CN109385402A

CN109385402A - A kind of equal proportion mixes the preparation method and application of two kinds of target spot CART cells

Info

Publication number: CN109385402A
Application number: CN201710686416.7A
Authority: CN
Inventors: 黄飞; 金涛; 王海鹰; 何凤; 史子啸
Original assignee: Shanghai Hrain Biotechnology Co Ltd
Current assignee: Shanghai Hrain Biotechnology Co Ltd
Priority date: 2017-08-11
Filing date: 2017-08-11
Publication date: 2019-02-26

Abstract

The invention discloses the preparation methods and purposes of equal proportion mixing CD19-CART cell and CD22-CART cell.Specifically, CART cell prepared by the present invention is equal proportion mixing CD19-41BB CART cell and CD22-41BB CART cell.Mixing CART cell prepared by the present invention all has strong killing ability to specific tumor cell.Mixing CART cell prepared by the present invention also has survival period long other than having the characteristics that the strong killing ability of tumour cell.It includes leukaemia and lymthoma that mixing CART cell, which can be used for the immunization therapy for the malignant tumour that two target spots of CD19 and CD22 are mediated, prepared by the present invention.Solid foundation has been established in research and clinical trial based on mixing CART cell prepared by the present invention.

Description

A kind of equal proportion mixes the preparation method and application of two kinds of target spot CART cells

Technical field

The invention belongs to Chimeric antigen receptor fields, and in particular to mixing CD19-41BB CART cell and CD22- 41BBCART cell and application thereof.

Background technique

Chimeric antigen receptor (Chimeric Antigen Receptor-T cell, CART) T cell refers to repairs through gene After decorations, specific purpose antigen, and the T cell of continuous activation amplification can be identified with MHC non-limiting way.It is 2012 international thin Born of the same parents, which treat association's annual meeting middle finger and go out biological immune cell therapy, has become operation, radiotherapy, the 4th kind for the treatment of tumour outside chemotherapy Means, and future tumors will be become and treat essential means.CART cell adoptive therapy is that most clearly have in current cancer therapies The immunotherapeutic form of effect.A large number of studies show that CART cell can effectively identify tumour antigen, cause the anti-swollen of specificity Tumor immune response significantly improves the survival state of patient.

Chimeric antigen receptor (CAR) is the core component of CART, assigns the non-dependent mode of T cell HLA and identifies that tumour is anti- Former ability, this, which enables, identifies wider mesh compared to nave T cell surface receptor TCR by the T cell of CAR transformation Mark.It include that tumor associated antigen (tumor-associated antigen, a TAA) combined area is (logical in the basic engineering of CAR Often derive from the scFV section of monoclonal antibody antigen bond area), an extracellular hinge area, a transmembrane region and a letter intracellular Number area.The selection of target antigen for the safety of the specificity of CAR, validity and genetic modification T cell itself all It is crucial determinant (Science.1986.233 (4770): p.1318-21.).

CD19 is the glycoprotein of the 95kDa on B cell surface a kind of, is expressed since the early stage that B cell is developed is, until its It is divided into thick liquid cell.CD19 is one of the member of immunoglobulin (Ig) superfamily, as B cell surface signal transduction compound One of component, participated in the signal transduction process of B-cell receptor.In the mouse model of CD19 defect, periphery The quantity of B cell will appear apparent reduction in lymphoid tissue, can also decline to the response of vaccine and mitogen, while with blood The attenuating of clear Ig level.Generally, it is considered that the expression of CD19 is only limited to B cell system (B-cell lineage), it is more without being expressed in It can hemopoietic stem cell surface.It is thin that CD19 is also expressed in most of B cell lymphomas, lymphoma mantle cell, ALLs, CLLs, crinosity The surface of born of the same parents' leukaemia and a part of acute myeloid leukemia cells in children.Therefore, in the treatment of leukaemia/lymthoma, CD19 It is a kind of very valuable immunotherapeutic targets.Importantly, CD19 will not be expressed in it is most of normal thin in addition to B cell Cellular surface, including pluripotential hemopoietic stem cell, this feature make CD19 can be used as a kind of safe therapy target, can send out patient Raw autoimmune disease or the risk of irreversibility bone marrow toxicity damage minimize.Currently, anti-CD19 is had been developed that Antibody or scFv segment, and demonstrate in mouse model and the mankind/primate the prospect of its application.

CD22 wide expression is in the acute lymphatic leukaemia (BCP-ALL) of B cell precursor, in 111 case loads of research In have and all express CD22 antigen in 109 initial cells, expression rate is greater than 90%.Lars Nitschke report CD22 is subordinate to In sialic acid binding domain-immunoglobulin sample agglutinin (Siglecs, sialic acid-binding immunoglobulin- Like lectins) family.The albumen of this family is only expressed in the cell of immune system, all autoimmunities and acquired Cell type in immune system at least expresses a kind of Siglecs family protein.B cell expresses the member of two this families, One of them is exactly CD22, another is Siglec-G.Most of Siglecs has the suppression of tyrosine immunity receptor dependence Structure (ITIMs, immunoreceptor tyrosine-based inhibitory motifs) processed carries out negative tune to immune Section.Tyrosine on ITIMs produces some containing SH2 (Src homology after Src family protein tyrosine phosphorylation 2) binding site of structural domain molecule.SHP1 (SHP, SH2-domain containing protein tyrosine Phosphatase) and the mostly important SH2 structural domain of SHP2 contains albumen, these albumen are enrolled into the receptor containing ITIMs Afterwards, the dephosphorylation of intracellular matter can be caused and inhibit several signal paths.For example CD22 is exactly by recruiting SHP-1 To the ITIMs of itself to inhibit calcium ion caused by the BCR (B-cell receptor, B-cell receptor) of normal B cells to believe Number access.CD22 combines the ligand with α 2-6 coupling sialic acid.This combination directly adjust the combination of CD22 and BCR from And regulate and control the inhibition function of CD22, the migration of B cell and the threshold value of BCR signal can be regulated and controled.Nitscheke reports CD22 length It is 140kDa, possesses seven immunoglobulin like domain, specific is expressed in B cell system, from pre B cell (pre-B Cell) period starts to express.CD22 is present in each period of B cell, B cell and memory B cell including activation；But exist Loss of expression in the thick liquid cell of terminal differentiation.CD22 becomes a very attractive in the developing wide spectrum expression of B cell Targeting B cell molecule.

Two generation CAR of two kinds of Chimeric antigen receptor different costimulatory molecules are clinically widely applied, the function between them There has also been many researchs for energy difference.In vitro, CD28 or 4-1BB CAR has similar anti-tumor capacity, but in vivo Preclinical study show 4-1BB CAR transformation T cell may have stronger proliferative capacity and survival ability.Particularly, The T cell being transformed researches show that two kind of two generation CAR clinically can continue to multiply in vivo, but include 4-1BB total Stimulation molecule CAR transformation T cell can be more long survival.In the clinical research for acute lymphoma leukaemia, The CD19-28Z CAR T cell of the reports such as Davila can survive in vivo 1 to 3 months.It is similar, from NCI's CD19-CAR T cell (costimulatory molecules of CD28), the longest time-to-live of report are 68 days.And CD19- is used in other In the research of the CAR T cell of 41BB, CART cell survival at 6 months up to 68%, and in this research, B cell development The highest patient of infull symptom sustainable 2 years, it is shown that the continuous lasting function of the CART cell of CD19-4-1BB.

The present invention carries out equal proportion mixing, costimulating factor choosing using the CART cell for targeting two kinds of target spots CD19 and CD22 41BB is selected, this process increases CART cell to the lethal effect of B cell malignant tumour.Because CD19 and CD22 expression covers Each period of B cell development.It is preferable functional to also demonstrate that the CART cell of mixing has for experiment in vitro simultaneously, of the invention Also it lays a good foundation for clinical trial.

Summary of the invention

First aspect present invention provides a kind of method for preparing CART cell, be will comprising CD19-CAR and CD22-CAR into The mixing of row equal proportion, obtains the CAR T cell.It is a feature of the present invention that the CART cell of the equal proportion mixing uses Following methods preparation: the CART cell first prepared respectively for CD19 and CD22 target spot carries out equal proportion mixing again.

Second aspect of the present invention provides CART cell, be the virus infection CD3 positive T cell comprising CAR is obtained it is described CAR T cell.

Third aspect present invention is the preparation of CD3 positive T cell, i.e., monocyte is separated from peripheral blood, then from monokaryon Enrichment obtains CD3 positive T cell in cell.

Fourth aspect present invention is the activation method of T cell, and CD3 positive T cell is carried out using CD3/CD28 antibody method Stimulation activation.

Fifth aspect present invention is the method for coating of retrovirus, and used retroviral vector uses Retronectin carries out coating processing.

Sixth aspect present invention is the culture medium of CART cell, and prepared CART cell is trained with the X-vivo 15 of LONZA Base+5%AB serum is supported to be cultivated.CART cell is cultivated with culture medium containing IL-2.

Seventh aspect present invention is the cultivated days of CART cell, and the time that CART cell is cultivated in vitro is 7-14 days.

Eighth aspect present invention is CART cell application range, and the T cell of gene modification treats CD19 and CD22 in preparation Purposes in the drug of the disease of mediation；Preferably, the disease that the CD19 and CD22 is mediated includes leukaemia and lymthoma；More Preferably, the disease that the CD19 and CD22 is mediated includes B cell lymphoma, lymphoma mantle cell, the white blood of acute lymphoblastic Disease, chronic lymphocytic leukemia, hairy cell leukemia and acute myeloid leukaemia.

Detailed description of the invention

Fig. 1 is RV-CD19-BBz CAR retrovirus expression vector (CD19-41BB) schematic diagram.SP: signal peptide；VL: Light chain variable region；Lk: connector (G₄S)₃；VH: heavy chain variable region；H:CD8 α hinge area；TM:CD8 transmembrane region.

Fig. 2 is RV-CD22-BBz CAR retrovirus expression vector (CD22-41BB) schematic diagram.SP: signal peptide；VL: Light chain variable region；Lk: connector (G₄S)₃；VH: heavy chain variable region；H:CD8 α hinge area；TM:CD8 transmembrane region.

Fig. 3 is 72 hours CAR expression efficiencies of FCM results show retroviral infection T cell.

Fig. 4 is FCM results show each target cell surface CD19 and CD22 expression.

Fig. 5 is that the CART cell for preparing 5 days and target cell co-culture 4 hours CD107a detection of expression.

Fig. 6 is that the CART cell for preparing 5 days and target cell co-culture IFN γ secretion in 4 hours and detect.

Fig. 7 is to prepare 5 days CART cells and detect after target cell co-cultivation 5 hours to the lethal effect of tumour cell.

Specific embodiment

Form each part mentioned above of fusion protein of the invention, the i.e. leader peptide of CD8 antigen, anti-CD19 single-chain antibody or anti- CD22 single-chain antibody, people CD8 α hinge area, people CD8 transmembrane region, people 4-1BB intracellular region people CD3 ζ intracellular region, between each other can be straight It connects, or can be connected by joint sequence in succession.Joint sequence can be the joint sequence suitable for antibody well known in the art, Such as the joint sequence containing G and S.In general, connector contains duplicate motif before and after one or more.For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG.Preferably, which is adjacent in joint sequence, is not had between repetition There is insertion amino acid residue.Joint sequence may include 1,2,3,4 or 5 repetition motif composition.The length of connector can be 3~ 25 amino acid residues, such as 3~15,5~15,10~20 amino acid residues.In certain embodiments, joint sequence is More glycine linlcers sequences.The quantity of glycine is not particularly limited in joint sequence, and usually 2~20, such as 2~15,2~ 10,2~8.Except glycine and serine come, other known amino acid residue, such as alanine are also contained in connector (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q) etc..As example Son, connector can be made of following amino acid sequence: G (SGGGG)₂SGGGLGSTEF、RSTSGLGGGS(GGGGS)₂G、 QLTSGLGGGS(GGGGS)₂G, GGGS, GGGGS, SSSSG, GSGSA, GGSGG, GGGGSGGGGSGGGGS etc..

In certain embodiments, between the light chain variable region and heavy chain variable region of the present invention anti-CD19 and CD22 by (GGGS)_nConnection, the integer that wherein n is 1~5.

It should be understood that in gene cloning operation, it is often necessary to design suitable restriction enzyme site, this certainly will be in expressed ammonia Base acid sequence end introduces one or more incoherent residues, and this has no effect on the activity of aim sequence.In order to construct Fusion protein, the expression for promoting recombinant protein obtain the recombinant protein being secreted into outside host cell automatically or are conducive to recombinant protein Purifying, it is often necessary to it is other suitable in the end N-, the end C- or the albumen of recombinant protein to be added to some amino acid In region, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extend etc..Therefore, of the invention The aminoterminal or c-terminus of fusion protein (the i.e. described CAR) can also be containing one or more polypeptide fragments, as protein tag.Appoint What suitable label may be used to herein.For example, the label can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7,4A6, ε, B, gE and Ty1.These labels can be used for carrying out albumen pure Change.

The present invention also includes by SEQ ID NO:1 (CD19-41BB CAR) and/or SEQ ID NO:2 (CD22-41BB CAR) it is sequentially connected in series the mutant for the CAR to be formed.These mutant include: with the CAR at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, the preferably at least 97% sequence identity and biology for retaining the CAR is living The amino acid sequence of property (such as activating T cell).The sequence between the sequence of BLASTp two comparisons of calculating of such as NCBI can be used The column phase same sex.

Mutant further include: there is one or several mutation (insertion, missing in the sequence shown in SEQ ID NO:1 and 2 Or replace), simultaneously still retain the CAR biological activity amino acid sequence.Several mutation be often referred to 1-10 with It is interior, such as 1-8,1-5 or 1-3.Replace and is preferably conservative replaces.For example, in the art, using similar performance Or similar amino acid does not usually change the function of protein or polypeptide when carrying out conservative replaces." similar performance is similar Amino acid " include for example, with similar side chain amino acid residue family, these families include the ammonia with basic side chain Base acid (such as lysine, arginine, histidine), has the amino acid (such as aspartic acid, glutamic acid) with acid side-chain Uncharged polar side chain amino acid (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, Cysteine), amino acid with non-polar sidechain (such as alanine, valine, leucine, isoleucine proline, phenylpropyl alcohol Propylhomoserin, methionine, tryptophan), with β-branched building block amino acid (such as threonine, valine, isoleucine) and tool There is the amino acid (such as tyrosine, phenylalanine, tryptophan, histidine) of aromatic side chain.Therefore, it is used in polypeptide of the present invention One or several sites are replaced from another amino acid residue of same side chain class, will not be in substantially influences its activity.

The present invention includes the polynucleotide sequence for encoding fusion protein of the present invention.Polynucleotide sequence of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can be it is single-stranded or Double-strand.DNA can be coding strand or noncoding strand.The present invention also includes the degeneracy of the polynucleotide sequence of encoding fusion protein Variant encodes identical amino acid sequence but the different nucleotide sequence of nucleotide sequence.

Polynucleotide sequence as described herein can usually be obtained with PCR amplification method.Specifically, can be public according to institute herein The nucleotide sequence opened, especially open reading frame sequence carry out design primer, and with the commercially available library cDNA or press art technology The library cDNA prepared by conventional method known to personnel expands as template and obtains related sequence.When sequence is longer, usually need Twice or repeatedly PCR amplification is carried out, then the segment that each time amplifies is stitched together by proper order again.For example, In certain embodiments, the polynucleotide sequence such as SEQ ID NO:1 (CD19-41BB CAR) of fusion protein described herein is encoded With shown in SEQ ID NO:2 (CD22-41BB CAR).

The present invention also relates to nucleic acid constructs, which contains the coded sequence of fusion protein as described herein, And the one or more regulating and controlling sequences being connect with these series of operations.The coded sequence of fusion protein of the present invention can It is operable to guarantee the expression of the albumen in many ways.It can be according to expression vector before nucleic acid constructs is inserted into carrier Difference or requirement and nucleic acid constructs is operated.The technology for changing polynucleotide sequence using recombinant DNA method is It is known in the art.

Regulating and controlling sequence can be suitable promoter sequence.Promoter sequence is usually grasped with the coded sequence of albumen to be expressed The property made connection.Promoter can be any nucleotide sequence that transcriptional activity is shown in selected host cell, including prominent Become, truncated and hybrid promoter, and can be from coding and the homologous or heterologous extracellular or intracellular polypeptide of the host cell Gene obtain.

Regulating and controlling sequence is also possible to suitable transcription terminator sequences, is identified by host cell to terminate the sequence of transcription. Terminator sequence is connect with 3 ' end effectors of the nucleotide sequence for encoding the polypeptide.It is functional in the host cell of selection Any terminator can be used in the present invention.

Regulating and controlling sequence is also possible to suitable leader sequence, the non-translational region of the mRNA important to host cell translation.Before It leads sequence and 5 ' ends of the nucleotide sequence for encoding the polypeptide is operatively connected.Functional in the host cell of selection What terminator can be used in the present invention.

In certain embodiments, the nucleic acid constructs is carrier.Usually the more of CAR are encoded by being operably connected Nucleotide sequence is incorporated to expression vector to promoter, and by construct, realizes the expression of the polynucleotide sequence of coding CAR.It should Carrier can be suitable for replicating and integrating eukaryocyte.Typical cloning vector includes that can be used for adjusting desired nucleic acid sequence Transcription and translation terminator, homing sequence and the promoter of expression.

The polynucleotide sequence for encoding CAR of the present invention can be cloned into the carrier of many types.For example, matter can be cloned into Grain, phasmid, phage-derived object, animal virus and clay.Further, carrier is expression vector.Expression vector can be with Viral vectors form is supplied to cell.Viral vector technology be well known in the present art and such as Sambrook etc. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) It is described in other virology and molecular biology manual.The virus that can be used as carrier includes but is not limited to reverse transcription disease Poison, adenovirus, adeno-associated virus, herpesviral and slow virus.

In general, suitable carrier includes the replication orgin to work at least one organism, promoter sequence, conveniently Restriction enzyme sites and one or more selectable label (for example, WO 01/96584；WO01/29058；And U.S. Patent number 6,326,193)。

For example, in certain embodiments, the present invention uses retroviral vector, which contains multiple Initiation site processed, 3 ' LTR, 5 ' LTR, polynucleotide sequence as described herein, and optional selectable label.

One example of suitable promoter is instant early stage cytomegalovirus (CMV) promoter sequence.The promoter sequence Column are can to drive the strong constitutive promoter sequence for being operably coupled to any polynucleotide sequence high level expression thereon Column.Another example of suitable promoter is -1 α of the elongation growth factor (EF-1 α).It is also possible, however, to use other composing types open Promoter sequences, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer viral (MMTV), people are immune scarce It is instant to fall into long end repetition (LTR) promoter of virus (HIV), MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus Early promoter, Rous sarcoma virus promoter and people's gene promoter, such as, but not limited to actin promoter, Myosin promoter, ferroheme promoter and creatine kinase promoter.Further, also it is contemplated that being started using induction type Son.The use of inducible promoter provides molecular switch, and the induction type that is operably connected can be opened when expressing in the time limit The expression of the polynucleotide sequence of promoter, and expression is being closed when expression is undesirable.The example of inducible promoter Including but not limited to metallothionein promoter, Glucocorticoid promoter, progesterone promoter and tetracycline promoter.

In order to assess the expression of CAR polypeptide or part thereof, the expression vector for being introduced into cell also may include selectable mark Any of gene or reporter or both are remembered, in order to from the cell mass for seeking to be transfected or infect by viral vectors Middle identification and selection expression cell.In other respects, selectable label can be carried on independent section of DNA and for corotation Contaminate program.The flank of selectable label and both reporters can all have adjusting sequence appropriate, so as in host It is expressed in cell.Useful selectable marker includes such as antibiotics resistance gene, such as neo etc..

Reporter is used to identify the cell of potential transfection and for evaluating the functionality for adjusting sequence.DNA by After introducing recipient cell, the expression of reporter is measured under the suitable time.Suitable reporter may include coding Luciferase, beta galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or Green Fluorescent Protein gene base Cause.Suitable expression system is well known and prepares using known technology or commercially obtain.

Gene is introduced into cell and is well known in the art the method that gene expression enters cell.Carrier can by Any method in this field is easily introduced into host cell, for example, mammal, bacterium, yeast or insect cell.For example, Expression vector can be transferred to host cell by physics, chemistry or biological means.

It include calcium phosphate precipitation by the physical method that polynucleotides introduce host cell, lipofection, particle bombardment, micro- Injection, electroporation etc..It include being carried using DNA and RNA by the biological method that interested polynucleotides introduce host cell Body.It include dispersion system of colloid by the chemical means that polynucleotides introduce host cell, such as macromolecular complex, nanometre glue Capsule, microballoon, pearl；With the system based on lipid, including oil in water emulsion, micella, mixed micelle and liposome.

It include using viral vectors, especially retrovirus vector by the biological method that polynucleotides introduce host cell Body, this has become the most widely used method by gene insertion mammal such as people's cell.Other viral vectors can source From slow virus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus etc..Many has been developed based on virus System, for gene transfer to be entered mammalian cell.For example, retrovirus provides the convenience for gene delivery system Platform.By the gene insertion vector of selection and retroviral particle is packaged into using technology as known in the art.It should Recombinant virus then can be separated and be transferred to internal or external subject cell.Many retroviral systems are in the art It is known.In some embodiments, using adenovirus vector.Many adenovirus vectors are well known in the art.? In one embodiment, slow virus carrier is used.

Therefore, in certain embodiments, the present invention also provides the retrovirus for activating T cell, which contains There are retroviral vector as described herein and corresponding packaging gene, such as gag, pol and vsvg.

Being suitable for the invention T cell can be various types of T cells in various sources.For example, T cell can derive from The PBMC of B cell malignant tumor patient.

It in certain embodiments, can be first with suitable (such as 30~80ng/ml, such as 50ng/ml) after obtaining T cell CD3 antibody stimulate activation, then containing suitable (such as 30~80IU/ml, such as 50IU/ml) IL2 culture medium carry out It cultivates spare.

CART cell of the invention can undergo firm internal T cell to extend and continue in blood and marrow with high level Extended time quantum, and form specific memory T cell.It is not intended to be fettered by any specific theory, encounter simultaneously then Eliminate expression Surrogate antigen target cell after, CART cell of the invention can differentiation in vivo at center remember sample state.

The invention also includes a kind of cell therapies, and wherein T cell is expressed CAR as described herein by gene modification, and CART cell is needed by injection in its recipient.The cell of injection can kill the tumour cell of recipient.Unlike antibody is treated Method, CART cell can replicate in vivo, generate the long-term persistence that can lead to continued tumor control.

The anti-tumor immune response as caused by CART cell can be active or passive immunity response.In addition, CAR mediation is exempted from Epidemic disease response can be a part of adoptive immunotherapy step, and wherein the induction of CART cell is special to the antigen-binding portion thereof in CAR The immune response of property.

Medicable cancer can be non-physical knurl, such as neoplastic hematologic disorder, such as leukaemia and lymthoma.Especially, it can adopt Disease with CAR of the invention, its coded sequence, nucleic acid constructs, expression vector, virus and CART cell therapy is preferably The neoplastic hematologic disorder that the disease that CD19 and CD22 is mediated, especially CD19 and CD22 are mediated.

Specifically, herein, " disease that CD19 and CD22 are mediated " includes but is not limited to leukaemia and lymthoma, such as The white blood of B cell lymphoma, lymphoma mantle cell, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell Disease and acute myeloid leukaemia.

The T cell of CAR- modification of the invention can be administered alone or as pharmaceutical composition and diluent and/or and its Such as relevant cell factor of his component or cell mass combine application.Briefly, pharmaceutical composition of the invention may include as CART cell as described herein, in conjunction with one or more pharmacy or physiologically acceptable carriers, diluent or excipient.This The composition of sample may include buffer such as neutral buffered saline, sulfate buffered saline etc.；Carbohydrate such as grape Sugar, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid such as glycine；Antioxidant；Chelating agent is all Such as EDTA or glutathione；Adjuvant (for example, aluminium hydroxide)；And preservative.

The mode that pharmaceutical composition of the invention can be suitable for the disease of (or prevention) to be treated is applied.The quantity of application It will be determined by such factor with frequency, such as the illness of patient and the type of patient disease and severity.

When pointing out " effective quantity in immunology ", " antitumor effective quantity ", " tumour-inhibition effective quantity " or " therapeutic dose ", The precise volume of the present composition to be administered can be determined that the age of consideration patient (object), weight, tumour are big by doctor The individual difference of small, infection or metastasis degree and illness.It can usually point out: the pharmaceutical composition including T cell described herein It can be with 10⁴To 10⁹A cell/kg weight dosage, preferably 10⁵To 10⁶A cell/kg weight dosage.T cell composition It can also be with these dosage multiple applications.Cell can be by using injection technique well known in immunotherapy (see for example Rosenberg etc., New Eng.J.of Med.319:1676,1988) application.Optimal dose and treatment for specific patient Scheme can be by monitoring the disease indication of patient and therefore adjustment for the treatment of is readily determined by medical domain technical staff.

The application of object composition object can carry out in any convenient manner, including pass through spray-on process, inject, swallow, is defeated Liquid, implantation or transplanting.Compositions described herein can by subcutaneous, intradermal, tumor, in knot, in spinal cord, intramuscular, pass through vein Patient is administered in interior injection or peritonaeum.In one embodiment, T cell composition of the invention passes through intradermal or subcutaneous note It penetrates and is administered to patient.In another embodiment, T cell composition of the invention preferably passes through intravenous injection application.T is thin The composition of born of the same parents can be injected directly into tumour, lymph node or infection position.

In some embodiments of the present invention, CART cell of the invention or combinations thereof object can with it is known in the art its Its therapy combines.The therapy includes but is not limited to chemotherapy, radiotherapy and immunosuppressor.For example, in combination with various radiotherapy agents Treated, these radiotherapy agents include: cyclosporin, imuran, methopterin, mycophenolate, FK506, fludarabine, Rapamycin and Mycophenolic Acid etc..In a further embodiment, cell composition of the invention and bone-marrow transplantation, utilize change Treat the T cell of agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide or antibody such as OKT3 or CAMPATH Burning-eroding therapy is administered to patient in conjunction with (prior to, concurrently with, or after for example).

Herein, " anti-tumor effect " refers to a kind of biological effect, can be by the reduction of gross tumor volume, tumor cell number Reduction, the increase of life expectancy or the improvement expression of various physiological signs relevant to cancer for reducing, shifting number.

" patient ", " object ", " individual " etc. are used interchangeably herein, and referring to can cause the work of immune response organic Body, such as mammal.Example includes but is not limited to people, dog, cat, mouse, rat and its genetically modified organism.

Embodiment

The present invention is described in further detail by reference to following EXPERIMENTAL EXAMPLE.These embodiments are merely for explanation Property purpose provide, be not intended to it is restrictive, unless otherwise prescribed.Therefore, the present invention should not be interpreted in any way as being limited to Lower embodiment, but should be interpreted as including since introduction provided herein will become apparent from any and whole variation.

The determination of embodiment 1:antiCD19scFv-CD8 α -41BB-CD3 ζ gene order

Anti-CD 19 antibodies light chain and heavy chain variable region (monoclonal FMC63), people are searched from NCBI site databases CD8 α hinge area, the CD8 transmembrane region of people and the CD3 ζ intracellular region gene sequence information with people 4-1BB intracellular region, people, these sequences Codon optimization is carried out on the http://sg.idtdna.com/CodonOpt of website, is guaranteed constant in encoding amino acid sequence In the case where be more suitable for human cell expression.

Above-mentioned sequence is successively pressed by anti-CD19scFv, people's CD8 α hinge area gene, people's CD8 transmembrane region base using over-lap PCR Cause, people's 41BB intracellular region gene, people's CD3 ζ intracellular region gene order are attached, and introduce different digestion positions in each sequence junction Point forms complete CD19-41BB CAR gene order.

With the nucleotide sequence of NotI (NEB) and EcoRI (NEB) double digestion the CAR molecule, connect through T4 ligase (NEB) The site NotI-EcoRI into retroviral vector MSCV (Addgene) is patched, competent E.coli (DH5 α) is transformed into.

Recombinant plasmid is served Hai Shenggong Bioisystech Co., Ltd be sequenced, by sequencing result be fitted to Whether mCD19-CAR sequence alignment is correct to verify sequence.Sequencing primer are as follows:

Sense sequences: AGCATCGTTCTGTGTTGTCTC (SEQ ID NO:3)

Antisense sequences: TGTTTGTCTTGTGGCAATACAC (SEQ ID NO:4)

After being sequenced correctly, the plasmid purification kit of Qiagen company is used to extract simultaneously plasmid purification, plasmid purification phosphorus Sour calcium method transfection 293T cell carries out retrovirus Packaging experimentation.

The plasmid map obtained constructed by the present embodiment is as shown in Figure 1.

The determination of embodiment 2:antiCD22scFv-CD8 α -41BB-CD3 ζ gene order

Anti-CD22 antibody light chain and heavy chain variable region (monoclonal m971), people are searched from NCBI site databases CD8 α hinge area, the CD8 transmembrane region of people and people 4-1BB intracellular region, people CD3 ζ intracellular region gene sequence information, these sequences Codon optimization is carried out on the http://sg.idtdna.com/CodonOpt of website, is guaranteed constant in encoding amino acid sequence In the case where be more suitable for human cell expression.

Above-mentioned sequence is successively pressed by anti-CD22scFv, people's CD8 α hinge area gene, people's CD8 transmembrane region base using over-lap PCR Cause, people's 4-1BB intracellular region gene, people's CD3 ζ intracellular region gene order are attached, and introduce different digestions in each sequence junction Site forms complete CD22-4-1BB CAR gene order.

Sense sequences: AGCATCGTTCTGTGTTGTCTC (SEQ ID NO:3)

Antisense sequences: TGTTTGTCTTGTGGCAATACAC (SEQ ID NO:4)

The plasmid map obtained constructed by the present embodiment is as shown in Figure 2.

Embodiment 3: retrovirus packaging

1. 293T cell should be less than for 20 generations in the 1st day, do not cover with excessively.With 0.6*10^6cells/ml bed board, 10cm Ware adds the DMEM culture medium of 10ml, mixes well cell, 37 degree of overnight incubations.

2. the 2nd day 293T cell fusion degree, which reaches 90% or so, is transfected (usually bed board 14-18h or so)；Prepare Plasmid composite, the amount of various plasmids are that Retro backbone is 12.5ug (MSCV), and Gag-pol 10ug, VSVg are 6.25ug CaCl₂250ul, H₂O is that 1ml total volume is 1.25ml；It is added in another pipe isometric with plasmid composite HBS, be vortexed concussion 20s when adding plasmid composite.Softly mixture is added in 293T ware along side, 37 degree of cultures 4h removes culture medium, and PBS is washed one time, rejoins the fresh culture of preheating.

3. the 4th day: collecting supernatant after transfection 48h and be stored in -80 degree with packing after the filtering of 0.45um filter, continue to add The fresh DMEM medium of preheating.

Embodiment 4: the T cell of retroviral infection people

1. purer CD3+T cell is obtained with Ficcol separating liquid (ocean Tianjin Hao) separation, with serum containing 5%AB (GEMINI) X-VIVO (LONZA) culture medium adjustment cell density is 1 × 10⁶/mL.Cell is inoculated into preparatory use with the hole 1ml/ Anti-human 50ng/ml CD3 antibody (Beijing is with vertical Hai Yuan) and 50ng/ml CD28 antibody (Beijing is with vertical Hai Yuan), add The interleukin 2 (the double aigrets in Beijing) of 100IU/ml, restrovirus infection in 48 hours is cultivated in stimulation.

After 2.T cell activation culture every other day, PBS is diluted to Retronectin (Takara) packet of final concentration of 15 μ g/ml By Non-tissue treated culture plate, the every 250 μ l of hole of 24 orifice plates.It is protected from light, 4 DEG C spare overnight.

The culture of 3.T cell activation two days later, takes out 2 pieces of 24 orifice plates being coated with, and inhales and abandons coating buffer, is added containing 2%BSA's HBSS room temperature closes 30min.Confining liquid volume is every 500 μ l of hole, inhales and abandons confining liquid, with the HBSS board-washing two containing 2.5%HEPES It is secondary.

4. in virus liquid adding hole, every hole adds 2ml virus liquid, 32 DEG C, 2000g, it is centrifuged 2h.

5. discarding supernatant liquid, the T cell 1 × 10 after activation is added in the every hole of 24 orifice plates⁶A, volume 1ml, culture medium is that T is thin IL-2 200IU/ml is added in born of the same parents' culture medium.30 DEG C, 1000g, it is centrifuged 10min.

6. culture plate is placed in 37 DEG C, is cultivated in 5%CO2 incubator after centrifugation.

7. after infection for 24 hours, cell suspension is sucked out, 1200rpm, 4 DEG C, it is centrifuged 7min.

8. after cell infection, the density of cell is observed daily, adds the T cell culture solution of the 100IU/ml containing IL-2 in due course, The density of T cell is set to maintain 5 × 10⁵/ ml or so, expands cell.

Thus to obtain the CART cell for having infected retrovirus shown in embodiment 3 respectively, it is respectively designated as CD19-41BB CART cell (the CD19-41BB CAR of expression embodiment 1) and the CD22-41BB CART cell (CD22- of expression embodiment 1 41BB CAR)。

Embodiment 5: the expression of T lymphocyte surface C AR albumen after flow cytomery infection

72 hours after infecting CART cells and NT cell (control group) are collected by centrifugation respectively, PBS is abandoned after washing 1 time Clearly, it pats test tube and mixes cell, PBS after corresponding antibody is protected from light 30min is added and washs, is resuspended, adds suitable Viability dyestuff (invitrogen) is protected from light incubation 15 minutes.Last flow cytomery CAR (CD19-41BB CAR It is detected with anti-mouse IgG F (ab') antibody (Jackson Immunoresearch), CD22-41BB CAR is used Anti-human IgG F (ab') antibody (Jackson Immunoresearch) detection).

The present embodiment result shows by Fig. 3, the retroviral infection T cell being prepared using embodiment 3 72 hours Afterwards, the expression efficiency of CD19-41BB CAR+ is up to the expression efficiency of 53%, CD22-41BB CAR+ up to 45%.

Embodiment 6: each target cell CD19 and CD22 expression is detected

K562-CD19, K562-CD22, Raji, NALM6, K562 dye anti-mouse CD19 and anti-simultaneously Human CD22, machine testing in streaming.

The present embodiment result shows by Fig. 4, and K562-CD19 only expresses CD19, and K562-CD22 only expresses CD22, Raji and NALM6 expresses CD19 and CD22 simultaneously, and then CD19 and CD22 is not expressed K562.

CD107a expression and IFN γ secretion detect after embodiment 7:CART cell and target cell co-culture

1. taking the CART cell prepared, it is resuspended in Lonza culture medium, adjustment cell concentration is 1 × 10⁶/mL。

2. the every hole of experimental group contains target cell (K562-CD19, K562-CD22, Raji, NALM6) or negative control cell (K562)2×10⁵It is a, CART/NT cell 2 × 10⁵A, 100 μ l are free of the Lonza culture medium of IL-2.96 are added after mixing well In orifice plate.BD GolgiStop (containing monesin, 1 μ l BD GolgiStop is added in every 1ml culture medium) is added, every hole is added 1ul anti-human CD107a, after mixing well, 37 DEG C are incubated for 4 hours.Cell is collected, as experimental group.

3. the PBS per effective 1mL is cleaned cell 1 time, 300g is centrifuged 5 minutes, carefully sucks or outwell supernatant.

4. padding Anti-human CD3 and CAR, the PBS cleaning of 4 DEG C of incubation 30min, every effective 1mL cell 1 time, 300g is centrifuged 5 minutes, carefully sucks or outwell supernatant.

After 5.PBS washes cell, 250 μ l/EP pipe Fixation/Permeabilization solution are added, 4 DEG C incubate 20 minutes are educated to fix cell and rupture of membranes.With 1 × BD Perm/Wash^TBuffer is cleaned cell 2 times, 1mL/ times.

6. carrying out factor dyeing intracellular, appropriate IFN-γ cell factor fluorescence antibody or negative control are taken, with BD Perm/ Wash^TMBuffer is diluted to 50 μ l.The cell for having fixed rupture of membranes is sufficiently resuspended with this antibody diluent, 4 DEG C are protected from light incubation 30min, 1 × BD Perm/Wash^TMThe cleaning cell 2 times of buffer 1mL/ times, is then resuspended with PBS.

7. flow cytomery.

As the result is shown in fig. 5 and fig., NT and each target cell co-culture and all hardly express CD107a the present embodiment； CD107a is not expressed in CD19-41BB CART and K562 or K562-CD22 co-cultivation, is trained altogether with K562-CD19, Raji, NALM6 Supporting CD107a expression has 70% or so；CD107a is not expressed in CD22-41BB CART and K562 or K562-CD19 co-cultivation, with K562-CD22, Raji, which co-culture CD107a expression, 70% or so, co-cultures CD107a expression 50% or so with NALM6； CD107a is co-cultured with K562 after CD19-41BB+CD22-41BB CART 1:1 mixing not express；It is co-cultured with Raji, NALM6 CD107a is expressed 60% or so；CD107a expression is co-cultured 20% or more with K562-CD19, K562-CD22.Explanation Single expression CD19 or CD22 and CD19 and CD22 is co-expressed after CD19-41BB+CD22-41BB CART 1:1 mixing Target cell is all functional.

NT and each target cell, which co-culture, all hardly secretes IFN γ；CD19-41BB CART and K562 or K562-CD22 is total IFN γ is not secreted in culture, co-cultures secretion IFN γ all 20% or more with K562-CD19, Raji, NALM6；CD22-41BB IFN γ is not secreted in CART and K562 or K562-CD19 co-cultivation, co-cultures secretion IFN γ all 30% with K562-CD22, Raji More than, secretion IFN γ 20% or so is co-cultured with NALM6；

IFN γ is not secreted with K562 co-cultivation after CD19-41BB+CD22-41BB CART 1:1 mixing；It is trained altogether with Raji Nutrient secretes IFN γ 30% or so；Secretion IFN γ is co-cultured 20% or so with K562-CD19, K562-CD22, NALM6.It says Single expression CD19 or CD22 and CD19 and CD22 is co-expressed after bright CD19-41BB+CD22-41BB CART 1:1 mixing Target cell it is all functional.

Embodiment 8:CART cell and target cell detect tumor specific cell lethal effect after co-culturing

1.K562 cell (negative control cell) is resuspended in serum free medium (1640), adjustment cell concentration be 1 × 10⁶/ ml, addition fluorescent dye BMQC (2,3,6,7-tetrahydro-9-bromomethyl-1H, 5Hquinolizino (9, 1-gh) coumarin) to final concentration of 5 μM.

2. mixing, 37 DEG C of incubation 30min.

3. room temperature, 1500rpm is centrifuged 5min, abandons supernatant, and cell is resuspended in cytotoxicity culture medium (without phenol red 1640+ 5%AB serum) in, 37 DEG C of incubation 60min.

4. fresh cells toxicity culture medium cleans twice of cell, and is resuspended in fresh cells toxicity culture medium, and density 1 × 10⁶/ml。

5.K562-CD19, K562-CD22, Raji or NALM6 cell (target cell) are suspended in the PBS containing 0.1%BSA In, adjustment concentration is 1 × 10⁶/ml。

6. fluorescein based dye CFSE (carboxyfluoresceindiacetatesuccinimidyl ester) is added to end Concentration is 1 μM.

7. mixing, 37 DEG C of incubation 10min.

8. after being incubated for, being added and being reacted with the isometric FBS of cell suspension, incubation at room temperature 2min with end mark.

9. cleaning cell is simultaneously resuspended in fresh cells toxicity culture medium, density 1 × 10⁶/ml。

10. cleaning effector T cell is simultaneously suspended in cytotoxicity culture medium, adjustment concentration is 5 × 10⁶/ml。

11. in all experiments, the cytotoxicity of the effector T cell (CART cell) of CAR is infected and has been uninfected by The cytotoxicity of negative control effector T cell (NT cell) compares, and these effector T cells come from the same patient.

12.CART and negative control effector T cell, according to T cell: target cell=10:1 3:1 1:1 ratio, in 5ml Sterility test pipe (BD Biosciences) is cultivated.In each co-cultivation group, target cell is Jeko1 cell 100,000 A (50 μ l), 100,000 K562 cell (50 μ l) of negative control cell.One group of setting only includes Jeko1 target cell simultaneously With K562 negative control cell.

13. co-cultured cell is placed in 37 DEG C of incubation 16h.

14. after the completion of being incubated for, PBS cleans cell, the concentration recommended to specifications immediately after rapidly joins 7-AAD (7-aminoactinomycin D), is incubated for 30min on ice.

15. being not required to clean, directly machine testing in progress streaming, data are analyzed with Flow Jo.

16. analysis uses the living cells gating of 7AAD feminine gender, measure target cell living after T cell and target cell co-culture with The ratio of negative control cell living.

Cytotoxic killer cell %=(1- (when containing effector cell the target cell viable count/K562 of when containing effector cell it is living Cell number)/(K562 viable count when target cell viable count when no effector cell/without effector cell)) * 100%.

The present embodiment is as the result is shown in Fig. 7.CD19-41BB CART has obviously K562-CD19, Raji, NALM6 Lethal effect does not kill K562-CD22；CD22-41BB CART has obvious killing to K562-CD22, Raji, NALM6 Effect, does not kill K562-CD19；CD19+CD22CART has obvious lethal effect to 4 kinds of target cells.Illustrate CD19- The target cell that single expression CD19 or CD22 and CD19 and CD22 is co-expressed after 41BB+CD22-41BB CART 1:1 mixing It is all functional.

Sequence table

<110>Shanghai Heng Run Da Sheng Biotechnology Co., Ltd

<120>a kind of equal proportion mixes the preparation method and application of two kinds of target spot CART cells

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1473

<212> DNA

<213>artificial sequence

<400> 1

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgacattc agatgactca gaccacaagc agcctcagtg cgagcctggg ggacagggtg 120

actatcagct gccgggccag ccaggacatt tccaagtacc tgaattggta ccagcagaag 180

cccgatggta ctgtgaaact cctgatatat catacttcta ggctccattc cggggttcca 240

agccgattca gtggctccgg ttccggtaca gattattccc tgaccattag caacttggaa 300

caggaggaca ttgcaacgta tttctgtcag caaggcaaca cattgcccta cacattcggg 360

ggcgggacta aactcgaaat aactggcggc gggggttctg gtggcggcgg cagcggcggt 420

ggaggatcag aagtgaagct gcaggaaagt ggccccgggc tggtagcccc aagtcagtcc 480

ctgagtgtaa cctgtacagt gagtggagtg tctcttcctg actacggggt aagttggatt 540

cggcaacctc cacgcaaggg cctggagtgg ctcggcgtga tttggggatc tgagacaact 600

tactacaatt ccgccctgaa gagcaggctg accatcatta aggacaatag caagtcacag 660

gtgtttctga agatgaactc actgcagacc gacgacaccg ccatctatta ctgcgccaaa 720

cattattatt atggcgggag ttatgctatg gactactggg gccagggcac tagcgtcacc 780

gtcagcagta ctacaactcc agcacccaga ccccctacac ctgctccaac tatcgcaagt 840

cagcccctgt cactgcgccc tgaagcctgt cgccctgctg ccgggggagc tgtgcatact 900

cggggactgg actttgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaggt tcagtgtcgt gaagagaggc 1020

cggaagaagc tgctgtacat cttcaagcag cctttcatga ggcccgtgca gactacccag 1080

gaggaagatg gatgcagctg tagattccct gaagaggagg aaggaggctg tgagctgaga 1140

gtgaagttct cccgaagcgc agatgcccca gcctatcagc agggacagaa tcagctgtac 1200

aacgagctga acctgggaag acgggaggaa tacgatgtgc tggacaaaag gcggggcaga 1260

gatcctgaga tgggcggcaa accaagacgg aagaaccccc aggaaggtct gtataatgag 1320

ctgcagaaag acaagatggc tgaggcctac tcagaaatcg ggatgaaggg cgaaagaagg 1380

agaggaaaag gccacgacgg actgtaccag gggctgagta cagcaacaaa agacacctat 1440

gacgctctgc acatgcaggc tctgccacca aga 1473

<210> 2

<211> 1485

<212> DNA

<213>artificial sequence

<400> 2

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctcaggttc agttgcagca gagcggcccc gggctggtga aaccttccca gacccttagc 120

ctgacgtgtg caatctccgg cgacagcgtc tcctctaata gcgccgcgtg gaactggata 180

agacagtctc cttcaagagg gctggagtgg cttggcagga cctattacag gagcaagtgg 240

tataacgact atgccgttag cgtgaaatcc cgcattacca ttaatcctga cacctccaaa 300

aaccagttca gtctgcagct gaactcagtc accccagagg acactgctgt ttactattgc 360

gccagagagg tgaccggcga tctggaagac gccttcgaca tatgggggca ggggaccatg 420

gtaaccgtga gcagtggcgg cgggggttct ggtggcggcg gcagcggcgg tggaggatca 480

gatattcaaa tgacacagag cccttcctcc ctgagtgcct ccgttgggga ccgcgttaca 540

ataacatgca gagcttccca aacaatttgg tcatatctta actggtacca gcagagacct 600

ggaaaggctc ctaacttgct tatctatgca gcatcctccc tccagtctgg tgttccatct 660

cggttcagtg ggcgaggcag cgggacagat ttcacactga ccatatccag cctccaggct 720

gaggactttg cgacctacta ctgccagcag tcatattcta taccacagac attcggtcag 780

ggaaccaaac tcgaaatcaa gactacaact ccagcaccca gaccccctac acctgctcca 840

actatcgcaa gtcagcccct gtcactgcgc cctgaagcct gtcgccctgc tgccggggga 900

gctgtgcata ctcggggact ggactttgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcag gttcagtgtc 1020

gtgaagagag gccggaagaa gctgctgtac atcttcaagc agcctttcat gaggcccgtg 1080

cagactaccc aggaggaaga tggatgcagc tgtagattcc ctgaagagga ggaaggaggc 1140

tgtgagctga gagtgaagtt ctcccgaagc gcagatgccc cagcctatca gcagggacag 1200

aatcagctgt acaacgagct gaacctggga agacgggagg aatacgatgt gctggacaaa 1260

aggcggggca gagatcctga gatgggcggc aaaccaagac ggaagaaccc ccaggaaggt 1320

ctgtataatg agctgcagaa agacaagatg gctgaggcct actcagaaat cgggatgaag 1380

ggcgaaagaa ggagaggaaa aggccacgac ggactgtacc aggggctgag tacagcaaca 1440

aaagacacct atgacgctct gcacatgcag gctctgccac caaga 1485

<210> 3

<211> 21

<212> DNA

<213>artificial sequence

<223>primer

<400> 3

agcatcgttc tgtgttgtct c 21

<210> 4

<211> 22

<212> DNA

<213>artificial sequence

<223>primer

<400> 4

tgtttgtctt gtggcaatac ac 22

Claims

1. a kind of method for preparing CART cell, which is characterized in that be that will carry out equal proportion comprising CD19-CAR and CD22-CAR Mixing, obtains the CART cell.

2. the method according to claim 1, wherein the CART cell of equal proportion mixing uses following methods Preparation: the CART cell first prepared respectively for CD19 and CD22 target spot carries out equal proportion mixing again.

3. a kind of mixed Chimeric antigen receptor is made of two kinds of independent Chimeric antigen receptors, targets different target spots respectively, Costimulating factor is all 41BB.

4. the Chimeric antigen receptor of claim 3, wherein the immunoreceptor tyrosine activating motif intracellular includes to be selected from CD3 ζ tyrosine activation motifs signal chains.

5. the Chimeric antigen receptor of claim 3 is co-expressed by a carrier, or by two identical or different carriers point It does not express.

6. a kind of method for preparing CART cell, which is characterized in that be to obtain the virus infection CD3 positive T cell comprising CAR The CART cell.

7. according to the method described in claim 2, it is characterized in that, the CD3 positive T cell is prepared in the following ways: from Monocyte is separated in peripheral blood, then enrichment obtains CD3 positive T cell from monocyte.

8. according to the method described in claim 3, it is characterized in that, from it is described from peripheral blood separate monocyte in be enriched with Stimulation activation is carried out using CD3/CD28 antibody method to CD3 positive T cell.

9. method according to claim 1, which is characterized in that used retroviral vector uses Retronectin Carry out coating processing.

10. method according to claim 1, which is characterized in that prepared CART cell is trained with the X-vivo 15 of LONZA Feeding base is cultivated.

11. method according to claim 2, which is characterized in that prepared CART cell is trained with culture medium containing IL-2 It supports.

12. method according to claim 2, which is characterized in that the time that prepared CART cell is cultivated in vitro is 7- 14 days.

13. the CART cell that claim 1-12 appoints method described in binomial to be prepared.

14. the composition containing the CART cell described in claim 13.

15. composition according to claim 14, which is characterized in that the composition also includes auxiliary material.

16. the T cell of gene modification described in claim 1-3 is in the drug of the preparation treatment CD19 and CD22 disease mediated In purposes；Preferably, the disease that the CD19 and CD22 is mediated includes leukaemia and lymthoma；It is highly preferred that the CD19 It include B cell lymphoma, lymphoma mantle cell, acute lymphoblastic leukemia, chronic lymphocytic with the CD22 disease mediated Leukaemia, hairy cell leukemia and acute myeloid leukaemia.