CN107058234B

CN107058234B - CAR.IL-33-T and its preparation and application

Info

Publication number: CN107058234B
Application number: CN201710334059.8A
Authority: CN
Inventors: 蒋敬庭; 卢斌峰; 陈与昂; 陈陆俊
Original assignee: First Peoples Hospital of Changzhou
Current assignee: First Peoples Hospital of Changzhou
Priority date: 2017-05-12
Filing date: 2017-05-12
Publication date: 2019-09-13
Anticipated expiration: 2037-05-12
Also published as: CN107058234A; WO2018205343A1

Abstract

The present invention relates to field of biotechnology, and in particular to a kind of CAR.IL-33-T cell and its preparation and application.Present invention firstly provides a kind of Chimeric antigen receptor T cell, expression targets the Chimeric antigen receptor (CAR) and interleukin-33 (IL-33) of tumour antigen.Compared with CAR-T cell, CAR.IL-33-T cell, which has, stronger kills tumor function.And confirm that CAR.IL-33-T cell is a kind of safe, special, effective CAR-T cell, there is important clinical value.

Description

CAR.IL-33-T and its preparation and application

Technical field

The present invention relates to field of biotechnology, and in particular to a kind of CAR.IL-33-T and its preparation and application.

Background technique

Chimeric antigen receptor (Chimeric antigen receptor, CAR) modifies T cell (CAR-T cell therapy skill Art) it is to develop a kind of very fast adoptive immunity cell therapy technology in recent years.Pass through genetic modification technology, effector T cell Targeting, killing activity and persistence be superior to the immunocyte routinely applied, and tumor by local immunosupress can be overcome micro- Environment and break host immune tolerance status, is targeted therapy mode new in tumor vaccine cells therapy field.CAR-T cell The characteristics for the treatment of technology, is going back to the nest the targeting specific of antibody and T cell, in conjunction with tissue penetration and targeting lethality Get up to be used for oncotherapy, and achieve rapid development in the past ten years, Several Kinds of Malignancy basic research and face Significant curative effect is achieved in bed conversion, multiple clinical test results are also exciting.

CAR-T technology will identify the antigen-binding portion of the antibody of certain tumour antigen and the born of the same parents of CD3- ζ chain or Fc ε RI γ Inner part is coupled in vitro makes it express Chimeric antigen receptor for a chimeric protein by gene transfer patient T cells, suffers from For the T cell of person by after " recodification ", the CAR-T for generating massive tumor specificity is thin.It is different according to the structure of intracellular region, it can incite somebody to action CAR-T is divided into three generations: first generation CAR-T (cFv⁺Signal transduction part) although the killing to tumour cell can be mediated to make With, but proliferation signal and inducing cytokine generation are not transduceed, and the continuous action time is not grown in vivo；Second generation CAR-T Intracellular domain by increasing costimulatory molecules (such as CD28) promotes it to expand energy rapidly to extend its internal time-to-live Power, the CAR-T by constructing scFv/Fc/CD28/CD3- ζ can crack target cell, and not need the effect of B7 and CD28, only Activation signals can be transmitted by single molecule, generate the cell factors such as a large amount of IFN-γ, IL-2；Third generation CAR-T increases More costimulatory signal structural domains (usually Tumor Necrosis Factor Receptors family members), such as 4-1BB is (also known as CD137), OX40 (also known as CD134), CD27, inducible co-stimulator (Inducible co-stimulatory Molecule, ICOS) etc., third generation CAR-T has preferably enhancing T cell activation, amplification, antitumor and rush compared with the second generation Into the energy of transgene expression.

At present Successful utilization in clinical CAR-T cell category mainly include CD19-CAR-T, HER-2/Neu-CAR-T Deng, in world wide multiple centers target different tumor associated antigens (Tumor associated antigen, TAA CAR-T cell therapy clinical research), including targeting CD133, CD138, CD20, CD30, EGFR, c-MET etc..It is real at present The target spot that body tumor has lacked, mainly due to the heterogeneity of solid tumor cell, so collecting and testing by a large amount of clinical samples Card, finds the important directions that more specific solid tumor target spot is current CAR-T cell therapy field.Our team are first The CAR-T cells such as target tumor antigens c EA, MUC-1 are constructed afterwards, tentatively establish the technology platform of CAR-T cell preparation, are carried out It is studied before range of clinical.

Immunotherapy of tumors by improve tumour cell immunogenicity and pairing effect cell killing sensibility, excitation or The immune system of body is transferred, enhancing tumor microenvironment anti-tumor immune response is horizontal, so that control and killing tumor cell, swell Tumor immunization therapy is also verified in more and more clinical applications as the antitumor best approach.However, tumour micro-loop The immunosupress in border is still to influence the principal element of immunization therapy curative effect, and tumour occurs to lack in progression appropriate " dangerous Signal " triggers and maintains oncological surveillance and anti-tumor immune response.

Summary of the invention

The case where in view of the prior art, the purpose of the present invention is to provide the inosculating antibodies that a kind of expression targets tumour antigen Original receptor and the T cell and its preparation and application that interleukin-33 can be secreted.

To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:

The first aspect of the present invention, provides a kind of Chimeric antigen receptor T cell, and expression targets the chimeric of tumour antigen Antigen receptor (CAR) and interleukin-33 (IL-33).

Preferably, the Chimeric antigen receptor (CAR) for targeting tumour antigen is expressed on T cell film.

Preferably, interleukin-33 (IL-33) expression secretion is in outside T cell film.

Preferably, the Chimeric antigen receptor for targeting tumour antigen includes the more of sequentially connected combination tumour antigen Peptide, CD28 are across touching area (CD28-TM), 4-1BB intracellular and CD3 ζ intracellular.

Preferably, the tumour antigen is carcinomebryonic antigen (CEA).

Preferably, the polypeptide of the combination carcinomebryonic antigen is the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA).

Preferably, the single-chain antibody (scFv) of the anti-carcinoembryonic antigen (CEA) includes light chain variable region and heavy chain variable region.

Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.11.

Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.12.

In some embodiments of the invention, the amino acid of the single-chain antibody (scFv) of the anti-carcinoembryonic antigen (CEA) is listed Shown in sequence SEQ ID NO.10.

Preferably, the amino acid sequence of the CD28 transmembrane region is as shown in SEQ ID NO.13.

Preferably, the amino acid sequence of the CD3 ζ intracellular is as shown in SEQ ID NO.14.

Preferably, the amino acid sequence of the 4-1BB intracellular is as shown in SEQ ID NO.15.

Preferably, pass through connection sheet between the polypeptide and CD28 transmembrane region (CD28-TM) of the combination tumour antigen (CEA) Section connection.The amino acid quantity of the junction fragment can be >=2.

Preferably, the junction fragment is the hinge area segment of Immunoglobulin IgD.It is further preferred that the connection Segment is Immunoglobulin IgG4 hinge area segment.

Preferably, the amino acid sequence of the Immunoglobulin IgG4 hinge area segment is as shown in SEQ ID NO.16.

The junction fragment can be interconnected to form dimer by disulfide bond.

Preferably, the N of the C-terminal of the polypeptide of the combination tumour antigen (CEA) and the CD28 transmembrane region (CD28-TM) End connection.

In some embodiments of the invention, the amino acid sequence of the Chimeric antigen receptor for targeting carcinomebryonic antigen is listed As shown in SEQ ID NO.9.

In some embodiments of the invention, the amino acid sequence such as SEQ ID NO.20 institute of interleukin-33 (IL-33) is listed Show.

The second aspect of the present invention provides a kind of method for preparing aforementioned Chimeric antigen receptor T cell, comprising steps of

(1) the Chimeric antigen receptor expression vector for targeting tumour antigen of interleukin-33 has been merged in building；

(2) step (1) described expression vector is converted into T cell inducing expression, obtains expression and targets tumour antigen Chimeric antigen receptor (CAR) and interleukin-33 (IL-33) Chimeric antigen receptor T cell.

Preferably, in step (1), the tumour antigen that targets for having merged interleukin-33 is contained in the expression vector Chimeric antigen receptor encoding polynucleotide sequence.

Preferably, the Chimeric antigen receptor coded polynucleotide for targeting tumour antigen for having merged interleukin-33, Be the Chimeric antigen receptor coded polynucleotide for targeting tumour antigen C-terminal successively merged t2A coded polynucleotide and Interleukin-33 (L-33) coded polynucleotide.

Preferably, the tumour antigen is carcinomebryonic antigen.

Preferably, the encoding polynucleotide sequence such as SEQ ID NO.8 institute of the Chimeric antigen receptor of carcinomebryonic antigen is targeted Show.

Preferably, the sequence of the coded polynucleotide of the t2A is as shown in SEQ ID NO.17.

Preferably, the sequence of the coded polynucleotide of the interleukin-33 is as shown in SEQ ID NO.18.

Preferably, the sequence of the Chimeric antigen receptor coded polynucleotide for targeting carcinomebryonic antigen of interleukin-33 has been merged Column are as shown in SEQ ID NO.19.

The third aspect of the present invention provides a kind of polynucleotides, targets tumour antigen for merged interleukin-33 Chimeric antigen receptor coded polynucleotide.

Preferably, the Chimeric antigen receptor coded polynucleotide for targeting tumour antigen for having merged interleukin-33, Be the Chimeric antigen receptor coded polynucleotide for targeting carcinomebryonic antigen C-terminal successively merged t2A coded polynucleotide and Interleukin-33 (L-33) coded polynucleotide.

Preferably, the tumour antigen is carcinomebryonic antigen.

Fourth aspect present invention provides a kind of expression vector, contains the polynucleotides.

Fifth aspect present invention provides a kind of host cell, is converted by the expression vector.

The sixth aspect of the present invention provides use of the aforementioned Chimeric antigen receptor T cell in preparation tumor therapeutic agent On the way.

The seventh aspect of the present invention provides a kind of tumor therapeutic agent, contains the Chimeric antigen receptor T cell.

The ninth aspect of the present invention provides a kind of tumor therapeuticing method, comprising steps of the Chimeric antigen receptor T is thin Born of the same parents are applied to tumor patient.

Preferably, the tumour is the highly expressed tumour of carcinomebryonic antigen.Such as: colon cancer, intestinal cancer, Gastric cancer with liver metastasis and pancreas Gland cancer.

Compared with prior art, the invention has the benefit that

The treatment of the malignant entity tumors such as intestinal cancer, Gastric cancer with liver metastasis and cancer of pancreas is still the difficult point of oncotherapy, although hand Art treatment, chemicotherapy, interventional therapy etc. have a certain curative effect, but survival is still without significantly improving.Currently, be concerned CAR-T cell therapy technology is expected to the breach as such oncotherapy.The present invention is expressed according to such tumour-specific height The characteristics of CEA tumour antigen, designs the specific antibody of targets identification CEA, using second generation CAR skeleton as carrier, design CEA.CAR and CEA.CAR.IL-33.And it cultivates and has expanded CEA.CAR and CEA.CAR.IL-33-T cell, and by a series of External and in vivo studies, it was demonstrated that the CEA.CAR-T cell of our designed, designeds can be with specific recognition and to kill CEA highly expressed Human tumor cell line and Transplanted tumor model, compared with CEA.CAR-T cell, CEA.CAR.IL-33-T cell, which has, stronger kills tumor Function.And confirm that CEA.CAR.IL-33-T cell is a kind of safe, special, effective CAR-T cell, there is important clinic Application value.

Detailed description of the invention

Fig. 1: CEA.CAR structural schematic diagram.

The entire expression mount structure schematic diagram of Fig. 2: CEA.CAR and CEA.CAR.IL-33.

Fig. 3 A: streaming the results show that IFN-γ stimulation before and after, all high expression CEA of LoVo cell.

For Fig. 3 B:ELISA the results show that with other tumour cells compared with CART co-cultures supernatant, LoVo is chimeric with CEA CD4+T cell or CEA are fitted into CD8+T cell and educate rear IFN-γ secretion highest altogether.

After Fig. 3 C:CEA.CAR-T (BFP+) and LoVo cell are educated altogether, T cell activation mark molecule (CD69) up-regulation.

Fig. 4: LoVo-NSG mouse model shows that CEA.CAR-T effectively inhibits tumour growth, with CEA.CAR-T cell phase Than CEA.CAR.IL-33-T cell has stronger cytotoxicity.

Specific embodiment

CAR-T

CAR-T, full name are Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen by Body T cell immunotherapy.

Chimeric antigen receptor Chimeric Antigen Receptor (CAR).

Interleukin-33 (Interleukin-33, IL-33)

An important member of the interleukin-33 (Interleukin-33, IL-33) as interleukin-1 (IL-1) family. IL-33 can be discharged by damage/infection histocyte or activated macrophage, be come as a kind of endogenic " danger signal " Trigger inflammation and cell-mediated immune response.The expression of IL-33 can significantly increase tumor group in up-regulation tumor microenvironment Knit middle infiltration CD8⁺T cell and the ratio of NK cell simultaneously enhance its effector function, and tumour immunity is escaped in the downward of IL33 expression It keeps away most important, is the important mechanisms for leading to tumour " immune ignore ".Early-stage study has proven to IL-33 receptor ST2 and is expressed in CD8⁺T cell, Th1 cell, on NK and NKT cell, this shows that IL-33/ST2 has important work in I type anti-tumor immune response With.At the same time, tumour growth can significantly be prevented by IL-33 being raised in TME, increased and infiltrated CD8 in tumor tissues⁺T cell and The ratio of NK cell, and promote tumor-infiltrated CD8⁺The IFN-γ secretion of T and NK cell enhances its effector function.

It is proposed that joint CAR and IL-33 effectively can convey IL-33 to tumor locus, the anti-of enhancing CAR-T cell swells Tumor function.

Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text In addition explicitly point out, singular "one", " one " and " this " include plural form.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc. MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

Embodiment 1 targets the building of the Chimeric antigen receptor expression vector of carcinomebryonic antigen and has merged interleukin-33 Target the building of the Chimeric antigen receptor expression vector of carcinomebryonic antigen

In the present invention, the Chimeric antigen receptor for targeting carcinomebryonic antigen may be simply referred to as CEA.CAR.

In of the invention, the Chimeric antigen receptor for targeting carcinomebryonic antigen for having merged interleukin-33 be may be simply referred to as CEA.CAR.IL-33。

In the present invention, as shown in Figure 1, CEA.CAR by the polypeptide of sequentially connected combination carcinomebryonic antigen (CEA), CD28 across Area (CD28-TM), 4-1BB intracellular and CD3 ζ intracellular is touched to merge.

It wherein, is the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA) in conjunction with the polypeptide of carcinomebryonic antigen (CEA).The list Chain antibody includes light chain variable region (V_L) and heavy chain variable region (V_H).Light chain variable region (the V_L) coding nucleotide sequence such as Shown in SEQ ID NO.1, specifically:

gacacactcctgctatgggtgctgctgctctgggttccaggttccacaggtgacattgtgctgaccca atctccagcttc

tttggctgtgtctcttgggcagagggccaccatgtcctgcagagccggtgaaagtgttgatatttttg gcgttgggtttt

tgcactggtaccagcagaaaccaggacagccacccaaactcctcatctatcgtgcatccaacctagaa tctgggatccct

gtcaggttcagtggcactgggtctaggacagacttcaccctcatcattgatcctgtggaggctgatga tgttgccaccta

ttactgtcagcaaactaatgaggatccgtacacgttcggaggggggaccaagctggaaataaaa。

Heavy chain variable region (the V_H) coding nucleotide sequence as shown in SEQ ID NO.2, specifically:

agcagcgaggttcagctacaacagtctggggcagagcttgtggagccaggggcctcagtcaagttgtc ctgcacagcttc

tggcttcaacattaaagacacctatatgcactgggtgaagcagaggcctgaacagggcctggaatgga ttggaaggattg

atcctgcgaatggtaatagtaaatatgtcccgaagttccagggcaaggccactataacagcagacaca tcctccaacaca

gcctacctgcagctcaccagcctgacatccgaggacactgccgtctattattgtgctccgtttggtta ctacgtgtctga

ctatgctatggcctactggggtcaaggaacctcagtcaccgtctcctca。

The coding nucleotide sequence of the single-chain antibody (scFv) of the anti-carcinoembryonic antigen (CEA) as shown in SEQ ID NO.3, Specifically:

ttactgtcagcaaactaatgaggatccgtacacgttcggaggggggaccaagctggaaataaaaggca gtactagcggcg

gtggctccgggggcggttccggtgggggcggcagcagcgaggttcagctacaacagtctggggcagag cttgtggagcca

ggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagacacctatatgcactgggt gaagcagaggcc

tgaacagggcctggaatggattggaaggattgatcctgcgaatggtaatagtaaatatgtcccgaagt tccagggcaagg

ccactataacagcagacacatcctccaacacagcctacctgcagctcaccagcctgacatccgaggac actgccgtctat

tattgtgctccgtttggttactacgtgtctgactatgctatggcctactggggtcaaggaacctcagt caccgtctcctca。

The coding nucleotide sequence of CD28 transmembrane region (CD28-TM) as shown in SEQ ID NO.4, specifically: Atgttctg ggtgctggtggtggtgggcggggtgctggcctgctacagcctgctggtgacagtggccttcatcatcttttgggtg。

The coding nucleotide sequence of 4-1BB intracellular as shown in SEQ ID NO.5, specifically:

CGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACC AGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG。

The coding nucleotide sequence of CD3 ζ intracellular as shown in SEQ ID NO.6, specifically:

cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacga gctgaacctgggcagaagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaag cctcggcggaagaacccccaggaaggcctgtataacgaactgcAgaaagacaagatggccgaggcctacagcgaga tcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatcagggcctgtccaccgccaccaagga tacctacgacgccctgcacatgcaggccctgcccccaagg。

In conjunction with the polypeptide of carcinomebryonic antigen (CEA), CD28 across touching Immunoglobulin IgG4 hinge area piece between area (CD28-TM) The connection of Duan Zuowei junction fragment.The coding nucleotide sequence of the Immunoglobulin IgG4 hinge area segment such as SEQ IDNO.7 institute Show, specifically:

Gagagcaagtacggccctccctgccccccttgccctgcccccgagttcctgggcggacccagcgtgtt cctgttcccccccaagcccaaggacaccctgatgatcagccggacccccgaggtgacctgcgtggtggtggacgtg agccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccca gagaggaacagttcaacagcacctaccgggtggtgtctgtgctgaccgtgctgcaccaggactggctgaacggcaa agaatacaagtgcaaggtgtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaaggccaagggccag cctcgcgagccccaggtgtacaccctgcctccctcccaggaagagatgaccaagaaccaggtgtccctgacctgcc tggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcctgagaacaactacaagac cacccctcccgtgctggacagcgacggcagcttcttcctgtacagccggctgaccgtggacaagagccggtggcag gaaggcaacgtctttagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgt ccctgggcaag。

For the ease of the identification to CEA.CAR, t2A-Tag BFP is connected in the C-terminal of CD3 ζ intracellular.

Authorized company synthesizes the entire expression cassette of CEA.CAR, as shown in Fig. 2, insertion pCART-EF1a-ScFv-car frame carries Body (is purchased from Shanghai Ji Kai company), after being sequenced correctly, is extracted using the plasmid purification kit of Qiagen company and purifies matter Grain, obtains the high-quality plasmid of recombinant expression carrier.

Through sequencing it is found that the full-length gene order of CEA.CAR is correct, it is consistent with expection.

Specifically, the coding nucleotide sequence of CEA.CAR is as shown in SEQ ID NO.8, specifically:

atggagacagacacactcctgctatgggtgctgctgctctgggttccaggttccacaggTgacacact cctgctatgggtgctgctgctctgggttccaggttccacaggtgacattgtgctgacccaatctccagcttctttg gctgtgtctcttgggcagagggccaccatgtcctgcagagccggtgaaagtgttgatatttttggcgttgggtttt tgcactggtaccagcagaaaccaggacagccacccaaactcctcatctatcgtgcatccaacctagaatctgggat ccctgtcaggttcagtggcactgggtctaggacagacttcaccctcatcattgatcctgtggaggctgatgatgtt gccacctattactgtcagcaaactaatgaggatccgtacacgttcggaggggggaccaagctggaaataaaaggca gtactagcggcggtggctccgggggcggttccggtgggggcggcagcagcgaggttcagctacaacagtctggggc agagcttgtggagccaggggcctcagtcaagttgtcctgcacagcttctggcttcaacattaaagacacctatatg cactgggtgaagcagaggcctgaacagggcctggaatggattggaaggattgatcctgcgaatggtaatagtaaat atgtcccgaagttccagggcaaggccactataacagcagacacatcctccaacacagcctacctgcagctcaccag cctgacatccgaggacactgccgtctattattgtgctccgtttggttactacgtgtctgactatgctatggcctac tggggtcaaggaacctcagtcaccgtctcctcaGagagcaagtacggccctccctgccccccttgccctgcccccg agttcctgggcggacccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagccggacccccga ggtgacctgcgtggtggtggacgtgagccaggaagatcccgaggtccagttcaattggtacgtggacggcgtggaa gtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctaccgggtggtgtctgtgctgaccgtgc tgcaccaggactggctgaacggcaaagaatacaagtgcaaggtgtccaacaagggcctgcccagcagcatcgaaaa gaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacaccctgcctccctcccaggaagagatgacc aagaaccaggtgtccctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacg gccagcctgagaacaactacaagaccacccctcccgtgctggacagcgacggcagcttcttcctgtacagccggct gaccgtggacaagagccggtggcaggaaggcaacgtctttagctgcagcgtgatgcacgaggccctgcacaaccac tacacccagaagagcctgagcctgtccctgggcaagAtgttctgggtgctggtggtggtgggcggggtgctggcct gctacagcctgctggtgacagtggccttcatcatcttttgggtgCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAA ACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGcgggtgaagttcagcagaagcgccgacgcccctgcctaccagc agggccagaatcagctgtacaacgagctgaacctgggcagaagggaagagtacgacgtcctggataagcggagagg ccgggaccctgagatgggcggcaagcctcggcggaagaacccccaggaaggcctgtataacgaactgcAgaaagac aagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatc agggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccccaagg。

Using the prior art, inducing expression in host cell is gone to the high-quality plasmid of recombinant expression carrier, from expression It is isolated and purified in product and obtains CEA.CAR.To the CEA.CAR of acquisition through N/C terminal sequence analysis, the results showed that expressed The equal frame of CEA.CAR is errorless, consistent with theoretical N/C terminal amino acid sequence.

Therefore, it can be seen that, the amino acid sequence of CEA.CAR as shown in SEQ ID NO.9, specifically:

METDTLLLWVLLLWVPGSTGDTLLLWVLLLWVPGSTGDIVLTQSPASLAVS

LGQRATMSCRAGESVDIFGVGFLHWYQQKPGQPPKLLIYRASNLESGIPVRFSG

TGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIKGSTSGGGSGGG

SGGGGSSEVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEW

IGRIDPANGNSKYVPKFQGKATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGY

YVSDYAMAYWGQGTSVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK

NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK

SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTV

AFIIFWVRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP

QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA

LPPRL。

The amino acid sequence of the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA) as shown in SEQ ID NO.10, specifically:

DTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATMSCRAGESVDIFGVGFLHWYQQKPGQPPKLL IYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIKGSTSGGGSGGGSGGGG SSEVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKATITADT SSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWGQGTSVTVSS。

The amino acid sequence such as SEQ ID NO.11 of the light chain variable region of the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA) It is shown, specifically:

DTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATMSCRAGESVDIFGVGFLHWYQQKPGQPPKLL IYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIK。

The amino acid sequence such as SEQ ID NO.12 of the heavy chain variable region of the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA) It is shown, specifically:

SSEVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQG KATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWGQGTSVTVSS。

The amino acid sequence of CD28 transmembrane region (CD28-TM) as shown in SEQ ID NO.13, specifically:

MFWVLVVVGGVLACYSLLVTVAFIIFWV。

The amino acid sequence of 4-1BB intracellular as shown in SEQ ID NO.14, specifically:

RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

The amino acid sequence of CD3 ζ intracellular as shown in SEQ ID NO.15, specifically:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

In conjunction with Immunoglobulin IgG4 hinge area piece between the polypeptide of carcinomebryonic antigen (CEA), CD28 transmembrane region (CD28-TM) The connection of Duan Zuowei junction fragment.The amino acid sequence of the Immunoglobulin IgG4 hinge area segment such as SEQ ID NO.16 institute Show, specifically:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK。

As shown in Fig. 2, CEA.CAR.IL-33 is to be connected to t2A-IL-33 in the C-terminal of the CD3 ζ intracellular of CEA.CAR..

The sequence of the coded polynucleotide of t2A as shown in SEQ ID NO.17, specifically:

gagggcagaggaagtcttctaacatgcggtgacGtggaggagaatcccggccctcgc。

The encoding polynucleotide sequence of Human secreting type IL-33 as shown in SEQ ID NO.18, specifically:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTAGCCTTGCTGCTCCACGCCGCCAGGCCGagtat cacaggaatttcacctattacagagtatcttgcttctctaagcacatacaatgatcaatccattacttttgctttg gaggatgaaagttatgagatatatgttgaagacttgaaaaaagatgaaaagaaagataaggtgttactgagttact atgagtctcaacacccctcaaatgaatcaggtgacggtgttgatggtaagatgttaatggtaaccctgagtcctac aaaagacttctggttgcatgccaacaacaaggaacactctgtggagctccataagtgtgaaaaaccactgccagac caggccttctttgtccttcataatatgcactccaactgtgtttcatttgaatgcaagactgatcctggagtgttta taggtgtaaaggataatcatcttgctctgattaaagtagactcttctgagaatttgtgtactgaaaatatcttgtt taagctctctgaaacttag。

Authorized company synthesizes the entire expression cassette of CEA.CAR.IL-33, as shown in Fig. 2, insertion slow virus carrier is (purchased from upper Hai Jikai company), after being sequenced correctly, simultaneously plasmid purification is extracted using the plasmid purification kit of Qiagen company, is weighed The high-quality plasmid of group expression vector.

By sequencing it is found that the overall length encoding gene of CEA.CAR.IL-33 is correct, it is consistent with expection.Specifically, The coding nucleotide sequence of CEA.CAR.IL-33 as shown in SEQ ID NO.19, specifically:

atggagacagacacactcctgctatgggtgctgctgctctgggttccaggttccacaggTgacacact cctgctatgggt

gctgctgctctgggttccaggttccacaggtgacattgtgctgacccaatctccagcttctttggctg tgtctcttgggc

agagggccaccatgtcctgcagagccggtgaaagtgttgatatttttggcgttgggtttttgcactgg taccagcagaaa

ccaggacagccacccaaactcctcatctatcgtgcatccaacctagaatctgggatccctgtcaggtt cagtggcactgg

gtctaggacagacttcaccctcatcattgatcctgtggaggctgatgatgttgccacctattactgtc agcaaactaatg

aggatccgtacacgttcggaggggggaccaagctggaaataaaaggcagtactagcggcggtggctcc gggggcggttcc

ggtgggggcggcagcagcgaggttcagctacaacagtctggggcagagcttgtggagccaggggcctc agtcaagttgtc

ctgcacagcttctggcttcaacattaaagacacctatatgcactgggtgaagcagaggcctgaacagg gcctggaatgga

ttggaaggattgatcctgcgaatggtaatagtaaatatgtcccgaagttccagggcaaggccactata acagcagacaca

tcctccaacacagcctacctgcagctcaccagcctgacatccgaggacactgccgtctattattgtgc tccgtttggtta

ctacgtgtctgactatgctatggcctactggggtcaaggaacctcagtcaccgtctcctcaGagagca agtacggccctc

cctgccccccttgccctgcccccgagttcctgggcggacccagcgtgttcctgttcccccccaagccc aaggacaccctg

atgatcagccggacccccgaggtgacctgcgtggtggtggacgtgagccaggaagatcccgaggtcca gttcaattggta

cgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctacc gggtggtgtctg

tgctgaccgtgctgcaccaggactggctgaacggcaaagaatacaagtgcaaggtgtccaacaagggc ctgcccagcagc

atcgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacaccctgcctccctc ccaggaagagat

gaccaagaaccaggtgtccctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagt gggagagcaacg

gccagcctgagaacaactacaagaccacccctcccgtgctggacagcgacggcagcttcttcctgtac agccggctgacc

gtggacaagagccggtggcaggaaggcaacgtctttagctgcagcgtgatgcacgaggccctgcacaa ccactacaccca

gaagagcctgagcctgtccctgggcaagAtgttctgggtgctggtggtggtgggcggggtgctggcct gctacagcctgc

tggtgacagtggccttcatcatcttttgggtgCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTC CTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGcgggtgaagttcagcagaagcgccgacgcccctgcctaccagcaggg ccagaatcagctgtacaacgagctgaacctgggcagaagggaagagtacgacgtcctggataagcggagaggccgg gaccctgagatgggcggcaagcctcggcggaagaacccccaggaaggcctgtataacgaactgcAgaaagacaaga tggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatcaggg cctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccccaaggctcgagggcggcgga gagggcagaggaagtcttctaacatgcggtgacGtggaggagaatcccggccctcgcATGGCCTTACCAGTGACCG CCTTGCTCCTGCCGCTAGCCTTGCTGCTCCACGCCGCCAGGCCGAGTATCACAGGAATTTCACCTATTACAGAGTA TCTTGCTTCTCTAAGCACATACAATGATCAATCCATTACTTTTGCTTTGGAGGATGAAAGTTATGAGATATATGTT GAAGACTTGAAAAAAGATGAAAAGAAAGATAAGGTGTTACTGAGTTACTATGAGTCTCAACACCCCTCAAATGAAT CAGGTGACGGTGTTGATGGTAAGATGTTAATGGTAACCCTGAGTCCTACAAAAGACTTCTGGTTGCATGCCAACAA CAAGGAACACTCTGTGGAGCTCCATAAGTGTGAAAAACCACTGCCAGACCAGGCCTTCTTTGTCCTTCATAATATG CACTCCAACTGTGTTTCATTTGAATGCAAGACTGATCCTGGAGTGTTTATAGGTGTAAAGGATAATCATCTTGCTC TGATTAAAGTAGACTCTTCTGAGAATTTGTGTACTGAAAATATCTTGTTTAAGCTCTCTGAAACTTAG。

Using the prior art, inducing expression in host cell is gone to the high-quality plasmid of recombinant expression carrier, from expression It is isolated and purified in product and obtains CEA.CAR and IL-33.To the CEA.CAR and IL-33 of acquisition through N/C terminal sequence analysis, show The expressed equal frame of CEA.CAR and IL-33 is errorless, consistent with theoretical N/C terminal amino acid sequence.

Wherein, the amino acid sequence of CEA.CAR is as shown in SEQ ID NO.9.

The amino acid sequence of Human secreting type IL-33 as shown in SEQ ID NO.20, specifically:

MALPVTALLLPLALLLHAARPSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDK VLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKT DPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET。

Embodiment 2 targets the Chimeric antigen receptor expression slow virus preparation of carcinomebryonic antigen and has merged interleukin-33 Target the Chimeric antigen receptor expression slow virus preparation of carcinomebryonic antigen

One, slow virus is prepared

1. purpose: introducing the method for production and concentration for the slow virus of subsequent mammalian cell transduction.

2. equipment and material

* DMEM " complete " culture medium is the DMEM culture medium that 10% heat inactivation FBS is added.In Silver Lab, we It also added 100U/ml Pen .- Strep.

If § inactivates FBS without heat, conventional FBS can be placed in 56 DEG C of water-baths 30 minutes, thaw completely and FBS is inactivated with heat.

3. step

Points for attention: traditional virus preparation needs 8 10 cm dishes.It can be adjusted as needed.This process In, while 3 kinds of viruses are prepared, every kind of virus needs 8 10 cm dishes.

3.1 prepare HEK 293T:

When 3.1.1. preparing preparation slow virus, the passage of HEK 293T cell was inoculated in T-150 culture bottle in 3 days in advance. HEK 293T cell culture processes it is following (note: as far as possible shortening cell in trypsase is concentrated and outside incubator when Between):

3.1.1.1. the culture medium in culture bottle is exhausted, is discarded.

3.1.1.2. 0.25% trypsase of 2ml is added into each T-150 culture bottle, and tilts culture bottle to ensure Trypsase covers entire bottom of bottle.

3.1.1.3. 37 DEG C incubation 2-3 minutes.

3.1.1.4. check whether cell has fallen off from bottom of bottle；Tiltable culture bottle is to promote cell dissociation to fall off.

3.1.1.5. after cell dissociation falls off, into each culture bottle be added 10ml DMEM complete medium, and with shifting Liquid pipe is sufficiently mixed, to ensure that cell is suspended in solution completely.

3.1.1.6. 10 μ l cell suspending liquids are transferred in 600 μ l centrifuge tubes, and the dilution of 90 μ l culture mediums is added, mixes It is even.

3.1.1.7. 15 μ l are taken out from the cell suspension that above-mentioned 1:10 diluted and are mixed with 15 μ l trypan blues, are counted simultaneously Calculate cell density.

3.1.1.8. with 5 × 10⁶The cell density of a/20ml culture medium is by cell inoculation (3 days in T-150 culture bottle The cell quantity in each T-150 culture bottle can reach 45-60 × 10 afterwards⁶)。

3.1.1.9. the CO that culture bottle is lain against₂In incubator.

3.1.2. after cultivating 72 hours, cell growth status is observed, if cell density reaches 80-90%, cell state is good Good (cell is bright, is closely attached to bottom of bottle, and cell edges are smooth etc.).It then can be used for subsequent slow virus preparation.

3.2. slow virus preparation the 1st day:

Note: due to need first equal cell adherences in culture dish bottom can just start to carry out CalPhos transfection, and this mistake Journey probably needs 4-6 hours.Therefore will slow virus prepare the 1st day as early as possible by cell inoculation in 10cm culture dish.

Note: alternatively, can before transfection 24 hours according to 3.5 × 10⁶A/9ml density is inoculated in 10cm culture dish.

3.2.1. a kind of slow virus is prepared, 8 10cm Tissue Culture Dish are needed, and indicates date, name and plasmid ID number.

3.2.2. as described in 3.1.1.1-3.1.1.7, HEK 293T is cells trypsinised, and DMEM is cultivated completely Base, which terminates, to be digested and counts.

3.2.3. cell quantity needed for determining slow virus preparation: 7 × 10⁶A cell/ware, 8 ware/viruses.

3.2.4. the cell actually collected is 3.5 × 10 more than the above required cell quantity⁶It is a, with loss-prevention (for example, system Standby 2 kinds of viruses need to actually collect 171.5 × 10⁶, the cell of 8 culture dishes of every kind of virus).

3.2.5. suitable culture medium, which is added, makes ultimate density 7 × 10⁶A cell/9ml.

3.2.6. 9ml cell suspending liquid is distributed in each 10cm culture dish marked.

3.2.7. each culture dish is lain against on Biohazard Safety Equipment, along all around lightly moving back and forth, so that carefully Born of the same parents are uniform

It is laid on ware bottom.

3.2.8. culture dish is put back into incubator.Multiple culture bottle sieve are not put in incubator.Due to the growth of cell Situation is different, and cell density reaches 80% and probably needs 3-6 hour.Later, can start to carry out CalPhos transfection.

3.2.9. the reagent volume needed for according to the form below calculates.

Reagent	The amount that each culture dish needs
		The Plasmid DNA of building	15μg
Viral vectors PCMV-Rev2 (pS013)	1μg
		Viral vectors PCHGP-2 (pS014)	10μg
Viral vectors PCMV-G (pS015)	2μg
		2M CaCl₂	62μl
Sterile water	Total volume is 500 μ l

3.2.10. in the following order by sterile water (coming from CalPhos transfection reagent box), Plasmid DNA and 2M CaCl 2 Solution (coming from CalPhos transfection reagent box) is added in 15ml centrifuge tube, and is sufficiently mixed.

3.2.11. according to averagely each 500 μ l 2X HBS of reaction system, 50ml centrifuge tube is added in suitable 2X HBS In, suitably increase volume, avoids loss (for example, 8 culture dish reaction systems can add 4100 μ l HBS altogether).

3.2.12. use 1ml liquid-transfering gun, isometric DNA/CaCl2 mixture is added dropwise in HBS, dropwise addition it is same When shake gently HBS pipe.

3.2.13. mixture is incubated at room temperature 20 minutes.When closing to an end wait be incubated for, the 293T completed morning is taken out Cell.

3.2.14. mixing HBS/CaCl2/DNA solution is blown and beaten up and down with 1ml liquid-transfering gun.

3.2.15. 1ml solution is added drop-wise to dropwise in each culture dish.

3.2.16. step 3.2.7 is repeated, is mixed well.

3.2.17. after having handled all culture dishes, culture dish is put into incubator and is incubated overnight, be no more than 18 hours Carry out next step experiment.

3.3. the 2nd day (taking out CalPhos ingredient) of slow virus preparation

3.3.1. prepare 0.6M butyric acid sodium solution according to following operation:

3.3.1.1. 3.3027g sodium butyrate (Sigma, article No. B5887-5G) is dissolved in 50ml H₂In O.0.22- μm of vacuum Filter sterilised.Room temperature storage.

3.3.2. CalPhos ingredient (toxic to cell) is removed, and replaces culture medium in 18 hours after transfection.

3.3.2.1. 2.45ml 0.6M butyric acid culture medium needed for preparing the 2nd day: is added in 245ml DMEM complete medium Sodium.(note: totally 24 culture dishes in this experiment, addition 10ml culture medium in each culture dish can appropriate adjustment.)

3.3.2.2. above-mentioned culture medium and 1X PBS without magnesium, calcium are preheated to 37 DEG C.

3.3.2.3. it is the overlong time for avoiding cell from staying outside incubator, can disposably takes out two pieces of culture dishes successively It is handled.

3.3.2.4. to distinguish removal and the position of culture medium is added in one line of the central marks of culture dish, with reduction pair The injury of other position cells.

3.3.2.6. it is placed slowly with 10ml pipette and pipettor, gently absorbs culture medium.

The position for absorbing culture medium is gone to opposite side by 3.3.2.7. rotating and culturing ware, with 10ml pipette along culture dish Upper limb edge is slowly added to the PBS of the above-mentioned preheating of 5ml.Flow down culture medium can along culture dish bottom surface slowly.

3.3.2.8. edge moves back and forth culture dish (as shown in step 3.2.7) all around with cleaning down.Then it rotates Culture dish absorbs PBS cleaning solution in the position for absorbing culture medium.

3.3.2.9. it is slowly added into the DMEM complete medium containing sodium butyrate prepared in 10ml step 3.3.2.1.

3.3.2.10. culture dish is returned into incubator.

3.3.2.11. handling remaining culture dish according to step 3.3.2.3-3.3.2.9.

The third day of 3.4 production slow virus (generation is collected and PEG processing)

3.4.1 viral supernatants (changing liquid after 24 hours) is collected

3.5 viruses are resuspended

3.5.8.1 the every disk of virus being collected into is added the 1640 of 50 μ serum-frees.It not mixed up and down with pipette.

3.5.8.2 ultra-clean effective sealed membrane is sealed, shakes several seconds on the oscillator for its resuspension completely.

3.5.8.3, it is stood to 30min-1h in 4 degrees Celsius of environment, every part of virus makes marks on its bottom V cryovial.

3.5.8.4, viral suspension is shaken to mixing on the oscillator to unobvious agglomerate.(solution is timely after ultracentrifugation It is also thick for not having obvious sediment).

3.5.8.5 the re-suspension liquid of same virus is added in the same pipe.

3.5.8.6 empty ultra-clean pipe is put into 50ml centrifuge tube, vibration 15sec moves on to tube bottom for viral supernatants are participated in.

3.5.8.7 the supernatant in 3.4.4.5 is transferred in a total collecting pipe.

3.5.8.8 supernatant concussion is made it uniformly.

3.5.8.9 a supernatant is transferred in the cryopreservation tube of the bottom V (every part of about 25-50 μ l).

3.5.9 every part of virus is stored in -80 DEG C.

Two, slow virus titrates

1. purpose: the method for introducing assessment lentiviruses transduction efficiency

2. equipment and material

3. step

1st day

Note: several holes are additionally transfected for long-term cultivation and freezen protective with highest virus input

Note: the cell of empty carrier transduction makees negative control

1. every hole is with 0.5ml RPMI complete medium by 0.1 × 10⁶A Jurkat cell is seeded in 24 orifice plates.

2. every hole is added polybrene (4mg/ml) according to 1:1000 and (such as 0.5 μ l 4ml/ml is added in 0.5ml culture medium Polybrene).

3. recovery virus

4. using RPMI complete medium, 1:1000 dilutes concentrating virus.

5. virus is added in each hole with following concentration.

A. diluted virus (the final viral volume: 5 × 10 of 50ul 1:1000 is added^-5ml)

B. undiluted virus (the final viral volume: 5 × 10 of 0.5ul is added^-4ml)

C. the undiluted virus of 1ul is added, and (final viral volume: being 1 × 10^-3ml)

D. undiluted virus (the final viral volume: 5 × 10 of 5ul is added^-3ml)

6.37 DEG C being incubated overnight.

2nd day

The fresh RPMI complete medium of 0.5ml is added in each hole to dilute polybrene.

3rd day

1. collecting sample and being detected using flow cytometry to analyze the expression of gene.

2. additionally transfecting several holes for long-term cultivation and freezen protective with highest virus input

Titre calculates:

Note: the range of linearity that titre calculates is 15%-45% carrier expression rate.

Finally, it is computed and obtains the Chimeric antigen receptor expression slow virus for targeting carcinomebryonic antigen and merged interleukin- The titre of the 33 Chimeric antigen receptor expression slow virus for targeting carcinomebryonic antigen meets the requirements.

In vitro culture, genetic modification and the amplification of embodiment 3T cell

The Chimeric antigen receptor expression slow virus slow virus for targeting carcinomebryonic antigen prepared using embodiment 2 and fusion The Chimeric antigen receptor expression lentiviruses transduction human T lymphocyte for targeting carcinomebryonic antigen of interleukin-33:

1. PBMC is inoculated in 3 holes of 24 orifice plates, 5,000,000, every hole cell, 1ml RPMI.

2. the diluted IL2 of 1ug/ml OKT3 and 1:1000, which is added, in every hole stimulates cell.

3. 37 DEG C are incubated for for 24 hours.

4. 6ul polybrene (4mg/ml) is added in 6ml vial supernatant, mix.

5. 2ml virus/polybrene mixture is added in every hole.

6. 1800r/s is centrifuged 50 minutes.

7. 2/3 supernatant is absorbed in every hole；And 2ml virus/polybrene mixture and 1:1000 dilution are added into each hole IL2.

8. 37 DEG C of overnight incubations.

9. absorbing 2/3 supernatant；And the fresh diluted IL2 of RPMI and 1:1000 of 2ml is added into each hole.

10. 37 DEG C of overnight incubations.

11. collecting sample and being detected (CD4, CD8, TIM3) using flow cytometry to analyze destination gene expression feelings Condition.

It is screened out from it the T cell for being successfully made genetic modification, that is, CEA.CAR-T and CEA.CAR.IL-33-T.

It the identification of following CEA overexpression cell line LoVo and is co-cultured with CEA-CAR-T, observes CEA-CAR-T cell The variation of effector function:

Under IFN-γ stimulation or inirritative situation, FACS testing result is shown and 293T, HT-29, CaCO2 Fig. 3 A. It is compared with A375, CEA is in high expression in LoVo cell.

Fig. 3 B. and 293T, HT-29, CaCO2 with A375 group is compared, when CEA.CAR-T and LoVo are co-cultured, CD4 and CD8 IFN-γ generation dramatically increases in subgroup.

Fig. 3 C. is constructed and high titre replication defect type slow virus carrier is concentrated.24 hours before transfection, by 11-12 × 10⁶ 293T human embryonic kidney cell 10cm culture plate bed board.Slow virus carrier uses liposome transfection 293T cell after purification, Transfection collected culture supernatant after 24 and 48 hours.Co-cultivation killing experiments are carried out using the Jurkat and LoVo of transfection CEA.CAR, The result shows that after co-culturing 8h, the horizontal significant up-regulation of CD69 developed by molecule in Jurkat cell.

The antitumor action of 4 in vivo studies of embodiment verifying CEA.CAR-T and CEA.CAR.IL-33-T cell

CEA.CAR-T cell and CEA.CAR.IL-33-T cell constructed in embodiment 3 are selected, it is thin using people's intestinal cancer Born of the same parents' strain LoVo constructs Transplanted tumor model in NSG mouse, and work of the CEA.CAR-T in the treatment of intestinal cancer model is inquired into vivo studies With.

As shown in figure 4,9 NSG mouse hypodermic inoculation 5x10⁶People's colon-cancer cell strain LoVo, 3 mouse were inoculated at the 14th day 6x10⁶CEA.CAR-T cell, 3 mouse were in the 14th day inoculation 6x10⁶CEA.CAR.IL-33-T cell, 3 mouse are 14 days inoculation Pbs, every 2 days measurement tumor sizes.LoVo-NSG mouse model shows that CEA CART effectively inhibits tumour growth. With CEA.CART cell ratio, CEA.CAR.IL-33-T cell more effectively inhibits tumour growth.

The present invention also attempted for the antibody of the anti-CEA on CEA.CAR to be substituted for other antibody for being directed to the antigen, to make Other standby CAR-T, as a result the effect of various aspects CEA.CAR-T effect not of the invention is good.

Above embodiment is can not to be interpreted as in order to illustrate embodiment disclosed by the invention to limit of the invention System.In addition, in various modifications and invention listed herein method, composition variation, do not departing from the scope of the present invention Be obvious for those skilled in the art under the premise of spirit.Although having combined of the invention a variety of specific Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments. In fact, various obviously modify as described above for those skilled in the art to obtain invention all should include Within the scope of the invention.

SEQUENCE LISTING

<110>Changzhou First People's Hospital

<120>CAR.IL-33-T and its preparation and application

<130> 172304

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 384

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of light chain variable region (VL)

<400> 1

gacacactcc tgctatgggt gctgctgctc tgggttccag gttccacagg tgacattgtg 60

ctgacccaat ctccagcttc tttggctgtg tctcttgggc agagggccac catgtcctgc 120

agagccggtg aaagtgttga tatttttggc gttgggtttt tgcactggta ccagcagaaa 180

ccaggacagc cacccaaact cctcatctat cgtgcatcca acctagaatc tgggatccct 240

gtcaggttca gtggcactgg gtctaggaca gacttcaccc tcatcattga tcctgtggag 300

gctgatgatg ttgccaccta ttactgtcag caaactaatg aggatccgta cacgttcgga 360

ggggggacca agctggaaat aaaa 384

<210> 2

<211> 369

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of heavy chain variable region (VH)

<400> 2

agcagcgagg ttcagctaca acagtctggg gcagagcttg tggagccagg ggcctcagtc 60

aagttgtcct gcacagcttc tggcttcaac attaaagaca cctatatgca ctgggtgaag 120

cagaggcctg aacagggcct ggaatggatt ggaaggattg atcctgcgaa tggtaatagt 180

aaatatgtcc cgaagttcca gggcaaggcc actataacag cagacacatc ctccaacaca 240

gcctacctgc agctcaccag cctgacatcc gaggacactg ccgtctatta ttgtgctccg 300

tttggttact acgtgtctga ctatgctatg gcctactggg gtcaaggaac ctcagtcacc 360

gtctcctca 369

<210> 3

<211> 801

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA)

<400> 3

gacacactcc tgctatgggt gctgctgctc tgggttccag gttccacagg tgacattgtg 60

ctgacccaat ctccagcttc tttggctgtg tctcttgggc agagggccac catgtcctgc 120

agagccggtg aaagtgttga tatttttggc gttgggtttt tgcactggta ccagcagaaa 180

ccaggacagc cacccaaact cctcatctat cgtgcatcca acctagaatc tgggatccct 240

gtcaggttca gtggcactgg gtctaggaca gacttcaccc tcatcattga tcctgtggag 300

gctgatgatg ttgccaccta ttactgtcag caaactaatg aggatccgta cacgttcgga 360

ggggggacca agctggaaat aaaaggcagt actagcggcg gtggctccgg gggcggttcc 420

ggtgggggcg gcagcagcga ggttcagcta caacagtctg gggcagagct tgtggagcca 480

ggggcctcag tcaagttgtc ctgcacagct tctggcttca acattaaaga cacctatatg 540

cactgggtga agcagaggcc tgaacagggc ctggaatgga ttggaaggat tgatcctgcg 600

aatggtaata gtaaatatgt cccgaagttc cagggcaagg ccactataac agcagacaca 660

tcctccaaca cagcctacct gcagctcacc agcctgacat ccgaggacac tgccgtctat 720

tattgtgctc cgtttggtta ctacgtgtct gactatgcta tggcctactg gggtcaagga 780

acctcagtca ccgtctcctc a 801

<210> 4

<211> 84

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of CD28 transmembrane region (CD28-TM)

<400> 4

atgttctggg tgctggtggt ggtgggcggg gtgctggcct gctacagcct gctggtgaca 60

gtggccttca tcatcttttg ggtg 84

<210> 5

<211> 141

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of 4-1BB intracellular

<400> 5

cgtttctctg ttgttaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 60

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 120

gaagaaggag gatgtgaact g 141

<210> 6

<211> 336

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of CD3 ζ intracellular

<400> 6

cgggtgaagt tcagcagaag cgccgacgcc cctgcctacc agcagggcca gaatcagctg 60

tacaacgagc tgaacctggg cagaagggaa gagtacgacg tcctggataa gcggagaggc 120

cgggaccctg agatgggcgg caagcctcgg cggaagaacc cccaggaagg cctgtataac 180

gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240

aggcggggca agggccacga cggcctgtat cagggcctgt ccaccgccac caaggatacc 300

tacgacgccc tgcacatgca ggccctgccc ccaagg 336

<210> 7

<211> 687

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of Immunoglobulin IgG4 hinge area segment

<400> 7

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgcg tggtggtgga cgtgagccag gaagatcccg aggtccagtt caattggtac 180

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gttcaacagc 240

acctaccggg tggtgtctgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagaa 300

tacaagtgca aggtgtccaa caagggcctg cccagcagca tcgaaaagac catcagcaag 360

gccaagggcc agcctcgcga gccccaggtg tacaccctgc ctccctccca ggaagagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcctgag aacaactaca agaccacccc tcccgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gaaggcaacg tctttagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaag 687

<210> 8

<211> 2109

<212> DNA

<213> Artificial

<220>

<223>coding nucleotide sequence of CEA.CAR

<400> 8

atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60

gacacactcc tgctatgggt gctgctgctc tgggttccag gttccacagg tgacattgtg 120

ctgacccaat ctccagcttc tttggctgtg tctcttgggc agagggccac catgtcctgc 180

agagccggtg aaagtgttga tatttttggc gttgggtttt tgcactggta ccagcagaaa 240

ccaggacagc cacccaaact cctcatctat cgtgcatcca acctagaatc tgggatccct 300

gtcaggttca gtggcactgg gtctaggaca gacttcaccc tcatcattga tcctgtggag 360

gctgatgatg ttgccaccta ttactgtcag caaactaatg aggatccgta cacgttcgga 420

ggggggacca agctggaaat aaaaggcagt actagcggcg gtggctccgg gggcggttcc 480

ggtgggggcg gcagcagcga ggttcagcta caacagtctg gggcagagct tgtggagcca 540

ggggcctcag tcaagttgtc ctgcacagct tctggcttca acattaaaga cacctatatg 600

cactgggtga agcagaggcc tgaacagggc ctggaatgga ttggaaggat tgatcctgcg 660

aatggtaata gtaaatatgt cccgaagttc cagggcaagg ccactataac agcagacaca 720

tcctccaaca cagcctacct gcagctcacc agcctgacat ccgaggacac tgccgtctat 780

tattgtgctc cgtttggtta ctacgtgtct gactatgcta tggcctactg gggtcaagga 840

acctcagtca ccgtctcctc agagagcaag tacggccctc cctgcccccc ttgccctgcc 900

cccgagttcc tgggcggacc cagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 960

atgatcagcc ggacccccga ggtgacctgc gtggtggtgg acgtgagcca ggaagatccc 1020

gaggtccagt tcaattggta cgtggacggc gtggaagtgc acaacgccaa gaccaagccc 1080

agagaggaac agttcaacag cacctaccgg gtggtgtctg tgctgaccgt gctgcaccag 1140

gactggctga acggcaaaga atacaagtgc aaggtgtcca acaagggcct gcccagcagc 1200

atcgaaaaga ccatcagcaa ggccaagggc cagcctcgcg agccccaggt gtacaccctg 1260

cctccctccc aggaagagat gaccaagaac caggtgtccc tgacctgcct ggtgaagggc 1320

ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcctga gaacaactac 1380

aagaccaccc ctcccgtgct ggacagcgac ggcagcttct tcctgtacag ccggctgacc 1440

gtggacaaga gccggtggca ggaaggcaac gtctttagct gcagcgtgat gcacgaggcc 1500

ctgcacaacc actacaccca gaagagcctg agcctgtccc tgggcaagat gttctgggtg 1560

ctggtggtgg tgggcggggt gctggcctgc tacagcctgc tggtgacagt ggccttcatc 1620

atcttttggg tgcgtttctc tgttgttaaa cggggcagaa agaaactcct gtatatattc 1680

aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1740

tttccagaag aagaagaagg aggatgtgaa ctgcgggtga agttcagcag aagcgccgac 1800

gcccctgcct accagcaggg ccagaatcag ctgtacaacg agctgaacct gggcagaagg 1860

gaagagtacg acgtcctgga taagcggaga ggccgggacc ctgagatggg cggcaagcct 1920

cggcggaaga acccccagga aggcctgtat aacgaactgc agaaagacaa gatggccgag 1980

gcctacagcg agatcggcat gaagggcgag cggaggcggg gcaagggcca cgacggcctg 2040

tatcagggcc tgtccaccgc caccaaggat acctacgacg ccctgcacat gcaggccctg 2100

cccccaagg 2109

<210> 9

<211> 704

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of CEA.CAR

<400> 9

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val

20 25 30

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

35 40 45

Ala Val Ser Leu Gly Gln Arg Ala Thr Met Ser Cys Arg Ala Gly Glu

50 55 60

Ser Val Asp Ile Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys

65 70 75 80

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu

85 90 95

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

100 105 110

Thr Leu Ile Ile Asp Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr

115 120 125

Cys Gln Gln Thr Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys

130 135 140

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

145 150 155 160

Gly Gly Gly Gly Ser Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu

165 170 175

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

180 185 190

Phe Asn Ile Lys Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu

195 200 205

Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser

210 215 220

Lys Tyr Val Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr

225 230 235 240

Ser Ser Asn Thr Ala Tyr Leu Gln Leu Thr Ser Leu Thr Ser Glu Asp

245 250 255

Thr Ala Val Tyr Tyr Cys Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr

260 265 270

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

275 280 285

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Ser Leu Gly Lys Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu

515 520 525

Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

530 535 540

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

545 550 555 560

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

565 570 575

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

<210> 10

<211> 267

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA)

<400> 10

Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr

1 5 10 15

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

20 25 30

Gly Gln Arg Ala Thr Met Ser Cys Arg Ala Gly Glu Ser Val Asp Ile

35 40 45

Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro

50 55 60

Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro

65 70 75 80

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

85 90 95

Asp Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr

100 105 110

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

115 120 125

Gly Ser Thr Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys

165 170 175

Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu

180 185 190

Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro

195 200 205

Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr

210 215 220

Ala Tyr Leu Gln Leu Thr Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr

225 230 235 240

Tyr Cys Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

260 265

<210> 11

<211> 128

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of the light chain variable region of the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA)

<400> 11

Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr

1 5 10 15

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

20 25 30

Gly Gln Arg Ala Thr Met Ser Cys Arg Ala Gly Glu Ser Val Asp Ile

35 40 45

Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro

50 55 60

Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro

65 70 75 80

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

85 90 95

Asp Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr

100 105 110

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

115 120 125

<210> 12

<211> 123

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of the heavy chain variable region of the single-chain antibody (scFv) of anti-carcinoembryonic antigen (CEA)

<400> 12

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

1 5 10 15

Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys

20 25 30

Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu

35 40 45

Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro

50 55 60

Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr

65 70 75 80

Ala Tyr Leu Gln Leu Thr Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 13

<211> 28

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of CD28 transmembrane region (CD28-TM)

<400> 13

Met 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> 14

<211> 47

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of 4-1BB intracellular

<400> 14

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

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40 45

<210> 15

<211> 112

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of CD3 ζ intracellular

<400> 15

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> 16

<211> 229

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of Immunoglobulin IgG4 hinge area segment

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly Lys

225

<210> 17

<211> 57

<212> DNA

<213> Artificial

<220>

<223>sequence of the coded polynucleotide of t2A

<400> 17

gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctcgc 57

<210> 18

<211> 543

<212> DNA

<213> Artificial

<220>

<223>encoding polynucleotide sequence of Human secreting type IL-33

<400> 18

atggccttac cagtgaccgc cttgctcctg ccgctagcct tgctgctcca cgccgccagg 60

ccgagtatca caggaatttc acctattaca gagtatcttg cttctctaag cacatacaat 120

gatcaatcca ttacttttgc tttggaggat gaaagttatg agatatatgt tgaagacttg 180

aaaaaagatg aaaagaaaga taaggtgtta ctgagttact atgagtctca acacccctca 240

aatgaatcag gtgacggtgt tgatggtaag atgttaatgg taaccctgag tcctacaaaa 300

gacttctggt tgcatgccaa caacaaggaa cactctgtgg agctccataa gtgtgaaaaa 360

ccactgccag accaggcctt ctttgtcctt cataatatgc actccaactg tgtttcattt 420

gaatgcaaga ctgatcctgg agtgtttata ggtgtaaagg ataatcatct tgctctgatt 480

aaagtagact cttctgagaa tttgtgtact gaaaatatct tgtttaagct ctctgaaact 540

tag 543

<210> 19

<211> 2724

<212> DNA

<213> Artificial

<220>

<223>the overall length encoding gene of CEA.CAR.IL-33

<400> 19

atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60

gacacactcc tgctatgggt gctgctgctc tgggttccag gttccacagg tgacattgtg 120

ctgacccaat ctccagcttc tttggctgtg tctcttgggc agagggccac catgtcctgc 180

agagccggtg aaagtgttga tatttttggc gttgggtttt tgcactggta ccagcagaaa 240

ccaggacagc cacccaaact cctcatctat cgtgcatcca acctagaatc tgggatccct 300

gtcaggttca gtggcactgg gtctaggaca gacttcaccc tcatcattga tcctgtggag 360

gctgatgatg ttgccaccta ttactgtcag caaactaatg aggatccgta cacgttcgga 420

ggggggacca agctggaaat aaaaggcagt actagcggcg gtggctccgg gggcggttcc 480

ggtgggggcg gcagcagcga ggttcagcta caacagtctg gggcagagct tgtggagcca 540

ggggcctcag tcaagttgtc ctgcacagct tctggcttca acattaaaga cacctatatg 600

cactgggtga agcagaggcc tgaacagggc ctggaatgga ttggaaggat tgatcctgcg 660

aatggtaata gtaaatatgt cccgaagttc cagggcaagg ccactataac agcagacaca 720

tcctccaaca cagcctacct gcagctcacc agcctgacat ccgaggacac tgccgtctat 780

tattgtgctc cgtttggtta ctacgtgtct gactatgcta tggcctactg gggtcaagga 840

acctcagtca ccgtctcctc agagagcaag tacggccctc cctgcccccc ttgccctgcc 900

cccgagttcc tgggcggacc cagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 960

atgatcagcc ggacccccga ggtgacctgc gtggtggtgg acgtgagcca ggaagatccc 1020

gaggtccagt tcaattggta cgtggacggc gtggaagtgc acaacgccaa gaccaagccc 1080

agagaggaac agttcaacag cacctaccgg gtggtgtctg tgctgaccgt gctgcaccag 1140

gactggctga acggcaaaga atacaagtgc aaggtgtcca acaagggcct gcccagcagc 1200

atcgaaaaga ccatcagcaa ggccaagggc cagcctcgcg agccccaggt gtacaccctg 1260

cctccctccc aggaagagat gaccaagaac caggtgtccc tgacctgcct ggtgaagggc 1320

ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcctga gaacaactac 1380

aagaccaccc ctcccgtgct ggacagcgac ggcagcttct tcctgtacag ccggctgacc 1440

gtggacaaga gccggtggca ggaaggcaac gtctttagct gcagcgtgat gcacgaggcc 1500

ctgcacaacc actacaccca gaagagcctg agcctgtccc tgggcaagat gttctgggtg 1560

ctggtggtgg tgggcggggt gctggcctgc tacagcctgc tggtgacagt ggccttcatc 1620

atcttttggg tgcgtttctc tgttgttaaa cggggcagaa agaaactcct gtatatattc 1680

aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1740

tttccagaag aagaagaagg aggatgtgaa ctgcgggtga agttcagcag aagcgccgac 1800

gcccctgcct accagcaggg ccagaatcag ctgtacaacg agctgaacct gggcagaagg 1860

gaagagtacg acgtcctgga taagcggaga ggccgggacc ctgagatggg cggcaagcct 1920

cggcggaaga acccccagga aggcctgtat aacgaactgc agaaagacaa gatggccgag 1980

gcctacagcg agatcggcat gaagggcgag cggaggcggg gcaagggcca cgacggcctg 2040

tatcagggcc tgtccaccgc caccaaggat acctacgacg ccctgcacat gcaggccctg 2100

cccccaaggc tcgagggcgg cggagagggc agaggaagtc ttctaacatg cggtgacgtg 2160

gaggagaatc ccggccctcg catggcctta ccagtgaccg ccttgctcct gccgctagcc 2220

ttgctgctcc acgccgccag gccgagtatc acaggaattt cacctattac agagtatctt 2280

gcttctctaa gcacatacaa tgatcaatcc attacttttg ctttggagga tgaaagttat 2340

gagatatatg ttgaagactt gaaaaaagat gaaaagaaag ataaggtgtt actgagttac 2400

tatgagtctc aacacccctc aaatgaatca ggtgacggtg ttgatggtaa gatgttaatg 2460

gtaaccctga gtcctacaaa agacttctgg ttgcatgcca acaacaagga acactctgtg 2520

gagctccata agtgtgaaaa accactgcca gaccaggcct tctttgtcct tcataatatg 2580

cactccaact gtgtttcatt tgaatgcaag actgatcctg gagtgtttat aggtgtaaag 2640

gataatcatc ttgctctgat taaagtagac tcttctgaga atttgtgtac tgaaaatatc 2700

ttgtttaagc tctctgaaac ttag 2724

<210> 20

<211> 180

<212> PRT

<213> Artificial

<220>

<223>amino acid sequence of Human secreting type IL-33

<400> 20

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 Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr

20 25 30

Leu Ala Ser Leu Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu

35 40 45

Glu Asp Glu Ser Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu

50 55 60

Lys Lys Asp Lys Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser

65 70 75 80

Asn Glu Ser Gly Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu

85 90 95

Ser Pro Thr Lys Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser

100 105 110

Val Glu Leu His Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe

115 120 125

Val Leu His Asn Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr

130 135 140

Asp Pro Gly Val Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile

145 150 155 160

Lys Val Asp Ser Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys

165 170 175

Leu Ser Glu Thr

180

Claims

1. a kind of Chimeric antigen receptor T cell, expression targets the Chimeric antigen receptor and interleukin-33 of tumour antigen, institute State target tumour Chimeric antigen receptor include sequentially connected combination tumour antigen polypeptide, CD28 transmembrane region, 4- intracellular 1BB and CD3 ζ intracellular, the polypeptide of the combination tumour antigen are the single-chain antibody of anti-carcinoembryonic antigen, the list of the anti-carcinoembryonic antigen Chain antibody includes light chain variable region and heavy chain variable region, the amino acid sequence of the light chain variable region such as SEQ ID NO.11 institute Show, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.12.

2. Chimeric antigen receptor T cell according to claim 1, which is characterized in that the single-stranded of the anti-carcinoembryonic antigen resists Shown in the amino acid sequence SEQ ID NO.10 of body.

3. Chimeric antigen receptor T cell according to claim 1, which is characterized in that further include any in following characteristics Item is multinomial: (1) amino acid sequence of the CD28 transmembrane region is as shown in SEQ ID NO.13；(2) ammonia of the CD3 ζ intracellular Base acid sequence is as shown in SEQ ID NO.14；(3) amino acid sequence of the 4-1BB intracellular is as shown in SEQ ID NO.15；Institute It states and is connected between polypeptide and CD28 transmembrane region in conjunction with tumour antigen by junction fragment.

4. Chimeric antigen receptor T cell according to claim 3, which is characterized in that the junction fragment is immune globulin The hinge area segment of white IgD.

5. Chimeric antigen receptor T cell according to claim 3, which is characterized in that the junction fragment is immune globulin White IgG4 hinge area segment.

6. Chimeric antigen receptor T cell according to claim 5, which is characterized in that the Immunoglobulin IgG4 hinge The amino acid sequence of area's segment is as shown in SEQ ID NO.16.

7. Chimeric antigen receptor T cell according to claim 3, which is characterized in that the polypeptide of the combination tumour antigen C-terminal connect with the N-terminal of the CD28 transmembrane region.

8. Chimeric antigen receptor T cell according to claim 1, which is characterized in that described to target the embedding of tumour antigen The amino acid sequence of antigen receptor is closed as shown in SEQ ID NO.9.

9. Chimeric antigen receptor T cell according to claim 1, which is characterized in that the amino acid sequence of the interleukin-33 Column are as shown in SEQ ID NO.20.

10. the preparation method of Chimeric antigen receptor T cell as described in any one of claim 1~9, which is characterized in that including step It is rapid:

(2) step (1) described expression vector is converted into T cell inducing expression, obtains expression and targets the embedding of tumour antigen Close the Chimeric antigen receptor T cell of antigen receptor and interleukin-33.

11. according to the method described in claim 10, having merged interleukin-33 it is characterized in that, containing in the expression vector The Chimeric antigen receptor encoding polynucleotide sequence for targeting tumour antigen, it is described that have merged targeting for interleukin-33 swollen The Chimeric antigen receptor coded polynucleotide of tumor antigen is in the Chimeric antigen receptor coded polynucleotide for targeting tumour antigen C-terminal successively merged t2A coded polynucleotide and interleukin-33 coded polynucleotide.

12. according to the method for claim 11, which is characterized in that including any one of following characteristics or multinomial: described Tumour antigen is carcinomebryonic antigen, targets the sequence such as SEQ ID of the coded polynucleotide of the Chimeric antigen receptor of carcinomebryonic antigen Shown in NO.8；The sequence of the coded polynucleotide of the t2A is as shown in SEQ ID NO.17；The coding of the interleukin-33 is more The sequence of nucleotide is as shown in SEQ ID NO.18.

13. according to the method for claim 11, which is characterized in that merged the tumour antigen of targeting of interleukin-33 The sequence of Chimeric antigen receptor coded polynucleotide is as shown in SEQ ID NO.19.

14. a kind of polynucleotides encode multicore glycosides to have merged the Chimeric antigen receptor for targeting tumour antigen of interleukin-33 Acid, the Chimeric antigen receptor coded polynucleotide for targeting tumour antigen for having merged interleukin-33, be target it is swollen The C-terminal of the Chimeric antigen receptor coded polynucleotide of tumor antigen has successively merged t2A coded polynucleotide and interleukin-33 coding Polynucleotides, the tumour antigen are carcinomebryonic antigen, target the coded polynucleotide sequence of the Chimeric antigen receptor of carcinomebryonic antigen Column are as shown in SEQ ID NO.8；The sequence of the coded polynucleotide of the t2A is as shown in SEQ ID NO.17；The interleukin- The sequence of 33 coded polynucleotide is as shown in SEQ ID NO.18.

15. polynucleotides according to claim 14, which is characterized in that the tumour that targets for having merged interleukin-33 resists The sequence of former Chimeric antigen receptor coded polynucleotide is as shown in SEQ ID NO.19.

16. a kind of expression vector contains the polynucleotides as described in any one of claim 14~15.

17. a kind of host cell is converted by expression vector as claimed in claim 16.

18. purposes of the Chimeric antigen receptor T cell in preparation tumor therapeutic agent as described in any one of claim 1~9.

19. a kind of tumor therapeutic agent contains the Chimeric antigen receptor T cell as described in any one of claim 1~9.