing its coded sequence Body pNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, described final concentration of with the mesothelin antigen and described The concentration formed after initial cell described in anti-CD28 antibody contact 1).

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 0.5 μ g/mL of the mesothelin Domain III antigen；The source of mouse anti-CD28 antibody it is final concentration of 1μg/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III passes through the PB transposon vector containing its coded sequence PNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, described final concentration of with the mesothelin antigen and described anti- The concentration formed after initial cell described in the contact 1) of CD28 antibody.

In certain embodiments, the contact is to resist mesothelin Domain III antigen and the anti-CD28 monoclonal of source of mouse Body is directly added to import the PBMC system of the Chimeric antigen receptor of targeting mesothelin III, is directly present in the form of a solution In the initial cell system, respectively with the initial cell surface targeting mesothelin Domain III CAR and CD28 antigen It combines, the final concentration of 1 μ g/mL of the mesothelin Domain III antigen；The source of mouse anti-CD28 antibody it is final concentration of 0.5μg/mL；Wherein the Chimeric antigen receptor of the targeting mesothelin III, which passes through, carries the PB transposons containing its coded sequence Body pNB328-Meso3CAR is imported in PBMC in such a way that electricity turns, described final concentration of with the mesothelin antigen and described The concentration formed after initial cell described in anti-CD28 antibody contact 1).

In the present invention, the culture can be the T cell condition of culture of this field routine.Preferably, the culture is made Culture medium also contains cell factor；It is highly preferred that the cell factor is IL-2；Even more preferably, the cell The culture medium is added when the culture carries out 0-6 hours in the factor；Most preferably, the cell factor is carried out in the culture The culture medium is added at 6 hours.

In certain embodiments, the temperature of the culture is 37 DEG C, the CO of the culture₂Concentration is 5%, the culture Culture medium used is the AIM-V containing 2% fetal calf serum, and the time of the culture is 5-10 days.

In certain embodiments, the temperature of the culture is 37 DEG C, the CO of the culture₂Concentration is 5%, the culture Culture medium used is the IL-2 of the AIM-V containing 2% fetal calf serum and final concentration of 500IU/mL, and the time of the culture is 5-7 days；The IL-2 is added when cultivating 6 hours.

The positive effect of the present invention is that: method of the invention will target mesothelium after sorting obtains T cell first Then the CAR channel genes T cell of element cooperatively stimulates T cell, and mesothelium with mesothelin antigen and anti-CD28 antibody again Plain antigen be directly added into the form of on-fixed together with anti-CD28 antibody in system to targeting mesothelin CAR-T cell into It assassinates and swashs, without first wrapping matrix that is fixed or suspending, greatly simplifie the behaviour of the CAR-T cell preparation of targeting mesothelin Make process while reducing costs；And the CAR-T cell for targeting mesothelin that this method prepares is horizontal in positive rate, proliferation It is all increased significantly, while having and mesothelin antigen and anti-CD28 antibody coating with ratio several respects of Effector memory T cell The tumor cell killing potential of the CAR-T cell phase same level of method targeting mesothelin obtained, and the expression of cell factor with Under mesothelin antigen has been compared significantly with the CAR-T cell of targeting mesothelin obtained after anti-CD28 antibody coating orifice plate stimulation Drop reduces the potential risk of cytokines release syndrome (CRS) generation under the premise of not reducing killing tumour ability.

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions (such as with reference to J. Pehanorm Brooker etc. writes, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press), or according to commodity Specification selection.Reagents or instruments used without specified manufacturer in the present invention is that can buy to obtain by market Conventional products.

The source of used part instrument and reagent is as follows in the present invention:

" room temperature " as described in the examples refers to the temperature for the operation room tested, generally 25 DEG C.

The synthesis commission Shanghai JaRa of gene order carries out；

It is commercialized PBMC and is purchased from attached Changhai hospital of army medical university of naval blood station, number 329；

FBS (fetal calf serum) is purchased from Gibco；

AIM-V is purchased from Gibco:

Electroporation is Lonza Nucleofactor 2b, is purchased from Lonza；

CCK8 detection kit is purchased from: Sigmaaldrich, article No.: 96992；

Mesothelin Domain III-Fc (Meso3-Fc) antigen is prepared as follows: composition sequence is SEQ ID NO:1 DNA fragmentation, the N-terminal that express amino acid sequence is SEQ ID NO:2 contains the Meso3-Fc fusion protein of light chain signal peptide, purport Meso3-Fc fusion protein is effectively secreted into outside eukaryocyte, subsequent purification is facilitated.In SEQ ID NO: After 5 ' ends are separately connected the restriction enzyme site connector of EcoRI and XbaI with 3 ' ends, it is connected on pCDNA3.4 carrier, building is obtained Express the expression vector of Meso3-Fc fusion protein.According to ExpiCHO^TMExpression system specification, uses ExpiCHO^TMExpression system After system is overexpressed above-mentioned fusion protein, the MabSelect affinity chromatography resin by specification of GE Healthcare is used Operating procedure purifies expression product, and the Meso3-Fc fusion protein of purifying is both made；

The preparation method of MUC1-Fc antigen is identical as the preparation method of Meso3-Fc antigen, and composition sequence is SEQ ID DNA fragmentation shown in NO:3, the Muc1-Fc fusion that the N-terminal that encoding amino acid sequence is SEQ ID NO:4 contains light chain signal peptide Albumen uses ExpiCHO after DNA fragmentation is connected on pCDNA3.4 carrier by aforesaid operations^TMExpression system expression and purification is made The MUC1-Fc fusion protein that must be purified；

The preparation method for recombinating mesothelin (rMSLN) antigen is identical as the preparation method of Meso3-Fc antigen, composition sequence For DNA fragmentation shown in SEQ ID NO:5, the N-terminal that encoding amino acid sequence is SEQ ID NO:6 contains between light chain signal peptide Skin element antigen, uses ExpiCHO after DNA fragmentation is connected on pCDNA3.4 carrier by aforesaid operations^TMExpression system expression and purification Obtain the recombination mesothelin antigen of purifying；

Mesothelin Domain III-Fc antigen-biotin (Meso3-Fc-biotin) and Muc1-Fc antigen-biotin (Muc1-Fc-biotin) the biotin labeling method of this field routine is pressed to above-mentioned 3 kinds of fusions egg obtained by Jin Sirui company Bai Kangyuan is marked obtained；

The band fluorescein-labeled streptavidin of PE (PE-streptavidin): limited purchased from Shanghai Jin Sirui biotechnology Company；

SKOV-3 cell line, PANC-1 cell line: (purchase is in American Type Culture Collecti ATCC)；

Anti-CD28 antibody is purchased from: Merck Millipore, article No.: CBL517；

No. 42, No. 43, No. 70 and No. 71 donor subjects are normal adults.

Abbreviation:

Meso3CAR: targeting the Chimeric antigen receptor of mesothelin, and extracellular antigen binding domain therein is for mesothelin egg The single-chain antibody of white Domain III；

Muc1CAR: targeting the Chimeric antigen receptor of MUC1, and extracellular antigen binding domain therein is for the single-stranded anti-of MUC1 Body；

PBMC: peripheral blood mononuclear cells；

RMSLN: recombination mesothelin antigen；

Meso3: mesothelin III antigen；

Muc1:Muc1 antigen；

ACD28: anti-CD28 antibody；

D70:70 donor subject；

D71:71 donor subject.

The building of expression vector of the embodiment 1 containing coding Meso3CAR and Muc1CAR gene

The coded sequence (SEQ ID NO:7) of artificial synthesized meso3CAR and Muc1CAR coded sequence (SEQ ID NO: 8), it is loaded between EcoRI the and SalI restriction enzyme site of the carrier pNB328 based on PB Transposon System (pNB328's respectively Entire contents are included in herein by structure and sequence by reference referring to CN201510638974.7, herein), the weight of building Group expression vector is named as pNB328-meso3CAR and pNB328-Muc1CAR, and the structural schematic diagram of the two is as shown in Figure 1.Fig. 1 Promoter sequence and polyA tailing signal sequence is not shown, is located between 5 ' LTR and signal peptide sequence and 3 ' LTR Before.

Embodiment 2 activates reagent fixation preparation Meso3CAR-T cell and Muc1CAR-T cell

Meso3 antigen and anti-CD28 antibody are coated with 6 orifice plates: 2mL DPBS being added in a hole of 6 orifice plates (Hyclone), the volume that respective 10 μ g is calculated according to the densimeter on Meso3 and anti-CD28 antibody packaging, is then inhaled with rifle It takes respectively and is added in hole, " cross mode of averaging " mixes, and making the peridium concentration of meso3 antigen and anti-CD28 antibody is 5 μ g/ mL.Overnight (or being incubated for 4h as 37 DEG C of cell incubators) as 4 DEG C of refrigerator coatings by 6 orifice plates.

Taking number is respectively 329 commercialization PBMC cell, adjusts concentration to 5 × 10 with physiological saline⁶A/mL.Each Number cell respectively takes 2 1.5mLEP to manage, number a and b, and 5 × 10 are added in every pipe⁶A cell, 1200rmp is centrifuged 3min, in abandoning Clearly, electricity is taken to turn kit (from Lonza company), a, b pipe are proportionally added into electricity and turn reagent totally 100 μ L.It is added into a pipe The 4 μ g of pNB328-meso3CAR plasmid that embodiment 1 constructs is added into b pipe, mixed liquor is shifted by 4 μ g of pNB328 empty plasmid Into electric revolving cup, it is put into Lonza Nucleofactor 2b electroporation, the program that number is U014 is chosen and shocks by electricity；It uses Micropipet in kit the cell suspension that electricity takes a turn for the better is transferred to be added culture solution, with meso3 antigen and anti-CD28 In six orifice plates that antibody has been coated with (culture solution is that the AIM-V containing 2%FBS trains liquid), mixes, be placed in 37 DEG C, 5%CO₂Cultivate 5-7 After it, then centrifuge cell, supernatant is abandoned, cell precipitation is resuspended with the fresh AIM-V culture medium containing 2%FBS, continue culture 3-5 days, Make culture total number of days reach 10 days, observation cell growth the case where, obtain be originated from business PBMC control T cell and Meso3CAR-T cell, cell are respectively designated as ConT-C329 and cMeso3CAR-T-C329.

The donor subject's blood for being 70 and 71 from number is fetched, obtains respective PBMC with the separation of Ficoll partition method. By PBMC adhere-wall culture 2-4h, wherein not adherent suspension cell is T cells, suspension cell is collected into 15ml centrifugation Guan Zhong, 1200rmp are centrifuged 3min, abandon supernatant, and physiological saline is added, and 1200rmp is centrifuged 3min, abandon physiological saline, and repeat this Step；Concentration is adjusted to 5 × 10 with physiological saline⁶A/mL.Each number cell respectively takes 2 1.5mLEP to manage, number a and b, often 5 × 10 are added in pipe⁶A cell, 1200rmp are centrifuged 3min, abandon supernatant, and electricity is taken to turn kit (purchased from Lonza company), a, b pipe It is proportionally added into electricity and turns reagent totally 100 μ L.4 μ g of pNB328 empty plasmid is added into a pipe, embodiment 1 is added into b pipe and constructs 4 μ g of pNB328-meso3CAR plasmid, mixed liquor is transferred in electric revolving cup, be put into Lonza Nucleofactor 2b electricity turn Instrument chooses the program that number is U014 and shocks by electricity；It is shifted using the cell suspension that the micropipet in kit takes a turn for the better electricity Into six orifice plates being coated with meso3 antigen and anti-CD28 antibody that culture solution has been added, (culture solution is containing 2%FBS's AIM-V trains liquid), it mixes, is placed in 37 DEG C, 5%CO₂After culture 5-7 days, then centrifuge cell, supernatant is abandoned, cell precipitation is contained with fresh The AIM-V culture medium of 2%FBS is resuspended, and continues culture 3-5 days, reaches total number of days of culture 10 days, the feelings of observation cell growth Condition obtains the control T cell and Meso3CAR-T cell for deriving from No. 70 and No. 71 donor subjects, is respectively designated as ConT- D70, ConT-D71, cMeso3CAR-T-D70 and cMeso3CAR-T-D71.

Similarly, donor subject's blood that number is 42 and 43 is taken, according to operation same as described above, preparation is derived from The Muc1CAR-T cell of No. 42 and No. 43 donor subjects, is respectively designated as ConT-D42, ConT-D43, cMuc1CAR-T- D42 and cMuc1CAR-T-D43.

Embodiment 3 activates reagent suspension method preparation Meso3CAR-T cell and Muc1CAR-T cell

It takes the commercialization PBMC cell that number is 329 to handle respectively, adjusts concentration to 5 × 10 with physiological saline⁶A/mL. Respectively take 4 1.5mLEP pipe, number c, d, e, f, addition 5 × 10 in every pipe⁶A cell, 1200rmp are centrifuged 3min, abandon supernatant, take Electricity turns kit (from Lonza company), and the every pipe of c-f pipe is proportionally added into electricity and turns reagent totally 100 μ L, and then reality is all added in every pipe The 4 μ g of pNB328-meso3CAR plasmid for applying the building of example 1, mixed liquor is transferred in electric revolving cup, Lonza is put into Nucleofactor 2b electroporation chooses the program that number is U014 and shocks by electricity, then using the micropipet in kit The cell suspension that takes a turn for the better of electricity is transferred in the hole for add 4 six orifice plates of training liquid to (culture solution is that the AIM-V containing 2%FBS is trained Liquid), it mixes, is placed in 37 DEG C, 5%CO₂Stimulating factor IL-2 is added to final concentration to the every hole in 4 holes after 6 hours in incubator culture 500IU/mL and meso3 antigen and anti-CD28 antibody: 0.1 μ g/mL+0.1 μ g/mL, 0.5 are added in combination by following final concentration respectively μ g/mL+0.5 μ g/mL, 1 μ g/mL+1 μ g/mL, 3 μ g/mL+3 μ g/mL, 37 DEG C, 5%CO₂After culture 5-7 days, then centrifuge cell, Supernatant is abandoned, cell precipitation is resuspended with the fresh AIM-V culture medium containing 2%FBS, continues culture 3-5 days, amounts to the time of culture The case where reaching 10 days, observing cell growth, the Meso3CAR-T cell of acquisition is named as fMeso3CAR-T-C329.

Take donor subject's blood that number is 70 and 71, according to operation same as described above, preparation from No. 70 and The Meso3CAR-T cell of No. 71 donor subjects, is respectively designated as fMeso3CAR-T-D70 and fMeso3CAR-T-D71.

Similarly, donor subject's blood that number is 42 and 43 is taken, according to operation same as described above, preparation is derived from The Muc1CAR-T cell of No. 42 and No. 43 donor subjects, is respectively designated as fMuc1CAR-T-D42 and fMuc1CAR-T-D43, Wherein fMuc1CAR-T-D42 and fMuc1CAR-T-D43 is suspended with+0.5 μ g/mL anti-CD28 antibody of 0.5 μ g/mL Muc1 antigen Stimulation.

Embodiment 4 activates reagent suspension method compared with activating Meso3CAR-T cell positive rate made from reagent fixation

1, Meso3-Fc-biotin and PE-stretavidin are taken and is dissolved in and is configured to concentration in PBS as 10.0mg/mL's 100 × stock solution dilutes 100 times with PBS using preceding up to the fluorescein-labeled Meso3-Fc dilution of PE

2, each Meso3CAR-T cell and respective control T cell each 1 × 10 made from difference Example 2,3,4⁶It is a, 1000rpm is centrifuged 3min, discards upper layer culture medium, is resuspended with 1mL fresh culture, 1,2,5 μ are separately added into all cells The fluorescein-labeled Meso3-Fc dilution of the PE of L step 1,37 DEG C of incubation 1h；

3, three times, washing is resuspended each Meso3CAR-T cell after being incubated for cold PBS washing step 2 with the cold PBS of 1mL every time Cell, 1000rpm are centrifuged 3min.The later fluorescence intensity for using flow cytomery cell three times, analysis ConT-C329, CMeso3CAR-T-C329 and fMeso3CAR-T-C329 and ConT-D70, ConT-D71, cMeso3CAR-T-D70, The positive rate of cMeso3CAR-T-D71 cell and fMeso3CAR-T-D70 and fMeso3CAR-T-D71, compares.

As a result as seen in figs. 2a-2c.Fig. 2A shows ConT-C329, cMeso3CAR-T-C329 and various concentration mesothelium The positive rate of fMeso3CAR-T-C329 cell obtained under conditions of the stimulation of plain III antigen+anti-CD28 antibody；Fig. 2 B is shown ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin III antigen+anti-CD28 antibody are obtained under conditions of stimulating The positive rate of fMeso3CAR-T-D70 cell；Fig. 2 C shows ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelium The positive rate of fMeso3CAR-T-D71 cell obtained under conditions of the stimulation of plain III antigen+anti-CD28 antibody.In Fig. 2A -2C 0.1+0.1,0.5+0.5 and 3+3 are respectively indicated with 0.1 μ g/mL Meso3+0.1 μ g/mL anti-CD28 antibody of final concentration, 0.5 μ g/ ML Meso3+0.5 μ g/mL anti-CD28 antibody and 3 μ g/mL Meso3+3 μ g/mL anti-CD28 antibodies stimulate cell in the form of a solution.

Fig. 2A -2C display the result shows that, with Meso3 antigen and anti-CD28 antibody coating orifice plate stimulation preparation Meso3CAR-T cell is compared, and the Meso3 antigen and anti-CD28 antibody for being directly added into each concentration combination suspend made from stimulation The positive rate of various Meso3CAR-T cells is obviously higher than the former, and the Meso3CAR-T cell in the commercialization source PBMC (329) there is same effect from the Meso3CAR-T cell of different donor subjects (donor 70 and donor 71) blood sources Fruit.

Embodiment 5 activates reagent suspension method and activates the horizontal ratio of proliferation of Meso3CAR-T cell made from reagent fixation Compared with

Activation reagent suspension method is detected as CCK8 proliferation test and Meso3CAR-T made from activation reagent fixation is thin The proliferation of born of the same parents is horizontal, and steps are as follows:

(1) T cell volume is bigger after T cell activation and under the molecular marker for increased proliferation phase, microscope, selects this shape T cell under state carries out proliferation experiment；

(2) it takes 10 μ L T cell suspensions to be added in disposable tally with Trypan Blue, counts；

(3) 6.4 × 10 are taken⁵A T cell, constant volume is in 800 μ L AIM-V complete mediums, so that cell number is 8 × 10⁴/ 100 μ L, then gradient dilution to 1250/100 μ L, totally 7 concentration gradients (8 × 10⁴、4×10⁴、2×10⁴、1×10⁴、0.5 ×10³、0.25×10³With 0.125 × 10³/100μL)；

(4) cell spreads 96 orifice plates, and each concentration spreads 3 holes, and every hole adds 100 μ L.Then, 10 μ L CCK8 are added in every hole, put Enter in cell incubator, be incubated for 4h, read the OD value under 450nm wavelength, calculates standard curve；

(5) 3 piece of 96 orifice plate, 6 multiple holes of every piece of plate middle berth, every hole 1 × 10 are taken again⁴A T cell, is placed in cell incubator Culture；

(6) for 24 hours, 48h and 72h when, takes out one block of plate respectively, and 10 μ L CCK8 are added, incubate in cell incubator 4h is educated, OD value is read under 450nm wavelength.Each period T cell number is calculated according to standard curve, makes growth curve.

As a result as shown in figs.3 a and 3b.Fig. 3 A shows ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin The growth curve of fMeso3CAR-T-D70 cell obtained under conditions of the stimulation of III antigen+anti-CD28 antibody；Fig. 3 B is shown ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody are obtained under conditions of stimulating The increment curve of fMeso3CAR-T-D71 cell.0.1+0.1,0.5+0.5,1+1 and 3+3 are respectively indicated with 0.1 μ g/ of final concentration ML Meso3+0.1 μ g/mL anti-CD28 antibody, 0.5 μ g/mL Meso3+0.5 μ g/mL anti-CD28 antibody, 1 μ g/mL Meso3+1 μ G/mL anti-CD28 antibody and 3 μ g/mL Meso3+3 μ g/mL anti-CD28 antibodies stimulate cell in the form of a solution.

It is that Fig. 3 A-3B is shown the result shows that, with Meso3 antigen and anti-CD28 antibody coating orifice plate stimulation preparation Meso3CAR-T cell is compared, made from the Meso3 antigen+anti-CD28 antibody stimulation for the addition that directly suspends under each concentration combination The proliferation level of Meso3CAR-T cell is bright with its respective control group T cell and compared with activating cell made from reagent fixation It is aobvious higher.Wherein ,+0.5 μ g/mL anti-CD28 antibody of 0.5 μ g/mL Meso3 antigen and+1 μ g/mL of 1 μ g/mL Meso3 antigen are anti- The raising of the proliferation level of Meso3CAR-T cell made from the combination of stimulation of two kinds of concentration of CD28 antibody is especially apparent.

Embodiment 6 activates reagent suspension method and activates the cell factor point of Meso3CAR-T cell made from reagent fixation Bleeding is flat to be compared

Take cMeso3CAR-T-D70, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody thorn FMeso3CAR-T-D70 and fMeso3CAR-T-D71 cell obtained under conditions of swashing, with the CBA Human Th1/ of BD Th2Cytokine Kit II detects the cytokine secretion profile of Meso3CAR-T cell made from various distinct methods, specifically Steps are as follows:

(1) the aforementioned recombination MSLN antigen (rMSLN) prepared is diluted to 5 μ g/mL with PBS, is coated with 6 orifice plates, often 3mL is added in hole, 4 DEG C overnight.It removes antigen coat liquid within second day, is cleaned 2~3 times with PBS；

(2) it is counted to various Meso3CAR-T cells, and takes 1 × 10⁶A Meso3CAR-T cell, 1500rpm, from Heart 5min abandons supernatant；

(3) T cell is resuspended with the AIM-V complete medium without IL-2, takes 1 × 10⁶Meso3CAR- cell, is laid on RMSLN is coated in hole；

(4) cell suspension is collected afterwards for 24 hours, 3000rpm is centrifuged 5min, collects supernatant；

(5) each sample takes 10 μ L.By the IL-2, IL-4, IL-6, IL- of source of people in Th1/Th2Cytokine Kit II 10, TNF-α and IFN-γ capture magnetic bead mix, and 40 μ L are added in every kind of capture magnetic bead, and 4 DEG C are protected from light incubation 3h；

(6) 3mL PBS is added into each reaction system to clean 1~2 time, 4000rpm, it is centrifuged 5min, abandons supernatant；

(7) magnetic bead that 300 μ L PBS are resuspended after reaction, upper machine testing is added.Finally calculated with FCAP Array v3 software Above-mentioned four kinds of cytokine concentrations.

As a result as shown in figs. 4 a-4d.Fig. 4 A-4D is shown in contacted with PANC-1 cell and SKOV-3 cell after, Under conditions of cMeso3CAR-T-D70, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulate FMeso3CAR-T-D70 and fMeso3CAR-T-D71 cell obtained secretes IL-2, IL-4, IL-10 and four kinds of IFN-γ respectively The level of cell factor.0.1+0.1,0.5+0.5 and 3+3 respectively indicate anti-with 0.1 μ g/mL Meso3+0.1 μ g/mL of final concentration CD28 antibody, 0.5 μ g/mL Meso3+0.5 μ g/mL anti-CD28 antibody and 3 μ g/mL Meso3+3 μ g/mL anti-CD28 antibodies are with molten Liquid form stimulates cell.

According to Fig. 4 A-4D is shown as a result, activation reagent is outstanding compared with the Meso3CAR-T cell made from coating well plate method The secretion level of part has apparent reduction in above-mentioned several cell factors in various Meso3CAR-T cells made from float glass process, The decline level of middle IL-10 and IFN-γ is especially apparent.Cytokines release syndrome (CRS) is the master of CAR-T cell therapy Want side effect, hence it is evident that reduced levels of cytokine secretion can greatly reduce the risk for generating side effect.

Embodiment 7 activates reagent suspension method and activates the exhaustion factor water of Meso3CAR-T cell made from reagent fixation It is flat to compare

Take ConT-C329, cMeso3CAR-T-C329 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation Under the conditions of fMeso3CAR-T-C329 cell obtained, with 1 × 10 after counting⁶A cell/pipe is separately added into the EP pipe of 1.5ml In, PBS is washed twice, and 1200rpm is centrifuged 5min, abandons supernatant；The streaming antibody anti-PD- of detection T cell failure phenotype is added 1-PE, flicking precipitating is uniformly mixed it；After room temperature is protected from light incubation 30min, PBS is cleaned one time, and 1200rpm is centrifuged 5min, is abandoned The physiological saline of 400 μ L is added in supernatant, cell is transferred in streaming pipe, upper machine testing.

As a result as shown in figure 5, with control group T cell and compared with being coated with Meso3CAR-T cell made from well plate method, activation The expression of fMeso3CAR-T-C329 cell PD-1 made from reagent suspension method is significantly higher, shows meso3 antigen and resists The two kinds of reagent dual suspension stimulations of CD28 antibody can more efficiently activate CAR-T cell.

Embodiment 8 activates reagent suspension method and activates the memory T cell of Meso3CAR-T cell made from reagent fixation The detection of phenotype

Take ConT-D70, ConT-D71, cMeso3CAR-T-D70, cMeso3CAR-T-D71 and various concentration mesothelin FMeso3CAR-T-D70 and fMeso3CAR-T-D71 obtained under conditions of the stimulation of III antigen+anti-CD28 antibody, after counting with 1×10⁶A cell/pipe is separately added into the EP pipe of 1.5ml, and PBS is washed twice, and 1200rpm is centrifuged 5min, abandons supernatant；Respectively The streaming antibody anti-CD197-FITC+anti-CD62L-PE of detection memory T cell phenotype is added, flicking precipitating makes its mixing Uniformly；After room temperature is protected from light incubation 30min, PBS is cleaned one time, and 1200rpm is centrifuged 5min, abandons the physiology salt that 400 μ l are added in supernatant Cell is transferred in streaming pipe by water, upper machine testing.

As a result as shown in figs 6 a-6 c.Fig. 6 A shows ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelin III Effector T cell flow cytometer detection knot under conditions of the stimulation of antigen+anti-CD28 antibody in fMeso3CAR-T-D70 cell obtained Fruit；Fig. 6 B shows ConT-D71, cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulation item Effector T cell flow cytometer detection result under part in fMeso3CAR-T-D71 cell obtained；Fig. 6 C is effect in Fig. 6 A and Fig. 6 B T cell percentage statistical result.In the Meso3CAR-T cell of the distinct methods preparation in No. 70 and No. 71 donor subject sources In, compared with control group T cell and activating T cell made from reagent fixation, activate Meso3CAR- made from reagent suspension method Effector memory T cell (T in T cell_EM) ratio it is significantly raised.

Embodiment 9 activates reagent suspension method and activates Meso3CAR-T cells against tumor cells made from reagent fixation Lethality compares

Using real-time n cell functional analysis instrument detection ConT-D70, ConT-D71, cMeso3CAR-T-D70, FMeso3CAR- obtained under conditions of cMeso3CAR-T-D71 and various concentration mesothelin III antigen+anti-CD28 antibody stimulate T-D70 and fMeso3CAR-T-D71 cell is in vitro to the lethal effect of tumour cell.

Specifically, choosing pancreatic carcinoma PANC-1 and ovarian cancer cell line SKOV3 as target cell, using Ai Sen The real-time n cell functional analysis instrument (RTCA) of company detects above-mentioned each Meso3CAR-T cell and its compares the body of T cell Outer killing activity, the specific steps are as follows:

(1) return to zero: 50 μ l DMEM or 1640 culture medium is added in every hole, is put into instrument, selects step 1, zeroing；

(2) target cell bed board: ovarian cancer cell SKOV-3, pancreatic cancer cell PANC-1 press 104, every hole cell/50 μ l It is layered in the plate containing detecting electrode, places several minutes, once to cytotostatic, place into instrument, start step2, culture Cell；

(3) effector cell is added: after target cell culture for 24 hours, suspending step 2, it is (i.e. above-mentioned to derive from that effector cell is added Each Meso3CAR-T of 71 blood of donor subject 70 and donor subject compares T cell with it), every 50 μ L of hole imitates target score It is not set as 1:1, respectively using ConT-D70, ConT-D71 cell as control, starts step 3, continues after co-culturing for 24 hours, sees Examine cell Proliferation curve.

As a result as shown in figures 7 a-7d.Fig. 7 A and 7B show ConT-D70, cMeso3CAR-T-D70 and various concentration mesothelium FMeso3CAR-T-D70 cell obtained is respectively to pancreatic carcinoma under conditions of the stimulation of plain III antigen+anti-CD28 antibody The killing curve of PANC-1 and ovarian cancer cell SKOV-3；Fig. 4 C and 4D show ConT-D71, cMeso3CAR-T-D71 and not With fMeso3CAR-T-D71 cell obtained under conditions of concentration mesothelin III antigen+anti-CD28 antibody stimulation respectively to pancreas The killing curve of cancerous cell line PANC-1 and ovarian cancer cell SK-OV-3.0.1+0.1,0.5+0.5,1+1 and 3+3 are respectively indicated With 0.1 μ g/mL Meso3+0.1 μ g/mL anti-CD28 antibody of final concentration, 0.5 μ g/mL Meso3+0.5 μ g/mL anti-CD28 antibody, 1 μ g/mL Meso3+1 μ g/mL anti-CD28 antibody and 3 μ g/mL Meso3+3 μ g/mL anti-CD28 antibodies stimulate carefully in the form of a solution Born of the same parents.

The structure that Fig. 7 A-7D is shown shows each Meso3CAR-T cell made from activation reagent suspension method to PANC-1 The lethality of cell and SK-OV-3 cell is substantially at compared with activating Meso3CAR-T cell made from reagent fixation substantially Same level does not differ significantly.Levels of cytokine secretion is significantly reduced as a result, with the present invention in 6 in conjunction with the embodiments Activate Meso3CAR-T cell made from reagent suspension method that there is higher peace under the premise of guaranteeing to tumor cell killing potential Quan Xing.

Comparative example 1 activates reagent suspension method compared with activating Muc1CAR-T cell positive rate made from reagent fixation

Referring to the step of embodiment 4, the Meso3-Fc-biotin in embodiment 4 is substituted for Muc1-Fc-biotin, is used The fluorescence intensity of flow cytomery cell analyzes ConT-D42, ConT-D43, cMuc1CAR- obtained in embodiment 2-3 The positive rate of T-D42, cMuc1CAR-T-D43 cell and fMuc1CAR-T-D42 and fMuc1CAR-T-D43, compares.

As a result as shown in figures 8 a-8b.Fig. 8 A shows the positive rate of cMuc1CAR-T-D42 and fMuc1CAR-T-D42 cell It is horizontal；Fig. 8 B shows that the positive rate of cMuc1CAR-T-D43 and fMuc1CAR-T-D43 cell is horizontal.It is shown according to Fig. 8 A-8B As a result, fMuc1CAR-T-D42 and fMuc1CAR-T-D43 cell made from activation reagent suspension method and the fixed hair system of activation reagent CMuc1CAR-T-D42, the cMuc1CAR-T-D43 obtained is compared, and positive rate does not improve, from donor subject No. 43 The positive rate of fMuc1CAR-T-D43 is even lower in Muc1CAR-T cell.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, those skilled in the art can be right after having read above content of the invention The present invention makes various changes or modifications, and these equivalent forms also fall within the scope of the appended claims of the present application.

Sequence table

<110>Shanghai cell therapy Engineering Technical Research Centre Group Co., Ltd

Shanghai cell therapy research institute

<120>a kind of method for the CAR-T cell for preparing targeting mesothelin

<130> 187080

<160> 8

<170> SIPOSequenceListing 1.0

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atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

aacgggtccg aatacttcgt gaagatccag tccttcctgg gtggggcccc cacggaggat 120

ttgaaggcgc tcagtcagca gaatgtgagc atggacttgg ccacgttcat gaagctgcgg 180

acggatgcgg tgctgccgtt gactgtggct gaggtgcaga aacttctggg accccacgtg 240

gagggcctga aggcggagga gcggcaccgc ccggtgcggg actggatcct acggcagcgg 300

caggacgacc tggacacgct ggggctgggg ctacagggcg gcatccccaa cggctacctg 360

gtcctagacc tcagcatgca agaggccctc tcggagtcca aatatggtcc cccatgccca 420

ccatgcccag ggcagccccg agagccacag gtgtacaccc tgcccccatc ccaggaggag 480

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc 540

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 600

ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg 660

caggagggga atgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 720

cagaagagcc tctccctgtc tctgggtaaa tga 753

<210> 2

<211> 250

<212> PRT

<213>artificial sequence (Artificial Sequence)

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Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe

20 25 30

Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn

35 40 45

Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val

50 55 60

Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val

65 70 75 80

Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile

85 90 95

Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln

100 105 110

Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu

115 120 125

Ala Leu Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly

130 135 140

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

145 150 155 160

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

165 170 175

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

180 185 190

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

195 200 205

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

210 215 220

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

225 230 235 240

Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

245 250

<210> 3

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<212> DNA

<213>artificial sequence (Artificial Sequence)

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atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

tctggtcatg caagctctac cccaggtgga gaaaaggaga cttcggctac ccagagaagt 120

tcagtgccca gctctactga gaagaatgct gtgagtatga ccagcagcgt actctccagc 180

cacagccccg gttcaggctc ctccaccact cagggacagg atgtcactct ggccccggcc 240

acggaaccag cttcaggttc agctgccctg tggggacagg atgtcacctc ggtcccagtc 300

accaggccag ccctgggctc caccaccccg ccagcccacg atgtcacctc agccccggac 360

aacaagccag ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 420

accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 480

accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 540

accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 600

accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 660

accaggccgg ccccgggctc caccgccccc ccagcccatg gtgtcacctc ggccccggac 720

aacaggcccg ccttgggctc caccgcccct ccagtccaca atgtcacctc ggcctcaggc 780

tctgcatcag gctcagcttc tactctggtg cacaacggca cctctgccag ggctaccaca 840

accccagcca gcaagagcac tccattctca attcccagcc accactctga tactcctacc 900

acccttgcca gccatagcac caagactgat gccagtagca ctcaccatag ctcggtacct 960

cctctcacct cctccaatca cagcacttct ccccagttgt ctactggggt ctctttcttt 1020

ttcctgtctt ttcacatttc aaacctccag tttaattcct ctctggaaga tcccagcacc 1080

gactactacc aagagctgca gagagacatt tctgaaatgt ttttgcagat ttataaacaa 1140

gggggttttc tgggcctctc caatattaag ttcaggccag gatctgtggt ggtacaattg 1200

actctggcct tccgagaagg taccatcaat gtccacgacg tggagacaca gttcaatcag 1260

tataaaacgg aagcagcctc tcgatataac ctgacgatct cagacgtcag cgtgagtgat 1320

gtgccatttc ctttctctgc ccagtctggg gagtccaaat atggtccccc atgcccacca 1380

tgcccagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1440

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1500

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1560

gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 1620

gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1680

aagagcctct ccctgtctct gggtaaatga 1710

<210> 4

<211> 569

<212> PRT

<213>artificial sequence (Artificial Sequence)

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Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys

20 25 30

Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys

35 40 45

Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly

50 55 60

Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala

65 70 75 80

Thr Glu Pro Ala Ser Gly Ser Ala Ala Leu Trp Gly Gln Asp Val Thr

85 90 95

Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala

100 105 110

His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr

115 120 125

Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala

130 135 140

Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp

145 150 155 160

Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr

165 170 175

Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala

180 185 190

His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr

195 200 205

Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala

210 215 220

Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp

225 230 235 240

Asn Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr

245 250 255

Ser Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn

260 265 270

Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro

275 280 285

Phe Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser

290 295 300

His Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro

305 310 315 320

Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly

325 330 335

Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn

340 345 350

Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg

355 360 365

Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu

370 375 380

Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu

385 390 395 400

Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr

405 410 415

Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr

420 425 430

Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln

435 440 445

Ser Gly Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln

450 455 460

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

465 470 475 480

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

485 490 495

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

500 505 510

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

515 520 525

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

530 535 540

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

545 550 555 560

Lys Ser Leu Ser Leu Ser Leu Gly Lys

565

<210> 5

<211> 972

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gaagtggaga agacagcctg tccttcaggc aagaaggccc gcgagataga cgagagcctc 120

atcttctaca agaagtggga gctggaagcc tgcgtggatg cggccctgct ggccacccag 180

atggaccgcg tgaacgccat ccccttcacc tacgagcagc tggacgtcct aaagcataaa 240

ctggatgagc tctacccaca aggttacccc gagtctgtga tccagcacct gggctacctc 300

ttcctcaaga tgagccctga ggacattcgc aagtggaatg tgacgtccct ggagaccctg 360

aaggctttgc ttgaagtcaa caaagggcac gaaatgagtc ctcaggtggc caccctgatc 420

gaccgctttg tgaagggaag gggccagcta gacaaagaca ccctagacac cctgaccgcc 480

ttctaccctg ggtacctgtg ctccctcagc cccgaggagc tgagctccgt gccccccagc 540

agcatctggg cggtcaggcc ccaggacctg gacacgtgtg acccaaggca gctggacgtc 600

ctctatccca aggcccgcct tgctttccag aacatgaacg ggtccgaata cttcgtgaag 660

atccagtcct tcctgggtgg ggcccccacg gaggatttga aggcgctcag tcagcagaat 720

gtgagcatgg acttggccac gttcatgaag ctgcggacgg atgcggtgct gccgttgact 780

gtggctgagg tgcagaaact tctgggaccc cacgtggagg gcctgaaggc ggaggagcgg 840

caccgcccgg tgcgggactg gatcctacgg cagcggcagg acgacctgga cacgctgggg 900

ctggggctac agggcggcat ccccaacggc tacctggtcc tagacctcag catgcaagag 960

gccctctcgt ga 972

<210> 6

<211> 323

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys

20 25 30

Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu

35 40 45

Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val

50 55 60

Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys

65 70 75 80

Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His

85 90 95

Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp

100 105 110

Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys

115 120 125

Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val

130 135 140

Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala

145 150 155 160

Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser

165 170 175

Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr

180 185 190

Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala

195 200 205

Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe

210 215 220

Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn

225 230 235 240

Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val

245 250 255

Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val

260 265 270

Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile

275 280 285

Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln

290 295 300

Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu

305 310 315 320

Ala Leu Ser

<210> 7

<211> 1485

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgga gtccggggga ggcctggtcc agcctggggg atccctgaga 120

ctctcctgcg cagcctctgg attcgacctc ggtttctact tttacgcctg ttgggtccgc 180

caggctccag ggaagggcct ggagtgggtc tcatgcattt atactgctgg tagtggtagc 240

acgtactacg cgagctgggc gaaaggccga ttcaccatct ccagagacaa ttcgaagaac 300

acgctgtatc tgcaaatgaa cagtctgaga gccgaggaca cggccgtgta ttactgtgcg 360

agatctactg ctaatactag aagtacttat tatcttaact tgtggggcca aggcaccctg 420

gtcaccgtct cctcaggcgg aggcggatca ggtggtggcg gatctggagg tggcggaagc 480

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 540

atcacttgcc aggccagtca gaggattagt agttacttat cctggtatca gcagaaacca 600

gggaaagttc ccaagctcct gatctatggt gcatccactc tggcatctgg ggtcccctcg 660

cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 720

gaagatgttg ccacttacta ctgtcagagt tatgcttatt ttgatagtaa taattggcat 780

gctttcggcg gagggaccaa ggtggagatc aaaaccacga cgccagcgcc gcgaccacca 840

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960

gcgcccctgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020

aaacggggca gaaagaagct cctgtatata ttcaaacaac catttatgag accagtacaa 1080

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1140

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1200

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1260

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1320

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1380

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1440

gacacctacg acgcccttca catgcaggcc ctgccccctc gctga 1485

<210> 8

<211> 1506

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagcgagg tccagctgca gcagtcagga ggaggcttgg tgcaacctgg aggatccatg 120

aaactctcct gtgttgcctc tggattcact ttcagtaact actggatgaa ctgggtccgc 180

cagtctccag agaaggggct tgagtgggtt gctgaaatta gattgaaatc taataattat 240

gcaacacatt atgcggagtc tgtgaaaggg aggttcacca tctcaagaga tgattccaaa 300

agtagtgtct acctgcaaat gaacaactta agagctgaag acactggcat ttattactgt 360

acctttggta actcctttgc ttactggggc caagggacca cggtcaccgt ctcctcaggt 420

ggttctggtt ctggcggctc cggttccggt ggatccggct ctgatatcgt tgtgactcag 480

gaatctgcac tcaccacatc acctggtgaa acagtcacac tcacttgtcg ctcaagtact 540

ggggctgtta caactagtaa ctatgccaac tgggtccaag aaaaaccaga tcatttattc 600

actggtctaa taggtggtac caacaaccga gcaccaggtg ttcctgccag attctcaggc 660

tccctgattg gagacaaggc tgccctcacc atcacagggg cacagactga ggatgaggca 720

atatatttct gtgctctatg gtacagcaac cattgggtgt tcggtggagg aaccaaactg 780

actgtcctag gatccgagtt cgtgccggtc ttcctgccag cgaagcccac cacgacgcca 840

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 900

gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 960

gatatctaca tctgggcgcc cctggccggg acttgtgggg tccttctcct gtcactggtt 1020

atcacccttt actgcaacca caaacggggc agaaagaagc tcctgtatat attcaaacaa 1080

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1140

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1200

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1260

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1320

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1380

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1440

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1500

cgctga 1506

Claims (11)

1. a kind of method for the CAR-T cell for preparing targeting mesothelin, which is characterized in that 1) it will be the following steps are included: will contain The nucleic acid for targeting the Chimeric antigen receptor coded sequence of mesothelin antigen imports peripheral blood mononuclear cells (PBMC), obtains initial Cell；2) with mesothelin antigen and anti-CD28 antibody contact 1) described in initial cell, culture, wherein the mesothelin antigen with The state of the anti-CD28 antibody is non-immobilization.

2. method according to claim 1, which is characterized in that the mesothelin antigen is overall length mesothelin antigen or mesothelin Domain III antigen, it is preferable that be mesothelin Domain III antigen；The mesothelin Domain III antigen preferably includes IgG Fc, the IgG Fc is preferably IgG4Fc；The mesothelin Domain III antigen preferably pass through hinge with it is described IgG Fc fusion is the preferably hinge CD8 hinge.

3. method according to claim 2, which is characterized in that the mesothelin antigen includes also signal peptide, the letter in N-terminal Number peptide is preferably light chain signal peptide；It is highly preferred that the mesothelin antigen is mesothelin Domain III antigen, in N-terminal and gently The fusion of chain signal peptide is merged by CD8 hinge with IgG4Fc in C-terminal, and amino acid sequence is shown in SEQ ID NO:2.

4. method according to claim 1, which is characterized in that the nucleic acid is nucleic acid construct or mRNA, it is therefore preferable to core Acid con-struct.

5. method according to claim 4, which is characterized in that the nucleic acid construct is expression vector, is optionally virus Carrier or Transposon System carrier；The viral vectors is preferably to be derived from the slow virus carrier of I type HIV, such as pWPT or pWPXL；The Transposon System be preferably the be transformed or Sleeping Beauty Transposon System carrier be not transformed or PiggyBac Transposon System carrier, PiggyBac Transposon System carrier being more preferably transformed or not being transformed are optimal Selection of land is the PiggyBac Transposon System carrier of transformation, and the pNB328 Transposon System as disclosed in CN105154473A carries Body.

6. method according to claim 4, which is characterized in that the nucleic acid is mRNA, contains 5 ' end cap minor structures, 5 ' UTR, the open reading frame (ORF) of the coding antigen, 3 ' UTR and polyA tails.

7. method according to claim 1, which is characterized in that the method for the importing be selected from infestation with virus particles, electricity turn, Any one of liposome transfection, calcium phosphate transfection and biolistic transformation, it is therefore preferable to which selected from infestation with virus particles, electricity turns and rouge Any one of plasmids, more preferably infestation with virus particles or electricity turn, and most preferably electricity turns.

8. method according to claim 1, which is characterized in that the anti-CD28 antibody is anti-CD28 monoclonal antibody or resists CD28 polyclonal antibody, it is therefore preferable to anti-CD28 monoclonal antibody, more preferably Humanized monoclonal antibodies, it is further more excellent Selection of land is the anti-CD28 monoclonal antibody of humanization source of mouse or rabbit source, the most preferably anti-CD28 monoclonal antibody of humanization source of mouse.

9. method according to claim 1, which is characterized in that the temperature of the culture is 37 DEG C；And/or the culture CO₂Concentration is 5%；And/or culture medium used in the culture is the AIM-V containing 2% fetal calf serum；And/or the training The feeding time is 5-10 days.

10. method according to claim 9, which is characterized in that in culture medium used in the culture also containing cell because Son；Preferably, the cell factor is IL-2, final concentration of 500IU/mL；And/or the cell factor is in the culture The culture medium is added when carrying out 0-6 hours.

11. any one of -10 the method according to claim 1, which is characterized in that the mesothelin antigen and the anti-CD28 The state of antibody is solution form；Preferably, the final concentration of 0.1-3 μ g/mL of the mesothelin antigen, the anti-CD28 are anti- The final concentration of 0.1-3 μ g/mL of body；It is highly preferred that the final concentration of 0.1-1 μ g/mL, the anti-CD28 of the mesothelin antigen The final concentration of 0.1-1 μ g/mL of antibody；Even more preferably, the final concentration of 0.5-1 μ g/mL of the mesothelin antigen, institute State the final concentration of 0.5-1 μ g/mL of anti-CD28 antibody；Most preferably, the final concentration of 0.5 μ g/mL of the mesothelin antigen, institute State the final concentration of 0.5 μ g/mL of anti-CD28 antibody.