CN108866088A - Target CLL-1 Chimeric antigen receptor and application thereof - Google Patents

Abstract

The present invention relates to the Chimeric antigen receptors and application thereof of targeting CLL-1.Specifically, the present invention provides a kind of polynucleotide sequence, it is selected from：(1) polynucleotide sequence of the coded sequence of the segment of the III containing extracellular domain and extracellular domain IV of the coded sequence containing sequentially connected anti-CLL-1 single-chain antibody, the coded sequence of people's CD8 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 optional EGFR；(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.CLL-1-BBz-tEGFR CAR-T cell prepared by the present invention has strong killing ability to specific tumor cell, and CD107a expression and IFN γ secretion are higher, and effect target ratio is 5:70% or so is all higher than to target cell killing-efficiency in the case where 1.

Description

Target CLL-1 Chimeric antigen receptor and application thereof

Technical field

The invention belongs to cell therapy fields, and in particular to target Chimeric antigen receptor of CLL-1 and application thereof.

Background technique

Chimeric antigen receptor (Chimeric Antigen Receptor-T cell, CAR-T) T cell refers to through gene After modification, specific purpose antigen, and the T cell of continuous activation amplification can be identified with MHC non-limiting way.State in 2012 Border cell therapy association annual meeting middle finger goes out biological immune cell therapy and has become operation, radiotherapy, the 4th kind for the treatment of outside chemotherapy The means of tumour, and future tumors will be become and treat essential means.CAR-T cell adoptive therapy be in current cancer therapies most Specify effective immunotherapeutic form.A large number of studies show that CAR-T cell can effectively identify tumour antigen, cause special The anti-tumor immune response of property, 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 tumour The ability of antigen, this, which enables, identifies more extensively by the T cell of CAR transformation compared to nave T cell surface receptor TCR Target.It include that a tumor associated antigen (tumor-associated antigen, TAA) combines in the basic engineering of CAR Area's (scFV section for being typically derived from monoclonal antibody antigen bond area), an extracellular hinge area, a transmembrane region and one Intracellular signal area.Safety of the selection of target antigen for the specificity of CAR, validity and genetic modification T cell itself For be all crucial determinant.

AML is the most common acute leukemia of adult, and with advancing age, disease incidence also increases, and has morbidity Suddenly, the features such as disease progression is fast, the death rate is high has heterogeneity in clinical manifestation and therapeutic response.With chemotherapy regimen Perfect, the survival rate of AML patient has had clear improvement, but still some people cannot reach the alleviation even later period and recur, and 5 years Disease-free survival rate only has 25% to 35%, and 60 years old or more patient is even lower.Although the chemotherapy of high doses applied, only 30% to 40% patients acuity marrow series leukemia (AML) existence, main cause are that AML is the pernicious of a stem cell Clonal disease, the LSC for having its source in remaining of AML recurrence.Studies have shown that when CD34+ cell number is greater than 1%, AML's LSC is present in the part CD34+CD38-.Experiment in vitro proves that these stem cells play an important role in chemotherapy resistance, and distinguishes Remaining pernicious CD34+CD38- cell is difficult in vivo, this eventually results in disease palindromia.In addition, early stage is sent out Existing LSC can provide the value of prognosis evaluation and palindromia prediction for patient.

CD19CART achieves good effect, but CART cell therapy AML mesh in terms for the treatment of acute lymphatic leukaemia Preceding to see surprising effect not yet, a main cause is because many antigens for AML are in normal cell and pernicious swollen Oncocyte expression is similar, then also to injure marrow while removing leukaemia cell thin for the CART cell of targeting AML Born of the same parents include normal myeloid tissue and myeloid progenitor, i.e., everybody is not intended to undershooting-effect " the on target, off seen tumor".Some target spot Lewis Y, CD33 and CD123 have been used to the clinical test for the treatment of marrow series leukemia, but without Lasting remission Case Report.Currently, being badly in need of a kind of cart cell of novel targets to treat marrow series leukemia.

C-type lectin-like molecule1 (also entitled CLL-1, KLRl and CLECl2A) is a kind of II type Transmembrane glycoprotein is the member of C- type agglutinin receptor large family relevant to immunological regulation.Normal person peripheral blood and Exist on the monocyte and granulocyte of marrow (BM), may be not present in non-blood tissue.The expression pattern of CLL-1 exists Hematopoietic cell is restrictive, the study found that CLL-1 is present in most CD34+ or CD34-AML, main expression is in periphery On myeloid cell and medullary system mother cell in blood and marrow.The expression of leukemia cell surface antigens c LL 1 helps to distinguish Marrow series leukemia and lymph series leukaemia.Mark antigen of the CLL-1 as LSC, facilitates normal stem cell and leukaemia is dry The identification asked and separation of cell have important value in the diagnosis of leukaemia immunophenotyping and the MRD detection of AML. Malcolm K.Brenner etc. nearest research achievement shows CLL-1CART cell in treatment marrow series leukemia animal model Aspect has preferable effect, and introducing the system with caspase-9 suicide gene is not desired to CLL-1CART simultaneously for they Cell can play the role of termination (Molecular Therapy Vol.25No 9 when playing a role in vivo September 2017)。

It is active medicine that the big advantage of the one of CAR-T cell, which is them, once input, physiological mechanism can regulate and control the flat of T cell Weighing apparatus, memory are formed and the amplification of antigen driving.However, this treatment is not perfect enough, T cell can miss the target and attack other groups It knits or amplification amount is excessively high, beyond needed for treatment.It has been included into standard care range in view of CAR-T cell, has designed patient or medicine The controllable starting of object or shutdown mechanism are highly useful come the presence for regulating and controlling CAR-T cell.Due to technical reason, machine is closed System is easier to be applied to T cell.As one of them, iCas9 system is just in clinical research.Cell when expressing iCas9, It can induce iCas9 precursor molecule using small molecule compound and form dimer, apoptosis pathway is activated, to realize scavenger-cell Purpose.In graft versus host disease(GVH disease), small molecule AP1903 has been used for inducing iCas9 dimer and removes T cell, table Feasibility (the Clin Cancer Res.2016 Apr 15 of this method is illustrated；22(8):1875-84.).

In addition, also make CAR-T cell using the scavenging antibody clinically used while expressing these antibody For albumen, such as tEGFR, treatment-related toxic reaction generate or treat after the completion of, by giving antibody medicine Object removes corresponding CAR-T cell (Sci Transl Med.2015； 7：275ra22.).Based on the considerations of safety we Car-t cell introduces safety switch i.e. tEGFR, the CD19-tEGFR that builds can actual time safety control its in vivo Expression.Our patents are while tEGFR structure to have also been introduced using the heavy chain of the scFV of CLL-1 and light chain as the structure of CAR. TEGFR lacks extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity, but remains natural ammonia Base acid sequence, belongs to the positioning of I type transmembrane cell surface, and space conformation can be with the monoclonal antibody against EGFR west of pharmaceutically grade Appropriate former times monoclonal antibody is combined closely (BLOOD.2011 Aug 4；118(5):1255-63.).The major function of tEGFR：It can be used as thin The label of cellular surface, while being also suitble to the internal tracking of T cell that can detect by streaming and immunohistochemistry；It can also be in vivo (cetuximab) is resisted to remove by appropriate Xidan.

The invention patent, which introduces tEGFR structure simultaneously using double targeting CLL-1CAR elements, can both keep CAR-T thin It carries out in cell space well by tracer, it is often more important that this structure can be used as the safety switch of CAR-T cell：It is not desired to CLL-1 can be added appropriate Xidan and resist when playing a role, safely and effectively the CAR-T cell for CLL-1 target spot of control infusion exists It plays a role in vivo.It lays a good foundation for CLL-1CART cell therapy marrow series leukemia clinical trial and clinical treatment.

Summary of the invention

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

(1) contain coded sequence, the coded sequence of people's CD8 hinge area, people of sequentially connected anti-CLL-1 single-chain antibody 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 optional The polynucleotide sequence of the coded sequence of the segment of the III containing extracellular domain and extracellular domain IV of EGFR；With

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

In one or more embodiments, code sequence of the polynucleotide sequence in the anti-CLL-1 single-chain antibody Coded sequence before arranging also containing CD8 signal peptide.In one or more embodiments, the amino acid sequence of the signal peptide Such as SEQ ID NO:Shown in 2 1-21 amino acids.In one or more embodiments, the anti-CLL-1 single-chain antibody The amino acid sequence of heavy chain variable region such as SEQ ID NO:Shown in 2 22-141 amino acids.In one or more embodiments In, the amino acid sequence such as SEQ ID NO of the light chain variable region of the anti-CLL-1 single-chain antibody:2 157-264 amino acids It is shown.In one or more embodiments, the amino acid sequence of the people CD8 hinge area such as SEQ ID NO:2 265- Shown in 311 amino acids.In one or more embodiments, the amino acid sequence of the people CD8 transmembrane region such as SEQ ID NO:Shown in 2 312-333 amino acids.In one or more embodiments, the amino acid sequence of the people 41BB intracellular region Column such as SEQ ID NO:Shown in 2 334-381 amino acids.In one or more embodiments, the people CD3 ζ intracellular region Amino acid sequence such as SEQ ID NO:Shown in 2 382-492 amino acids.It is described in one or more embodiments The segment of EGFR contains the extracellular domain III, extracellular domain IV and transmembrane region of EGFR, or the extracellular structure by EGFR Domain III, extracellular domain IV and transmembrane region composition.In one or more embodiments, the segment of the EGFR contains someone The 310-646 amino acids sequence of EGFR, or be made of the 310-646 amino acids sequence of Human epidermal growth factor receptor.Such as SEQ ID NO:Shown in 2 541-875 amino acids.

The signal peptide in one or more embodiments, before the coded sequence of the anti-CLL-1 single-chain antibody Coded sequence such as SEQ ID NO:Shown in 1 1-63 nucleotide sequences.It is described anti-in one or more embodiments The coded sequence of the heavy chain variable region of CLL-1 single-chain antibody such as SEQ ID NO:Shown in 1 64-423 nucleotide sequences.? In one or more embodiments, the coded sequence such as SEQ ID NO of the light chain variable region of the anti-CLL-1 single-chain antibody:1 Shown in 469-792 nucleotide sequences.In one or more embodiments, the coded sequence of the people CD8 hinge area is such as SEQ ID NO:Shown in 1 793-933 nucleotide sequences.In one or more embodiments, the people CD8 transmembrane region Coded sequence such as SEQ ID NO:Shown in 1 934-999 nucleotide sequences.It is described in one or more embodiments The coded sequence of people's 41BB intracellular region such as SEQ ID NO:Shown in 1 1000-1143 nucleotide sequences.In one or more In embodiment, the coded sequence such as SEQ ID NO of the people CD3 ζ intracellular region:1 1144-1476 nucleotide sequence institutes Show.In one or more embodiments, the coded sequence of the segment of the EGFR such as SEQ ID NO:1 1621-2625 Shown in nucleotide sequence.

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

(1) intracellular containing sequentially connected anti-CLL-1 single-chain antibody, people CD8 hinge area, people CD8 transmembrane region, people 41BB The segment of the III containing extracellular domain and extracellular domain IV of the fusion protein and optional EGFR of area and people's CD3 ζ intracellular region Coded sequence；With

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

Preferably, the anti-CLL-1 single-chain antibody is anti-CLL-1 monoclonal antibody M26；

Third aspect present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs contains multicore glycosides as described herein Acid sequence.

In one or more embodiments, the nucleic acid constructs is carrier.In one or more embodiments, The nucleic acid constructs is retroviral vector, contains replication origin, 3 ' LTR, 5 ' LTR, multicore glycosides as described herein Acid sequence, and optional selectable label.

Fourth aspect present invention provides a kind of retrovirus, and the retrovirus contains nucleic acid structure as described herein Object is built, the carrier is preferably comprised, 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 herein The piece of the III containing extracellular domain of the fusion protein and optional EGFR, extracellular domain IV and optional transmembrane region Section.

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 Purposes of the T cell or its pharmaceutical composition of virus or gene modification in the drug of the preparation treatment CLL-1 disease mediated.

In one or more embodiments, the disease that CLL-1 is mediated is marrow series leukemia.

Detailed description of the invention

Fig. 1 is RV-CLL-1-BBz-tEGFR retrovirus expression vector schematic diagram.

Fig. 2 is the CLL-1-BBz-tEGFRCART expression in 72 hours of flow cytomery retroviral infection T cell Efficiency.

Fig. 3 is flow cytomery each target cell surface CD123 and CLL-1 expression.

Fig. 4 is that the CLL-1-BBz-tEGFR CART cell for preparing 5 days and each target cell co-culture 5 hours CD107a tables It reaches.

Fig. 5 is that the CLL-1-BBz-tEGFR CART cell for preparing 5 days and each target cell co-culture 5 hours INF- γ Secretion.

Fig. 6 is to prepare 5 days CLL-1-BBz-tEGFR CART cells and after the co-cultivation of each target cell 5 hours to tumour The lethal effect of cell.

Specific embodiment

The present invention provides a kind of Chimeric antigen receptor (CAR) for targeting CLL-1.The CAR contains sequentially connected anti-CLL- 1 single-chain antibody, people CD8 hinge area, people CD8 transmembrane region, people 41BB intracellular region, people's CD3 ζ intracellular region and optional EGFR contain The segment of extracellular domain III and extracellular domain IV.

Various anti-CD8 monoclonal antibodies well known in the art can be derived from by being suitable for the invention anti-CD8 single-chain antibody.

Optionally, the light chain variable region and heavy chain variable region can be linked together by joint sequence.In certain implementations In scheme, the monoclonal antibody is the monoclonal antibody that clone number is M26.In certain embodiments, the anti-CLL-1 The amino acid sequence of the heavy chain variable region of single-chain antibody such as SEQ ID NO:Shown in 2 22-141 amino acids residue.At it In its embodiment, the amino acid sequence such as SEQ ID NO of the light chain variable region of the anti-CLL-1 single-chain antibody:The of 2 Shown in 157-264 amino acids residue.

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

Being suitable for the invention 41BB can be the various 41BB for CAR known in the art.As exemplary example Son, the present invention use SEQ ID NO:41BB shown in 2 334-381 amino acids sequences.

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 such as SEQ ID NO of the people CD3 ζ intracellular region:2 382-492 amino acids It is shown.

The each part mentioned above of fusion protein of the invention is formed, such as the light chain variable region and again of anti-382-492 single-chain antibody Chain variable region, people CD8 hinge area, people CD8 transmembrane region, 41BB and people's CD3 ζ intracellular region etc., can be directly connected between each other, or Person can be connected by joint sequence.Joint sequence can be the joint sequence suitable for antibody well known in the art, such as containing G With the joint sequence of S.In general, connector contains duplicate motif before and after one or more.For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG.Preferably, which is adjacent in joint sequence, without inserting between repetition Enter amino acid residue.Joint sequence may include 1,2,3,4 or 5 repetition motif composition.The length of connector can be 3~25 A amino acid residue, such as 3~15,5~15,10~20 amino acid residues.In certain embodiments, joint sequence is More glycine linlcers sequences.The quantity of glycine is not particularly limited in joint sequence, and usually 2~20, such as 2~15,2 ~10,2~8.Except glycine and serine come, other known amino acid residue, such as alanine are also contained in connector (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q) etc..Certain In embodiment, by (GGGGS) between the light chain variable region and heavy chain variable region of the anti-CLL-1 single-chain antibody of the present invention_nConnection, The wherein integer that n is 1~5.

It in certain embodiments, also include containing for EGFR as described below in the amino acid series of CAR of the invention The segment of extracellular domain III and extracellular domain IV, signal peptide and joint sequence.

It should be understood that in gene cloning operation, it is often necessary to design suitable restriction enzyme site, this certainly will be expressed Amino acid sequence end introduces one or more incoherent residues, and this has no effect on the activity of aim sequence.For structure Fusion protein, the expression for promoting recombinant protein are built, the recombinant protein being secreted into outside host cell automatically is obtained or is conducive to recombination The purifying of albumen, it is often necessary to be added to some amino acid other in the end N-, the end C- or the albumen of recombinant protein In appropriate area, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extend etc..Therefore, this hair The aminoterminal or c-terminus of bright fusion protein (the i.e. described CAR) can also be containing one or more polypeptide fragments, as albumen mark Label.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 albumen into Row purifying.

The present invention also includes SEQ ID NO:CAR, SEQ ID NO shown in 2 21-875 amino acids sequences:2 CAR shown in 21-875 amino acids sequence, SEQ ID NO:CAR or SEQ ID shown in 2 1-613 amino acids sequences NO:The mutant of CAR shown in 2.These mutant include：With the CAR at least 80%, preferably at least 85%, preferably extremely Few 90%, preferably at least 95%, preferably at least 97% sequence identity simultaneously retains the biological activity of the CAR (such as activation T Cell) amino acid sequence.The sequence identity between the sequence of BLASTp two comparisons of calculating of such as NCBI can be used.

Mutant further includes：In SEQ ID NO:2 amino acid sequences, SEQ ID NO shown in 21-875:2 Amino acid sequence shown in 21-875, SEQ ID NO:2 amino acid sequence or SEQ ID NO shown in 1-875:2 institutes With one or several biology for being mutated (insertion, deletion or substitution) while still retaining the CAR in the amino acid sequence shown Active amino acid sequence.Several mutation are often referred within 1-10, such as 1-8,1-5 or 1-3.It takes Generation preferably conservative replaces.For example, in the art, when carrying out conservative replaces with amino acid similar in performance, The function of protein or polypeptide is not usually changed." amino acid similar in performance " includes for example, having similar side chain Amino acid residue family, these families include have basic side chain amino acid (such as lysine, arginine, group ammonia Acid), amino acid (such as aspartic acid, glutamic acid), the amino with uncharged polar side chain with acid side-chain Sour (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) has nonpolar side Amino acid (such as alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, the color ammonia of chain Acid), the amino acid with β-branched building block amino acid (such as threonine, valine, isoleucine) and with aromatic side chain (such as tyrosine, phenylalanine, tryptophan, histidine).Therefore, with from the another of the same side chain class in polypeptide of the present invention Amino acid residue 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 with It is DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded Or double-strand.DNA can be coding strand or noncoding strand.The present invention also includes the polynucleotide sequence of encoding fusion protein Degeneracy variant, that is, encode 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 according to this paper institute Disclosed nucleotide sequence, especially open reading frame sequence carry out design primer, and with the commercially available library cDNA or press this field skill The library cDNA prepared by conventional method known to art personnel expands as template and obtains related sequence.When sequence is longer, often It often needs to carry out twice or repeatedly PCR amplification, then the segment that each time amplifies is stitched together by proper order again.Example Such as, in certain embodiments, the polynucleotide sequence such as SEQ ID NO of fusion protein described herein is encoded:1 60- Shown in 1218 nucleotide, or such as SEQ ID NO:Shown in 1 1-1218 nucleotide.

In certain embodiments, polynucleotide sequence of the invention also includes the nucleotides sequence for encoding the segment of EGFR Column.

Being suitable for the invention EGFR can be EGFR well known in the art, such as the EGFR from people.EGFR contains N Terminal, extracellular domain I and II, extracellular domain III, extracellular domain IV, transmembrane region, membrane-proximal region structural domain and tyrosine Kinase domain.Present invention preferably uses truncated EGFR (" tEGFR ", i.e., the segments of EGFR as described herein), especially not Truncated EGFR including its intracellular region (membrane-proximal region structural domain and tyrosine kinase domain).In certain embodiments, It can also further will not include that be further truncated to not include extracellular domain I and II by the EGFR of intracellular region.Therefore, exist In certain embodiments, the tEGFR that the present invention uses contains the extracellular domain III, extracellular domain IV and cross-film of EGFR Area, or be made of the extracellular domain III, extracellular domain IV and transmembrane region of EGFR.In certain embodiments, described TEGFR contains the 310-646 amino acids sequence of Human epidermal growth factor receptor, or is made of the 310-646 amino acids sequence of Human epidermal growth factor receptor, Wherein 310-480 amino acids sequence is the extracellular domain III of Human epidermal growth factor receptor, and 481-620 is the extracellular structure of Human epidermal growth factor receptor Domain IV, 621-646 amino acids are the transmembrane region of Human epidermal growth factor receptor.In certain embodiments, the amino acid sequence of the tEGFR Extracellular domain III and IV such as SEQ ID NO:Amino acid sequence shown in 2 541-875 amino acids.

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 be in many ways It is operable to guarantee the expression of the fusion protein (CAR and/or tEGFR).It can root before nucleic acid constructs is inserted into carrier Nucleic acid constructs is operated according to the difference or requirement of expression vector.Change polynucleotides sequence using recombinant DNA method The technology of column is known in the art.

Regulating and controlling sequence can be suitable promoter sequence.Coded sequence of the promoter sequence usually with albumen to be expressed It is operatively connected.Promoter can be any nucleotide sequence that transcriptional activity is shown in selected host cell, including Mutation, truncated and hybrid promoter, and can be from coding and homologous or heterologous extracellular or intracellular more of the host cell The gene of peptide obtains.Regulating and controlling sequence is also possible to suitable transcription terminator sequences, is identified by host cell to terminate transcription Sequence.Terminator sequence is connect with 3 ' end effectors of the nucleotide sequence for encoding the polypeptide.In the host cell of selection Functional any terminator can be used in the present invention.Regulating and controlling sequence is also possible to suitable leader sequence, turns over to host cell Translate the non-translational region of important mRNA.5 ' ends of leader sequence and the nucleotide sequence for encoding the polypeptide are operatively connected.? Functional any terminator can be used in the present invention in the host cell of selection.

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 load Body 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.

Polynucleotide sequence of the invention can be cloned into the carrier of many types.For example, plasmid can be cloned into, bitten Bacterium grain, phage-derived object, animal virus and clay.Further, carrier is expression vector.Expression vector can be with virus Carrier format 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 Virus, adenovirus, adeno-associated virus, herpesviral and slow virus.

In general, suitable carrier includes the replication orgin, promoter sequence, side to work at least one organism Just the selectable label of restriction enzyme sites and one or more is (for example, WO 01/96584；WO01/29058；It is special with the U.S. Benefit number is 6,326,193).

For example, in certain embodiments, the present invention uses retroviral vector, which contains Replication origin, 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 Promoter sequence, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer viral (MMTV), people exempt from The long end of epidemic disease defective virus (HIV) repeats (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, EB disease Malicious early promoter, Rous sarcoma virus promoter and people's gene promoter, such as, but not limited to actin immediately Promoter, Myosin promoter, ferroheme promoter and creatine kinase promoter.Further, also it is contemplated that using induction Type promoter.The use of inducible promoter provides molecular switch, can open when expressing in the time limit and operationally connect The expression of the polynucleotide sequence of inducible promoter is connect, and is closing expression when expression is undesirable.Induction type starting The example of son includes but is not limited to metallothionein promoter, Glucocorticoid promoter, progesterone promoter and tetracycline starting Son.

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 for seeking to be transfected or infect by viral vectors Expression cell is identified and selected in group.In other respects, selectable label can be carried on independent section of DNA and be used for Cotransfection program.The flank of selectable label and both reporters can all have adjusting sequence appropriate, so as to It is expressed in host 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 Gene.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 pass through Any method in the art is easily introduced into host cell, for example, mammal, bacterium, yeast or insect cell.Example Such as, expression vector can be transferred to host cell by physics, chemistry or biological means.

By polynucleotides introduce host cell physical method include calcium phosphate precipitation, lipofection, particle bombardment, Microinjection, electroporation etc..It include using DNA and RNA by the biological method that interested polynucleotides introduce host cell Carrier.It include dispersion system of colloid by the chemical means that polynucleotides introduce host cell, such as macromolecular complex, nanometer 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 by the biological method that polynucleotides introduce host cell Carrier, this has become the most widely used method by gene insertion mammal such as people's cell.Other viral vectors can From slow virus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus etc..It has developed many based on disease The system of poison, for gene transfer to be entered mammalian cell.For example, retrovirus is provided for gene delivery system Convenient platform.By the gene insertion vector of selection and retrovirus is packaged into using technology as known in the art Particle.The recombinant virus then can be separated and be transferred to internal or external subject cell.Many retroviral systems exist It is known in this field.In some embodiments, using adenovirus vector.Many adenovirus vectors are in the art It is known.In one embodiment, using slow virus carrier.

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 source In 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, and the T of the gene modification is thin Born of the same parents contain polynucleotide sequence as described herein, or contain retroviral vector as described herein, or infected described herein Retrovirus, or be prepared using method described herein, or stablize and express fusion protein as described herein and optionally TEGFR.

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 appointed TEGFR the and CAR-T cell of choosing is needed by injection in its recipient.The tumour that the cell of injection can kill recipient is thin Born of the same parents.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 thereof in CAR The immune response of specificity.

Therefore, CAR of the invention, its coded sequence, nucleic acid constructs, expression vector, virus and CAR-T can be used The disease of cell therapy is preferably the disease that CLL-1 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 number of application Amount and frequency will be determined by such factor, 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：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) it applies.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 Injection is administered to patient.In another embodiment, T cell composition of the invention preferably passes through intravenous injection application. The composition of T cell 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 CLL-1 mediate disease radiotherapy or chemotheraping preparation treated.

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

" 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 beneficial effects of the invention are as follows：The present invention uses CLL-1 scFV gene order, and from NCBI GenBank data People CD8 hinge area, people CD8 transmembrane region, people 41BB intracellular region and people's CD3 ζ intracellular region and people are searched in library Truncated EGFR (tEGFR) gene sequence information, full genome synthesize Chimeric antigen receptor CLL-1 scFV-41BB-CD3 The genetic fragment of ζ-tEGFR is inserted into retroviral vector RV, can be used for recombinating introducing purpose nucleic acid sequence, that is, compiles The nucleic acid sequence of code CAR.Recombinant plasmid packaging virus in 293T cell infects T cell, T cell is made to express the inosculating antibody Original receptor.In one embodiment of the invention, the conversion side of the T lymphocyte of Chimeric antigen receptor gene modification is realized Method is based on Retroviral Transformation method.This method has high conversion efficiency, and foreign gene can stablize expression, and can be with Shorten the advantages that in vitro culture T lymphocyte reaches the time of clinical number of stages.On the transgenosis T lymphocyte surface, conversion Nucleic acid by transcription, accurate translation on it.The retrovirus of present invention expression CAR obtained passes through Retronectin method prepares CAR-T cell, the efficiency of infection of the CAR-T cell flow cytometer detection CAR after preparation 3 days, preparation 5 CAR-T cell co-cultures 5 hours with CLL-1 or the tumour cell (HL-60, MOLM-13) of the CD123 positive examine in vitro after it The secretion of CD107a expression and IFN γ is surveyed, CAR-T cell is thin with the tumour of CLL-1 or the CD123 positive in vitro after preparation 5 days Born of the same parents (HL-60, MOLM-13) co-culture the specific killing action (cell toxicant of 5 hours method detection CAR-T cells against tumor cells Property).Therefore CLL-1-BBz-tEGFR CART of the present invention can be applied in marrow series leukemia.

The present invention is described in further detail by reference to following EXPERIMENTAL EXAMPLE.These embodiments are merely for saying The purpose of bright property provides, 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 Following embodiment, but should be interpreted as including since introduction provided herein will become apparent from any and whole change Change.Method used in embodiment and reagent, unless otherwise stated, being the method and reagent of this field routine.

Embodiment 1：The determination of CLL-1 scFV-41BB-CD3 ζ-tEGFR gene order

1.1 search people CD8 hinge area, people CD8 transmembrane region, people 41BB intracellular region, Yi Jiren from NCBI site databases Truncated EGFR (tEGFR) gene order of CD3 ζ intracellular region and people, anti-CLL-1 single-chain antibody clone number is M26, this A little sequences are in websitehttp://sg.idtdna.com/siteUpper carry out codon optimization, guarantee encoding amino acid sequence not It is more suitable for human cell's expression in the case where change.Each amino acid and gene sequence information are shown in SEQENCE LISTING (SEQUNCE ID NO.1-2)。

Above-mentioned sequence is successively attached, different restriction enzyme sites is introduced in each sequence junction, forms complete CLL- 1 scFV-41BB-CD3 ζ-tEGFR gene sequence information.

The sequencing of 1.2 recombinant plasmids

Recombinant plasmid is served Hai Sheng work Bioisystech Co., Ltd be sequenced, by sequencing result be fitted to Whether CLL-1 scFV-41BB-CD3 ζ-tEGFR sequence alignment is correct to verify sequence.Sequencing primer is：

Sense sequences:AGCATCGTTCTGTGTTGTCTC(SEQUNCE ID NO.3)

Antisense sequences:TGTTTGTCTTGTGGCAATACAC(SEQUNCE ID NO.4)

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

Embodiment 2：The building of the viral vectors of nucleic acid sequence comprising CAR molecule

By the nucleotide sequence of the CAR molecule prepared in embodiment 1 through NotI (NEB) and EcoRI (NEB) double digestion, warp The site NotI-EcoRI of T4 ligase (NEB) connection insertion retrovirus RV carrier, is transformed into competence E.coli (DH5 α), after being sequenced correctly, the plasmid purification kit of Qiagen company is used to extract simultaneously plasmid purification, the plasmid of plasmid purification Calcium phosphate method transfects 293T cell and carries out retrovirus Packaging experimentation.

Embodiment 3：Retrovirus packaging

1. 293T cell should be less than for 20 generations in the 1st day, do not cover with excessively.With 0.6*10^6cells/ml bed board, 10cm Ware adds the DMEM culture medium of 10ml, mixes well cell, 37 degree of overnight incubations；2. 293T cell fusion degree reached in the 2nd day 90% or so is transfected (usually bed board 14-18h or so)；Prepare plasmid composite, the amount of various plasmids is RV-CLL-1 ScFV-41BB-CD3 ζ-tEGFR is 12.5ug, Gag-pol 10ug, VSVg 6.25ug, CaCl₂250ul, H₂O is 1ml Total volume is 1.25ml；The HBS isometric with plasmid composite is added in another pipe, is vortexed when adding plasmid composite Shake 20s.Softly mixture being added in 293T ware, 37 degree of culture 4h along side, removes culture medium, PBS is washed one time, Rejoin the fresh culture of preheating；

3. the 4th day：Supernatant is collected after transfection 48h and is stored in -80 degree with packing after the filtering of 0.45um filter, continues to add Add the fresh DMEM medium of preheating.

Embodiment 4：The T cell of retroviral infection people

1. purer CD3+T cell is obtained with Ficcol separating liquid (ocean Tianjin Hao) separation, with the X- of serum containing 5%AB It is 1 × 10 that VIVO (LONZA) culture medium, which adjusts cell density,⁶/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 41BB antibody (Beijing is with vertical Hai Yuan), add the white of 100IU/ml Cytokine 2 (the double aigrets in Beijing), restrovirus infection in 48 hours is cultivated in stimulation.

After 2.T cell activation culture every other day, PBS is diluted to the Retronectin (Takara) of final concentration of 15 μ g/ml It is coated with non-tissue treated (corning) 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 and contains 2%BSA HBSS room temperature close 30min.Confining liquid volume is every 500 μ l of hole, inhales and abandons confining liquid, is washed with the HBSS containing 2.5%HEPES Plate is twice.

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

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

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

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

8. after cell infection, observing the density of cell daily, adding the T cell culture of the 100IU/ml containing IL-2 in due course Liquid makes the density of T cell maintain 5 × 10⁵/ ml or so, expands cell.

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

72 hours after infecting CLL-1 scFV-41BB-CD3 ζ-tEGFR cells are collected by centrifugation respectively, PBS is washed 1 time After abandon supernatant, corresponding antibody be added be protected from light PBS after 30min and wash, be resuspended, last flow cytomery.CAR+ is by APC- Anti EGFR (Biolegend) antibody test.

The present embodiment result as shown in Fig. 2, CLL-1 scFV-41BB-CD3 ζ-tEGFR CART positive rate 30% with On.

Embodiment 6：Detect each target cell CLL-1 and CD123 expression

HL-60, MOLM-13, K562 dye anti-CLL-1 and anti-CD123 simultaneously, in streaming machine testing CD123 and CLL-1 expression.

The present embodiment result is as shown in figure 3, HL-60 cell CLL-1 expression quantity is higher than CD123, MOLM-13 cell CD123 Expression quantity is higher than CLL-1.

Embodiment 7：CD107a detection of expression after 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 (HL-60, MOLM-13)/it is right Photo cell (K562) 2*10⁵It is a, the X-VIVO complete medium for being free of IL-2 for 200ul is resuspended, BD GolgiStop is added (containing monesin, 1 μ l BD GolgiStop is added in every 1ml culture medium), 2ul CD107a antibody (1 is added in every hole:50), 37 DEG C are incubated for 4 hours, collect 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 CAR, CD3, 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.

Appropriate PBS is resuspended, flow cytomery CAR, CD3, CD107a.

The present embodiment result is as shown in figure 4, CLL-1 scFV-41BB-CD3 ζ-tEGFR CART cell and target cell HL- 60 co-culture CD107a expression 50% or so；CD107a expression is co-cultured 10% or so with target cell (MOLM13)；With it is right Photo cell (K562) is then almost without CD107a expression.

Embodiment 8：IFN γ secretion detects after 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 (HL-60, MOLM-13) or negative control cell (K562) 2 × 10⁵It is a, CAR- T/NT cell 2 × 10⁵A, 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 GolgiStop (contains monesin, 1 μ l BD GolgiStop is added in every 1ml culture medium), and after mixing well, 37 DEG C of incubations 5 are small When.Cell is collected, as experimental group.

3. the PBS per effective 1mL is cleaned cell 1 time, 300g is centrifuged 5 minutes.Supernatant is abandoned, appropriate specificity is added in every pipe Surface antibody CAR, CD3, resuspension volume 100ul are protected from light incubation 30 minutes on ice.

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 CAR, CD3, IFN-γ.

The present embodiment result is as shown in figure 5, CLL-1 scFV-41BB-CD3 ζ-tEGFR CART cell and target cell HL- 60 co-culture IFN-γ expression 20% or so；IFN-γ expression is co-cultured 5% or so with target cell (MOLM13)；With it is right Photo cell (K562) is then almost without IFN-γ expression.

Embodiment 9：CAR-T cell and target cell detect tumor specific cell lethal effect after co-culturing

1.K562 cell (being free of CD123 or CLL-1, be negative control cell) is resuspended in serum free medium (1640) In, adjustment cell concentration is 1 × 10⁶Fluorescent dye BMQC (2,3,6,7-tetrahydro-9- is added in/ml 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, density 1 ×10⁶/ml。

5.HL-60, MOLM-13 cell (containing CD123 or CLL-1, be target cell) are suspended in the PBS containing 0.1%BSA In, adjustment concentration is 1 × 10⁶/ml。

6. fluorescein based dye CFSE (carboxyfluoresceindiacetatesuccinimidyl ester) is added extremely Final concentration of 1 μM.

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

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

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

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

11. having infected the effect T cell of CLL-1 scFV-41BB-CD3 ζ-tEGFR CAR in all experiments The cytotoxicity of (CAR-T cell) and the cytotoxicity of the negative control effector T cell being uninfected by (NT cell) compare, And these effector T cells come from the same patient.

12.CLL-1 scFV-41BB-CD3 ζ-tEGFR CAR-T and negative control effector T cell, according to T cell：Target Cell=5:1,1:1, ratio, cultivated in 5ml sterility test pipe (BD Biosciences).Each co-cultivation group In, target cell is 100,000, Raji cell (50 μ l), 100,000 K562 cell (50 μ l) of negative control cell.Together When to be arranged one group only include Raji 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, the target cell to live after T cell and target cell co-cultivation is measured With the ratio of negative control cell living.

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

The present embodiment result is as shown in fig. 6, effect target ratio E:T=5:When 1, CLL-1 scFV-41BB-CD3 ζ-tEGFR CART is both greater than 70% to target cell (HL-60) killing rate；To target cell (MOLM-13) killing rate all 10% or so.Show CLL-1 scFV-41BB-CD3 ζ-tEGFR CART has manifest function to the target cell of expression CLL-1.

Sequence table

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

<120>Target CLL-1 Chimeric antigen receptor and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2625

<212> DNA

<213>Artificial sequence (Homo sapiens)

<400> 1

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgaggtcc agctgcagca gtctggacct gagctggtaa agcctggggc ttcagtgaag 120

atgtcctgca aggcttctgg atacacattc actagctatt ttatacactg ggtgaagcag 180

aagcctggac agggccttga gtggattgga tttattaatc cttacaatga tggttctaag 240

tacaatgaga agttcaaagg caaggccaca ctgacttcag acaaatcctc cagcacagcc 300

tacatggagc tcagcagcct gacctctgaa gactcagcgg tctattactg tacaagagat 360

gatggttatt acggctatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420

tcaggcggcg ggggttctgg tggcggcggc agcggcggtg gaggatcaga catccagatg 480

acccagtctc catcctcctt atctgcctct ctgggagaaa gagtcagtct cacttgtcgg 540

gcaactcagg aacttagtgg ttacttaagc tggcttcagc agaaaccaga tggaactatt 600

aaacgcctga tctacgccgc atccacttta gattctggtg tcccaaaaag gttcagtggc 660

aataggtctg ggtcagatta ttctctcacc atcagcagcc ttgagtctga agattttgca 720

gactattact gtctacaata tgctatttat ccgtacacgt tcggaggggg gaccaagctg 780

gaaataaaac ggactacaac 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 tcccatgttg 1560

ctccttgtga cgagcctcct gctctgcgag ctgccccatc cagccttcct cctcatcccg 1620

cggaaggtgt gcaatggcat aggcattggc gagtttaaag attctctgag cataaatgct 1680

acgaatatta agcatttcaa gaattgtact tctattagtg gcgacctcca tattcttccg 1740

gttgccttca ggggtgactc tttcacccac acacctccat tggatccaca agaacttgac 1800

atcctgaaga cggttaaaga gattacaggc ttcctcctta tccaagcgtg gcccgagaac 1860

agaacggact tgcacgcctt tgagaacctc gaaataatac ggggtcggac gaagcaacac 1920

ggccaattta gccttgcggt tgttagtctg aacattactt ctctcggcct tcgctctttg 1980

aaagaaatca gcgacggaga tgtcatcatt agtggaaaca agaacctgtg ctacgcgaac 2040

acaatcaact ggaagaagct cttcggtact tcaggccaaa agacaaagat tattagtaac 2100

agaggagaga atagctgtaa ggctaccgga caagtttgtc acgccttgtg tagtccagag 2160

ggttgctggg gaccggaacc aagggattgc gtcagttgcc ggaacgtgag tcgcggacgc 2220

gagtgtgtgg ataagtgcaa tcttctggaa ggggaaccgc gagagtttgt agaaaattcc 2280

gaatgtatac agtgtcatcc cgagtgtctt ccacaagcaa tgaatatcac atgtacaggg 2340

aggggtcctg ataactgtat ccaatgtgca cactacatag atggtcctca ctgtgtaaag 2400

acgtgccccg ccggagtaat gggtgaaaac aacaccctcg tgtggaagta cgccgatgcc 2460

gggcatgtct gtcatttgtg tcatcccaac tgcacatatg gctgtaccgg tcctggattg 2520

gagggctgtc caacaaacgg gccgaaaata ccgagtatcg caacaggcat ggtgggagca 2580

cttttgcttc tcctcgttgt cgccctgggc atcggcttgt tcatg 2625

<210> 2

<211> 875

<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 Gly Val Gly Leu Gly Gly Ser Gly Pro Gly Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ala Val Thr Thr Cys Thr Ala Ala Ala Gly Thr Thr Gly Thr Ala Met

115 120 125

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

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gly Met

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

His Ala Pro Gly Ala Leu Gly Ile Ile Ala Gly Ala Thr Leu Gly His

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Pro Ala Gly Pro Val Gly Ala Ser Gly Cys Ile Gly Cys His Pro Gly

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

Leu Val Val Ala Leu Gly Ile Gly Leu Pro Met

865 870 875

<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-CLL-1 single-chain antibody, the coded sequence of people's CD8 hinge area, people CD8 across The coded sequence in film area, the coded sequence of people's 41BB intracellular region, the coded sequence of people's CD3 ζ intracellular region and optional EGFR contain The polynucleotide sequence of the coded sequence of the segment of extracellular domain III and extracellular domain IV；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 such as SEQ ID NO of the CD8 signal peptide before the coded sequence of the anti-CLL-1 single-chain antibody:1 Shown in 1-63 nucleotide sequences；And/or

The coded sequence such as SEQ ID NO of the heavy chain variable region of the anti-CLL-1 single-chain antibody:1 64-423 nucleotides sequences Shown in column；And/or

The coded sequence such as SEQ ID NO of the light chain variable region of the anti-CLL-1 single-chain antibody:1 469-792 nucleotides sequences Shown in column；And/or

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

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

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

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

The coded sequence of the segment of the EGFR such as SEQ ID NO:1 1621-2625

Shown in the nucleotide sequence of position.

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

(1) contain sequentially connected anti-CLL-1 single-chain antibody, people CD8 hinge area, people CD8 transmembrane region, people 41BB intracellular region and people The code sequence of the segment of the fusion protein of CD3 ζ intracellular region and the III containing extracellular domain of optional EGFR and extracellular domain IV Column；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-CLL-1 single-chain antibody is anti-CLL-1 monoclonal antibody M26.

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 CD8 signal peptide in the N-terminal of the anti-CLL-1 single-chain antibody, it is preferable that the signal peptide Amino acid sequence such as SEQ ID NO:Shown in 2 1-21 amino acids；

The amino acid sequence such as SEQ ID NO of the heavy chain variable region of the anti-CLL-1 single-chain antibody:2 22-141 amino acids It is shown；

The amino acid sequence of the light chain variable region of the anti-CLL-1 single-chain antibody can be such as SEQ ID NO:2 157-264 bit aminos Shown in acid；

The amino acid sequence of the people CD8 hinge area such as SEQ ID NO:Shown in 2 265-311 amino acids；

The amino acid sequence of the people CD8 transmembrane region such as SEQ ID NO:Shown in 2 312-333 amino acids；

The amino acid sequence of the people 41BB intracellular region such as SEQ ID NO:Shown in 2 334-381 amino acids；

The amino acid sequence such as SEQ ID NO of the people CD3 ζ intracellular region:Shown in 2 382-492 amino acids；With

The segment of the EGFR contains the extracellular domain III, extracellular domain IV and transmembrane region of EGFR, or the born of the same parents by EGFR Extracellular portion III, extracellular domain IV and transmembrane region composition；Preferably, the segment contains 310-646 of Human epidermal growth factor receptor Amino acid sequence, or be made of the 310-646 amino acids sequence of Human epidermal growth factor receptor；It is highly preferred that the amino acid sequence of the segment Column such as SEQ ID NO:Shown in 2 541-875 amino acids.

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 With the segment of the I containing extracellular domain II of optional EGFR, extracellular domain IV and optional transmembrane region.

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 CLL-1 disease mediated；

Preferably, the disease that the CLL-1 is mediated is marrow series leukemia.