CN109336980A

CN109336980A - A kind of Chimeric antigen receptor modification T cell and application thereof targeting Muc1

Info

Publication number: CN109336980A
Application number: CN201710623293.2A
Authority: CN
Inventors: 钱其军; 金华君; 何周; 王超; 李林芳
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2017-07-27
Filing date: 2017-07-27
Publication date: 2019-02-15
Anticipated expiration: 2037-07-27
Also published as: WO2019020087A1; CN109336980B

Abstract

The present invention relates to a kind of Chimeric antigen receptor modification T cells and application thereof for targeting Muc1.Specifically, Chimeric antigen receptor provided by the invention is from N-terminal to C-terminal successively containing hinge area, transmembrane region, costimulatory signal molecule intracellular domain and immunoreceptor tyrosine activating motif more than memebrane protein signal peptide, the single-chain antibody at the nearly film end anti-Muc1, long 50 amino acid residues.The invention also includes the T cell for expressing Chimeric antigen receptor described herein, which can identify and combine the nearly film end of Muc1 antigen, and by efficient activation and proliferation, secrete cytokines kill the tumour cell of the Muc1 positive.T cell provided by the invention can be used for the treatment of a variety of Muc1 positive malignancies.

Description

A kind of Chimeric antigen receptor modification T cell and application thereof targeting Muc1

Technical field

The invention belongs to genetic engineerings and oncology, are related to a kind of Chimeric antigen receptor of selectively targeted Muc1 antigen Modified cells and its antitumous effect.

Background technique

Treatment technology is undoubtedly for Chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) It is a superstar to rise up slowly in tumor vaccine cells therapy field.CAR-T technology will be identified by technique for gene engineering After the antibody variable gene sequence of certain antigen molecule and the intracellular region sequence assembly of T lymphocyte immunity receptor, pass through reverse In record virus or slow virus carrier, transposons or transposase system or direct mRNA transduction to lymphocyte, and express fusion egg It is white so that T lymphocyte is identified specific antigen by the restrictive mode of non-MHC in cell surface, enhance its identification and killing The ability of tumour.

After the structure of CAR is put forward for the first time from Eshhar research group of Israel in 1989, by nearly development in 30 years, It is proved have having a better effect in immunotherapy of tumors by the T cell of CAR structural modification.First generation CAR receptor includes The segment of extracellular specific recognition tumour antigen (single-chain variable fragment, scFv), activation letter intracellular It number is transmitted by CD3 ζ signal chains.But the costimulatory signal of first generation CAR receptor deficiency T cell, cause T cell that can only play Moment effect, has that the time is short, cytokine secretion is few in vivo.Second generation CAR receptor increases costimulatory signal molecule Intracellular domain, including such as CD28, CD134/OX40, CD137/4-1BB, Lymphocyte-specific protein-tyrosine kinase (LCK), the structural domains such as derivable T cell costimulation agent (ICOS) and DNAX activating protein 10 (DAP10), it is thin to enhance T The proliferative capacity of born of the same parents and the secreting function of cell factor, IL-2, IFN-γ and GM-CSF increase, to break through tumor microenvironment Immunosupress, extend AICD (cell death of activation-inducing, AICD).Third generation CAR receptor is then in costimulation structure A second level costimulatory molecules such as 4-1BB is merged between CD28 and ITAM signal chains again, thus produces a triple signals The T cell of CAR receptor, the transformation of third generation CAR receptor has better effector function and internal time-to-live.It is common classical CAR-T structure is two generation CAR receptors, and structure particularly may be divided into following 4 part: identifying that the single chain antibody of tumour antigen is variable Area (scFv), hinge area, transmembrane region, stimulus signal structural region intracellular.Wherein, CAR structure hinge area is responsible for forming correct structure As forming dimer.The length and amino acid sequence feature of hinge area determine the space conformation of CAR, also determine it and swell The ability that oncocyte surface antigen combines.

Solid tumor has the heterogeneity of height, different patients, same patient's difference lesion, same lesion difference tumour cell Between there is the difference of height.The heterogeneity of this height causes neoplasm targeted therapy to lack ideal general, wide spectrum target spot, Limit the curative effect of CAR-T cell therapy solid tumor.Thus, finding effective CAR-T cell therapy target spot already becomes CAR-T The most important thing of cell therapy.Muc1 (mucins, mucoprotein) is I type transmembrane glycoprotein of a kind of high molecular weight (> 200kD) (being mostly connected with O-glycosides key with the Ser/Thr on polypeptide backbone), be mainly expressed in Various Tissues under normal circumstances, in organ on The nearly lumen of chrotoplast or lumen of gland face are expressed, polarity distribution in top.When tumour occurs, Muc1 albumen can be in tumour cell table Face unconventionality expression, expression quantity up to it is normal when 100 times or more.Also, it loses in the polarity distribution of cell surface, can be whole A cell surface is uniformly distributed.In addition, incomplete due to glycosylating, the structure of Muc1 albumen also changes, and new sugar chain occurs And peptide epitopes.Although these new sugar chains and peptide epitopes for occurring can become the highly desirable target spot of CAR-T cell therapy, by In that can show different sugar chain and peptide epitopes in different tumor cell surfaces, the CAR-T of certain selectively targeted glycopeptide is thin Perhaps, born of the same parents cannot be applied in a plurality of types of tumours.

Summary of the invention

The extracellular region of Muc1 antigen is one-stage serial repetitive sequence, and N-terminal can be sheared, and close to the piece of cell membrane surface Duan Buhui is cut, thus can be existed in the highly expressed tumor cell surface of any Muc1 antigen, and CAR-T can be become The suitable target spot of cell therapy.Since this epitope is present in nearly film end, the present invention devises 2 kinds of different hinge lengths CAR structure, one is CD8 hinge areas, and the cross-film hinge area to be formed is combined with CD8 transmembrane region, and one is by IgG4Fc CH2CH3 Hinge area combines the cross-film hinge area to be formed with CD8 transmembrane region.IgG4Fc segment is easy by monokaryon/huge as present on CAR Phagocyte identifies and T cell AICD is caused to react, and the present invention is transformed with full IgG4Fc CH2CH3 hinge area on CAR The requirement that foot CAR segment of the present invention not only can be very good identification antigen but also AICD is not caused to react, to reach good identification Thus antigen and killing functions of immunocytes complete the present invention.

Therefore, provided herein is a kind of IgG4Fc CH2CH3 hinge area, the amino acid sequence of the hinge area such as SEQ ID Shown in NO:5.

The coded sequence of the IgG4Fc CH2CH3 hinge area, including its complementary series is also provided herein.

In one or more embodiments, the coded sequence of IgG4Fc CH2CH3 hinge area such as SEQ ID NO:12 Shown in 796-1479 bit base.

A kind of Chimeric antigen receptor (CAR) is also provided herein, from N-terminal to C-terminal, which successively contains signal Peptide, anti-Muc1 single-chain antibody, the hinge area for growing 50 amino acid residues or more, transmembrane region, costimulatory signal molecule structure intracellular Domain and immunoreceptor tyrosine activating motif.

In one or more embodiments, the signal peptide is memebrane protein signal peptide.

In one or more embodiments, the signal peptide is selected from CD8 signal peptide, CD28 signal peptide and CD4 signal peptide.

In one or more embodiments, the signal peptide is CD8 signal peptide, amino acid sequence such as SEQ ID NO: Shown in 1.

In one or more embodiments, the anti-Muc1 single-chain antibody is the special of the nearly film terminal amino acid sequence of Muc1 Property antibody.

In one or more embodiments, the amino acid sequence at the nearly film end the Muc1 is as shown in SEQ ID NO:10.

In one or more embodiments, the amino acid sequence of the anti-Muc1 single-chain antibody such as SEQ ID NO:3 institute Show.

In one or more embodiments, the hinge area is CD8 α hinge area, amino acid sequence such as SEQ ID Shown in NO:4.

In one or more embodiments, the amino acid sequence of the IgG4Fc CH2CH3 hinge area such as SEQ ID Shown in NO:5.

In one or more embodiments, the transmembrane region be selected from CD28 transmembrane region, CD8 transmembrane region, CD3 ζ transmembrane region, One of CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region are a variety of.

In one or more embodiments, the transmembrane region is CD8 transmembrane region, and amino acid sequence is preferably such as SEQ ID Shown in NO:6.

In one or more embodiments, the costimulatory signal molecule intracellular domain is selected from CD28, CD134/ One of OX40, CD137/4-1BB, LCK, ICOS and DAP10 intracellular domain is a variety of.

In one or more embodiments, the costimulatory signal molecule intracellular domain is CD28 intracellular domain, Its amino acid sequence is as shown in SEQ ID NO:7.

In one or more embodiments, the costimulatory signal molecule intracellular domain is CD137 intracellular domain, Its amino acid sequence is as shown in SEQ ID NO:8.

In one or more embodiments, the immunoreceptor tyrosine activating motif is CD3 ζ and/or Fc ε RI γ's Tyrosine activation motifs.

In one or more embodiments, the immunoreceptor tyrosine activating motif is CD3 ζ tyrosine activation base Sequence, amino acid sequence is as shown in SEQ ID NO:9.

In one or more embodiments, the Chimeric antigen receptor successively contains CD8 signal peptide from N-terminal to C-terminal, resists Muc1 single-chain antibody, CD8 hinge area or IgG4Fc CH2CH3 hinge area, CD8 transmembrane region, CD137 intracellular domain and CD3 ζ Tyrosine activation motifs.

In one or more embodiments, the Chimeric antigen receptor successively contain from N-terminal to C-terminal CD8 signal peptide, The tyrosine work of anti-Muc1 single-chain antibody, IgG4Fc CH2CH3 hinge area, CD8 transmembrane region, CD137 intracellular domain and CD3 ζ Change motif.

In one or more embodiments, the amino acid sequence of the Chimeric antigen receptor such as SEQ ID NO:11 institute Show.

The coded sequence of this paper Chimeric antigen receptor, including its complementary series is also provided herein.

In one or more embodiments, the nucleotide sequence of the coded sequence is as shown in SEQ ID NO:12.

A kind of nucleic acid constructs is also provided herein, the coded sequence containing Chimeric antigen receptor described herein.

In one or more embodiments, the nucleic acid constructs is expression vector, as described herein embedding for expressing Close antigen receptor.

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

In one or more embodiments, the transposon vector contains selected from piggybac, sleeping The transposable element of beauty, frog prince, Tn5 and Ty.

A kind of recombinant host cell is also provided herein, the recombinant host cell contains Chimeric antigen receptor described herein Coded sequence or nucleic acid constructs as described herein.

In one or more embodiments, the host cell is mammalian cell.

In one or more embodiments, the host cell is T cell.

In one or more embodiments, the host cell is originally culture T cell.

In one or more embodiments, the cell expresses chimeric antibody receptor as described herein.

Following purposes are also provided herein:

(1) the IgG4Fc CH2CH3 hinge area or its coded sequence prepare Chimeric antigen receptor described herein or its Purposes in coded sequence；

(2) purposes of the coded sequence of Chimeric antigen receptor described herein in preparation and reorganization expression vector；

(3) purposes of the nucleic acid constructs as described herein in preparation and reorganization host cell；And

(4) purposes of the recombinant host cell as described herein in the drug that preparation treats or prevents cancer.

In one or more embodiments, the cancer is selected from: gland cancer, lung cancer, colon cancer, colorectal cancer, breast cancer, ovum Nest cancer, cervical carcinoma, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, cancer of pancreas or prostate cancer.

A kind of pharmaceutical composition is also provided herein, described pharmaceutical composition contains recombinant host cell as described herein.

Detailed description of the invention

Fig. 1: pNB328-Muc1CAR1, the expression cassette mode of pNB328-Muc1CAR2, pNB328-Muc1CAR3.

Fig. 2: the PBMCs in different patient sources detects Muc1CAR gene expression after Muc1CAR gene modification Western blotting detection figure.

Expression of the genome of Fig. 3: Muc1CAR gene in T cell genome.

Fig. 4: the lethal effect of the Muc1CAR-T cell of different hinge areas compares.

Fig. 5: the killing activity of T cell can be enhanced in the T cell for expressing Muc1CAR3 in vitro.A, B: flow cytometer detection is indirect Reflect the label CD107 α of T cell killing activity, the mark of activation label CD69 and aging label KLRG1 and memory t cell Remember CD62L and CCR7.C, Muc1CAR3T cell and Mock T cell D: are detected by Muc1 antigen by multiple-factor detection kit IL-2 after stimulation, IL-4, IL-6, IL-10, the variation of TNF-α and IFN-γ cell factor.

The internal killing ability testing result of Fig. 6: Muc1CAR3T cell.

Specific embodiment

The present inventor passes through a large amount of preliminary experiment and creative labor, establishes mature CAR expression system, and pass through The screening combination of hinge area and transgenosis modification optimization, building produce the high efficiency stable expression CAR gene of targeting Muc1 antigen And the Muc1CAR-T cell (abbreviation Muc1CAR-T) with killing activity, the T cell can the stable expression CAR bases of high level Cause.The CAR gene of heterogenous expression can accurately target Muc1 antigen, enhance the proliferative capacity of T cell and point of cell factor It secretes, enhances the killing of CAR-T cells against tumor cells, and by enhancing immune response, play antitumor action.External source simultaneously CAR gene can be integrated into the genome of T cell through PB transposase system, thus the steady and sustained expression in T cell.This hair The Muc1CAR-T cell that the high level of bright acquisition stablizes expression CAR gene can be used for the highly expressed malignant tumour of a variety of Muc1 Treatment.

The Chimeric antigen receptor (CAR) of this paper, from N-terminal to C-terminal, successively containing signal peptide, anti-Muc1 single-chain antibody, long 50 More than a amino acid residue hinge area, transmembrane region, costimulatory signal molecule intracellular domain and immunity receptor tyrosine activation Motif.

Signal peptide is the short peptide chain (5-30 amino acid of length) that the newly synthesized protein of guidance is shifted to secretion access, often Refer in new synthesis polypeptide chain for instruct protein transmembrane process (positioning) the end N- amino acid sequence (sometimes not necessarily In N-terminal), it is responsible in subcellular organelle of the protein priming to cell containing different membrane structures.The signal peptide of this paper is memebrane protein Signal peptide can be selected from CD8 signal peptide, CD28 signal peptide and CD4 signal peptide.In preferred embodiments, the present invention uses CD8 Signal peptide.The amino acid sequence of exemplary CD8 signal peptide can be as shown in SEQ ID NO:1.

Single-chain antibody (scFv) refers to by antibody's light chain variable region (V_LArea) amino acid sequence and heavy chain variable region (V_HArea) ammonia Base acid sequence is formed by connecting through hinge, has the antibody fragment in conjunction with antigenic capacity.Anti- Muc1 single-chain antibody as described herein can To be the single-chain antibody for Muc1 antigen well known in the art.Preferably, the chain variable region amino acid of the single-chain antibody Sequence and heavy chain variable amino acid sequence are from the antibody for being directed to the nearly film terminal amino acid sequence of Muc1.In certain embodiments In, the nearly film terminal amino acid sequence of the Muc1 is as shown in SEQ ID NO:10.The amino acid sequence of illustrative anti-Muc1 single-chain antibody Column are as shown in SEQ ID NO:3.

Hinge area refers to the region between the functional areas heavy chain immunoglobulin CHl and CH2, which does not form α Spiral easily occurs stretching, extension and distorts to a certain degree, is conducive to the complementary knot between the antigen-binding site of antibody and epitope It closes.Hinge area suitable for this paper can be selected from the extracellular hinge area of CD8, IgG1Fc CH2CH3 hinge area, IgD hinge area, CD28 Extracellular hinge area, IgG4Fc CH2CH3 hinge area and CD4 extracellular hinge area any one or more, preferably long 50 Hinge area more than a amino acid residue, more than more preferably long 80 amino acid.In certain embodiments, CD8 α is used herein Hinge area and IgG4Fc CH2CH3 hinge area.The amino acid sequence of illustrative CD8 α hinge area is as shown in SEQ ID NO:4. The amino acid sequence of illustrative IgG4FcCH2CH3 hinge area is as shown in SEQ ID NO:5.

Transmembrane region can be selected from CD28 transmembrane region, CD8 transmembrane region, CD3 ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, One of ICOS transmembrane region and DAP10 transmembrane region are a variety of.Preferably, the transmembrane region for the Chimeric antigen receptor of this paper is CD8 transmembrane region.The amino acid sequence of illustrative CD8 transmembrane region can be as shown in SEQ ID NO:6.

Costimulatory signal molecule intracellular domain can be selected from CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS and One of DAP10 intracellular domain is a variety of.In certain embodiments, the intracellular domain or CD137 of CD28 are applicable in Intracellular domain.The amino acid sequence of illustrative CD28 intracellular domain can be as shown in SEQ ID NO:7；Illustratively The amino acid sequence of CD137 intracellular domain can be as shown in SEQ ID NO:8.

Immunoreceptor tyrosine activating motif can be the tyrosine activation motifs of CD3 ζ and/or Fc ε RI γ.Illustratively The amino acid sequence of CD3 ζ tyrosine activation motifs can be as shown in SEQ ID NO:9.

The each part mentioned above for forming this paper Chimeric antigen receptor, such as CD8 signal peptide, the light chain variable of anti-Muc1 single-chain antibody Area and heavy chain variable region, hinge area, transmembrane region, costimulatory signal molecule intracellular domain and immunity receptor tyrosine activation base Sequence etc. can be directly connected between each other, or can be connected by joint sequence.Joint sequence can be well known in the art be applicable in In the joint sequence of antibody, such as the joint sequence containing G and S.The length of connector can be 3~25 amino acid residues, such as 3 ~15,5~15,10~20 amino acid residues.In certain embodiments, joint sequence is more glycine linlcers sequences.It connects The quantity of glycine is not particularly limited in header sequence, and usually 2~20, such as 2~15,2~10,2~8.Except glycine Come with serine, other known amino acid residue, such as alanine (A), leucine (L), threonine are also contained in connector (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q) etc..

It should be understood that in gene cloning operation, it is often necessary to design suitable restriction enzyme site, this certainly will be in expressed ammonia Base acid sequence end introduces one or more incoherent residues, and this has no effect on the activity of aim sequence.In order to construct Fusion protein, the expression for promoting recombinant protein obtain the recombinant protein being secreted into outside host cell automatically or are conducive to recombinant protein Purifying, it is often necessary to it is other suitable in the end N-, the end C- or the albumen of recombinant protein to be added to some amino acid In region, it may for example comprise but be not limited to, suitable joint peptide, signal peptide, leader peptide, end extend etc..Therefore, the CAR of this paper Aminoterminal or c-terminus can also be containing one or more polypeptide fragments, as protein tag.Any suitable label is ok For 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.

In certain embodiments, the Chimeric antigen receptor of this paper successively contains CD8 signal peptide, anti-Muc1 from N-terminal to C-terminal ScFv, CD8 hinge area, CD8 transmembrane region, CD137 intracellular domain and the CD3 ζ tyrosine activation motifs at nearly film end.

In other embodiments, the Chimeric antigen receptor of this paper successively contains CD8 signal peptide, anti-Muc1 from N-terminal to C-terminal ScFv, IgG4FcCH2CH3 hinge area, CD8 transmembrane region, CD137 intracellular domain and the CD3 ζ tyrosine activation base at nearly film end Sequence.

The amino acid sequence of illustrative Chimeric antigen receptor can be as shown in SEQ ID NO:11.

It herein further include the polynucleotide sequence for encoding the Chimeric antigen receptor.The polynucleotide sequence of this paper 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.

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 carrys out design primer, and made with the commercially available library cDNA or by conventional method well known by persons skilled in the art The standby library cDNA obtains related sequence as template, amplification.When sequence is longer, it is often necessary to which PCR expands twice or repeatedly for progress Increase, then the segment that each time amplifies is stitched together by proper order again.For example, in certain embodiments, code book The polynucleotide sequence of the text fusion protein is as shown in SEQ ID NO:12.

It herein further include nucleic acid constructs, the polynucleotides sequence containing the coding Chimeric antigen receptor as described herein Column, and the one or more regulating and controlling sequences being connect with these series of operations.

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

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

In certain embodiments, the nucleic acid constructs is carrier.Specifically, can be by the coded sequence of this paper CAR Be cloned into the carrier of many types, for example, these types carrier include but is not limited to plasmid, phasmid, phage-derived object, Animal virus and clay.Carrier can be expression vector.Expression vector can be supplied to cell in the form of viral vectors.It can be used as The virus of carrier includes but is not limited to retrovirus, 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 labels.For example, in certain embodiments, the present invention uses reverse transcription disease Poisonous carrier, the retroviral vector contain replication origin, 3 ' LTR, 5 ' LTR, the coded sequence of CAR described herein, with And optional selectable label.

Suitable promoter includes but is not limited to 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.Certain In embodiment, the various promoter sequences that CN201510021408.1 can be used to be announced, including but not limited to this application SEQ The CCEF promoter of enhancer containing mCMV shown in ID NO:1, hCMV enhancer and EF1 α promoter；Shown in SEQ ID NO:2 Enhancer containing CD3e and EF1 α promoter TEF promoter；Enhancer containing CD3e shown in SEQ ID NO:3, mCMV enhancing The TCEF promoter of son, hCMV enhancer and EF1 α promoter；Enhancer containing mCMV shown in SEQ ID NO:4, hCMV enhancing The CCEFI promoter of son and the EF1 α promoter containing introne；Enhancer containing CD3e shown in SEQ ID NO:5 and contain introne EF1 α promoter TEFI promoter；And enhancer containing CD3e shown in SEQ ID NO:5, mCMV enhancer, hCMV increase The TCEFI promoter of hadron and the EF1 α promoter containing introne.All the contents of the application are received by reference herein Enter herein.

Selectable label includes any of selectable marker gene or reporter or both, in order to from quilt Expression cell is identified and selected in the cell mass of viral vector infection.Useful selectable marker gene is anti-including such as antibiotic Property gene, neo etc..Suitable reporter may include coding fluorescence element enzyme, beta galactosidase, chloramphenicol acetyl transfer The gene of enzyme, secreted alkaline phosphatase or Green Fluorescent Protein gene.

In certain embodiments, the expression vector carrier for expression of eukaryon of this paper, specifically transposon vector.In certain realities It applies in scheme, the transposon vector is containing selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty The carrier for expression of eukaryon of transposable element.This kind of transposon vector contains 5 ' inverted terminal repeats (5 ' of corresponding transposons ) and 3 ' inverted terminal repeats of corresponding transposons (3 ' ITR) ITR.For example, in certain embodiments, the nucleic acid of this paper Construction or expression vector from 5 ' to 3 ' are successively encoded containing 5 ' inverted terminal repeat of transposons (5 ' ITR), CD8 signal peptide Sequence, anti-Muc1scFv coded sequence, CD8 hinge area coded sequence or IgG4Fc CH2CH3 coded sequence, CD8 transmembrane region are compiled Code sequence, 3 ' inverted terminal repeat (3 ' of CD137 coded sequence, CD3 ζ intracellular signal area's coded sequence and transposons ITR).The transposon vector also promoter containing transposase coding sequence and control transposase coding sequence expression.? In certain embodiments, the carrier for expression of eukaryon is pNB328 carrier.

Conventional method can be used by the vector introduction host cell of this paper, these methods include microinjection, base Because of marksmanship, electroporation, virus-mediated conversion method, electron bombardment method, calcium phosphate precipitation etc..In certain embodiments, The nucleic acid constructs of this paper is imported in host cell using electroporation herein.Specifically, by the plasmid of recombination through electroporation The effect of high-voltage electricity is gone in interested host cell.

Host cell suitable for this paper can be mammalian cell well known in the art, preferably T cell, including each Various types of T cells in kind source.For example, T cell can derive from the PBMC of B cell malignant tumor patient.In certain implementations In scheme, T cell is originally culture T cell.

It therefore, herein also include a kind of recombinant host cell, the recombinant host cell contains chimeric antigen described herein The coded sequence of receptor or nucleic acid constructs as described herein；And/or the recombinant host cell expresses as described herein be fitted into Antigen receptor.The recombinant host cell can be the previously described host cell for being transferred to carrier described herein.

For in this paper Chimeric antigen receptor IgG4Fc CH2CH3 hinge area and its coded sequence be also included within this paper's Within the scope of.It more specifically, herein include its amino acid sequence IgG4Fc CH2CH3 hinge as shown in SEQ ID NO:7 Area and its coded sequence (including complementary series).

It herein further include the purposes of various amino acid sequences, nucleic acid sequence, recombinant host cell being mentioned above etc..Specifically For, it herein include that the IgG4Fc CH2CH3 hinge area and/or its coded sequence are preparing Chimeric antigen receptor described herein And/or the purposes in its coded sequence；Purposes of the coded sequence of the Chimeric antigen receptor in preparation and reorganization expression vector； Purposes of the nucleic acid constructs in preparation and reorganization host cell；And the recombinant host cell is treated or prevented in preparation Purposes in the drug of cancer.In certain embodiments, herein include the IgG4Fc CH2CH3 hinge area and/or its Coded sequence, the Chimeric antigen receptor and/or its coded sequence and the nucleic acid constructs are in preparation for treating or pre- Application in the recombinant host cell of anti-cancer.

Suitable for the preferred Muc1 positive cancer of cancer that CAR described herein or its expression cell are treated or prevented, tool Body includes the cancer of cancer cell surfaces unconventionality expression Muc1, such as Muc1 cancer cell surfaces expression quantity be it is normal when 100 Times or more and Muc1 in the equally distributed cancer of entire cell surface.Specifically, this kind of cancer can be selected from: gland cancer, lung Cancer, colon cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, cancer of pancreas or forefront Gland cancer.

A kind of kit is also provided herein, the kit contains recombinant expression carrier as described herein.Kit may be used also Containing suitable for the recombinant expression carrier is transferred to the reagent cell, and optional instruct those skilled in the art by institute State the specification that recombinant expression carrier is transferred to cell.

A kind of pharmaceutical composition is also provided herein, described pharmaceutical composition contains recombinant host cell and medicine as described herein Acceptable carrier on.The pharmaceutically acceptable carrier can be the load suitable for cell administration well known in the art Body, including but not limited to pNB328 carrier.

The method for treating or preventing cancer is also included within this context, and the method includes will be as described herein heavy Group host cell or pharmaceutical composition give the step of individual in need.The method given can be common in cell therapy Method.The dosage given can take according to factors such as sufferer gender, age, illnesses, physical conditions.

The CART cell of selectively targeted Muc1 antigen provided herein can promote the proliferation of T cell, enhance T cell External function plays the double action of cellular immunity and humoral immunity in terms of tumor vaccine cells treatment or treatment of cancer.This A little cells can play killing toxicity by non-MHC restrictive approach specific recognition tumor surface antigen, thus efficiently special Property kill tumour cell.

Embodiment according to the present invention is described in detail below in conjunction with case study on implementation.Those skilled in the art It will be understood that case study on implementation below is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.In case study on implementation Particular technique or condition person is not specified, described technology or conditions are (such as with reference to J. Pehanorm cloth according to the literature in the art Luke etc. writes, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or according to product description It carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.In addition, with reason Solution, " containing " used herein also include " by ... form ".

Embodiment 1: the building of recombinant plasmid pNB328-Muc1CAR1, pNB328-Muc1CAR2, pNB328-Muc1CAR3 With the acquisition of Muc1CAR gene modification T cell

1, artificial synthesized to contain signal peptide (CD8 signal peptide or light chain signal peptide), antigen recognizing single-chain antibody (anti- Muc1scFv), hinge area (CD8 hinge area or IgG4Fc CH2CH3 hinge area), CD8 transmembrane region, costimulatory signal molecule are intracellular Structural domain and immunoreceptor tyrosine activating motif Muc1CAR foreign gene (be respectively designated as Muc1CAR1, Muc1CAR2 and Muc1CAR3 polyclone enzyme enzyme site (BglII-XbaI-EcoRI-BamHI)), and at its upstream is introduced, is being inserted into enzyme downstream Enzyme site (SalI-NheI-HindIII-SpeI), commission Shanghai JaRa biotech firm synthesis, is loaded into and uses EcoR1+SalI In the pNB328-EF1 α carrier of double digestion (pNB328 of the promoter of α containing EF1, pNB328 carrier are shown in CN 201510812654.9) Recombinant plasmid is constituted, pNB328-Muc1CAR1, pNB328-Muc1CAR2 and pNB328-Muc1CAR3 are respectively designated as, is tied Structure difference is as shown in Figure 1.Wherein, the amino acid sequence of CD8 signal peptide is as shown in SEQ ID NO:1；The amino of light chain signal peptide Acid sequence is as shown in SEQ ID NO:2；The sequence of antigen recognizing single-chain antibody is as shown in SEQ ID NO:3；The ammonia of CD8 hinge area Base acid is as shown in SEQ ID NO:4；The amino acid sequence of IgG4Fc CH2CH3 hinge area is as shown in SEQ ID NO:5；CD8 across The amino acid sequence in film area is as shown in SEQ ID NO:6；The amino acid sequence such as SEQ of costimulatory signal molecule intracellular domain Shown in ID NO:8；The amino acid sequence of immunoreceptor tyrosine activating motif is as shown in SEQ ID NO:9.

2, the T cell of Muc1CAR gene modification is obtained

It is separated from donor's blood using Filcoll partition method and obtains peripheral blood mononuclear cells (PBMCs).PBMC is pasted Suspension cell is collected into 15ml centrifuge tube by wall culture 2-4h wherein not adherent suspension cell is T cells, 1200rmp is centrifuged 3min, abandons supernatant, and physiological saline is added, and 1200rmp is centrifuged 3min, abandons physiological saline, and repeats this step Suddenly.

For each recombinant plasmid, two 1.5ml centrifuge tubes are taken respectively, and every pipe is added 5 × 10⁶A cell, number a, B, 1200rmp are centrifuged 3min, abandon supernatant.Electricity is taken to turn kit (from Lonza company), a, b pipe are proportionally added into electricity and turn reagent The recombinant plasmid pNB328-Muc1CAR1 or pNB328-Muc1CAR2 or pNB328- that 6ug is built is added in total 100ul, a pipe Muc1CAR3, is resuspended and mixes cell, and 6ug control plasmid is added in b pipe.Mixed liquor is transferred in electric revolving cup, electroporation is put into, is selected Required program is taken, is shocked by electricity.The cell suspension that electricity takes a turn for the better is transferred to using the micropipet in kit and adds good culture solution Six orifice plates in (V culture solution of AIM- containing 2%FBS), mix, be placed in 37 DEG C, 5%CO₂Incubator culture.Add after six hours Enter stimulating factor IL-2 and comprising CD3 antibody, CD28 antibody, IL-15 antibody trophocyte SH385 (5 × 10⁴/ hole), 37 DEG C, 5%CO₂The growing state of T cell is observed in culture 3~4 days, obtains the T cell of expression Muc1CAR gene.

Embodiment 2: the PBMCs cell in different patient sources T cell after the modification activation of Muc1CAR foreign gene is expressed Muc1CAR gene expression identification

After recombinant plasmid pNB328-Muc1CAR1 or pNB328-Muc1CAR2 or pNB328-Muc1CAR3 electricity is turned not Activating T cell with patient presses 2 × 10⁶Cell number collects cell precipitation, and the 2X SDS-PAGE Loading of 80ul is added 100 DEG C of buffer are boiled 10min, and -20 DEG C save backup.Western blotting (use CD3 ζ antibody as primary antibody, The artificial secondary antibody of HRP- goat-anti) test the expression for detecting Muc1CAR.

As a result as shown in Figure 2.The results show that T cell can stablize expression Muc1CAR1 and Muc1CAR3 recombinant protein, but Muc1CAR2 recombinant protein cannot be expressed.

Embodiment 3: the PBMCs cell in different patient sources is after the modification of Muc1CAR foreign gene in T cell genome The detection of Muc1CAR genomic expression level

Extract the genomic DNA (examination of Mock T cell (T cell that electricity turns pNB328 empty carrier) and Muc1CAR-T cell Agent box method), for experimental procedure referring to specification subsidiary in kit, the DNA for measuring Mock T cell and Muc1CAR-T cell is dense Degree, using the expression of the method detection Muc1CAR genome of real-time fluorescence quantitative PCR, response procedures are as follows: 50 DEG C, 2min → 95 DEG C, 10min → 95 DEG C, 15s → 60 DEG C, 1min, 40 circulations.

As a result as shown in Figure 3.The results show that through PB transposase system, Muc1CAR1, Muc1CAR2 and Muc1CAR3 gene Group is integrated into T cell genome.

Embodiment 4: the Muc1CAR-T cell of the different cross-film hinge areas of Muc1CAR is expressed in vitro to the tumour of culture The killing experiments of cell

The matched effector cell of MHC class I parting and target cell are chosen, using real-time n cell functional analysis The cytotoxicity of instrument (RTCA) detection cell, the specific steps are as follows:

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

(2) target cell bed board: cervical cancer cell Hela, breast cancer cell MCF7, (purchase is in U.S. bacterium for lung cell A549 Kind collection ATCC) press every hole 10⁴A cell/50 μ l are layered in the plate containing detecting electrode, are placed several minutes, steady to cell It is fixed once to place into instrument, start step 2, cultivates cell；

(3) effector cell is added: after target cell culture for 24 hours, effector cell is added in pause step 2, and every 50 μ l of hole imitates target Than being respectively set to 8:1,4:1,2:1, using the Mock T cell of non-Pignus pignoris grain as control, starts step 3, continue to co-culture After for 24 hours, cell Proliferation curve is observed；

As a result as shown in Figure 4.The results show that the Muc1CAR3T cells against tumor cells of expression IgG4 cross-film hinge area Lethal effect is better than the Muc1CAR1T cell of expression CD8 α cross-film hinge area.

Embodiment 5: the experiment of flow cytometer detection Muc1CAR3T cell phenotype and cytokine secretion.

1, the Muc1CAR3T cell and Mock T cell that collection part suspends, with 1 × 10 after counting⁶A cell/pipe difference It is added in the EP pipe of 7 1.5ml, PBS is cleaned twice, and 1200rpm is centrifuged 5min, is separately added into the isotype control Ab of 2ul IgG1-PE, fluorescence flow antibody anti-CD69-PE, anti-KLRG1-PE, isotype control Ab IgG1-PC5, fluorescence flow Antibody anti-CD107 α-PC5, isotype control Ab IgG1FITC, fluorescence flow antibody anti-CD62L-FITC, Isotype control IgG antibody 1-PC5, fluorescence flow antibody anti-CD45RO-PC5, isotype control Ab IgG1-PE, fluorescence flow antibody Anti-CCR7-PE, flicking precipitating is uniformly mixed it, and room temperature, which is protected from light, is incubated for 30min, and PBS is cleaned one time, and 400ul PBS is added Cell is transferred in streaming pipe, upper machine testing.

As a result as shown in Figure 5 A and 5B.The results show that Muc1CAR3T cell has stronger killing activity, while also can Promote the formation of memory T, and activates that label CD69 is apparently higher than Mock T cell and aging label KLRG1 will be significantly lower than Mock T cell.

2, with 24 orifice plate of Muc1 antigen coat of 5ug/ml, overnight, PBS is cleaned 3 times 4 DEG C of coatings, is added 3 × 10⁵'s Cell conditioned medium is collected in Muc1CAR3T cell and Mock T cell, culture afterwards for 24 hours.Use BD^TMCBA Human Th1/ The secretion situation of Th2Cytokine Kit II detection Muc1CAR3T cell cell factor after by Muc1 antigenic stimulus:

(1) IL-2, IL-4, IL-6, IL-10, TNF of people are mixed, IFN-γ captures magnetic bead, and vortex oscillation mixes capture magnetic The capture magnetic bead after 50ul is mixed is added in pearl, every pipe；

(2) be added 50ul people Th1/Th2 cytokine standards product (doubling dilution 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml, 20pg/ml, 0pg/ml) and 50ul Sample to be tested (through 2 times of dilution dilute)；

(3) the detection antibody of the Th1/Th2-II-PE of the people of 50ul is added in every pipe；

(4) room temperature, which is protected from light, is incubated for 3h；

(5) washing buffer of 1ml is added in every pipe, and 200 centrifugation 5min abandon supernatant；

(6) cell is resuspended in the washing buffer that 300ul is added in every pipe, and is transferred in streaming pipe, is examined with flow cytometer Survey fluorescent value；

As a result as shown in Figure 5 C, Muc1CAR3T cell secretion IL-2, IL-6 and IFN-γ compared to Mock T cell and Speech is greatly improved, and there is no what differences by the IL-10 and TNF-α that two kinds of cells are secreted.

3,1 × 10 is collected⁶A Muc1CAR3T cell and Mock T cell, is added in the EP pipe of 1.5ml, PBS cleaning two Time, 1200rpm is centrifuged 5min, and the α-CD3CD4CD8 antibody of 2ul is added, and room temperature, which is protected from light, is incubated for 30min, and PBS is cleaned one time, added Enter 400ulPBS cell is transferred in streaming pipe, upper machine testing.

As a result as shown in Figure 5 D, CD3 in Muc1CAR3T cell and Mock T cell⁺CD4⁺, CD3⁺CD8⁺Shared by cell There is no very big differences for percentage.

Embodiment 6: the in vivo functionality experiment of expression Muc1CAR3T cell

Step 1: the complete immunodeficient mouse of 4~6 week old NSG 15,22~27g of average weight, ties up sensible life by Beijing Object Technology Co., Ltd. provides, SPF grades of Animal Lab. raisings.

Step 2: extracorporeal culture human cervical carcinoma cell Hela, logarithmic growth phase adherent growth cell, 0.25% pancreatin disappear Change, centrifugation is resuspended after collecting cell with PBS liquid, and 3000g room temperature is centrifuged 2 minutes, abandons supernatant, then be centrifuged and receive after being resuspended with PBS liquid Collect cell, adjustment concentration of cell suspension to 5 × 10⁷A/ml.

Step 3: inoculating Hela-luc cell in the right ribbed back portion of mouse, 0.1ml/ is only.After inoculation 10 days or so, lead to Living imaging instrument observation tumor size is crossed, NSG immunodeficient mouse is randomly divided into 3 groups, every group of 5 mouse are given respectively PBS, Mock T cell and Muc1CAR3T cell (1 × 10⁷A/only) 1 time.Administration route is multi-point injection in direct tumor.

Mouse tumor change is observed step 4: observing the animation of mouse daily and passing through living imaging instrument every 10 days Change.

As a result as shown in Figure 6.There is a dead mouse at PBS group 50 days.

Although a specific embodiment of the invention has obtained detailed description.It will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence table

<110>Shanghai cell therapy research institute

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

<120>a kind of Chimeric antigen receptor modification T cell and application thereof for targeting Muc1

<130> 174382

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> PRT

<213>artificial sequence

<220>

<223>CD8 signal peptide

<400> 1

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

1 5 10 15

His Ala Ala Arg Pro Ser

20

<210> 2

<211> 20

<212> PRT

<213>artificial sequence

<220>

<223>light chain signal peptide

<400> 2

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

1 5 10 15

Asp Thr Thr Gly

20

<210> 3

<211> 243

<212> PRT

<213>artificial sequence

<220>

<223>anti-Muc1 single-chain antibody

<400> 3

Asp Ile Val Ile Thr Gln Thr Thr Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Glu Val Thr Leu Thr Cys Ser Ala Thr Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Arg Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

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

50 55 60

Gly Tyr Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Glu Glu Ser

115 120 125

Gly Gly Asp Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Arg Tyr Gly Met Ser Trp Val Arg Gln

145 150 155 160

Thr Pro Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly

165 170 175

Thr Tyr Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys

195 200 205

Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp Asn Tyr Gly Ser

210 215 220

Ser Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

225 230 235 240

Val Ser Ser

<210> 4

<211> 83

<212> PRT

<213>artificial sequence

<220>

<223>CD8 α hinge area

<400> 4

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

50 55 60

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn

65 70 75 80

His Arg Asn

<210> 5

<211> 228

<212> PRT

<213>artificial sequence

<220>

<223>IgG4 Fc CH2CH3 hinge area

<400> 5

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

1 5 10 15

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

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

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

180 185 190

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Leu Gly Lys

225

<210> 6

<211> 81

<212> PRT

<213>artificial sequence

<220>

<223>CD8 transmembrane region

<400> 6

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

50 55 60

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn

65 70 75 80

His

<210> 7

<211> 28

<212> PRT

<213>artificial sequence

<220>

<223>CD28 intracellular domain

<400> 7

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

1 5 10 15

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 8

<211> 42

<212> PRT

<213>artificial sequence

<220>

<223>CD137 intracellular domain

<400> 8

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 9

<211> 112

<212> PRT

<213>artificial sequence

<220>

<223>CD3 ζ tyrosine activation motifs

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

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

100 105 110

<210> 10

<211> 233

<212> PRT

<213>artificial sequence

<220>

<223>the nearly film terminal amino acid sequence of Muc1

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

His Asn Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser

50 55 60

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

65 70 75 80

Ala Ser His Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser

85 90 95

Val Pro Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser

100 105 110

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

115 120 125

Phe Asn Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Leu Thr Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser

210 215 220

Ala Gln Ser Gly Ala Gly Val Pro Gly

225 230

<210> 11

<211> 728

<212> PRT

<213>artificial sequence

<220>

<223>Chimeric antigen receptor amino acid sequence

<400> 11

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

1 5 10 15

His Ala Ala Arg Pro Ser Asp Ile Val Ile Thr Gln Thr Thr Ala Ile

20 25 30

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

35 40 45

Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Arg Pro Gly Thr Ser

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg

100 105 110

Ser Ser Ser Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

115 120 125

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

130 135 140

Val Lys Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly Ser

145 150 155 160

Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr Gly

165 170 175

Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala

180 185 190

Thr Ile Ser Ser Gly Gly Thr Tyr Ile Tyr Tyr Pro Asp Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

210 215 220

Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

225 230 235 240

Arg Asp Asn Tyr Gly Ser Ser Tyr Asp Tyr Ala Met Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro

260 265 270

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

275 280 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

290 295 300

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

305 310 315 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

325 330 335

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

340 345 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

355 360 365

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

370 375 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385 390 395 400

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

405 410 415

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

420 425 430

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

435 440 445

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

450 455 460

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465 470 475 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Phe Val Pro

485 490 495

Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro

500 505 510

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

515 520 525

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

530 535 540

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

545 550 555 560

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Lys Arg

565 570 575

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

580 585 590

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

595 600 605

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

610 615 620

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

625 630 635 640

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

645 650 655

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

660 665 670

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

675 680 685

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

690 695 700

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

705 710 715 720

His Met Gln Ala Leu Pro Pro Arg

725

<210> 12

<211> 2184

<212> DNA

<213>artificial sequence

<220>

<223>coded sequence of Chimeric antigen receptor

<400> 12

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagcgata ttgtgatcac ccagactaca gcaatcatgt ctgcatctcc aggggaggag 120

gtcaccctaa cctgcagtgc cacctcaagt gtaagttaca tacactggtt ccagcagagg 180

ccaggcactt ctcccaaact ctggatttat agcacatcca acctggcttc tggagtccct 240

gttcgcttca gtggcagtgg atatgggacc tcttactctc tcacaatcag ccgaatggag 300

gctgaagatg ctgccactta ttactgccag caaaggagta gttccccatt cacgttcggc 360

tcggggacaa agttggaaat aaaaggtgga ggcggttcag gcggaggtgg cagcggcggt 420

ggcgggtcgg aggttaagct ggaggagtct gggggagact tagtgaagcc tggagggtcc 480

ctgaaactct cctgtgcagc ctctggattc actttcagta gatatggcat gtcttgggtt 540

cgccagactc cagacaagag gctggagtgg gtcgcaacca ttagtagtgg tggtacttac 600

atctactatc cagacagtgt gaaggggcga ttcaccatct ccagagacaa tgccaagaac 660

accctgtacc tgcaaatgag cagtctgaag tctgaggaca cagccatgta ttactgtgca 720

agggataact acggtagtag ctacgactat gctatggact actggggtca aggaacctca 780

gtcaccgtct cctcagagtc caaatatggt cccccatgcc caccatgccc agcacctccc 840

gtggccggac catcagtctt cctgttcccc ccaaaaccca aggacactct catgatctcc 900

cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc aggaagaccc cgaggtccag 960

ttcaactggt acgtggatgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 1020

cagttccaga gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1080

aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccgtcctc catcgagaaa 1140

accatctcca aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc 1200

caggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1260

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1320

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaggctaac cgtggacaag 1380

agcaggtggc aggaggggaa tgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1440

cactacacac agaagagcct ctccctgtct ctgggtaaat tcgtgccggt cttcctgcca 1500

gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1560

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 1620

agggggctgg acttcgcctg tgatatctac atctgggcgc ccctggccgg gacttgtggg 1680

gtccttctcc tgtcactggt tatcaccctt tactgcaacc acaaacgggg cagaaagaag 1740

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1800

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1860

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1920

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1980

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 2040

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 2100

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 2160

cacatgcagg ccctgccccc tcgc 2184

Claims

1. a kind of Chimeric antigen receptor, which is characterized in that from N-terminal to C-terminal, which successively contains memebrane protein signal More than the single-chain antibody at the nearly film end peptide, anti-Muc1, long 50 amino acid residues hinge area, transmembrane region, costimulatory signal molecule Intracellular domain and immunoreceptor tyrosine activating motif.

2. Chimeric antigen receptor as described in claim 1, which is characterized in that the amino acid sequence at the nearly film end the Muc1 is such as Shown in SEQ ID NO:10；Preferably, the amino acid sequence of the anti-Muc1 single-chain antibody is as shown in SEQ ID NO:3.

3. Chimeric antigen receptor as claimed in claim 1 or 2, which is characterized in that the Chimeric antigen receptor has with next A or multiple features:

The memebrane protein signal peptide is selected from CD8 signal peptide, CD28 signal peptide and CD4 signal peptide；Preferably, the signal peptide is CD8 signal peptide, amino acid sequence is as shown in SEQ ID NO:1；

Hinge areas more than 50 amino acid residues of the length is selected from CD8 α hinge area, IgD hinge area, IgG1Fc CH2CH3 hinge Sequence and IgG4Fc CH2CH3 hinge area；Preferably, the hinge area is CD8 α hinge area or IgG4Fc CH2CH3 hinge area； It is highly preferred that the amino acid sequence of CD8 α hinge area is as shown in SEQ ID NO:4；The ammonia of the IgG4Fc CH2CH3 hinge area Base acid sequence is as shown in SEQID NO:5；

The transmembrane region be selected from CD28 transmembrane region, CD8 transmembrane region, CD3 ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, One of ICOS transmembrane region and DAP10 transmembrane region are a variety of；Preferably, the transmembrane region is CD8 transmembrane region, amino acid sequence Column are as shown in SEQ ID NO:6；

The costimulatory signal molecule intracellular domain be selected from CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS and One of DAP10 intracellular domain is a variety of；Preferably, the costimulatory signal molecule intracellular domain is CD28 knot intracellular Structure domain, amino acid sequence is as shown in SEQ ID NO:7 or the costimulatory signal molecule intracellular domain is CD137 intracellular Structural domain, amino acid sequence is as shown in SEQ ID NO:8；With

The immunoreceptor tyrosine activating motif is the tyrosine activation motifs of CD3 ζ and/or Fc ε RI γ；Preferably, described Immunoreceptor tyrosine activating motif is CD3 ζ tyrosine activation motifs, and amino acid sequence is as shown in SEQ ID NO:9.

4. Chimeric antigen receptor as claimed in any one of claims 1-3, which is characterized in that

The Chimeric antigen receptor from N-terminal to C-terminal successively containing CD8 signal peptide, the nearly film end anti-Muc1 scFv, CD8 hinge area, CD8 transmembrane region, CD137 intracellular domain and CD3 ζ tyrosine activation motifs；Or

The Chimeric antigen receptor from N-terminal to C-terminal successively containing CD8 signal peptide, the nearly film end anti-Muc1 scFv, IgG4FcCH2CH3 hinge area, CD8 transmembrane region, CD137 intracellular domain and CD3 ζ tyrosine activation motifs；

Preferably, the amino acid sequence of the Chimeric antigen receptor is as shown in SEQ ID NO:11.

5. a kind of polynucleotide sequence, is selected from:

(1) polynucleotide sequence of Chimeric antigen receptor described in any one of claim 1-4 is encoded；With

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

Preferably, the polynucleotide sequence is selected from polynucleotide sequence or its complementary series shown in SEQ ID NO:12.

6. a kind of nucleic acid constructs contains the polynucleotide sequence described in claim 5；

Preferably, the nucleic acid constructs is expression vector；

It is highly preferred that the expression vector is carrier for expression of eukaryon, preferably comprise selected from piggybac, sleepingbeauty, The transposable element of frog prince, Tn5 and Ty.

7. a kind of recombinant host cell, the recombinant host cell contains nucleic acid constructs as claimed in claim 6, or expression power Benefit requires Chimeric antigen receptor described in any one of 1-4；

Preferably, the host cell is mammalian cell；It is highly preferred that the host cell is T cell；It is highly preferred that The host cell is originally culture T cell.

8. a kind of IgG4Fc CH2CH3 hinge area, which is characterized in that the amino acid sequence of the hinge area such as SEQ ID NO:5 It is shown.

9. a kind of polynucleotide sequence, is selected from:

(1) sequence of IgG4Fc CH2CH3 hinge area described in claim 8 is encoded；With

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

Preferably, (1) described sequence is as shown in SEQ ID NO:12 796-1479 bit base.

10. embedding described in IgG4Fc CH2CH3 hinge area according to any one of claims 8 and/or its coded sequence, claim 1-4 Antigen receptor and/or its coded sequence and/or nucleic acid constructs as claimed in claim 6 are closed in preparation for treating or preventing cancer Application in the recombinant host cell of disease；And recombinant host cell as claimed in claim 7 treats or prevents cancer in preparation Application in drug.

11. application as claimed in claim 10, which is characterized in that the cancer is its cancer cell surfaces unconventionality expression Muc1's Cancer, preferably 100 times or more and Muc1 in Muc1 when the expression quantity of cancer cell surfaces is normal are in entire cell surface Equally distributed cancer；Preferably, the cancer is selected from: gland cancer, lung cancer, colon cancer, colorectal cancer, breast cancer, oophoroma, uterine neck Cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, the cancer of the esophagus, cancer of pancreas or prostate cancer.

12. a kind of pharmaceutical composition, which is characterized in that it is thin that described pharmaceutical composition contains recombinant host as claimed in claim 7 Born of the same parents and pharmaceutically acceptable carrier.