CN109503721A

CN109503721A - Target the Chimeric antigen receptor and application thereof of CD19

Info

Publication number: CN109503721A
Application number: CN201910012631.8A
Authority: CN
Inventors: 李启靖; 金涛; 何凤; 史子啸
Original assignee: Shanghai Hrain Biotechnology Co Ltd
Current assignee: Shanghai Hrain Biotechnology Co Ltd
Priority date: 2016-05-31
Filing date: 2016-05-31
Publication date: 2019-03-22
Anticipated expiration: 2036-05-31
Also published as: CN107446051B9; CN107446051B; CN109503721B; CN107446051A

Abstract

The present invention relates to the Chimeric antigen receptors and application thereof of targeting CD19.Specifically, the present invention provides a kind of fusion protein, and the fusion protein is selected from: (1) fusion protein of the leader peptide containing sequentially connected CD8 antigen, anti-CD19 single-chain antibody, people CD8 α hinge area, people CD28 transmembrane region, people CD28 intracellular region and people's CD3 ζ intracellular region；(2) by replacing, missing or adding one or several amino acid and retaining the active fusion protein as derived from (1) of activating T cell in the amino acid sequence that (1) limits.The present invention also provides the purposes of the coded sequence of the fusion protein, the carrier containing the coded sequence and the fusion protein, coded sequence, carrier.

Description

Target the Chimeric antigen receptor and application thereof of CD19

Present patent application be entitled the Chimeric antigen receptor and application thereof of CD19 " targeting ", application No. is 201610377871.4 application for a patent for invention divisional application.

Technical field

The invention belongs to Chimeric antigen receptor fields, and in particular to target Chimeric antigen receptor of CD19 and application thereof.

Background technique

Chimeric antigen receptor (Chimeric Antigen Receptor-T cell, CAR-T) 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.CAR-T cell adoptive therapy is that most clearly have in current cancer therapies The immunotherapeutic form of effect.A large number of studies show that CAR-T cell can effectively identify tumour antigen, cause the anti-of specificity Tumor immune response significantly improves the survival state of patient.

Chimeric antigen receptor (CAR) is the core component of CAR-T, 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.

With the continuous development of Chimeric antigen receptor T cell technology, CAR-T can mainly be divided into for four generations at present.

First generation CAR-T cell by extracellular combined area-single-chain antibody (single-chain fragment variable, ScFV), transmembrane region (transmembrane region, TM) and intracellular signal area --- immunoreceptor tyrosine activating motif (immunoreceptortyrosine-based activation motif, ITAM) is formed, wherein each portion of Chimeric antigen receptor Divide and connected by following form: scFv-TM-CD3 ζ.Although first generation CAR it can be seen that some specificity cytotoxicity, Have been found that curative effect is barely satisfactory when carrying out clinical test summary to it within 2006.Trace it to its cause is because of first generation CAR-T Cell will soon exhaust that persistence (persistence) is very poor in patient body, so that CAR-T cell comes not yet And just apoptosis when touching a large amount of tumour cell.This kind of CAR-T cell can excite antitumoral cytotoxic Effect, but cytokine secretion is fewer, but its survival period in vivo is shorter cannot excite lasting anti-tumor effect (Zhang T etc., Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma Growth in a manner involving multiple cytokines and cytotoxic pathways, Cancer Res 2007,67 (22): 11029-11036).

Second generation CAR-T cell optimizes T cell activation signaling zone in CAR design, is still the hot spot of research.T cell The effect for depending on dual signal and cell factor is activated completely.Wherein the first signal is specific signals, is passed by TCR identification antigen Started in the Antigenic Peptide-MHC compound of cell surface；Second signal is costimulatory signal.Early in 1998 there have been Second generation CAR (Finney HM etc., J Immunol.1998,161 (6): 2791-7).2nd generation CAR adds in intracellular signal peptide area A costimulatory molecules are added, i.e., costimulatory signal have been assembled into inside CAR, can preferably be provided for CAR-T cell Activation signals can activate costimulatory molecules and intracellular signal after such CAR tumor cell simultaneously, realize dual work Change, T cell proliferation secretion capacity and anti-tumor effect can be significantly improved.First T cell costimulatory signal studied in detail Receptor is CD28, it can be in conjunction with the B7 family member of target cell surface.The costimulation of CD28 can promote the increasing of T cell It grows, the synthesis and expression of IL-2 and the ability of enhancing T cell resistance apoptosis.Then occurs CD134 (OX40) and CD137 again Costimulatory molecules such as (4-1BB) maintain t cell response to improve cytotoxicity, the proliferation activity of T cell, extend T cell and deposit Live time etc..Such second generation CAR produces unexpected effect in subsequent clinical test, is based on from 2010 The clinical report of second generation CAR repeatedly causes vibration, especially for recurrent, intractable patient ALL, complete remission rate Up to 90% or more.

Third generation CAR signal peptide area integrates 2 or more costimulatory molecules, T cell continuous activation can be made to be proliferated, cell The ability of factor continuous release, T cell killing tumor cell is more significant, i.e., CAR of new generation can get stronger antitumor Response (Pule MA etc., Mol Ther.2005,12 (5): 933-941).Most typical is exactly UPen Carl June in CD28 The stimulating factor of a CD137 (4-1BB) is added under the action of stimulating factor again.

The CAR-T cell of forth generation then joined cell factor or costimulation ligand, such as four generation CAR can produce IL- 12, immune microenvironment can be adjusted, the activation of T cell is increased, while activating inherent immunity cell that it is made to play a role and coming clearly Except the cancer cell of target antigen feminine gender, to have the function that bidirectional modulation (Chmielewski M, Abken H.TRUCKs:the fourth generation of CARs.Expert Opin Biol Ther.2015；15 (8): 1145-54).

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.

Summary of the invention

First aspect present invention provides a kind of fusion protein, and the fusion protein is selected from:

(1) containing the leader peptide of sequentially connected CD8 antigen, anti-CD19 single-chain antibody, people CD8 α hinge area, people CD28 across Film area, people CD28 intracellular region and people's CD3 ζ intracellular region fusion protein；With

(2) it lives in the amino acid sequence that (1) limits by replacing, missing or adding one or several amino acid and retaining Change the active fusion protein as derived from (1) of T cell.

In one or more embodiments, the amino acid sequence of the CD8 antigen leader peptide such as SEQ ID NO:1 1- Shown in 21 amino acids.

In one or more embodiments, the anti-CD19 single-chain antibody is anti-CD19 monoclonal antibody FMC63.

In one or more embodiments, the anti-CD19 single-chain antibody contains light chain variable region and heavy chain variable region.

In one or more embodiments, the light chain variable region passes through joint sequence phase with the heavy chain variable region Even.

In one or more embodiments, the anti-CD19 single-chain antibody contains anti-CD19 monoclonal antibody FMC63's Light chain variable region and heavy chain variable region, the light chain variable region and heavy chain variable region are connected optionally by connector and are connected.

In one or more embodiments, the amino acid sequence such as SEQ ID NO:1 of the anti-CD19 single-chain antibody Shown in 22-263 amino acids.

In one or more embodiments, the amino acid sequence of the people CD8 α hinge area such as SEQ ID NO:3 1- Shown in 47 amino acids.

In one or more embodiments, the amino acid sequence of the people CD28 transmembrane region such as SEQ ID NO:3 48- Shown in 74 amino acids.

In one or more embodiments, the amino acid sequence of the people CD28 intracellular region such as SEQ ID NO:3 75- Shown in 115 amino acids.

In one or more embodiments, the amino acid sequence such as SEQ ID NO:3 of the people CD3 ζ intracellular region Shown in 116-226 amino acids.

In one or more embodiments, the polypeptide sequence is sequentially connected in series by SEQ ID NO:1 and SEQ ID NO:3 It is formed.

Second aspect of the present invention provides a kind of polynucleotide sequence, which is selected from:

(1) polynucleotide sequence of fusion protein described in this paper first aspect is encoded；With

(2) complementary series of (1) described polynucleotide sequence.

In one or more embodiments, the polynucleotide sequence contains nucleotides sequence shown in SEQ ID NO:2 Column.

In one or more embodiments, the polynucleotide sequence contains nucleotides sequence shown in SEQ ID NO:4 Column.

In one or more embodiments, the polynucleotide sequence contains nucleotide shown in SEQ ID NO:2 and 4 Sequence.

Third aspect present invention provides a kind of nucleic acid constructs, described to contain polynucleotides sequence described in this paper second aspect Column.

In one or more embodiments, the nucleic acid constructs is carrier.

In one or more embodiments, the nucleic acid constructs is expression vector.

In one or more embodiments, the expression vector is retroviral vector.

In one or more embodiments, the retroviral vector contains replication origin, 3 ' LTR, and 5 ' Polynucleotide sequence described in LTR, this paper second aspect and resistant gene.

Fourth aspect present invention provides a kind of retrovirus, and the retrovirus contains described in this paper third aspect Nucleic acid constructs.

In one or more embodiments, the retrovirus contains carrier, the preferably described expression vector.

In one or more embodiments, the retrovirus contains the retroviral vector.

Fifth aspect present invention provides a kind of T cell of gene modification, which contains the multicore glycosides of this paper second aspect Acid sequence.

In one or more embodiments, the T cell contains retroviral vector described in this paper third aspect.

In one or more embodiments, the T cell has infected retrovirus described in this paper fourth aspect.

Sixth aspect present invention provides a kind of method of ex vivo activation T cell, and the method includes using four directions herein The step of T cell described in retroviral infection described in face.

The T that seventh aspect present invention provides the gene modification being prepared using method described in sixth aspect present invention is thin Born of the same parents.

Eighth aspect present invention provides fusion protein, polynucleotide sequence, carrier or retrovirus described herein and is making Application in the T cell of standby activation.

Ninth aspect present invention provides fusion protein, polynucleotide sequence, carrier, retrovirus or base as described herein T cell because of modification is preparing the purposes in the drug for treating the disease that CD19 is mediated.

In one or more embodiments, the disease that the CD19 is mediated includes leukaemia and lymthoma.

In one or more embodiments, the disease that the CD19 is mediated includes B cell lymphoma, jacket cell lymph Tumor, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and acute myeloid leukaemia.

Tenth aspect present invention also provides a kind of pharmaceutical composition, and the composition contains the T of gene modification as described herein Cell.

Detailed description of the invention

Fig. 1 is MSCV-CAR retrovirus expression vector schematic diagram.

Fig. 2 is the part sequencing result peak value figure of MSCV-CAR retrovirus expression plasmid.

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

Fig. 4 is the secretion of 5 hours INF- γ of CAR-T cell and target cell co-cultivation of preparation 3 days.

Fig. 5 is to prepare 3 days CAR-T cells and after target cell co-cultivation 5 hours to the lethal effect of tumour cell.

Fig. 6 prepares the NOG mouse survivorship curve of CAR-T cell therapy inoculation RAJI tumour cell, and wherein solid line represents CAR- T, dotted line represent NT.

Specific embodiment

The present invention provides a kind of CAR for targeting CD19 antigen.The CAR contains the leader peptide, anti-of sequentially connected CD8 antigen CD19 single-chain antibody, people CD8 α hinge area, people CD28 transmembrane region, people CD28 intracellular region and people's CD3 ζ intracellular region fusion protein.

It is suitable for the invention the amino acid sequence such as SEQ ID NO:1 1-21 amino acids institute of CD8 antigen leader peptide Show.

Being suitable for the invention anti-CD19 single-chain antibody is the various anti-CD19 single-chain antibodies commonly used in the art in CAR.? In certain embodiments, affiliated anti-CD19 single-chain antibody is anti-CD19 monoclonal antibody FMC63.In general, being suitable for the invention Anti- CD19 single-chain antibody can contain light chain variable region and heavy chain variable region, or be made of light chain variable region and heavy chain variable region.Gently Chain variable region is connected with the heavy chain variable region by joint sequence.In certain embodiments, the anti-CD19 single-chain antibody The amino acid sequence of light chain variable region can be as shown in SEQ ID NO:1 22-128 amino acids.In other embodiments, The amino acid sequence of the heavy chain variable region of the anti-CD19 single-chain antibody can be such as SEQ ID NO:1 144-263 amino acids institute Show.

The amino acid sequence for being suitable for the invention people's CD8 α hinge area can be such as SEQ ID NO:3 1-47 amino acids institute Show.

Being suitable for the invention people's CD28 transmembrane region can be the various people CD28 transmembrane region sequences commonly used in the art in CAR Column.In certain embodiments, the amino acid sequence of the people CD28 transmembrane region such as SEQ ID NO:3 48-74 amino acids It is shown.

Being suitable for the invention people's CD28 intracellular region can be the various people CD28 intracellular region sequences commonly used in the art in CAR Column.In certain embodiments, the amino acid sequence of the people CD28 intracellular region such as SEQ ID NO:3 75-115 amino acids It is shown.

It is suitable for the invention the various people CD3 ζ intracellular regions that people's CD3 ζ intracellular region can be this field conventionally used for CAR. In certain embodiments, the amino acid sequence of the people CD3 ζ intracellular region such as SEQ ID NO:3 116-226 amino acids institute Show.

Form each part mentioned above of fusion protein of the invention, the i.e. leader peptide of CD8 antigen, anti-CD19 single-chain antibody, people CD8 α hinge area, people CD28 transmembrane region, people CD28 intracellular region and people's CD3 ζ intracellular region, can be directly connected to, Huo Zheke between each other It is connected by joint sequence.Joint sequence can be the joint sequence suitable for antibody well known in the art, such as containing G's and S Joint sequence.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, and amino acid is not inserted between repetition Residue.Joint sequence may include 1,2,3,4 or 5 repetition motif composition.It is residual that the length of connector can be 3~25 amino acid Base, such as 3~15,5~15,10~20 amino acid residues.In certain embodiments, joint sequence is more glycine linlcers Sequence.The quantity of glycine is not particularly limited in joint sequence, and usually 2~20, such as 2~15,2~10,2~8.It removes Glycine and serine come, and also contain other known amino acid residue in connector, for example, alanine (A), leucine (L), Threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q) etc..As an example, connector can be by following Amino acid sequence composition: G (SGGGG)₂SGGGLGSTEF(SEQ ID NO:7)、RSTSGLGGGS(GGGGS)₂G(SEQ ID NO: 8)、QLTSGLGGGS(GGGGS)₂G(SEQ ID NO:9)、GGGS(SEQ ID NO:10)、GGGGS(SEQ ID NO:11)、 SSSSG(SEQ ID NO:12)、GSGSA(SEQ ID NO:13)、GGSGG(SEQ ID NO:14)、GGGGSGGGGSGGGGS (SEQ ID NO:15), SSSSGSSSSGSSSSG (SEQ ID NO:16), GSGSAGSGSAGSGSA (SEQ ID NO:17) and GGSGGGGSGGGGSGG (SEQ ID NO:18) etc..

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

In certain embodiments, the amino acid sequence of CAR of the present invention by SEQ ID NO:1 and SEQ ID NO:3 successively Series connection is formed.

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 the mutant that the CAR formed is sequentially connected in series by SEQ ID NO:1 and SEQ ID NO:3.These Mutant includes: with the CAR at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably extremely Lack 97% sequence identity and retains the amino acid sequence of the biological activity (such as activating T cell) of the CAR.It can be used for example The BLASTp of NCBI calculates the sequence identity between the sequence of two comparisons.

Mutant further include: there is one or several mutation (insertion, missing in the sequence shown in SEQ ID NO:1 and 3 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, benzene Alanine, methionine, tryptophan), with β-branched building block amino acid (such as threonine, valine, isoleucine) and Amino acid (such as tyrosine, phenylalanine, tryptophan, histidine) with aromatic side chain.Therefore, it is used in polypeptide of the present invention Another amino acid residue from same side chain class replaces one or several sites, and 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 of fusion protein described herein is encoded as shown in SEQ ID NO:2 and 4.

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.

CAR-T cell of the invention can undergo firm internal T cell to extend and be held in blood and marrow with high level Continue extended time quantum, and forms specific memory T cell.Be not intended to be fettered by any specific theory, encounter and with Afterwards eliminate expression Surrogate antigen target cell after, CAR-T 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 CAR-T cell is needed by injection in its recipient.The cell of injection can kill the tumour cell of recipient.Unlike antibody is treated Method, CAR-T 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 CAR-T cell can be active or passive immunity response.In addition, what CAR was mediated Immune response can be a part of adoptive immunotherapy step, and wherein the induction of CAR-T cell is to the antigen-binding portion dtex in CAR Anisotropic immune response.

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 CAR-T cell therapy is preferred The neoplastic hematologic disorder mediated for the disease that CD19 is mediated, especially CD19.

Specifically, herein, " disease that CD19 is mediated " includes but is not limited to leukaemia and lymthoma, such as B cell Lymthoma, lymphoma mantle cell, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and urgency Property myelogenous leukemia.

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 CAR-T cell as described herein, in conjunction with one or more pharmacy or physiologically acceptable carriers, diluent or excipient. Such composition may include buffer such as neutral buffered saline, sulfate buffered saline etc.；Carbohydrate such as Portugal Grape sugar, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid such as glycine；Antioxidant；Chelating agent 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, CAR-T cell of the invention or combinations thereof object can with it is known in the art Other therapies combine.The therapy includes but is not limited to chemotherapy, radiotherapy and immunosuppressor.For example, in combination with various radiotherapy systems Agent is treated, these radiotherapy agents include: cyclosporin, imuran, methopterin, mycophenolate, FK506, fluorine up to draw Shore, rapamycin and Mycophenolic Acid etc..In a further embodiment, cell composition of the invention and bone-marrow transplantation, benefit With the T of chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide or antibody such as OKT3 or CAMPATH Cell 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:CD8 leader sequence-mCD19scFv-CD8 α-CD28-CD3 ζ gene order and reverse transcription disease The building of poisonous carrier

From NCBI site databases search the CD8 α hinge area of people, the CD28 transmembrane region of people, people CD28 intracellular region and The CD3 ζ intracellular region gene sequence information of people, anti-CD19 single-chain antibody clone number is FMC63, these sequences are in website http: // Codon optimization is carried out on sg.idtdna.com/site, guarantees to be more suitable for the mankind in the case where encoding amino acid sequence is constant Cell expression.

Above-mentioned sequence is successively pressed by anti-CD19scFv, people's CD8 α hinge area gene, people's CD28 transmembrane region base using over-lap PCR Cause, people's CD28 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 mCD19-CAR gene order, obtains CAR molecule.

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 retrovirus MSCV (Addgene) carrier is patched, competent E.coli (DH5 α) is transformed into.

Retroviral vector obtained is served Hai Shenggong Bioisystech Co., Ltd to be sequenced, by sequencing result It is whether correct to verify sequence with the mCD19-CAR sequence alignment that is fitted to.Sequencing primer are as follows:

Ariyoshi: AGCATCGTTCTGTGTTGTCTC (SEQ ID NO:5)

Antisense: TGTTTGTCTTGTGGCAATACAC (SEQ ID NO:6)

After being sequenced correctly, the plasmid purification kit of Qigene company is used to extract simultaneously purification of retrovirus carrier.

The plasmid map obtained constructed by the present embodiment is as shown in Figure 1.Fig. 2 shows the MSCV-CAR retrovirus expression The part sequencing result peak value figure of plasmid.

Embodiment 2: retrovirus packaging

The retroviral vector that the purifying of embodiment 1 obtains is subjected to reverse transcription disease using calcium phosphate method transfection 293T cell Malicious Packaging experimentation, the specific steps are as follows:

1st day: selection was less than 20 generations, the 293T cell not covered with excessively, with 0.6 × 10⁶A cell/ml bed board, 10cm Ware adds 10ml DMEM culture medium, mixes well cell, 37 degree of overnight incubations.

2nd day: 293T cell fusion degree reaches 90% or so and is transfected (usually bed board 14-18h or so)；Prepare matter Grain compound, the amount of various plasmids are as follows: MSCV skeleton carrier 12.5ug, the Gag-pol 10ug that embodiment 1 is prepared, VSVg6.25ug, CaCl₂250ul, H₂O 1ml, total volume 1.25ml；It is added in another pipe isometric with plasmid composite HBSS (Hank ' s balanced salt solution), be vortexed when adding plasmid composite concussion 20s.Softly mixture is added along side Enter into 293T ware, 37 DEG C of culture 4h, remove culture medium, PBS is washed one time, rejoins the fresh culture of preheating.

4th day: collecting supernatant after transfection 48h and be stored in -80 DEG C with packing after the filtering of 0.45um filter, it is pre- to continue addition The fresh DMEM medium of heat.

Embodiment 3: retroviral infection human T-cell

1, purer CD3+T cell is obtained with Ficcol separating liquid (ocean Tianjin Hao) separation, with the X-VIVO of serum containing 5%AB (LONZA) culture medium adjustment cell density is 1 × 10⁶/mL.Cell is inoculated into advance with the hole 1ml/ with anti-human 50ng/ml On CD3 antibody (Beijing is with vertical Hai Yuan) and 50ng/ml CD28 antibody (Beijing is with vertical Hai Yuan) coated tissue culture plate, then plus Enter the interleukin 2 (the double aigrets in Beijing) of 100IU/ml, stimulation culture carried out virus infection after 48 hours.

2, after 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 treatment culture plate, the every 250 μ l of hole of 24 orifice plates.It is protected from light, 4 DEG C spare overnight.

3, T cell activation culture 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, is centrifuged 2h.

5, liquid is discarded supernatant, 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, after being centrifuged, culture plate is placed in 37 DEG C, 5%CO₂It is cultivated in incubator.

7, after infecting for 24 hours, cell suspension is sucked out, 1200rpm, 4 DEG C, 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.

Embodiment 4: the ratio of T lymphocyte and the expression of surface C AR albumen after flow cytomery infection

72 hours after infecting CAR-T cells and NT cell (control group) are collected by centrifugation respectively, PBS is abandoned after washing 1 time Clearly, PBS after corresponding antibody is protected from light 30min is added to wash, is resuspended, last flow cytomery.CAR+ is by anti-mouse IgG F (ab') antibody (Jackson Immunoresearch) detects.

As a result as shown in Figure 3.It is shown in figure, after retroviral infection T cell 72 hours, CAR+ expression efficiency reaches 68.2%.This efficiency of infection significantly beyond many research institutions efficiency (J Immunother., in September, 2009,32 (7): 689-702, doi:10.1097/CJI.0b013e3181ac6138).

INF- γ secretion detects after embodiment 5:CAR-T cell and target cell (Raji) are co-cultured

1, the CAR-T cell that Example 3 prepares, is resuspended in Lonza culture medium, adjustment cell concentration be 1 × 10⁶/mL。

2, the culture plate of positive controls uses CD3 monoclonal antibody 500ng/mL that CD28 monoclonal antibody 500ng/ml is added to be coated in advance, training It supports in base and does not add IL-2.It is added after mixing well in 24 orifice plates, every hole 1mL cell suspension.BD GolgiPlug is added simultaneously (containing BFA, 1 μ l BD GolgiPlug is added in every 1ml cell culture medium), after mixing well, 37 DEG C incubation 5-6 hours.It collects Cell is compareed as CAR-T cell positive.

3, the every hole cell containing k562-CD19+ of experimental group or Raji cell 2 × 10⁵It is a, CD19-CAR-T cell 2 × 10⁵It is a, 200 μ l are free of the Lonza culture medium of IL-2.It is added after mixing well in 96 orifice plates.Be added simultaneously BD GolgiPlug (containing BFA, 1 μ l BD GolgiPlug is added in every 1ml cell culture medium), after mixing well, 37 DEG C incubation 5-6 hours.Cell is collected, is made For experimental group.

4, the PBS per effective 1mL is cleaned cell 1 time, and 300g is centrifuged 5 minutes.Carefully suck or outwell supernatant.

5, after PBS washes cell, 250 μ l/EP pipes is added and fix/penetrating fluid, 4 DEG C are incubated for 20 minutes to fix cell and break Film.With 1 × BD Perm/Wash^TMBuffer solution for cleaning cell 2 times, 1mL/ times.

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

7, flow cytomery.

Fig. 4 show preparation 3 days CAR-T cell and target cell co-culture 5 hours after INF- γ secretion situation.CAR- CAR-T cell is largely activated (54.5%) after T cell and target cell secretion, and activation amount is more than sun ginseng (45.3%).

Embodiment 6:CAR-T cell and target cell (Raji) detect tumor specific cell lethal effect after co-culturing

1, K562 cell (being free of CD19 target protein, be the negative control cell of target cell) is resuspended in serum free medium (1640) in, adjustment cell concentration is 1 × 10⁶Fluorescent dye BMQC (2,3,6,7- tetrahydro -9- bromomethyl -1H, 5H are added in/ml Quinolizino (9,1-gh) cumarin) to final concentration of 5 μM.

2, it mixes, 37 DEG C of incubation 30min.

3, room temperature, 1500rpm are centrifuged 5min, abandon 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, Raji cell is suspended in the PBS containing 0.1%BSA, and adjustment concentration is 1 × 10⁶/ml。

6, fluorescein based dye CFSE (carbox fluorescenceindiacetate succinimidyl ester) is added to final concentration of 1 μM.

7, it mixes, 37 DEG C of incubation 10min.

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

9, it cleans cell and is resuspended in fresh cells toxicity culture medium, density 1 × 10⁶/ml。

10, it cleans effector T cell (i.e. embodiment 3 be prepared CAR-T cell) and is suspended in cytotoxicity culture medium In, adjustment concentration is 5 × 10⁶/ml。

11, in all experiments, infected the effector T cell (CAR-T cell) of anti-CD19CAR cytotoxicity and The cytotoxicity for having infected the negative control effector T cell (NT cell) being uninfected by compares, and these effector T cells come from The same patient.

12, for having infected the effector T cell and negative control effector T cell of anti-CD19CAR, according to T cell: target is thin Born of the same parents=10:1, the ratio of 3:1,1:1 are cultivated in 5ml sterility test pipe (BD Biosciences), and every group of setting two is multiple Hole.In each co-cultivation group, target cell is 50,000, Raji cell (50 μ l), 50,000 K562 of negative control cell Cell (50 μ l).One group of setting only includes Raji target cell and K562 negative control cell simultaneously.

13, co-cultured cell is placed in 37 DEG C of incubation 4h.

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

15, it is 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, and it is thin to measure the Raji target lived after T cell and target cell co-cultivation The ratio of born of the same parents and K562 negative control cell living.

A) for the T cell of each group of co-cultivation and target cell,

Target cell survival %=Raji viable count/K562 viable count.

B) the target cell survival % of cytotoxic killer cell %=100- calibration, i.e., (Raji is living thin when no effector cell Born of the same parents' number-when containing effector cell Raji viable count)/K562 viable count ratio.

As a result as shown in Figure 5.After CAR-T cell and target cell (Raji cell) co-culture, with effect target ratio increase its Killing ability also increases, and dosage is presented and relies on type.

Embodiment 7:CART cell induces Raji the therapeutic effect evaluation of the NOG mouse of raw tumor

1, with the female NOG mouse of 8 week old, on the day before injection T cell, tail vein injection 0.2x10⁶Raji cell, Raji cell used is with 2x10⁶The density of/ml is dissolved in physiological saline, the cell re-suspension liquid of every mouse injection 100ul；

2, corresponding CAR-T cell and control cell (NT) are injected by each experimental group, cell dosage is 1x10⁷.T used Cell is with 5x10⁷The density of/ml is dissolved in physiological saline, the cell re-suspension liquid of every mouse injection 200ul.

3, routine observation mouse twice a week records mouse survival situation.

4, mouse survival curve is drawn.

As a result as shown in Figure 6.With the NOG mouse (lymthoma mouse) of CAR-T cell therapy inoculation Raji (with solid line in Fig. 6 Indicate) relative to control group (NT, untreated fish group；It is represented by dotted lines in Fig. 6), significantly extend the life span of mouse.

Sequence table

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

<120>Chimeric antigen receptor and application thereof of CD19 is targeted

<130> 163639F1

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 263

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>amino acid sequence of CD8 leader sequence-mCD19scFv

<400> 1

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser

260

<210> 2

<211> 789

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>coded sequence of CD8 leader sequence-mCD19scFv

<400> 2

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

gtcagcagt 789

<210> 3

<211> 226

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>amino acid sequence of CD8 α-CD28-CD3 ζ

<400> 3

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Phe

35 40 45

Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu

50 55 60

Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg

65 70 75 80

Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro

85 90 95

Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala

100 105 110

Tyr Arg Ser Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

115 120 125

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

130 135 140

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

145 150 155 160

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

165 170 175

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

180 185 190

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

195 200 205

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

210 215 220

Pro Arg

225

<210> 4

<211> 540

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>coded sequence of CD8 α-CD28-CD3 ζ

<400> 4

ttctgggtgc tggtcgtggt cggaggggtg ctggcctgtt atagcctgct ggtgactgtc 60

gccttcatta tcttctgggt gcggagcaag aggtctcgcg gtgggcattc cgactacatg 120

aacatgaccc ctagaaggcc tggcccaacc agaaagcact accagccata cgcccctccc 180

agagatttcg ccgcttatcg aagcgtgaag ttctcccgaa gcgcagatgc cccagcctat 240

cagcagggac agaatcagct gtacaacgag ctgaacctgg gaagacggga ggaatacgat 300

gtgctggaca aaaggcgggg cagagatcct gagatgggcg gcaaaccaag acggaagaac 360

ccccaggaag gtctgtataa tgagctgcag aaagacaaga tggctgaggc ctactcagaa 420

atcgggatga agggcgaaag aaggagagga aaaggccacg acggactgta ccaggggctg 480

agtacagcaa caaaagacac ctatgacgct ctgcacatgc aggctctgcc accaagatga 540

<210> 5

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer

<400> 5

agcatcgttc tgtgttgtct c 21

<210> 6

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer

<400> 6

tgtttgtctt gtggcaatac ac 22

<210> 7

<211> 21

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 7

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Leu

1 5 10 15

Gly Ser Thr Glu Phe

20

<210> 8

<211> 21

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 8

Arg Ser Thr Ser Gly Leu Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly

20

<210> 9

<211> 21

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 9

Gln Leu Thr Ser Gly Leu Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly

20

<210> 10

<211> 4

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 10

Gly Gly Gly Ser

1

<210> 11

<211> 5

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 11

Gly Gly Gly Gly Ser

1 5

<210> 12

<211> 5

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 12

Ser Ser Ser Ser Gly

1 5

<210> 13

<211> 5

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 13

Gly Ser Gly Ser Ala

1 5

<210> 14

<211> 5

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 14

Gly Gly Ser Gly Gly

1 5

<210> 15

<211> 15

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 15

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 16

<211> 15

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 16

Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly

1 5 10 15

<210> 17

<211> 15

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 17

Gly Ser Gly Ser Ala Gly Ser Gly Ser Ala Gly Ser Gly Ser Ala

1 5 10 15

<210> 18

<211> 15

<212> PRT

<213>artificial sequence (Artificial Sequence)

<220>

<223>joint sequence

<400> 18

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Claims

1. a kind of fusion protein, the fusion protein is selected from:

(1) contain leader peptide, anti-CD19 single-chain antibody, the people CD8 α hinge area, people's CD28 cross-film of sequentially connected CD8 antigen Area, people CD28 intracellular region and people's CD3 ζ intracellular region fusion protein；With

(2) by replacing, missing or adding one or several amino acid and retaining activation T in the amino acid sequence that (1) limits The fusion protein as derived from (1) of cell activity；

Preferably, the anti-CD19 single-chain antibody is anti-CD19 monoclonal antibody FMC63.

2. fusion protein as described in claim 1, which is characterized in that the fusion protein has following one or more special Sign:

The amino acid sequence of the CD8 antigen leader peptide is as shown in SEQ ID NO:1 1-21 amino acids；

The amino acid sequence of the light chain variable region of the anti-CD19 single-chain antibody such as SEQ ID NO:1 22-128 amino acids institute Show；

The amino acid sequence of the heavy chain variable region of the anti-CD19 single-chain antibody can be such as SEQ ID NO:1 144-263 bit amino Shown in acid；

The amino acid sequence of the people CD8 α hinge area is as shown in SEQ ID NO:3 1-47 amino acids；

The amino acid sequence of the people CD28 transmembrane region is as shown in SEQ ID NO:3 48-74 amino acids；

The amino acid sequence of the people CD28 intracellular region is as shown in SEQ ID NO:3 75-115 amino acids；With

The amino acid sequence of the people CD3 ζ intracellular region is as shown in SEQ ID NO:3 116-226 amino acids.

3. fusion protein as described in claim 1, which is characterized in that the amino acid sequence of the fusion protein is by SEQ ID NO:1 and SEQ ID NO:3 is sequentially connected in series to be formed.

4. a kind of polynucleotide sequence, which is selected from:

(1) polynucleotide sequence of fusion protein described in any one of claim 1-3 is encoded；With

(2) complementary series of (1) described polynucleotide sequence；

Preferably, the polynucleotide sequence contains shown in nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:4 Nucleotide sequence, or the nucleotide sequence as shown in SEQ ID NO:2 and 4 are connected in series according to this.

5. a kind of nucleic acid constructs, the nucleic acid constructs contains polynucleotide sequence as claimed in claim 4；

Preferably, the nucleic acid constructs is carrier；

It is highly preferred that the nucleic acid constructs is retroviral vector, and containing replication origin, 3 ' LTR, 5 ' LTR, and Polynucleotide sequence as claimed in claim 4.

6. a kind of retrovirus, the retrovirus contains nucleic acid constructs described in claim 5, preferably comprises described Carrier, the further preferably described retroviral vector.

7. a kind of method of ex vivo activation T cell, the method includes using retroviral infection institute as claimed in claim 6 The step of stating T cell.

8. the pharmaceutical composition of a kind of T cell of gene modification or the T cell containing the gene modification, which is characterized in that described thin Born of the same parents contain polynucleotide sequence as claimed in claim 4 herein, or containing the retroviral vector described in claim 5, or Retrovirus as claimed in claim 6 has been infected, or has been prepared using method of claim 7.

9. fusion protein of any of claims 1-3, polynucleotide sequence as claimed in claim 4, claim The application of carrier described in 5 or retrovirus as claimed in claim 6 in the T cell of preparation activation.

10. fusion protein of any of claims 1-3, polynucleotide sequence as claimed in claim 4, right are wanted It is prepared by the T cell of carrier described in asking 5, reverse transcription disease as claimed in claim 6 or gene modification according to any one of claims 8 Treat the purposes in the drug of the disease of CD19 mediation；

Preferably, the disease that the CD19 is mediated includes leukaemia and lymthoma；

It is highly preferred that the disease that the CD19 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.