CN109320615A - Target the Chimeric antigen receptor and application thereof of novel B CMA - Google Patents

Abstract

The present invention relates to the Chimeric antigen receptors and application thereof of targeting BCMA.Specifically, the present invention provides a kind of polynucleotide sequence, is selected from: (1) polynucleotide sequence of the polynucleotide sequence coding sequence containing the coded sequence of sequentially connected anti-BCMA single-chain antibody, the coded sequence of people's CD8 α hinge area, the coded sequence of people's CD8 transmembrane region, the coded sequence of people's 41BB intracellular region, the coded sequence of people's CD3 ζ intracellular region and mbIL15 structural coding sequence；(2) complementary series of (1) described polynucleotide sequence.The present invention also provides the purposes of relevant 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 novel B CMA

Technical field

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

Background technique

Huppert's disease is a kind of malignant plasma cell disease, shows as bone marrow plasma cells malignant clone hyperplasia, secretion Monoclonal immunoglobulin or its segment (M albumen) lead to the related target organ such as bone, kidney or tissue damage, common clinical Show as ostalgia, anaemia, renal insufficiency, infection etc. [Multiple myeloma.N Engl J Med, 2011.364 (11): p.1046-60.].Huppert's disease is the second largest malignant tumour of hematological system at present, accounts for Malignancy 10%, frequently-occurring disease increases year by year with advancing age in male, disease incidence, is in recent years even more to have the tendency that rejuvenation [Siegel,R.,et al.,Cancer statistics,2014.CA Cancer J Clin,2014.64(1):p.9- 29.]。

B cell maturation antigen (B-cell maturation antigen, BCMA), also known as CD269, by 184 amino acid Residue composition, intracellular region contain 80 amino acid residues, and extracellular domain sequence is very short, only one carbohydrate recognition domain is that B is thin Cellular surface molecule.BCMA belongs to the I type transmembrane signaling proteins for lacking signal peptide, is Tumor Necrosis Factor Receptors family (TNFR) one Member, it can respectively with B cell activity factor BAFF or proliferation-inducing ligand (a proliferation induced ligand, APRIL) two kinds of ligands combine [Tumor necrosis factor family ligand-receptor binding.Curr Opin Struct Biol,2004.14(2):p.154-60.].In the normal tissue, BCMA is expressed in into Ripe B cell and thick liquid cell surface, BCMA gene knockout mice immune system are acted normally, and have normal spleen structure, bone-marrow-derived lymphocyte Development it is normal, but thick liquid cell quantity significantly reduces, it was demonstrated that BCMA has played important role in the survival for maintaining thick liquid cell, Mechanism mainly includes BCMA and BAFF protein binding, and raises anti-apoptotic genes expression Bcl-2, Mcl-1 and Bclw etc., maintains cell raw Long [BCMA is essential for the survival of long-lived bone marrow plasma cells.J Exp Med,2004.199(1):p.91-8.].Similarly, which has also played function in myeloma cell, Important facilitation [BAFF and APRIL protect myeloma cells has been risen to the neoplasm of myeloma cell from apoptosis induced by interleukin 6 deprivation and dexamethasone.Blood, 2004.103(8):p.3148-57.].Studies have shown that BCMA is generally expressed in multiple myeloma cell line, in multiple bone Detection in myeloma patient has also obtained result [Expression of BCMA, TACI, the and BAFF-R in of consistency multiple myeloma:a mechanism for growth and survival.Blood,2004.103(2):p.689- 94.].Kochenderfer etc. is on the basis of having been reported, and use in conjunction Q-PCR, Flow Cytometry and immune group Change method has extensively studied the expression characteristic of BCMA, confirm normal human tissue of the BCMA except mature B cell, thick liquid cell without Expression, and also without expression [B-cell maturation antigen is a promising in CD34+ hematopoietic cell target for adoptive T-cell therapy of multiple myeloma.Clin Cancer Res, 2013.19(8):p.2048-60.].In conjunction with the high similarity and anti-CD19CAR T cell of BCMA expression characteristic and CD19 The successful progress for the treatment of, the cell for prompting our BCMA to can be used as one of target spot of CAR-T cell for Huppert's disease Immunization therapy.

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.

As technology is or not Chimeric antigen receptor T cell (Chimeric Antigen Receptor-T cell, CAR-T) Disconnected development, 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 (immunoreceptor tyrosine based activation motif, ITAM) composition, wherein Chimeric antigen receptor is each Part is 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 is very poor in patient body, so that CAR-T cell has enough time touching largely not yet Tumour cell when just this kind of CAR-T cell of apoptosis can excite antitumoral cytotoxic effect, but cell because Son secretion is fewer, but its survival period in vivo is shorter cannot excite lasting anti-tumor effect (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〕。

T cell activation signaling zone is still the hot spot of research in the optimization CAR design of second generation CAR-T cell.T cell it is complete Full activation depends on the effect of dual signal and cell factor.Wherein the first signal is specific signals, identifies antigen submission by TCR Antigenic Peptide-MHC the compound of cell surface is started；Second signal is costimulatory signal.Early in 1998, there have been Two generation CAR (J Immunol.1998；161 (6): 2791-7).2nd generation CAR is added to a collaboration thorn in intracellular signal peptide area Swash molecule, i.e., costimulatory signal is assembled into inside CAR, preferably can provide activation signals for CAR-T cell, in this way Costimulatory molecules and intracellular signal can be activated after CAR tumor cell simultaneously, realize dual activation, T can be significantly improved Cell Proliferation secretion capacity and anti-tumor effect.First T cell costimulatory signal receptor studied in detail is CD28, its energy Enough in conjunction with the B7 family member of target cell surface.The costimulation of CD28 can promote the proliferation of T cell, the synthesis of IL-2 and table Reach and enhance the ability that T cell resists apoptosis.Then occur the costimulations such as CD134 (OX40) and 41BB (4-1BB) point again Son maintains t cell response to improve cytotoxicity, the proliferation activity of T cell, extends T cell time-to-live etc..Such Two generation CAR produce unexpected effect in subsequent clinical test, the clinical report from 2010 based on second generation CAR Road repeatedly causes vibration, and especially for recurrent, intractable patient ALL, complete remission rate is 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 (Mol Ther., 2005,12 (5): 933-941).Most typical is exactly U Pen Carl June in CD28 stimulating factor Under the action of added the stimulating factor of a 41BB again.

The CAR-T cell of forth generation then joined cell factor or costimulation ligand, such as four generation CAR can produce IL- 12, the activation of immune microenvironment-increase T cell can be adjusted, 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 (TRUCKs:the fourth generation of CARs, Expert Opin Biol Ther., 2015；15 (8): 1145-54).

Interleukin 15 (interleukin 15, IL-15) is a kind of cell factor of discovery in 1994, is by 114 The glycoprotein of the 14-15KD size of a amino acid composition, belongs to a member in four-helix bundle cytokine family.IL-15 is being tied There is homology with interleukin 2 (interleukin 2, IL-2) on structure, receptor is by the IL-15 receptor a with high-affinity Chain, IL-2/15 receptor β chain and public chain γ chain composition.Therefore IL-15 has the function of similar in some and IL-2, for example pierces Swash T cell activation and proliferation, enhance NK cell killing activity and B cell is promoted to generate immunoglobulin.The IL- of research discovery recently 15 have played important function in NK cell, the differentiation of NKT cell and enterocyte, proliferation, activation process, IL-15 with IL-17 is extremely important to the steady-state adjustment of CD8 memory t cell.Research also confirms that IL-15 passes through a kind of special " anti-submission " Mechanism come regulate and control CD8 memory t cell proliferation and NK cell life cycle, i.e., it is a kind of express IL-15 α chain receptor cell It can be by IL-15 submission to the cell of expression IL-15B chain and γ chain.More research shows that IL-15 is that one kind has extensive biology The powerful cell factor for learning function, in addition to there is important adjustment effect to immune system, in non-immunological systems No less important, for example important regulative is also played to skeletal muscle anabolism.

Invention introduces IL15 structure, IL15 is powerful cell factor, there is important tune to immune system Section effect, has the addition of this structure, the CAR-T cell of building will have stronger strong function.

Summary of the invention

First aspect present invention provides a kind of polynucleotide sequence, and the polynucleotide sequence is selected from:

(1) contain the coded sequence of sequentially connected anti-BCMA single-chain antibody, the coded sequence of people's CD8 α hinge area, people The coded sequence of CD8 transmembrane region, the coded sequence of people's 41BB intracellular region, the coded sequence of people's CD3 ζ intracellular region and IL15 structure are compiled The polynucleotide sequence of the polynucleotide sequence coding sequence of code sequence；With

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

In one or more embodiments, the signal peptide before the coded sequence of the anti-BCMA single-chain antibody Coded sequence is as shown in SEQ ID 1-63 nucleotide sequences of NO:1.In one or more embodiments, the anti-BCMA The coded sequence of the light chain variable region of single-chain antibody is as shown in SEQ ID 64-396 nucleotide sequences of NO:1.At one or In multiple embodiments, the coded sequence of the heavy chain variable region of the anti-BCMA single-chain antibody such as SEQ ID NO:1 442-792 Shown in the nucleotide sequence of position.In one or more embodiments, the coded sequence such as SEQ ID of the people CD8 α hinge area Shown in 793-933 nucleotide sequences of NO:1.In one or more embodiments, the code sequence of the people CD8 transmembrane region Column are as shown in SEQ ID 934-999 nucleotide sequences of NO:1.In one or more embodiments, the people 41BB born of the same parents The coded sequence of inner region is as shown in SEQ ID 1000-1143 nucleotide sequences of NO:1.In one or more embodiments In, the coded sequence of the people CD3 ζ intracellular region is as shown in SEQ ID 1144-1476 nucleotide sequences of NO:1.At one Or in multiple embodiments, the polynucleotide sequence of the IL-15 intracellular region such as 1609-2739 multicores of SEQ ID NO:1 Shown in thuja acid.

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

(1) contain sequentially connected anti-BCMA single-chain antibody, people CD8 α hinge area, people CD8 transmembrane region, people's 41BB intracellular region With people's CD3 ζ intracellular region and with IL15 structural coding sequence；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；

Preferably, the anti-BCMA single-chain antibody is anti-BCMA monoclonal antibody C11D5.3.

In one or more embodiments, the fusion protein also contains letter in the N-terminal of the anti-BCMA single-chain antibody Number peptide.In one or more embodiments, the amino acid sequence of the signal peptide such as SEQ ID NO:2 1-21 amino acids It is shown.In one or more embodiments, the amino acid sequence such as SEQ of the light chain variable region of the anti-BCMA single-chain antibody Shown in ID NO:2 22-132 amino acids.In one or more embodiments, the heavy chain of the anti-BCMA single-chain antibody can The amino acid sequence for becoming area can be as shown in SEQ ID NO:2 148-264 amino acids.In one or more embodiments, The amino acid sequence of the people CD8 α hinge area is as shown in SEQ ID NO:2 265-311 amino acids.One or more real It applies in scheme, the amino acid sequence of the people CD8 transmembrane region is as shown in SEQ ID NO:2 312-333 amino acids.At one Or in multiple embodiments, the amino acid sequence of the people 41BB intracellular region such as SEQ ID NO:2 334-381 amino acids institute Show.In one or more embodiments, the amino acid sequence of the people CD3 ζ intracellular region such as SEQ ID NO:2 382-492 Shown in amino acids.In one or more embodiments, the coded sequence of the segment of the IL-15 such as SEQ ID NO:2 Shown in 537-913 amino acids sequence.

Third aspect present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs contains polynucleotides as described herein Sequence.

In one or more embodiments, the nucleic acid constructs is carrier.In one or more embodiments, institute Stating nucleic acid constructs is retroviral vector, contains replication origin, 3 ' LTR, 5 ' LTR, polynucleotides as described herein Sequence, and optional selectable label.

Fourth aspect present invention provides a kind of retrovirus, and the retrovirus contains nucleic acid construct as described herein Object preferably comprises the carrier, the further preferably described retroviral vector.

Fifth aspect present invention provides a kind of T cell of gene modification, and the cell contains polynucleotides as described herein Sequence, or contain nucleic acid constructs as described herein, or infected retrovirus as described herein, or stablize expression this paper institute The fusion protein stated.

Sixth aspect present invention provides a kind of pharmaceutical composition of T cell containing gene modification as described herein.

Seventh aspect present invention provides polynucleotide sequence, fusion protein, nucleic acid constructs or reverse transcription as described herein Application of the virus in the T cell of preparation activation.

Eighth aspect present invention offer polynucleotide sequence as described herein, fusion protein, nucleic acid constructs, reverse transcription disease Purposes of the T cell or its pharmaceutical composition of poison or gene modification in the drug of the preparation treatment BCMA disease mediated.

In one or more embodiments, the disease that the BCMA is mediated is Huppert's disease.

Detailed description of the invention

Fig. 1 is BCMA-CAR retrovirus expression vector (BCMA-41BBz-mbIL15) schematic diagram.

Fig. 2 is 72 hours BCMA-41BBz-mbIL15-CAR+ of FCM results show retroviral infection T cell Expression efficiency.

Fig. 3 is that the BCMA-41BBz-mbIL15-CART cell for preparing 5 days and target cell co-culture 5 hours CD107a tables It reaches.

Fig. 4 is point of 5 hours INF- γ of BCMA-41BBz-mbIL15-CART cell and target cell co-cultivation of preparation 5 days It secretes.

Fig. 5 is to prepare 5 days BCMA-41BBz-mbIL15-CART cells and after target cell co-cultivation 20 hours to tumour The lethal effect of cell.

Specific embodiment

The present invention provides a kind of Chimeric antigen receptor (CAR) for targeting BCMA.It is mono- that the CAR contains sequentially connected anti-BCMA Chain antibody, people CD8 α hinge area, people CD8 transmembrane region, the segment of people 41BB intracellular region, people CD3 ζ intracellular region and IL-15.

Various anti-BCMA monoclonal antibodies well known in the art can be derived from by being suitable for the invention anti-BCMA single-chain antibody.

Optionally, the light chain variable region and heavy chain variable region can be linked together by joint sequence.Can illustrate this Class single-chain antibody includes but is not limited to C11D5.3, J22.9.In certain embodiments, the monoclonal antibody is that clone number is The monoclonal antibody of C11D5.3.In certain embodiments, the amino acid sequence of the light chain variable region of the anti-BCMA single-chain antibody Column are as shown in the 22-132 amino acids residue of SEQ ID NO:2.In other embodiments, the anti-BCMA single-chain antibody Heavy chain variable region amino acid sequence as shown in the 148-264 amino acids residue of SEQ ID NO:2.

The amino acid sequence for being suitable for the invention people's CD8 α hinge area can be such as SEQ ID NO:2 265-311 bit amino Shown in acid.

Being suitable for the invention people's CD8 transmembrane region can be the transmembrane domain various people CD8 commonly used in the art in CAR. In certain embodiments, the amino acid sequence of the people CD8 transmembrane region such as SEQ ID NO:2 312-333 amino acids institute Show.

Being suitable for the invention 41BB can be the various 41BB for CAR known in the art.As illustrative example, The present invention uses 41BB shown in SEQ ID NO:2 334-381 amino acids sequence.

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:2 382-492 amino acids institute Show.

The each part mentioned above of fusion protein of the invention is formed, such as the light chain variable region of anti-BCMA single-chain antibody and heavy chain can Become area, people CD8 α hinge area, people CD8 transmembrane region, 41BB and people's CD3 ζ intracellular region etc., can be directly connected between each other, Huo Zheke 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..It should be understood that being operated in gene cloning In, it is often necessary to suitable restriction enzyme site is designed, this certainly will introduce one or more in expressed amino acid sequence end Incoherent residue, and this has no effect on the activity of aim sequence.In order to construction of fusion protein, promote recombinant protein expression, It obtains and is secreted into the recombinant protein outside host cell or the purifying conducive to recombinant protein automatically, it is often necessary to by some amino acid It is added in other appropriate areas in the end N-, the end C- or the albumen of recombinant protein, it may for example comprise but be not limited to, it fits Joint peptide, signal peptide, leader peptide, end extension of conjunction etc..Therefore, the aminoterminal of fusion protein of the invention (the i.e. described CAR) Or c-terminus can also be containing one or more polypeptide fragments, as protein tag.Any 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 purifying albumen.

The present invention also includes CAR, SEQ ID NO:2 22- shown in SEQ ID NO:2 22-492 amino acids sequence CAR shown in 913 amino acids sequences, CAR or SEQ ID NO:2 shown in SEQ ID NO:2 1-492 amino acids sequence Shown in CAR mutant.These mutant include: with the CAR at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity simultaneously retains the biological activity of the CAR (as activation T is thin Born of the same parents) amino acid sequence.The sequence identity between the sequence of BLASTp two comparisons of calculating of such as NCBI can be used.

Mutant further include: amino acid sequence, SEQ ID NO:2 22- shown in SEQ ID NO:2 22-492 Amino acid sequence shown in 913, shown in amino acid sequence or SEQ ID NO:2 shown in SEQ ID NO:2 1-492 Still retain the biological activity of the CAR in amino acid sequence with one or several mutation (insertion, deletion or substitution), simultaneously Amino acid sequence.Several mutation are often referred within 1-10, such as 1-8,1-5 or 1-3.Substitution is preferably Conservative replaces.For example, in the art, when carrying out conservative replaces with amino acid similar in performance, usually will not Change the function of protein or polypeptide." amino acid similar in performance " includes for example, the amino acid with similar side chain The family of residue, these families include the amino acid (such as lysine, arginine, histidine) with basic side chain, have acid The amino acid (such as aspartic acid, glutamic acid) of property side chain, amino acid (such as the sweet ammonia with uncharged polar side chain Acid, asparagine, glutamine, serine, threonine, tyrosine, cysteine), the amino acid (example with non-polar sidechain Such as alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), have β-branch side The amino acid (such as threonine, valine, isoleucine) of chain and amino acid (such as tyrosine, phenylpropyl alcohol with aromatic side chain Propylhomoserin, tryptophan, histidine).Therefore, with another amino acid residue replacement one from the same side chain class in polypeptide of the present invention A or several sites, 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, polynucleotide sequence such as 64-1476 cores of SEQ ID NO:1 of fusion protein described herein are encoded Shown in thuja acid, or as shown in SEQ ID 1-1476 nucleotide of NO:1.

The present invention also relates to nucleic acid constructs, which contains polynucleotide sequence as described herein, Yi Jiyu One or more regulating and controlling sequences of these series of operations connection.Polynucleotide sequence of the present invention can in many ways by Operate the expression to guarantee the fusion protein (CAR).It can be according to expression vector before nucleic acid constructs is inserted into carrier Difference is required and is operated to nucleic acid constructs.The technology for changing polynucleotide sequence using recombinant DNA method is this Known to field.

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 Column.Terminator sequence is connect with 3 ' end effectors of the nucleotide sequence for encoding the polypeptide.Have in the host cell of selection Any terminator of function can be used in the present invention.Regulating and controlling sequence is also possible to suitable leader sequence, to host cell translation The non-translational region of important mRNA.5 ' ends of leader sequence and the nucleotide sequence for encoding the polypeptide are operatively connected.It is selecting Functional any terminator can be used in the present invention in the host cell selected.

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

Polynucleotide sequence of the invention can be cloned into the carrier of many types.For example, plasmid, phagocytosis can be cloned into Grain, 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.

Therefore, in certain embodiments, the present invention provides a kind of T cell of gene modification, the T cell of the gene modification Containing polynucleotide sequence as described herein, or contain retroviral vector as described herein, or infected as described herein Retrovirus, or be prepared using method described herein.

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 therapy, wherein T cell is expressed CAR as described herein and optionally by gene modification TEGFR and CAR-T cell needed in its recipient by injection.The cell of injection can kill the tumour cell of recipient. Unlike antibody therapy, 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.

Therefore, it is thin that CAR of the invention, its coded sequence, nucleic acid constructs, expression vector, virus and CAR-T can be used The disease of born of the same parents' treatment is preferably the disease that BCMA is mediated.

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 well known in the art Treatment BCMA mediate disease radiotherapy or chemotheraping preparation treated.

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.

The present invention using the anti-BCMA antibody scFV of clone C11D5.3 (specifically be derived from) gene order, and from The CD8 α hinge area of people, the CD8 transmembrane region of people, the 41BB intracellular region of people and people are searched in NCBI GenBank database CD3 ζ intracellular region and IL15 gene sequence information, full genome synthesize the anti-BCMA scFv-CD8 hinge area-of Chimeric antigen receptor The genetic fragment of CD8TM-41BB-CD3 ζ-IL15, is inserted into retroviral vector.Recombinant plasmid wraps in 293T cell Pretend to be sick poison, infects T cell, T cell is made to express the Chimeric antigen receptor.The T of present invention realization Chimeric antigen receptor gene modification The method for transformation of lymphocyte is based on Retroviral Transformation method.This method has high conversion efficiency, and foreign gene can Stablize expression, and the advantages that in vitro culture T lymphocyte reaches the time of clinical number of stages can be shortened.It is drenched in transgenosis T Bar cell surface, the nucleic acid of conversion by transcription, accurate translation on it.CAR-T cell prepared by the present invention is swollen to specificity Oncocyte has strong killing ability, and in the case that effect target ratio is 10 to 1, killing-efficiency is more than 70%.

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. Method used in embodiment and reagent, unless otherwise stated, being the method and reagent of this field routine.

The determination of embodiment 1:BCMA-scFv-CD8 α-CD28-41BB-CD3 ζ-mbIL15 gene order

From NCBI site databases search people CD8 α hinge area, people CD8 α transmembrane region, 41BB intracellular region, people CD3 ζ and MbIL15 intracellular region gene sequence information, anti-BCMA single-chain antibody clone number is C11D5.3, 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-BCMA scFv, people's CD8 α hinge area gene, people's CD8 α transmembrane region using over-lap PCR Gene, 41BB intracellular region gene, people's CD3 ζ intracellular region and mbIL15 gene order are attached, and are introduced in each sequence junction Different restriction enzyme sites form complete BCMA-CAR gene sequence information.

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) is patched, competent E.coli (DH5 α) is transformed into.

Recombinant plasmid is served Hai Sheng work Bioisystech Co., Ltd be sequenced, by sequencing result be fitted to Whether BCMA-mbIL15CAR sequence alignment is correct to verify sequence.Sequencing primer are as follows:

Justice: AGCATCGTTCTGTGTTGTCTC (SEQ ID NO:3)；

Antisense: TGTTTGTCTTGTGGCAATACAC (SEQ ID NO:4).

After being sequenced correctly, simultaneously plasmid purification is extracted using the plasmid purification kit of Qiagen company, plasmid purification Plasmid calcium phosphate method transfects 293T cell and carries out retrovirus Packaging experimentation.

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

Embodiment 2: retrovirus packaging

1, the 1st day: 293T cell should be less than for 20 generations, not cover with excessively.With 0.6 × 10⁶Cell/ml bed board, 10cm Ware adds the DMEM culture medium of 10ml, mixes well cell, 37 DEG C of overnight incubations；

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

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

Embodiment 3: the T cell of retroviral infection people

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 CD3 antibody (Beijing is with vertical Hai Yuan) and 50ng/ml CD28 antibody (Beijing is with vertical Hai Yuan), add the leucocyte of 100IU/ml Interleukin 2 (the double aigrets in Beijing), the virus infection that stimulation culture is prepared after 48 hours with embodiment 3；

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 treatment 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. every hole adds 2ml virus liquid by the virus liquid adding hole being prepared with embodiment 3,32 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, 5%CO after centrifugation₂It is cultivated in incubator.

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.

9. being named as BCMA- thus to obtain the CART cell for having infected retrovirus shown in embodiment 3 respectively MbIL15CART cell (BCMA-mbIL15CAR of expression embodiment 1).

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

CAR-T cell and NT cell (control group) that 72 hours after infecting embodiments 4 are prepared are collected by centrifugation respectively, PBS abandons supernatant after washing 1 time, and PBS after corresponding antibody is protected from light 30min is added and washs, is resuspended, last flow cytomery. CAR+ is detected by anti-mouse IgG F (ab') antibody (Jackson Immunoresearch).

Fig. 2 shows, behind retroviral infection T cell 72 hours be prepared using embodiment 3, BCMA- The expression efficiency of mbIL15CAR+ is up to 20%.

CD107a detection of expression after embodiment 5:CAR-T cell and target cell co-culture

1. taking one piece of 96 orifice plate of the bottom V, every hole adds CART/NT cell 2*10⁵A and target cell (H929 and L363)/control is thin Born of the same parents (K562) 2*10⁵It is a, the X-VIVO complete medium for being free of IL-2 for 100ul is resuspended, BD GolgiStop is added and (contains 1 μ l BD GolgiStop is added in every 1ml culture medium in monesin), 2ul CD107a antibody (1:50) is added in every hole, and 37 DEG C It is incubated for 4 hours, collects cell.

2. sample is centrifuged removal culture medium, it is primary that PBS washes cell, 400g, and 4 DEG C are centrifuged 5 minutes.Supernatant is abandoned, every pipe adds Enter appropriate specific surfaces antibody CD3, CD4, CD8, resuspension volume 100ul is protected from light incubation 30 minutes on ice.

3. the PBS per effective 3mL is cleaned cell 1 time, 400g is centrifuged 5 minutes.Carefully suck supernatant.

4. appropriate PBS is resuspended, flow cytomery CD107a.

It is shown in Fig. 3.BCMA-mbIL15CART cell in H929 cell CD107a secrete percentage be 34.3%, BCMA-mbIL15 cell are 38.7% in the percentage that L363 cell CD107a secretes.

INF- γ secretion detects after embodiment 7:CAR-T cell and target cell co-culture

1. taking the CAR-T cell prepared, it is resuspended with Lonza culture medium, adjustment cell concentration is 1 × 106/mL.

2. the every hole of experimental group contains target cell (H929 and L363) or negative control cell (K562) 2 × 105, CAR-T is thin Born of the same parents 2 × 105,200 μ l are free of the Lonza culture medium of IL-2.It is added after mixing well in 96 orifice plates.BD is added simultaneously GolgiPlug (contains BFA, 1 μ l BD GolgiPlug is added in every 1ml cell culture medium), after mixing well, 37 DEG C of incubation 5-6 Hour.Cell is collected, as experimental group.

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

After 4.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^TMBuffer is cleaned cell 2 times, 1mL/ times.

5. 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.

6. flow cytomery.

It is shown in Fig. 4.BCMA-mbIL15 CART cell percentage of IFN-γ secretion in H929 cell is 24.7%, BCMA-mbIL15 cell are 42.8% in the percentage of L363 cell IFN-γ secretion.

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

1.K562 cell (being free of BCMA target protein, be the negative control cell of target cell) is resuspended in serum free medium (1640) in, adjustment cell concentration is 1 × 106/ml, and fluorescent dye BMQC (2,3,6,7-tetrahydro-9- is added 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.L363 cell (target protein containing BCMA is target cell) is suspended in the PBS containing 0.1%BSA, and 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.

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

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 CAR-T cell and the negative control effector T cell (NT being uninfected by Cell cytotoxicity) compares, and these effector T cells come from the same patient.

12.CAR-T and NT, according to effector cell: target cell=10:1,2:1, ratio, in 5ml sterility test pipe (BD Biosciences it) is cultivated, every group of two multiple holes of setting.In each co-cultivation group, target cell is 50,000, L363 cell (50 μ l), 50,000 K562 cell (50 μ l) of negative control cell.Simultaneously setting one group only include L363 target cell and K562 negative control cell.

13. co-cultured cell is placed in 37 DEG C of incubation 5h.

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, it is thin to measure the L363 target lived after T cell and target cell co-cultivation The ratio of born of the same parents and K562 negative control cell living.

17. T cell and target cell for each group of co-cultivation

The % 18. target cell of cytotoxic killer cell %=100- calibration survives, i.e., (L363 is living thin when no effector cell Born of the same parents' number-when containing effector cell L363 viable count)/K562 viable count ratio.

As the result is shown in Fig. 5.Fig. 5 is shown, when imitating target ratio is 10:1, BCMA-tEGFR CART cell pair The killing rate of L363 cell is 70%.

Sequence table

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

<120>Chimeric antigen receptor and application thereof of novel B CMA is targeted

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2739

<212> DNA

<213>artificial sequence (Homo sapiens)

<400> 1

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgacatcg ttttgacaca atctcctgcg tcattggcca tgagtctcgg gaagcgcgca 120

acaatatcct gtcgcgccag tgaatctgtg tctgtgatag gagcgcactt gatccattgg 180

tatcagcaga aacctggaca acctcccaag ctgctcatct acctcgccag taaccttgaa 240

acaggagtac ctgctcggtt ttcaggttcc gggtcaggga cggatttcac tttgactatc 300

gacccagttg aggaagacga cgtagccata tatagctgcc tgcagtctcg gatcttcccg 360

cgcacgttcg ggggaggaac taagctggag attaagggcg gcgggggttc tggtggcggc 420

ggcagcggcg gtggaggatc acaaatccaa ctggttcagt ccggtccaga actgaaaaag 480

ccgggggaga cggtgaaaat ctcctgtaag gcctcaggtt ataccttcac cgattacagc 540

atcaattggg taaagcgggc tccagggaaa ggtctgaaat ggatgggttg gatcaacaca 600

gaaacccgag aaccagccta tgcttacgac tttcgaggtc gattcgcttt ttccttggaa 660

acttccgcaa gcacagccta tctgcaaatc aacaatctca agtacgaaga tacggccacg 720

tatttttgtg ccctggatta cagctatgca atggattact ggggtcaggg gacgtctgtt 780

acagtttcta gtactacaac tccagcaccc agacccccta cacctgctcc aactatcgca 840

agtcagcccc tgtcactgcg ccctgaagcc tgtcgccctg ctgccggggg agctgtgcat 900

actcggggac tggactttgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960

ggggtccttc tcctgtcact ggttatcacc ctttactgca ggttcagtgt cgtgaagaga 1020

ggccggaaga agctgctgta catcttcaag cagcctttca tgaggcccgt gcagactacc 1080

caggaggaag atggatgcag ctgtagattc cctgaagagg aggaaggagg ctgtgagctg 1140

agagtgaagt tctcccgaag cgcagatgcc ccagcctatc agcagggaca gaatcagctg 1200

tacaacgagc tgaacctggg aagacgggag gaatacgatg tgctggacaa aaggcggggc 1260

agagatcctg agatgggcgg caaaccaaga cggaagaacc cccaggaagg tctgtataat 1320

gagctgcaga aagacaagat ggctgaggcc tactcagaaa tcgggatgaa gggcgaaaga 1380

aggagaggaa aaggccacga cggactgtac caggggctga gtacagcaac aaaagacacc 1440

tatgacgctc tgcacatgca ggctctgcca ccaagacgag ctaaacgagg ctcaggcgcg 1500

acgaacttta gtttgctgaa gcaagctggg gatgtagagg aaaatccggg tcccatggat 1560

tggacttgga ttttgttcct cgttgccgca gcgactcgcg tccatagtaa ttgggtgaac 1620

gtaattagtg acttgaaaaa aattgaggac cttatacaaa gtatgcatat cgatgcaaca 1680

ctgtacacgg agtccgacgt gcacccaagc tgcaaggtca ccgcaatgaa atgctttttg 1740

ctcgaattgc aagttatctc acttgagtca ggggacgctt caatccatga tactgtggag 1800

aatttgataa tcctggcgaa caatagcctt agttcaaatg gcaacgtcac tgagtcaggc 1860

tgcaaggaat gtgaggaatt ggaagaaaaa aatatcaagg aatttttgca atcttttgtt 1920

cacatagttc agatgttcat taacactagt tccgggggcg gcagtggagg tggcggtagc 1980

ggcgggggtg gctctggtgg aggcggctct gggggcggaa gtctgcagat aacatgcccc 2040

ccacctatga gtgttgaaca tgctgatatc tgggttaaat cttactccct ttacagtcga 2100

gaaaggtaca tttgcaactc cggctttaaa cgcaaagccg ggactagttc actgactgaa 2160

tgtgtattga ataaagcgac aaatgtcgca cactggacta ccccttccct caaatgcatt 2220

cgcgatcctg ccttggtgca tcagcgacca gcaccgccgt ccacggtaac taccgcagga 2280

gtaacaccgc agcccgagag cctttccccc tcaggcaaag agccggccgc atcctcccca 2340

tcttccaata ataccgcagc taccaccgca gcaatcgtac ccgggtccca gctgatgccc 2400

agcaaaagtc cgagtactgg aacgactgaa atctccagtc acgagtcttc tcatggaact 2460

ccgagtcaaa ctacagcaaa gaattgggag ctgactgctt ccgcttcaca ccagccgcca 2520

ggcgtttatc ctcagggaca ctcagatacc acggtggcga ttagcacaag caccgtcctc 2580

ctgtgtgggc tgagtgcagt gtcacttctc gcctgctacc ttaagtccag acagacaccc 2640

cctttggcaa gcgttgaaat ggaagccatg gaagccttgc ctgtcacatg ggggacttca 2700

tcccgcgatg aagacttgga gaactgctca caccatctt 2739

<210> 2

<211> 913

<212> PRT

<213>artificial sequence (Homo sapiens)

<400> 2

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

1 5 10 15

His Ala Ala Ala Pro Ala Ile Val Leu Thr Gly Ser Pro Ala Ser Leu

20 25 30

Ala Met Ser Leu Gly Leu Ala Ala Thr Ile Ser Cys Ala Ala Ser Gly

35 40 45

Ser Val Ser Val Ile Gly Ala His Leu Ile His Thr Thr Gly Gly Leu

50 55 60

Pro Gly Gly Pro Pro Leu Leu Leu Ile Thr Leu Ala Ser Ala Leu Gly

65 70 75 80

Thr Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro

85 90 95

Thr Leu Thr Ile Ala Pro Val Gly Gly Ala Ala Val Ala Ile Thr Ser

100 105 110

Cys Leu Gly Ser Ala Ile Pro Pro Ala Thr Pro Gly Gly Gly Thr Leu

115 120 125

Leu Gly Ile Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Ile Gly Leu Val Gly Ser Gly Pro Gly Leu Leu Leu

145 150 155 160

Pro Gly Gly Thr Val Leu Ile Ser Cys Leu Ala Ser Gly Thr Thr Pro

165 170 175

Thr Ala Thr Ser Ile Ala Thr Val Leu Ala Ala Pro Gly Leu Gly Leu

180 185 190

Leu Thr Met Gly Thr Ile Ala Thr Gly Thr Ala Gly Pro Ala Thr Ala

195 200 205

Thr Ala Pro Ala Gly Ala Pro Ala Pro Ser Leu Gly Thr Ser Ala Ser

210 215 220

Thr Ala Thr Leu Gly Ile Ala Ala Leu Leu Thr Gly Ala Thr Ala Thr

225 230 235 240

Thr Pro Cys Ala Leu Ala Thr Ser Thr Ala Met Ala Thr Thr Gly Gly

245 250 255

Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Ala Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro

275 280 285

Gly Ala Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu

290 295 300

Ala Pro Ala Cys Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr Cys

305 310 315 320

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Ala Pro Ser

325 330 335

Val Val Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro

340 345 350

Pro Met Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys

355 360 365

Ala Pro Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro

370 375 380

Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly Leu

385 390 395 400

Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala

405 410 415

Leu Ala Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Ala Ala Leu

420 425 430

Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala

435 440 445

Gly Ala Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu

450 455 460

Gly His Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr

465 470 475 480

Thr Ala Ala Leu His Met Gly Ala Leu Pro Pro Ala Ala Ala Leu Ala

485 490 495

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

500 505 510

Gly Gly Ala Pro Gly Pro Met Ala Thr Thr Thr Ile Leu Pro Leu Val

515 520 525

Ala Ala Ala Thr Ala Val His Ser Ala Thr Val Ala Val Ile Ser Ala

530 535 540

Leu Leu Leu Ile Gly Ala Leu Ile Gly Ser Met His Ile Ala Ala Thr

545 550 555 560

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

565 570 575

Leu Cys Pro Leu Leu Gly Leu Gly Val Ile Ser Leu Gly Ser Gly Ala

580 585 590

Ala Ser Ile His Ala Thr Val Gly Ala Leu Ile Ile Leu Ala Ala Ala

595 600 605

Ser Leu Ser Ser Ala Gly Ala Val Thr Gly Ser Gly Cys Leu Gly Cys

610 615 620

Gly Gly Leu Gly Gly Leu Ala Ile Leu Gly Pro Leu Gly Ser Pro Val

625 630 635 640

His Ile Val Gly Met Pro Ile Ala Thr Ser Ser Gly Gly Gly Ser Gly

645 650 655

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

660 665 670

Gly Ser Leu Gly Ile Thr Cys Pro Pro Pro Met Ser Val Gly His Ala

675 680 685

Ala Ile Thr Val Leu Ser Thr Ser Leu Thr Ser Ala Gly Ala Thr Ile

690 695 700

Cys Ala Ser Gly Pro Leu Ala Leu Ala Gly Thr Ser Ser Leu Thr Gly

705 710 715 720

Cys Val Leu Ala Leu Ala Thr Ala Val Ala His Thr Thr Thr Pro Ser

725 730 735

Leu Leu Cys Ile Ala Ala Pro Ala Leu Val His Gly Ala Pro Ala Pro

740 745 750

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

755 760 765

Ser Pro Ser Gly Leu Gly Pro Ala Ala Ser Ser Pro Ser Ser Ala Ala

770 775 780

Thr Ala Ala Thr Thr Ala Ala Ile Val Pro Gly Ser Gly Leu Met Pro

785 790 795 800

Ser Leu Ser Pro Ser Thr Gly Thr Thr Gly Ile Ser Ser His Gly Ser

805 810 815

Ser His Gly Thr Pro Ser Gly Thr Thr Ala Leu Ala Thr Gly Leu Thr

820 825 830

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

835 840 845

Ala Thr Thr Val Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu

850 855 860

Ser Ala Val Ser Leu Leu Ala Cys Thr Leu Leu Ser Ala Gly Thr Pro

865 870 875 880

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

885 890 895

Thr Gly Thr Ser Ser Ala Ala Gly Ala Leu Gly Ala Cys Ser His His

900 905 910

Leu

<210> 3

<211> 21

<212> DNA

<213>artificial sequence (Homo sapiens)

<400> 3

agcatcgttc tgtgttgtct c 21

<210> 4

<211> 22

<212> DNA

<213>artificial sequence (Homo sapiens)

<400> 4

tgtttgtctt gtggcaatac ac 22

Claims (9)

1. a kind of polynucleotide sequence, the polynucleotide sequence is selected from:

(1) containing the coded sequence of sequentially connected anti-BCMA single-chain antibody, the coded sequence of people's CD8 α hinge area, people CD8 across The coded sequence in film area, the coded sequence of people's 41BB intracellular region, people's CD3 ζ intracellular region coded sequence and IL15 structured coding sequence The polynucleotide sequence of the polynucleotide sequence coding sequence of column；With

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

2. polynucleotide sequence as described in claim 1, which is characterized in that

The coded sequence of the signal peptide before the coded sequence of the anti-BCMA single-chain antibody such as SEQ ID NO:1 1-63 Shown in the nucleotide sequence of position；And/or

The coded sequence of the light chain variable region of the anti-BCMA single-chain antibody such as 64-396 nucleotide sequences of SEQ ID NO:1 It is shown；And/or

The coded sequence of the heavy chain variable region of the anti-BCMA single-chain antibody such as 442-792 nucleotides sequences of SEQ ID NO:1 Shown in column；And/or

The coded sequence of the people CD8 α hinge area is as shown in SEQ ID 793-933 nucleotide sequences of NO:1；And/or

The coded sequence of the people CD8 transmembrane region is as shown in SEQ ID 934-999 nucleotide sequences of NO:1；And/or

The coded sequence of the people 41BB intracellular region is as shown in SEQ ID 1000-1143 nucleotide sequences of NO:1；And/or

The coded sequence of the people CD3 ζ intracellular region is as shown in SEQ ID 1144-1476 nucleotide sequences of NO:1；And/or

The polynucleotide sequence of the IL-15 intracellular region is as shown in SEQ ID 1609-2739 polynucleotides of NO:1；.

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

(1) contain sequentially connected anti-BCMA single-chain antibody, people CD8 α hinge area, people CD8 transmembrane region, people 41BB intracellular region and people CD3 ζ intracellular region and with IL15 structural coding sequence；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-BCMA single-chain antibody is anti-BCMA monoclonal antibody C11D5.3.

4. fusion protein as claimed in claim 3, which is characterized in that the fusion protein has following one or more special Sign:

The fusion protein also contains signal peptide in the N-terminal of the anti-BCMA single-chain antibody, it is preferable that the amino of the signal peptide Acid sequence is as shown in SEQ ID NO:2 1-21 amino acids；And/or

The amino acid sequence of the light chain variable region of the anti-BCMA single-chain antibody such as SEQ ID NO:2 22-132 amino acids institute Show；And/or

The amino acid sequence of the heavy chain variable region of the anti-BCMA single-chain antibody such as SEQ ID NO:2 148-264 amino acids It is shown；And/or

The amino acid sequence of the people CD8 α hinge area is as shown in SEQ ID NO:2 265-311 amino acids；And/or

The amino acid sequence of the people CD8 transmembrane region is as shown in SEQ ID NO:2 312-333 amino acids；And/or

The amino acid sequence of the people 41BB intracellular region is as shown in SEQ ID NO:2 334-381 amino acids；And/or

The amino acid sequence of the people CD3 ζ intracellular region is as shown in SEQ ID NO:2 382-492 amino acids；And/or

The coded sequence of the segment of the IL-15 is as shown in SEQ ID NO:2 537-913 amino acids sequence.

5. a kind of nucleic acid constructs, the nucleic acid constructs contains polynucleotide sequence of any of claims 1-2；

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 of any of claims 1-2.

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. 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 of any of claims 1-2, or containing the nucleic acid constructs described in claim 5, Or retrovirus as claimed in claim 6 is infected, or stablize and express fusion protein described in any one of claim 3-4 Segment.

8. fusion egg described in any one of polynucleotide sequence of any of claims 1-2, claim 3-4 Nucleic acid constructs white, described in claim 5 or retrovirus as claimed in claim 6 are in the T cell of preparation activation Using.

9. fusion egg described in any one of polynucleotide sequence of any of claims 1-2, claim 3-4 Nucleic acid constructs, retrovirus as claimed in claim 6 or gene according to any one of claims 8 white, described in claim 5 The purposes of the T cell of modification or its pharmaceutical composition in the drug of the preparation treatment BCMA disease mediated；

Preferably, the disease that the BCMA is mediated is Huppert's disease.