CN110229236A

CN110229236A - Induce tumour cell up-regulation antigen MUC1 expression CAR and its application

Info

Publication number: CN110229236A
Application number: CN201910525834.7A
Authority: CN
Inventors: 张毅; 张凯; 梅子; 乔彬
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2019-06-13
Filing date: 2019-06-13
Publication date: 2019-09-13
Anticipated expiration: 2039-06-13
Also published as: CN110229236B

Abstract

The application belongs to tumour cell immunotherapy techniques field, and in particular to a kind of to can induce the CAR and its apply patent application matters that tumour cell up-regulation antigen MUC1 is expressed.The Chimeric antigen receptor is the amino acid sequence as the connection of dried albumen segment, specifically: sequentially connected people CD8a molecular signal peptide, humanization MUC1 single-chain antibody, people CD8 molecule across membranes area and 41BB molecule intracellular region, people's CD3z molecule intracellular region；In preferred design, people's CD3z molecule intracellular region further passes through catenation sequence and is attached with IL22 CDS region sequence, further to promote the expression of MUC1 by this cell factor of IL-22.The application shows good technical effect by the further structure optimization for CAR, for the generation for effectively inducing tumor cell surface antigen MUC1 expression, reducing undershooting-effect, and the application effect of raising CAR-T cell.

Description

Induce tumour cell up-regulation antigen MUC1 expression CAR and its application

Technical field

The application belongs to tumour cell immunotherapy techniques field, and in particular to one kind can induce tumour cell up-regulation antigen The CAR(Chimeric Antigen Receptor of MUC1 expression, Chimeric antigen receptor) and its using patent application matters.

Background technique

CAR-T(Chimeric Antigen Receptor T-Cell, Chimeric antigen receptor T cell) it is used as tumour immunity A kind of effective therapeutic modality for the treatment of was ratified listing by food and drug administration (FDA) on August 30th, 2017 and is used In treatment acute lymphoblastic leukemia (ALL), CD19 can be effectively removed by target spot of CD19⁺Tumour cell is facing Show before bed and in clinical trial it is encouraging as a result, and it is verified they with intractable and recurrent The effective anti-leukemia effect carried out in b- precursor acute lymphatic leukemia Children and teenager is safe and feasible, institute Some toxicity is reversible and there is no lasting B cell hypoplasia.But its application in solid tumor is current still not to the utmost People's will needs to solve there are many more problem, and main cause is to be in solid tumor do not have the effective tumour similar to CD19 special The inhibiting effect of Specific Antigen and tumor microenvironment.

Lentivirus is in a kind of RNA virus of Retroviridae, wherein most known is human immunodeficiency Malicious (HIV-1).HIV-1 diameter about 120nm contains two positive chain RNAs, can effectively enter in nucleus, to division cells and Nondividing phase cell all has very high efficiency of infection.It can be by entrained gene integration to cell base after slow virus infected cell It is expressed because in group, and long lasting for stablizing, while heredity can be stablized with cell division and gone down, therefore become and import external source The effective tool of gene.Slow virus carrier is gene therapy vector made of one kind is transformed based on HIV-1, passes through removal Disease-causing gene, while the homology of helper plasmid and vector plasmid is reduced, so that improved slow virus is provided with titre higher, raw The characteristics such as object safety is more preferable, importing exogenous sequences abilities are stronger.Slow virus system has been widely applied to gene mistake at present In the cells and zoopery of functional studies such as expression, RNA interference, miRNA research and gene knockout.

MUC1(Mucin1, mucin 1) it is a kind of macromolecular transmembrane glycoprotein, extensively and height is expressed in breast cancer, ovary In the malignant cells such as cancer, lung cancer, prostate cancer, colon cancer, liver cancer and cancer of pancreas, and relative to normal cell, tumour is thin The MUC1 of cellular surface has different glycosylation structures.For theory, the expression characteristic based on MUC1 can relatively be used on tumour The differentiation of cell and non-tumor cell, and it is overexpressed and glycosylates abnormal but also MUC1 can become various immunization therapies especially The promising target of CAR-T treatment, but in practical application, the reasons such as MUC1 expression quantity are limited to, has not yet to see and preferably answers Use effect.

Summary of the invention

The application is designed to provide a kind of CAR that can induce tumour cell up-regulation antigen MUC1 expression, thus for correlation CAR-T establishes certain technical foundation in the treatment of solid tumor.

Details are as follows for the technical solution that the application is taken.

A kind of CAR can induce tumour cell up-regulation antigen MUC1 expression, which is as dried albumen piece The amino acid sequence of section connection, is named with MUC1-41BB-CD3z, specifically: sequentially connected people CD8a molecular signal peptide (CMV), humanization MUC1 single-chain antibody (scFV.MUC1), people CD8 molecule across membranes area and 41BB molecule intracellular region (41BB), people CD3z molecule intracellular region (CD3Zeta), it may be assumed that CMV-scFV.MUC1-41BB-CD3Zeta(CD3 ζ)；

In preferred design, people's CD3z molecule intracellular region (CD3Zeta) further (is specifically connected for example, by using TA2 by catenation sequence Meet sequence (T2A)) it is attached with IL22 CDS region sequence (IL22), further to be promoted by this cell factor of IL-22 Into the expression of MUC1, i.e. overall structure are as follows: CMV-scFV.MUC1-41BB-CD3Zeta(CD3 ζ)-T2A-IL22, this is excellent Choosing design is named with MUC1-41BB-CD3z-IL22；

The people CD8a molecular signal peptide (CMV), including 21 amino acid, sequence as shown in SEQ ID No.1, specifically:

MALPVTALLLPLALLLHAARP；

The humanization MUC1 single-chain antibody (scFV.MUC1), including 244 amino acid, sequence as shown in SEQ ID No.2, Specifically:

EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTI SRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVVTQESALTTSPGE TVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCAL WYSNHWVFGGGTKLTVLGSE；

The people CD8 molecule across membranes area and 41BB molecule intracellular region (41BB), including people CD8 molecule across membranes area and 41BB molecule born of the same parents Two parts of inner region, in which:

People's CD8 molecule across membranes area, including 71 amino acid, sequence as shown in SEQ ID No.3, specifically:

LETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC；

41BB molecule intracellular region, including 42 amino acid, sequence as shown in SEQ ID No.4, specifically:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

The people CD3z molecule intracellular region (CD3Zeta), including 112 amino acid, sequence is as shown in SEQ ID No.5, specifically Are as follows:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

The TA2 catenation sequence (T2A), including 18 amino acid, sequence as shown in SEQ ID No.6, specifically:

EGRGSLLTCGDVEENPGP；

The IL22 CDS region sequence (IL22), including 179 amino acid, sequence as shown in SEQ ID No.7, specifically:

MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDV RLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKL KDTVKKLGESGEIKAIGELDLLFMSLRNACI；

Accordingly, it is preferred that MUC1-41BB-CD3z-IL22 amino acid sequence, including 730 amino acid, sequence such as SEQ ID Shown in No.8, specifically:

MALPVTALLLPLALLLHAARPGSMALPVTALLLPLALLLHAARPEVQLQQSGGGLVQPGGSMKLSCVASGFT FSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSF AYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTG LIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEMALPVTALLLPL ALLLHAARPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPREGRGSLLTCGDVEENPGPMAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYIT NRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRL STCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI。

Utilize slow virus expression plasmid constructed by the CAR that can induce tumour cell up-regulation antigen MUC1 expression, tool Body includes the following steps:

(1) according to central dogma, the coded sequence DNA of the CAR is obtained using the prior art；

(2) using Lenti-2G plasmid as expression vector, BsrGI, Not I double digestion are carried out to it, then utilize T4 DNA connection Enzyme enters the coded sequence DNA integration recombination in step (1) in Lenti-2G plasmid；

(3) connection product in step (2) is converted into STABL3 competent cell, screening expands and cultivates laggard onestep extraction matter Grain, slow virus expression plasmid (divides after can be obtained the recombination that can express MUC1-41BB-CD3z or MUC1-41BB-CD3z-IL22 It does not name are as follows: Lenti-2G-MUC1-41BB-CD3z, Lenti-2G-MUC1-41BB-CD3z-IL22).

The slow virus expression plasmid is specifically, for example, HNSCC cancer preparing the application in anti-tumor agents, the tumour (head and neck squamous cell carcinoma refers mainly to the cell carcinogenesis of the tissue sites such as nasal cavity, nasal sinus, oral cavity, tonsillotome, pharyngeal and throat) Related neoplasms, further for example, Human tongue cancer cell line system CAL33, population swallow the relevant tumours such as squamous cell carcinoma system HN4；Using When, CAR-T cell is prepared by transfection and is applied, for improving the MUC1 expression quantity of tumour cell；

Concrete application step includes the following steps:

(1) slow virus is packed；Specifically refer to following operation:

Using 293T cell as aim cell to be transfected, progress bed board first is incubated for for 24 hours；

Then the slow virus expression plasmid of constructed acquisition is mixed with packaging plasmid, using lipofectamine to be transfected Aim cell 293T cell carry out transfection 48h；

After transfection, supernatant is collected, it is spare, to prepare to be used for the infection of T cell；

(2) T cell of preparation purifying

Firstly, obtaining mononuclearcell from human peripheral separation (specifically for example, by using density gradient centrifugation method)；

Then, separation obtains the CD of purifying³⁺T cell (is specifically isolated and purified for example, by using T cell separation magnetic bead),

Finally, be added appropriate CD3/CD28 magnetic bead activate 2 days it is spare；

(3) T cell infects

In cell in step (2) after activation 2 days, it is poly- that collected viral supernatants and polybrene(in step (1) is added Solidifying amine, also known as be hexadimethrine bromide), it is incubated overnight to be infected；

(4) it expands T cell and detects

To being incubated for T cell after infection in step (3), after eccentric cleaning (general no less than 3 times), be added IL-2 containing 1000U and The RPMI1640 culture medium of 5% fetal calf serum, further to expand T cell；

To T cell after amplification can using Flow Cytometry with to T cell surface C AR expression or cell Proliferation into Row detection determines, can be used to the MUC1 expression that stimulation improves tumour cell after further feeding back according to testing result.

IL-22 is a kind of cell factor mainly secreted by Th22 cell, belongs to IL-10 superfamily, existing research is thought Body inherent immunity can be enhanced in IL-22, protects body cell from damaging and promoting histiocytic Regeneration and Repair, and join With the regulation of a variety of diseases and verifying；And studies have reported that thinking that IL-22 can promote tumor cell surface antigen MUC1 expression Up-regulation.And the application is using tumour antigen MUC1 as target spot, and by CAR structure optimization, especially by series connection IL22 molecule, Tumour antigen MUC1 is further effectively enhanced in the expression of tumor cell surface, for improving CAR-T cell for swollen The identification of oncocyte, the ability for improving targets neoplastic cells, and then improve CAR-T cell killing to the tumour cell of the MUC1 positive Hurt effect and establishes good technical foundation.

Generally, the immunologic escape problem of miss the target problem and the tumour in view of existing CAR-T cell in treatment of solid tumors, The application is taken off by the further structure optimization for CAR for effectively inducing tumor cell surface antigen MUC1 expression, reducing The generation of targeted effect, and the application effect of raising CAR-T cell all show good technical effect, therefore have preferable Practical value and popularization and application meaning.

Detailed description of the invention

Fig. 1 be the upper figure of MUC1-41BB-CD3z(), the MUC1-41BB-CD3z-T2A-IL22(following figure) CAR structural representation Figure；

Fig. 2 is Lenti-2G-MUC1-41BB-CD3z-T2A-IL22 structural schematic diagram；

Fig. 3 is Flow cytometry CAR-T transfection efficiency figure；

Fig. 4 is Transfected cells Multiplying culture situation statistical result；

Fig. 5 ~ Fig. 7 is killing-efficiency result figure of the CAR-T cell to different tumour cells；Wherein Fig. 5 is for OME cell, Fig. 6 For for Cal33 cell, Fig. 7 is for HN4 cell；

Fig. 8 summarizes for tumour cell effects different in the case of different effect target ratios (E/T)；

Fig. 9 is that Flow cytometry IL-22 can induce tumour cell MUC1 to raise；

Figure 10 is 22 secreting, expressing situation of IL；

Figure 11 is that bioluminescence imaging technology monitors tumour growth and fluorescence.

Specific embodiment

Explanation is further explained to the application below with reference to embodiment.Before introducing specific embodiment, with regard to following realities Situations such as applying part experimental material, laboratory apparatus in example is briefly introduced and is described as follows.

Experimental material:

Non-obese patients with type Ⅰ DM/severe combined immunodeficient (nod/scid) mouse, 6-8 weeks female mice are tieed up purchased from Beijing Co., Ltd, tonneau China (BeiJing, China), gnotobasis culture；

Human tongue cancer cell line system (CAL33), population pharynx squamous cell carcinoma system (HN4), are purchased from Medical College, Shanghai Communication Univ. Shanghai City 9th the People's Hospital；

Relevant cell ties up to culture in Dulbecco's Modified Eagle culture medium (DMEM-F12), contains in the culture medium There are 10% fetal calf serum (FBS, HyClone, Chicago, IL, USA) and 100 U/ml penicillin, 100 μ g/ml streptomysins (Invitrogen, Carlsbad, CA, USA)；

Before experiment starts, with the vial supernatant transfection tumor cell of-GFP containing luciferase, with FACS AriaTM cell sorting Instrument (BD Biosciences, San Jose, CA, USA) sorts the channel GFP, and final obtain stablizes expression fluorescein The cell strain HN4(DMED-F12 culture medium culture of enzyme-green fluorescent protein (GFP))；

Immortalized cell line OME is the oral mucosa epithelial cell system that hTERT is overexpressed, which is MUC1 negative, DMED- It is cultivated in F12 culture medium.

Embodiment 1

On existing CAR Research foundation, to improve CAR-T cell therapy effect, the effect of missing the target of current CAR-T cell therapy is reduced It answers, inventor has carried out further Optimizing Reconstruction for CAR structure, to determine CAR technical effect after Optimizing Reconstruction, inventor Two kinds of CAR are prepared for, concrete structure schematic diagram is as shown in Figure 1.

Specifically:

MUC1-41BB-CD3z are as follows: sequentially connected people CD8a molecular signal peptide (CMV), humanization MUC1 single-chain antibody (scFV.MUC1), people CD8a molecular flexibility segment (CD8aTM), people CD8 molecule across membranes area and 41BB molecule intracellular region (41BB), People's CD3z molecule intracellular region (CD3Zeta), it may be assumed that CMV-scFV.MUC1-41BB-CD3Zeta(CD3 ζ)；

MUC1-41BB-CD3z-IL22 are as follows: sequentially connected people CD8a molecular signal peptide (CMV), humanization MUC1 single-chain antibody (scFV.MUC1), people CD8 molecule across membranes area and 41BB molecule intracellular region (41BB), people's CD3z molecule intracellular region (CD3Zeta), TA2 catenation sequence (T2A), IL22 CDS region sequence (IL22)；That is: CMV-scFV.MUC1-41BB-CD3Zeta(CD3 ζ)-T2A-IL22.

The people CD8a molecular signal peptide (CMV), including 21 amino acid, sequence is as shown in SEQ ID No.1, specifically Are as follows:

MALPVTALLLPLALLLHAARP。

The humanization MUC1 single-chain antibody (scFV.MUC1), including 244 amino acid, sequence such as SEQ ID No.2 institute Show, specifically:

EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTI SRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVVTQESALTTSPGE TVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCAL WYSNHWVFGGGTKLTVLGSE。

The people CD8 molecule across membranes area and 41BB molecule intracellular region (41BB), including people CD8 molecule across membranes area and 41BB points Two parts of sub- intracellular region, in which:

LETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

The people CD3z molecule intracellular region (CD3Zeta), including 112 amino acid, sequence as shown in SEQ ID No.5, Specifically:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

EGRGSLLTCGDVEENPGP。

The IL22 CDS region sequence (IL22), including 179 amino acid, sequence is as shown in SEQ ID No.7, specifically Are as follows:

MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDV RLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKL KDTVKKLGESGEIKAIGELDLLFMSLRNACI。

MUC1-41BB-CD3z amino acid sequence, including 490 amino acid, particular sequence are as follows:

MALPVTALLLPLALLLHAARPEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEI RLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGS GGGGSDIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGD KAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSELETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

MUC1-41BB-CD3z-IL22 amino acid sequence, including 730 amino acid, sequence as shown in SEQ ID No.8, Specifically:

Embodiment 2

On that basis of example 1, inventor further constructs Lentiviral, and specific building process can refer to following step Suddenly.

(2) the bis- enzymes of BsrGI, Not I are carried out to it for expression vector with Lenti-2G plasmid (Lenti-2G-MCS-conGFP) It cuts, is then entered the coded sequence DNA integration recombination in step (1) in Lenti-2G plasmid using T4 DNA ligase；

(3) connection product in step (2) is converted into STABL3 competent cell, screening expands and cultivates laggard onestep extraction matter Grain, slow virus expression plasmid (divides after can be obtained the recombination that can express MUC1-41BB-CD3z or MUC1-41BB-CD3z-IL22 It does not name are as follows: Lenti-2G-MUC1-41BB-CD3z, Lenti-2G-MUC1-41BB-CD3z-IL22).Constructed Lenti- 2G-MUC1-41BB-CD3z-IL22 plasmid construct schematic diagram is as shown in Figure 2.

It should be noted that plasmid employed in the present embodiment is by PPL (Public Protein/Plasmid Library, China) company according to above-mentioned steps prepare provide.

Embodiment 3

On the basis of embodiment 2, it is thin that constructed slow virus expression plasmid is further prepared into CAR-T by transfection by inventor Born of the same parents have carried out preliminary experiment, and detailed process is briefly discussed below.

(1) slow virus is packed

Using 293T cell as aim cell to be transfected, to cultivate 293T cell, (DMEM is complete for 24 hours for the incubation of six orifice plate bed boards first 37 DEG C of culture medium)；

Culture medium is changed to OPTI-MEM(dosage 2ml when transfection), then by the slow virus expression plasmid of constructed acquisition 1.5g, packaging plasmid psPAX2 and Pmd2.G distinguish 1.5g and 1g, and lipofectamine 8ul mixed, to turn to treating The aim cell 293T cell of dye is transfected；

Normal DMEM culture medium is changed to after 12 hours；

Viral supernatants are collected after continuing culture 48 hours, 1500rpm is centrifuged 10 minutes, takes -80 DEG C of supernatant to save backup, to be used for Infect T cell.

(2) the CD3+T cell of preparation purifying

Firstly, obtaining mononuclearcell from human peripheral separation (specifically using density gradient centrifugation method)；

Then, separation obtains the CD3 of purifying⁺T cell (is specifically isolated and purified using T cell separation magnetic bead)；

After again, will the obtained T cell of purifying in 24 porocyte culture plates with containing IL-2 (100 IU/ml, PeproTech, Suzhou, Jiangsu, China) and L-Glutamine (2 mM, Gibco/Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) RPMI-1640 cultivated；

Finally, before use, by T cell CD3/CD28 activating antibodies (1 ul/10⁷Cells stimulation activation 2 days) is carried out.

(3) T cell infects

Using 24 porocyte culture plates, every hole (10⁶/ hole) T cell in step (2) after activation 2 days is added, while every hole is added Collected viral supernatants 1ml and polybrene(8 μ g/mL in step (1)), it is incubated overnight to be infected；

After 24 hours, be changed to normally containing IL-2 (100 IU/ml, PeproTech, Suzhou, Jiangsu, China) and L-Glutamine (2 mM, Gibco/Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) RPMI-1640 cultivated.

(4) it expands T cell and detects

To T cell after infection is incubated in step (3), after eccentric cleaning (3 times), IL-2 containing 1000U and 5% fetal calf serum is added RPMI1640 culture medium (every 2-3 days one subcultures of replacement), further to expand T cell, prepared cell is remembered respectively Are as follows: MUC1-T(contains slow virus carrier plasmid MUC1-41BB-CD3z), MUC1-IL22-T(contain can stimulate MUC1 express Slow disease.

It should be noted that, with reference to aforesaid operations, inventor packs the Lenti-2G plasmid containing GFP as control At direct infection T cell after slow virus, prepared cell is denoted as: GFP-T(is equivalent to the blank control T cell for only expressing GFP Group).

(5) experiment detection

(1) viral Packing Condition is detected and is evaluated T cell proliferative conditions

After infecting 5 days T cell, detected using expression of the flow cytometry to T cell surface C AR.As a result such as Fig. 3 It is shown, it will thus be seen that CAR the positive expression rate reaches 50% ~ 70%, this result shows that constructed CAR expression plasmid by success It is packaged into lentiviral particle, and transfects success.

Cell proliferation situation is counted, as a result as shown in Figure 4.Analysis can be seen that GFP-T group cell Proliferation the most Slowly, and other two experimental group cell Proliferation is very fast, but due to being influenced by slow-virus transfection, when with proliferation Between extension, the cell quantity of each processing group gradually decreases；In other words, for cell Proliferation standpoint of efficiency, proliferation The cell Proliferation amount of culture 5 days or so is highest.

(2) different apoptosis of tumor cells situations are detected

After CAR-T cell is expanded 15 days, using the tumour cell of different MUC1 expressions as experimental subjects, to detect evaluation Apoptosis of tumor cells situation, specific experiment setting are as follows:

Immortalization oral mucous epithelia respectively with carcinoma of mouth tumour cell Cal33, HN4 and MUC1 feminine gender of high expression MUC1 is thin Born of the same parents OME is as target cell, using the T cell of different disposal as effector cell (GFP-T, MUC1-T, MUC1-IL22-T), according to Different effect target ratios (1:1,10:1,20:1) by T cell and target cell according to being incubated for altogether in 96 orifice plates 6 hours, every group of setting Three multiple holes.

The supernatant after cell is incubated for altogether is collected, by cell precipitate Annexin V- binding buffer (BioLegend, San Diego, CA, USA) is resuspended, by 1ul CD326 antibody and 1ul Annexin V Antibody (BioLegend) is separately added into cell suspension, is incubated for 15 minutes in 4 DEG C of light protected environment；Propidium (propidium iodide, Sigma) is added in cell suspension before upper machine；All sample standard deviations use FACSCanto II or C6 fluidic cell Instrument (Becton Dickinson) is analyzed, data using FlowJo software (FlowJo, LLC, Ashland, Covington, KY, USA) it is analyzed.

As a result as shown in Fig. 5, Fig. 6, Fig. 7, Fig. 8, it will thus be seen that even if different tumour cells is directed to, compared to GFP-T Cell, MUC1-T cell can the effective specific killing MUC1 positive tumour cell, and the MUC1- under same ratio The fragmentation effect ratio MUC1-T cell of IL22-T cell is more preferable, and the result shows MUC1-IL22-T cells for tumour cell Elimination effect it is more preferable.Such as: when imitating target ratio is 20:1, differential effect is become apparent, and MUC1-T cell is to MUC1 The elimination effect of positive tumour cell is in 50-60%, and removing of the MUC1-IL22-T cell to the tumour cell of the MUC1 positive Effect can reach 90% or more.

(3) IL22 secretion and the detection of MUC1 expression

In above-mentioned (2) different apoptosis of tumor cells situation detection evaluation procedures, inventor is further to IL22 secretion and MUC1 Expression is tested and analyzed.

It should be noted that MUC1 detection of expression method is with specific reference to as follows:

Firstly, extracting mRNA in TRIzol reagent (Invitrogen) respectively tumor sample；

Then, using the PrimeScript RT Reagent Kit of TaKaRa company by RNA reverse transcription at cDNA；

Finally, carrying out qRT-PCR analysis on Agilent Mx3005P using SYBR premix Ex Taq II (TaKaRa), have When body analyzes MUC1, design of primers is as follows:

It is positive: 5 '-TTTCCAGCCCGGGATACCTA-3 ',

It is reversed: 5 '-AGAGGCTGCTGCCACCATTA-3 '；

Using glyceraldehyde 3 phosphate hydrogen enzyme (GAPDH) as internal reference, 2- is utilized^△△CtData are analyzed.

In experimentation, for the stimulation that verifying external source IL22 expresses tumor cell surface MUC1, inventor will not IL22 (20ng/ is added in six porocyte culture plates cultures with carcinoma of mouth tumour cell and immortalization oral mucosa epithelial cell Ml it after) being incubated for 72 hours altogether, is expressed with Flow cytometry MUC1.

Concrete outcome is as shown in Figure 9.As can be seen that tumor cell surface MUC1 is obviously raised, and immortalize oral mucosa Epithelial cell OME surface MUC1 expression is without significant change, and the result shows IL22 can effectively and specificity induction tumour is thin Cellular surface antigen MUC1 expression up-regulation, to other cells without obvious effect.

Further, in the case of different effect target ratios, treated to Cal33 cell for different effect T cell group IL22, MUC1 expression are detected and are counted, and the results are shown in Figure 10.

As a result it counts and analyzes it can be seen that E/T ratio is smaller, the cytotoxicity function of T cell is weaker, T cell proliferation It is slower, while the ability of secreting function cell factor is weaker, and the CAR-T cell for secreting IL22 compare other cells have compared with Strong cytotoxic effect, therefore, it is a kind of more feasible for improving T cell toxicity by enhancing the secreting, expressing amount of IL22 Approach.With this result to corresponding, the MUC1 expression of Cal33 cell is by being adjusted to 95% or so on original 70%, also further table It is bright, it is feasible by improving IL-22 expression quantity come the expression for raising MUC1 by being coupled IL-22 gene.

(4) mice tumors grew situation detects

On above-mentioned experiment basis, inventor's further progress mouse animal experiment, detailed process are briefly discussed below.

Using NOD/SCID immunodeficient mouse, PBS is resuspended 10 are inoculated⁶HN4-fluc cell (100ul), structure The subcutaneous lotus knurl model of mouse is built, after 12 days, injects 5 × 10 respectively through tail vein⁶The other T cell of a difference group (PBS, GFP-T, MUC1-T, MUC1-IL22-T) treated (every group 5).

Electronic scale and vernier caliper detection mouse weight, tumor volume change are distinguished in experimentation and utilize petty action Object living imaging equipment detects the change in fluorescence that tumour is expressed, and (the bioluminescence photo of shooting in every 10 days is put to death small on the 40th day Mouse is analyzed).

When the change in fluorescence expressed using small animal living body imaging device detection tumour, first with 3% Isoflurane (RWD life Science, Shenzhen, China) anesthetized mice in induction room；Then d-fluorescence of 100ul is injected intraperitoneally with syringe for every mouse Plain solution (0.15mg/ml, Yeasen Biotech Co., Ltd., Shanghai, China), it is living using animal after ten minutes III spectrometer of body imaging device IVIS Lumina, Series (Caliper Life Science) detection is glimmering Light is finally analyzed using live image 4.3.1 software (PerkinElmer, Waltham, MA, USA).

Experimental result is as shown in figure 11.As a result counting (measurement result based on the 40th day last time) and analysis can see Out:

In terms of tumour fluorescence intensity: in PBS and GFP-T treatment group, the tumour fluorescence of mouse is all 10⁹Rank, the two do not have Difference, and the mouse tumor fluorescence for passing through CAR-MUC1-T cell therapy drops to 10⁸Rank, it is thin by CAR-MUC1-IL22-T The mouse tumor fluorescence of born of the same parents' treatment drops to 10⁷Rank compares between each group, does not have difference before first two groups, latter two groups with First two groups have notable difference, and fluorescence is substantially reduced, and CAR-MUC1-IL22-T cell therapy group is relative to CAR-MUC1-T cell Treatment group's fluorescence further declines an order of magnitude, and difference is obvious；

In terms of gross tumor volume: preceding two groups of gross tumor volume is all in 1500mm³The mouse of left and right, CAR-MUC1-T cell therapy group is swollen Knurl product drops to 350mm³, and the mouse tumor volume for passing through CAR-MUC1-IL22-T cell therapy is reduced to 150mm³, it may be assumed that CAR-MUC1-IL22-T cell therapy group has apparent removing and inhibiting effect to mouse tumor, and function and effect are clearly better than First three groups.

To sum up experimental result can be seen that CAR-T cell therapy effect it is related with the expression of tumor cell surface antigen, The face of the present invention CAR structure for targeting MUC1 existing at present optimizes, and the CAR-MUC1-IL22 structure of optimization is by slow disease Poison packaging and the CAR-MUC1-IL22-T cell of T cell infection building can have the table of effective induction tumor cell surface MUC1 Up to up-regulation to play the role of preferably killing and controlling tumour, the appearance of undershooting-effect can be effectively avoided.

SEQUENCE LISTING

<110>the first affiliated hospital, Zhengzhou University

<120>induction tumour cell up-regulation antigen MUC1 expression CAR and its application

<130> none

<160> 8

<170> PatentIn version 3.5

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

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Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

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Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu

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

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Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

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Tyr Cys Thr Phe Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr Thr

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

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Gly Gly Gly Ser Asp Ile Val Val Thr Gln Glu Ser Ala Leu Thr Thr

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

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Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His

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

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Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr

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

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Leu Gly Ser Glu

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

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Leu Val Ile Thr Leu Tyr Cys

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

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

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Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn Phe Gln

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Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe

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Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln Ser Asp Arg Phe Gln

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Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu

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Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Met Lys Leu

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Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp

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

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Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly

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

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Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Phe

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Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

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

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Asp Ile Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

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

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

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

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Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

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

260 265 270

His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Glu

275 280 285

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

290 295 300

His Ala Ala Arg Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

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

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

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

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Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys

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Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

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

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

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

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

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

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

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

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

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Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

530 535 540

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

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Ser Phe Leu Met Gly Thr Leu Ala Thr Ser Cys Leu Leu Leu Leu Ala

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Leu Leu Val Gln Gly Gly Ala Ala Ala Pro Ile Ser Ser His Cys Arg

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Leu Asp Lys Ser Asn Phe Gln Gln Pro Tyr Ile Thr Asn Arg Thr Phe

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Met Leu Ala Lys Glu Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg

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Leu Ile Gly Glu Lys Leu Phe His Gly Val Ser Met Ser Glu Arg Cys

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Tyr Leu Met Lys Gln Val Leu Asn Phe Thr Leu Glu Glu Val Leu Phe

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Pro Gln Ser Asp Arg Phe Gln Pro Tyr Met Gln Glu Val Val Pro Phe

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Leu Ala Arg Leu Ser Asn Arg Leu Ser Thr Cys His Ile Glu Gly Asp

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Asp Leu His Ile Gln Arg Asn Val Gln Lys Leu Lys Asp Thr Val Lys

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Lys Leu Gly Glu Ser Gly Glu Ile Lys Ala Ile Gly Glu Leu Asp Leu

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Leu Phe Met Ser Leu Arg Asn Ala Cys Ile

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Claims

1. it is a kind of can induce tumour cell up-regulation antigen MUC1 expression CAR, which is characterized in that the Chimeric antigen receptor be as The amino acid sequence of dried albumen segment connection, is named with MUC1-41BB-CD3z, specifically: sequentially connected people CD8a molecule letter Number Peptide C MV, humanization MUC1 single-chain antibody scFV.MUC1, people CD8 molecule across membranes area and 41BB molecule intracellular region 41BB, people CD3z molecule intracellular region CD3Zeta, it may be assumed that CMV-scFV.MUC1-41BB-CD3Zeta；

The people CD8a molecular signal Peptide C MV, including 21 amino acid, sequence as shown in SEQ ID No.1, specifically:

MALPVTALLLPLALLLHAARP；

The humanization MUC1 single-chain antibody scFV.MUC1, including 244 amino acid, sequence is as shown in SEQ ID No.2, tool Body are as follows:

The people CD8 molecule across membranes area and 41BB molecule intracellular region 41BB, including people CD8 molecule across membranes area and 41BB molecule it is intracellular Two, area part, wherein

LETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC；

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；

The people CD3z molecule intracellular region CD3Zeta, including 112 amino acid, sequence as shown in SEQ ID No.5, specifically:

2. can induce the CAR of tumour cell up-regulation antigen MUC1 expression as described in claim 1, which is characterized in that people CD3z points Sub- intracellular region CD3Zeta further passes through catenation sequence and is attached with IL22 CDS region sequence IL22；

The IL22 CDS region sequence IL22, including 179 amino acid, sequence as shown in SEQ ID No.7, specifically:

3. can induce the CAR of tumour cell up-regulation antigen MUC1 expression as claimed in claim 3, which is characterized in that the connection Sequence is TA2 catenation sequence T2A；

The TA2 catenation sequence T2A, including 18 amino acid, sequence as shown in SEQ ID No.6, specifically:

EGRGSLLTCGDVEENPGP；

Therefore, the CAR overall structure constituted are as follows: CMV-scFV.MUC1-41BB-CD3Zeta-T2A-IL22, with MUC1-41BB-CD3z-IL22 name；

The MUC1-41BB-CD3z-IL22, amino acid sequence include 730 amino acid, sequence as shown in SEQ ID No.8, Specifically:

4. using slow constructed by the CAR that can induce tumour cell up-regulation antigen MUC1 expression described in any one of claim 1 ~ 3 Viral expression plasmids, which is characterized in that be prepared especially by following steps:

(1) according to central dogma, the coded sequence DNA of the CAR is obtained；

(3) connection product in step (2) is converted into STABL3 competent cell, screening expands and cultivates laggard onestep extraction matter Grain can be obtained slow virus expression plasmid after recombination.

5. slow virus expression plasmid described in claim 4 is preparing the application in anti-tumor agents, which is characterized in that in application, It is prepared into CAR-T cell by transfection to be applied, for improving the MUC1 expression quantity of tumour cell；Concrete application step includes Following steps:

(1) packaging is prepared into slow virus；

(2) CD of purifying is obtained³⁺T cell

(3) T cell infects, and CD is activated in step (2)³⁺In T cell, institute's virus and polybrene in step (1) is added, incubates It educates overnight to be infected；

(4) it expands T cell and detects spare, T cell after infecting incubation in step (3), after recovery and rinsing, culture medium is added, Further to expand T cell；It is spare after qualified to T cell detection after amplification, or can be used to stimulation after further feeding back and mention The MUC1 expression of high tumour cell.

6. slow virus expression plasmid as claimed in claim 5 is preparing the application in anti-tumor agents, which is characterized in that step (1) in, when packaging prepares slow virus, using 293T cell as aim cell to be transfected.

7. slow virus expression plasmid as claimed in claim 5 is preparing the application in anti-tumor agents, which is characterized in that described swollen Tumor is specially HNSCC cancer related neoplasms.

8. slow virus expression plasmid as claimed in claim 7 is preparing the application in anti-tumor agents, which is characterized in that described HNSCC cancer is specially Human tongue cancer cell line system CAL33 or the population pharynx corresponding cancer of squamous cell carcinoma system HN4.