CN107523549A - A kind of CAR T cells of high efficiency stable expression activated form antibody and application thereof - Google Patents

Abstract

The present invention provides a kind of CAR T cells of high efficiency stable expression activated form antibody and application thereof.Specifically, containing encoding chimeric antigen acceptor of stable integration in the genome of the transgenic T cells of the present invention, and the expression cassette of the nucleotide sequence of the activated form antibody of co-stimulators or its acceptor, and the both ends of expression cassette include the inverted terminal repeat of transposons.In the multipotency CAR T cell genomes of the present invention by Transposon System stable integration the expression cassette of activated form antibody, so as on the premise of its original CAR T cells killing activity is kept, possess the activity of stability and high efficiency expression activated form antibody, enable the cell of feedback in tumor locus fast breeding.Meanwhile to ensure the security of multipotency CAR T cells treatment, present invention introduces molecule brake system, can remove the multipotency CAR T cells of feedback in time in the case of necessary.

Description

A kind of CAR-T cells of high efficiency stable expression activated form antibody and application thereof

Technical field

The invention belongs to cell biology and oncology, is related to a kind of CAR- of high efficiency stable expression activated form antibody T cell and application thereof.

Background technology

Immunization therapy for malignant tumour is quickly grown in recent years, obtains the clinical efficacy to attract people's attention.Wherein, it is immunized Checkpoint (such as CTLA4, PD1/PDL1) monoclonal antibody is treated, and the tumor specific T cells remained by activation patient in situ, which play, to be made With reaching 30% to the overall efficiency of Several Kinds of Malignancy, and many patients are once play efficiency long-term surviving；Transgenosis CAR- T/TCR-T cell therapies, be by ex vivo gene modify means quickly obtain tumor specific T cells after, adopt feed back into Row treatment plays a role, to the complete remission rate of recurrent and refractory B cell leukemia more than 90%.

Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) is typically by a scFv (single- Chain variable Fragment) (antibody light and weight chain variable region connects and composes single-chain antibody through Linker, is responsible for combining film Antigen), transmission (is responsible for transmembrane region, intracellular signal structure by hinge arrangement (being responsible for forming correct conformation, form dimer) T cell activation signal) connect and compose.T cell (CAR-T) through CAR genetic modifications is endowed tumor cell membranous antigen peptide Molecule, start the ability of killing or propagation.What it is due to CAR-T cell recognitions is antigen of the expression on cell membrane, rather than and MHC By the antigen of submission to cell surface after molecule combination, thus can be restricted around T cell MHC, avoid due to tumour cell Lower or lack immunologic escape caused by MHC molecule.

CAR design is worked together with it by Israel scholar Eshhar proposed in 1989 earliest, by its developing stage to mesh Before untill can be divided into three generations.First generation CAR acceptors contain the svFC fragments of extracellular specific recognition tumour antigen, intracellular activation letter Number by CD3 ζ or Fc ε RI γ ITAM (immunoreceptor tyrosine-based activation motifs) signal Chain transmits.But the costimulatory signal of first generation CAR receptor deficiency T cells, cause T cell to play moment effect, The shortcomings of internal existence time is short, cytokine secretion is few.Second generation CAR acceptors add the intracellular knot of costimulatory signal molecule Structure domain, including such as CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine Kinase (LCK), inducible T-cell co-stimulator (ICOS) and DNAX-activation protein The domains such as 10 (DAP10), enhance the multiplication capacity of T cell and the secreting function of cell factor, IL-2, IFN-γ and GM-CSF increases, so as to break through the immunosupress of tumor microenvironment, extend AICD (activation induced cell death,AICD).Third generation CAR acceptors are then to merge a two level costimulatory molecules such as 4- again on the basis of second generation CAR 1BB, so as to produce the CAR acceptors of a triple signals, the T cell of third generation CAR acceptors transformation has more preferable effector function With the internal time-to-live.In recent years, some scholars propose forth generation CAR acceptor concepts, they are on the basis of third generation CAR Adding IL12, IL15 etc. has the cell factor of positive regulatory T-cell function, further improve the propagation of T cell, killing, Internal survival ability.

At present, the CAR-T cells of existing multiple targets are carrying out the clinical test for the treatment of of solid tumors, including GD2, FR- α、L1-CAM、HER2、EGFR、EGFRvIII、VEGFR-2、IL-13Rα2、FAP、Mesothelin、c-MET、PSMA、CEA、 GPC3, EphA2, MUC1, CAIX (carbonic anhydrase IX) etc..Wherein, partial clinical experiment obtains relatively good As a result, for example, the clinical test (19 patients) of the CAR-T cell therapy high risk neuroblastomas for GD2,8 Patient feed back after completed tumor regression, 3 patients do not disappeared after feedback the 6th week observing time point display complete response, 1 Name complete response patient remains to detect CAR-T cells for 192 weeks；For HER2 CAR-T cell therapy HER2 positive solid tumors Clinical test (totally 19, wherein osteosarcoma 16), 4 maintain without progression of disease state 12 weeks to 14 months, wherein 3 tumours disappear Move back, 1 regression more than 90%.But in general, the CAR-T of solid tumor is treated relative to neoplastic hematologic disorder, curative effect still suffer from compared with Big gap, to find out its cause, mainly including：

1st, immune microenvironment is suppressed

Immunosuppressive microenvironment, including Treg cells, tumour associated fibroblast cell, marrow be present in solid tumor mass Source immature DC cell, M2 type macrophages etc. and its cell factor that they secrete, such as IL-6, IL-10, IDO, VEGF, TGF The cell factor of β etc., these cells and its secretion can suppress the function of T cell by direct or indirect mode.By putting Chemotherapy destroys tumor microenvironment, immunologic test point antibody (such as PD1/PDL1) or immune negative growth factor (such as IDO little molecules in inhibiting Agent) specific blockage associated signal paths, the immune microenvironment of inhibitor integrated regulation of epigenetic modification relevant enzymes, it is overexpressed Immune positive regulating factor (such as IL-12), it is directly targeted and removes stromal cells (such as targeting FAP positive tumor associated fibroblasts The CAR-T of cell) etc. strategy, can act on the immunosuppressive microenvironment of solid tumor, the CAR-T cells for improving infiltration are deposited Viability and fragmentation effect.

2nd, suitable CAR-T therapy targets are lacked

Solid tumor has the heterogeneity of height, different patients, same patient's difference focus, same focus difference tumour cell Between the difference of height be present.The heterogeneity of this height causes neoplasm targeted therapy to be absorbed in shortage preferably general, wide spectrum The unfavorable condition of target spot, the effect of limiting CAR-T cell therapy solid tumors.Thus, to expand the killing model of CAR-T cells Enclose, the design concept for thering is scholar to propose TanCAR, the scFv of the different tumor associated antigens of two kinds of combinations is cascaded, formed One new to identify and with reference to the CAR of two target spots simultaneously, the effect of effectively increasing CAR-T cells.

3rd, it is difficult to reach effective quantity in vivo

The required cluster effect of T cell killing tumor cell, killing a tumour cell needs the cooperation of several T cells, because And after only effector cell reaches certain amount, tumour cell just can be killed effectively.So when T cell contacts specific target spot Tumour cell after, can fast breeding, and by directly contacting and paracrine approach, amplify killing ability.CAR-T cells pass through Intravenously administrable, under the conditions of neoplastic hematologic disorder, contact tumour cell is very easy to, the quantity of CAR-T cells can quickly amplify, even Excessive amplification forms cytokine storm, thus curative effect is relatively preferable；And under the conditions of solid tumor, tumor cell number in blood circulation Measure limited, CAR-T cells, which need to reach tumor locus, could receive stimulation, it is difficult to reach effective dose.

Thus, especially can be on the CAR-T cell bases of wide spectrum identification tumour membranous antigen if in CAR-T, expression has The antibody of the functions such as T cell propagation, survival is directly or indirectly stimulated, then can effectively overcome the problem of solid tumor CAR-T treatments, greatly Width improves curative effect.

Foreign gene is transduceed into the report of such as T cell although having had, commonly uses Gene transfer vector system at present It is low to the effect immunocyte transfection efficiency with cell killing toxicity, or be difficult to make foreign gene intracellular at a high level at it Expression.Can be with the expression in short-term of mediate foreign gene more efficient in T cell using adenovirus vector (circles), but activate T cell growth rate it is very fast, the exogenous gene expression frame of carrying will quickly be lost in passage, expression be difficult to hold Long；Integration that can be with mediate foreign gene in T cell genome using retrovirus or slow virus, it can realize in theory steady Fixed expression, but antibody includes light chain and heavy chain, and coded sequence is long, molecular weight is big, causes the reverse for carrying full length antibody expression cassette Larger difficulty be present with preparation, be difficult to high efficient expression antibody (for example, being expressed using CAR-T cells in record virus or slow virus packaging Antibody, concentration only 200ng/ml, Oncotarget.2016Apr 29.doi:10.18632/oncotarget.9114), typically For the simple single-chain antibody of expression structure (lacking constant segments, insufficiency and half-life short).Thus, before this, It there is no the report of CAR-T cell high-efficients expression antibody.

The content of the invention

First aspect present invention provides a kind of transgenic T cells, and stable integration is embedding containing encoding in the T cell genome Conjunction antigen receptor, and the expression cassette of the nucleotide sequence of the activated form antibody of co-stimulators or its acceptor, and expression cassette Both ends include the inverted terminal repeat of transposons.

In one or more embodiments, the activated form antibody is selected from：Antibody full length sequence or its functional fragment.

In one or more embodiments, the activated form antibody is selected from：Fab, Fab ', F (ab ') 2, Fv, scFv and scFv-Fc。

In one or more embodiments, the transposons is selected from：piggybac、sleeping beauty、frog Prince, Tn5 and Ty；Preferably, the transposons is piggybac.

In one or more embodiments, the activated form antibody of the T cell expression is secreting type or film anchor type；It is excellent Selection of land, the antibody are film anchor type.

In one or more embodiments, the Chimeric antigen receptor is directed to the one or more in following antigen： CD19, CD20, CEA, GD2 (also known as B4GALNT1, β Isosorbide-5-Nitraes-acetyl group-galactosaminyl transferase 1), (Flavin is reduced FR Enzyme), PSMA (PSMA), PMEL premelanosomes albumen), CA9 (carbonic anhydrase IX), CD171/L1- CAM, IL-13R α 2, MART-1 (also known as mucoprotein-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma associated antigen E1) family protein, BAGE (B melanoma antigens family) family protein, GAGE (release by growth hormone Put the factor) family protein, AFP (α-fetoprotein), MUC1 (mucin 1, cell surface related), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R α, EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8 α-N- Acetyl group-ceramide α -2,8- sialyltransferases 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, the related peptase 3 of kallikrein), HMGA2, fetal type acetylcholinergic receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16, Cell surface is related), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25- dihydrovitamin D3) acceptor), (β -2- are micro- by β 2-MG Globulin) and PROGRP (GRP gastrin releasing peptides)；Preferably, the Chimeric antigen receptor is for the chimeric of EGFR families Antigen receptor.

In one or more embodiments, the transgenic T cells are also comprising brake molecule.

In one or more embodiments, the brake molecule is the membranous antigen that can have been listed antibody drug identification； Preferably, the membranous antigen be selected from CD11a, CD15, CD19, CD20, CD25, CD44, CD47, CD52, EGFR, ERBB2, The integrin of ERBB3, ERBB4, VEGFR1, VEGFR2, EpCAM, MSLN, GPIIb/IIIa, α 4 and the integrins of 4 β of α 7；Preferably, The membranous antigen is CD20.

In one or more embodiments, the activated form antibody is directed to the one or more in following antigen：CD28、 CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、 CD80、CD86；Preferably, the activated form antibody is the scFv of anti-CD28 antibody.

In one or more embodiments, the lethal cell of transgenosis has been transferred to following nucleic acid constructs C：

Nucleic acid constructs C：Containing the inverted terminal repeat of transposons 5 ' (5 ' ITR), optional brake molecule, embedding is encoded The promoter closed the nucleotide sequence of antigen receptor (CAR) and activated form antibody and control the nucleotide sequence to express, polyA tailings letter Number sequence, the inverted terminal repeat of transposons 3 ' (3 ' ITR), transposase coding sequence and control transposase coding sequence expression Promoter.

In one or more embodiments, using viral transduction, microinjection, particle bombardment, via Particle Bombardment Transformation and electricity The nucleic acid constructs is transferred in the cell by one or more methods in turning, and is turned preferably by electricity.

Second aspect of the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition contains transgenosis T as described herein Cell and pharmaceutically acceptable auxiliary material.

Third aspect present invention provides the purposes of transgenic T cells as described herein or pharmaceutical composition, it is characterised in that The purposes is selected from：

Prepare for suppress growth of tumour cell medicine, prepare for suppress viral growth medicine, prepare for controlling Treat the medicine of tumour, prepare for treat disease of viral infection medicine, prepare medicine for treating bacterial infection disease Thing and prepare the medicine for treating autoimmune disease；

Wherein, the tumour is selected from：Liver cancer, lung cancer, colon cancer, cancer of pancreas, stomach cancer, breast cancer, nasopharyngeal carcinoma, lymthoma, Oophoroma, carcinoma of urinary bladder, prostate cancer and head and neck neoplasm.

Fourth aspect present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs contains the opposing end weight of transposons 5 ' Complex sequences (5 ' ITR), encode optional brake molecule, Chimeric antigen receptor (CAR) and co-stimulators or its acceptor The nucleotide sequence of activated form antibody and the promoter for controlling the nucleotide sequence to express, polyA tailing signal sequences, transposons 3 ' are anti- The promoter of terminad repetitive sequence (3 ' ITR), transposase coding sequence and control transposase coding sequence expression.

In one or more embodiments, the activated form antibody is selected from：Antibody full length sequence or its functional fragment.

In one or more embodiments, the activated form antibody is selected from：Fab, Fab ', F (ab ') 2, Fv, scFv and scFv-Fc。

In one or more embodiments, the nucleic acid constructs also includes the volume of the extracellular hinge areas of CD28 and transmembrane region Code sequence.

In one or more embodiments, the transposase is the transposase from piggybac transposon systems, described 5 ' inverted terminal repeats (5 ' ITR) and 3 ' inverted terminal repeats are that 5 ' opposing ends of piggybac transposons repeat Sequence and 3 ' inverted terminal repeats.

In one or more embodiments, the brake molecule is CD20.

In one or more embodiments, the Chimerical receptor antigen is the Chimeric antigen receptor for EGFR families.

In one or more embodiments, the activated form antibody is directed to the one or more in following antigen：CD28、 CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、 CD80、CD86；Preferably, the activated form antibody is the scFv of anti-CD28 antibody.

In one or more embodiments, the nucleic acid constructs contains SEQ ID NO:Nucleotide sequence shown in 2.

Brief description of the drawings

Fig. 1：The expression cassette ideograph of antibody.ITR is transposon ends repetitive sequence, and HyPB is piggybac transposases.

Fig. 2：The CD28 film anchor type antibody flow cytometer detection figures of herinCAR-CD28 cells.

Fig. 3：The CD28 molecule flow cytometer detection figures of herinCAR-CD28 cells.

Fig. 4：The in-vitro multiplication detection of herinCAR-CD28 cells.

Fig. 5：HerinCAR-CD28 cells detect to the killing activity of tumour cell in vitro.

Fig. 6：HerinCAR-CD28 cells detect to inhibitory action inside transplantable tumor.

Fig. 7：Propagation detection of the herinCAR-CD28 cells in transplantable tumor.

Fig. 8：The Function detection of herinCAR-CD28 cellular elements brake system.

Embodiment

Part term of the present invention is explained below.

In the present invention, term " expression cassette " refers to express the completed element needed for a gene, including promoter, gene Coded sequence, PolyA tailing signal sequences.

Term " coded sequence " be defined as directly determining in nucleotide sequence in the text its protein product (such as the molecule that brakes, CAR, single-chain antibody, hinge area and transmembrane region) amino acid sequence part.The border of coded sequence is typically by close to mRNA 5 ' hold the ribosome bind site (for prokaryotic) of opening code-reading frame upstream and hold opening code-reading frame downstream close to mRNA 3 ' Transcription terminator determine.Coded sequence can include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.

Term " activated form antibody " can activate the antibody of a certain specific immune response in the presence of referring to.The activated form antibody Can be the full length sequence or its functional fragment of antibody.In one or more embodiments, the activated form antibody is selected from： Fab, Fab ', F (ab ') 2, Fv, scFv and scFv-Fc.In certain embodiments, activated form antibody as described herein is single-stranded Antibody.

Term " antigen-binding fragment " (antigen-binding fragment, Fab) refers to tie positioned at antibody molecule " Y " Structure two-arm end, is made up of hypervariable region amino acid sequence, determines the specific peptide fragment of antibodies bind antigen.

Term " Fc " is the crystallizable fragment (fragment crystallizable, Fc) of antibody, is referred to positioned at antibody point The shank end of sub " Y " structure, include the peptide fragment of heavy chain constant region CH2 and CH3 domain, be antibody with effector molecule or The position of person's cell interaction.

Term " epitope " is also known as antigenic determinant (antigenic determinant, AD), refers in antigen molecule certainly Determine the special chemical group of antigentic specificity.Generally, a polypeptide epitope contains 5~6 amino acid residue epitopes, It can be identified by specific antibody.Property, number and the steric configuration of epitope determine the specificity of antigen.And according to antigen The successional difference of amino acid of epitope, can be divided into linear epitope and predicting space epitope, and linear epitope is that one section of sequence is adjacent The epitope of amino acid composition, and predicting space epitope is several non-conterminous, but the epitope of the composition of adjacent amino acid on space structure.

Term " costimulatory molecules " refers to be present in antigen presenting cell surface, can with the costimulatory molecules on Th cells by Body combines, and produces the molecule of costimulatory signal.The propagation of lymphocyte not only needs the combination of antigen, it is also necessary to receives thorn altogether Swash the signal of molecule.It is mainly the costimulation point by expression in Antigen Presenting Cell surface that costimulatory signal, which passes to T cell, Sub- CD80, CD86 are combined with the CD28 molecules on T cell surface.B cell receives costimulatory signal can be by general pathogen Composition such as LPS, either pass through complement component or the CD40L of the Th cell surfaces of the antigentic specificity by have activated.

Term " joint " or hinge are the polypeptide fragments between the different albumen of connection or polypeptide, and the purpose is to make what is connected Albumen or polypeptide keep respective space conformation, to maintain the function of albumen or polypeptide or activity.Exemplary joint includes containing There are G and/or S joint, and such as Furin 2A peptides.

Term " specific binding " refers to the reaction between antibody or antigen-binding fragment and its targeted antigen. In some embodiments, the antibody (or having specific antibody to certain antigen) for specifically binding certain antigen refers to, antibody with Less than about 10^-5M, it is, for example, less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or smaller affinity (K_D) combine and be somebody's turn to do Antigen." specific recognition " has similar implication.

Term " pharmaceutically acceptable auxiliary material " refer in pharmacology and/or physiologically with subject and active component phase The carrier and/or excipient of appearance, it is well known in the art (see, for example, Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company, 1995), and include but is not limited to：PH adjusting agent, surfactant, adjuvant, ionic strength reinforcing agent.For example, pH adjusting agent Including but not limited to phosphate buffer；Surfactant includes but is not limited to cation, anion or non-ionic surface Activating agent, such as Tween-80；Ionic strength reinforcing agent includes but is not limited to sodium chloride.

Term " effective dose ", which refers to realize in subject, treats, prevents, mitigates and/or alleviates disease of the present invention Or the dosage of illness.

Term " disease and/or illness " refers to a kind of condition of the subject, the condition and institute of the present invention State disease and/or illness is relevant.

Term " subject " can refer to patient or it is other receive pharmaceutical composition of the present invention with treat, prevent, mitigate and/ Or alleviate the animal of disease or illness of the present invention, particularly mammal, such as people, dog, monkey, ox, horse etc..

Provided herein is a kind of nucleic acid constructs (herein also referred to as " nucleic acid constructs C "), such nucleic acid constructs, which contains, to be turned The inverted terminal repeat of stand 5 ' (5 ' ITR), encode optional brake molecule, Chimeric antigen receptor (CAR) and immune thorn altogether Swashing the nucleotide sequence of molecule or the activated form antibody (such as single-chain antibody interested) of its acceptor and controlling the nucleotide sequence to express Promoter, polyA tailing signal sequences, the inverted terminal repeat of transposons 3 ' (3 ' ITR), transposase coding sequence and control The promoter of transposase coding sequence expression.

" Chimeric antigen receptor " (CAR) is artificial reconstructed acceptor, can be by the specificity of tumor cell surface antigen Molecule (such as antibody) is anchored on immunocyte (such as T cell), makes immunocyte identification tumour antigen or viral antigen and kill Tumour cell or the cell of virus infection.

Chimeric antigen receptor suitable for this paper can be various CAR well known in the art.Generally, CAR is successively comprising letter Number peptide, the polypeptide with reference to tumour cell membranous antigen, hinge area, transmembrane region and intracellular signal area.Use well known in the art can be used The CAR of the present invention is built in structure CAR signal peptide, hinge area, transmembrane region and intracellular signal area.Generally, it is thin with reference to tumour The polypeptide of membrane antigenses can be typically inserted into antigen with medium affinity combination tumour cell wide expression membranous antigen, the polypeptide Epitope, any 1,2 or 3 in following 3 positions of the position of insertion：Signal peptide is with combining tumour cell membranous antigen Polypeptide between, inside the polypeptide with reference to tumour cell membranous antigen, and combine tumour cell membranous antigen polypeptide and hinge area Between.The polypeptide of the combination tumour cell membranous antigen is natural polypeptides or artificial synthetic polypeptide；Preferably, artificial synthetic polypeptide For single-chain antibody or Fab fragments.

In certain embodiments, the Chimeric antigen receptor is directed to the one or more in following antigen：CD19、 CD20, CEA, GD2 (also known as B4GALNT1, β Isosorbide-5-Nitraes-acetyl group-galactosaminyl transferase 1), FR (Flavin reductases), PSMA (PSMA), PMEL premelanosomes albumen), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL- 13R α 2, MART-1 (also known as mucoprotein-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanocytes Knurl related antigen E1) family protein, BAGE (B melanoma antigens family) family protein, GAGE (somatotropin releasing factor) family Race's albumen, AFP (α-fetoprotein), MUC1 (mucin 1, cell surface related), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R α, EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8 α-N- acetyl group-god Through acid amides α -2,8- sialyltransferases 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, the related peptase 3 of kallikrein), HMGA2, fetal type acetylcholinergic receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16, cell surface phase Close), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25- dihydrovitamin D3) acceptor), β 2-MG (β -2- microballoon eggs In vain) and PROGRP (GRP gastrin releasing peptides).Preferably, the CAR is the Chimeric antigen receptor for EGFR families.

In certain embodiments, the herinCAR from CN 201510812654.9 is used herein (herein by this application Full content include by reference herein).

In certain embodiments, the polypeptide of the combination tumour cell membranous antigen is natural polypeptides, is mankind's Her2 bases Because of the amino acid sequence HERIN of the 8th introne Herin coding；Preferably, its amino acid sequence such as CN 201510812654.9 SEQ ID NO:Shown in 5.

In certain embodiments, the amino acid sequence of CAR signal peptides of the present invention such as CN 201510812654.9 SEQ ID NO:Shown in 3.

In certain embodiments, CAR of the present invention hinge area is selected from CD8 extracellular hinge area, CD28 extracellular hinge Any one or more of area and CD4 extracellular hinge area；Preferably CD8 extracellular hinge area.In certain embodiments, The extracellular hinge area of the CD8 such as CN 201510812654.9 SEQ ID NO:Shown in 7.

In certain embodiments, CAR of the present invention transmembrane region is selected from CD8 transmembrane region, CD28 transmembrane region and CD4 Any one or more of transmembrane region；Preferably, it is CD8 transmembrane regions；Preferably, the amino acid sequence of the CD8 transmembrane regions is such as CN 201510812654.9 SEQ ID NO:Shown in 8.

In certain embodiments, CAR of the present invention intracellular signal area may be selected from CD28, CD134/OX40, CD137/4- The intracellular signal area of any one or more in 1BB, LCK, ICOS, DAP10, CD3 ζ and Fc ε RI γ, preferably 4-1BB born of the same parents Interior signaling zone and CD3 ζ intracellular signals area, or CD28 intracellular signals area and CD3 ζ intracellular signals area；Preferably, the 4-1BB The amino acid sequence in intracellular signal area and CD3 ζ intracellular signals area is respectively such as CN 201510812654.9 SEQ ID NO:9 Hes SEQ ID NO:Shown in 10；Preferably, the amino acid sequence in the CD28 intracellular signals area and CD3 ζ intracellular signals area is respectively such as CN 201510812654.9 SEQ ID NO:11 and SEQ ID NO:Shown in 10.

In certain embodiments, the polypeptide of the epitope and combination tumour cell membranous antigen is joined directly together or led to Albumen joint is crossed to be connected.Generally, the joint is at least two glycine, such as 2,3,4,5,6,7,8,9 or 10 glycine.

In certain embodiments, Chimeric antigen receptor of the invention is by signal peptide, CD20 epitopes, mankind's Her2 bases Because of amino acid sequence HERIN, CD20 epitope, CD8 hinge areas, CD8 transmembrane regions, the 4- of the 8th introne Herin coding 1BB costimulation peptide fragments are formed successively.Preferably, its amino acid sequence such as CN 201510812654.9 SEQ ID NO:1 institute Show.

Herein, " single-chain antibody " (scFv) interested refers to by antibody light chain variable region (V_LArea) amino acid sequence and Weight chain variable district (V_HArea) amino acid sequence forms through hinge connection, there is the antibody fragment with reference to antigenic capacity.In some realities Apply in scheme, single-chain antibody (scFv) interested carrys out self-interested antibody.It should be understood that this paper nucleic acid constructs can be compiled Two kinds of single-chain antibodies of code, a kind of single-chain antibody are present among shown CAR, and another single-chain antibody is the list being connected with CAR Chain antibody.Both single-chain antibodies can be identical, also can be different, it is preferred that two kinds of single-chain antibodies perform different antibody functions.

Antibody interested can be human antibody, including human mouse chimeric antibody and humanized antibody.Antibody interested is excellent Activated form antibody is elected as, in particular for co-stimulators and its activated form antibody of acceptor.Antibody can be secreting type Or film anchor type；Preferably film anchor type.

In certain embodiments, the antibody is directed to the one or more in following antigen：CD28、CD137、CD134、 CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226.Preferably, the antibody For the scFv of anti-CD28 antibody.

Herein, term " CD28 " refers to human leukocytes differentiation antigen 28, and its official name in NCBI GeneBank is CD28, ID number 940 have 3 isomers (cDNA sequence/protein sequence), respectively NM_006139.3/NP_006130.1, NM_001243077.1/NP_001230006.1, NM_001243078.1/NP_001230007.1.

Single-chain antibody can contain the respective weight chain variable district of each antibody interested and light chain variable district, or by weight chain variable district Formed with light chain variable district and optional joint.It can be connected between weight chain variable district and light chain variable district by well known joint Connect, such as the joint containing G and/or S.Joint length is usually 15~20 amino acid.In certain embodiments, joint is (GGGS)_n, n is 1-5 integer.

In certain embodiments, in nucleic acid constructs C of the present invention, may be used also after the nucleotide sequence of ciphering activation type antibody Including from same or different antibodies hinge areas and transmembrane domain.For example, when using anti-CD28 antibody, it can be used and derive From the scFv of the antibody, and the extracellular hinge areas of the CD28 and transmembrane domain are being come from connection after the scFv.

" brake molecule " used herein and " molecule brake " is used interchangeably, and refers to the film that can have been listed antibody drug identification Antigen.The antibody drug of " brake molecule " is somebody's turn to do by adding identification, the cell that " brake molecule " is somebody's turn to do in carrying can be removed quickly, from And improve Therapeutic safety.Suitable brake molecule (membranous antigen) may be selected from：CD11a、CD15、CD19、CD20、CD25、CD44、 CD47, CD52, EGFR, ERBB2, ERBB3, ERBB4, VEGFR1, VEGFR2, EpCAM, MSLN (mesothelin), GPIIb/IIIa, The integrins of α 4 and the integrins of 4 β of α 7.In certain embodiments, membranous antigen CD20.In certain embodiments, institute can be used State the linear epitope or predicting space epitope of membranous antigen.

When exist brake molecule when, brake molecule can by joint sequence commonly used in the art (as previously described containing G and S joint sequence) it is connected with CAR, or directly can be connected with CAR.CAR can be joined directly together with single-chain antibody, also can be by connecing Header sequence (such as Furin 2A peptides) is attached.

" CD20 " refers to human leukocytes differentiation antigen 20, and it is MS4A1 in NCBI GeneBank official name, ID number For 931, there are 2 isomers (cDNA sequence/protein sequence), respectively NM_021950.3/NP_068769.2, NM_ 152866.2/NP_690605.1.When referring to CD20 amino acid sequence, it includes, and the total length of CD20 albumen or has The CD20 of CD20 functions fragment；Also include the fusion protein of the total length or fragment.Also, it will be appreciated by those skilled in the art that In CD20 amino acid sequence, can it is naturally-produced or be artificially introduced mutation or variation (including but not limited to replace, missing and/ Or addition), without influenceing its biological function.Also, when describing CD20 protein sequence fragments, in addition to its natural or people Corresponding sequence fragment in work variant.

Corresponding promoter sequence can be selected according to selected CAR coded sequence.The example of this kind of promoter include but It is not limited to EF1 α promoters.(entire contents are included to this by reference herein as described in CN201510021408.1 Text), promoter upstream may also include enhancer, such as one in mCMV enhancers, hCMV enhancers and CD3e enhancers, arbitrarily Two or all three.

Therefore, in certain embodiments, the various promoter sequences announced herein using CN201510021408.1, Including but not limited to this application SEQ ID NO:The CCEF of enhancer containing mCMV, hCMV enhancers and EF1 α promoters shown in 1 Promoter；SEQ ID NO:The TEF promoters of enhancer containing CD3e and EF1 α promoters shown in 2；SEQ ID NO:Shown in 3 Enhancer containing CD3e, mCMV enhancers, the TCEF promoters of hCMV enhancers and EF1 α promoters；SEQ ID NO:Shown in 4 The CCEFI promoters of enhancer containing mCMV, hCMV enhancers and the EF1 α promoters containing introne；SEQ ID NO:Shown in 5 The TEFI promoters of enhancer containing CD3e and the EF1 α promoters containing introne；And SEQ ID NO:Increasing containing CD3e shown in 5 Hadron, mCMV enhancers, the TCEFI promoters of hCMV enhancers and the EF1 α promoters containing introne.

Herein, transposase can be turned from piggybac, sleeping beauty, frog prince, Tn5 or Ty The transposase of base system.When using the transposase from different transposon systems, the 5 ' ITR and 3 ' in nucleic acid constructs of the present invention ITR sequence also accordingly changes into the sequence being adapted to the transposon system, and this can be readily determined by those skilled in the art.

In certain embodiments, transposase is the transposase from piggybac transposon systems.Therefore, implement at these In scheme, the inverted terminal repeat of transposons 5 ' and 3 ' inverted terminal repeats are respectively the 5 ' anti-of piggybac transposons Terminad repetitive sequence and 3 ' inverted terminal repeats.In certain embodiments, the inverted terminal repeat of transposons 5 ' As CN 201510638974.7 (herein includes its content herein) SEQ ID NO by reference:Shown in 1.In some realities Apply in scheme, the inverted terminal repeat of transposons 3 ' such as CN201510638974.7SEQ ID NO:Shown in 4.In some implementations In scheme, piggybac transposases are the transposase of the coded sequence of nuclear localization signal containing c-myc.In certain embodiments, The coded sequence of piggybac transposases such as CN 201510638974.7SEQ ID NO:Shown in 5.

The promoter of transposase coding sequence can be known in the art for controlling transposase coding sequence to express Various promoters.In certain embodiments, the expression of transposase coding sequence is controlled using CMV promoter.CMV promoter Sequence can be such as CN 201510638974.7SEQ ID NO:Shown in 6.

PolyA tailing signal sequences well known in the art can be used.In certain embodiments, the polyA comes from SV40.In some embodiments, CN 201510638974.7SEQ ID NO can be used:Sequence shown in 3.

Selected membranous antigen can be directed to and select suitable promoter, to control the expression of membranous antigen.In some embodiments In, promoter is EF1 α promoters.In certain embodiments, the sequence of EF1 α promoters such as CN 201510638974.7 SEQ ID NO:Shown in 8.

In certain embodiments, this paper nucleic acid constructs C contains the opposing end of transposons 5 ' being sequentially connected and repeated Sequence (5 ' ITR), the activation of control coding brake molecule, Chimeric antigen receptor (CAR) and co-stimulators or its acceptor The promoter of the nucleotide sequence expression of type antibody (single-chain antibody such as interested), coding brake molecule, Chimeric antigen receptor (CAR) With the nucleotide sequence of the activated form antibody of co-stimulators or its acceptor (single-chain antibody such as interested), polyA tailings letter Number sequence, the inverted terminal repeat of transposons 3 ' (3 ' ITR), transposase coding sequence and control transposase coding sequence expression Promoter.

In certain embodiments, this paper nucleic acid constructs C contains the opposing end of transposons 5 ' being sequentially connected and repeated Sequence (5 ' ITR), control encode the activated form of brake molecule, Chimeric antigen receptor (CAR), co-stimulators or its acceptor What the extracellular hinge area of the targeted antigen of antibody (single-chain antibody such as interested), antibody and the nucleotide sequence of transmembrane region were expressed opens Mover, the activated form antibody for encoding brake molecule, Chimeric antigen receptor (CAR), co-stimulators or its acceptor (are such as felt emerging Interesting single-chain antibody), the targeted extracellular hinge area of antigen and the nucleotide sequence of transmembrane region of antibody, polyA tailing signal sequences, The startup of the inverted terminal repeat of transposons 3 ' (3 ' ITR), transposase coding sequence and control transposase coding sequence expression Son.

In certain embodiments, this paper nucleic acid constructs C contains the opposing end of transposons 5 ' being sequentially connected and repeated Sequence (5 ' ITR), EF1 α promoters, brake molecule (CD20), CAR, anti-CD28 single-chain antibodies, CD28 extracellular hinge area and across Film area, polyA tailing signal sequences, the inverted terminal repeat of transposons 3 ' (3 ' ITR), transposase coding sequence and control turn The promoter (CMV) of seat enzyme coded sequence expression.

In certain embodiments, the nucleic acid constructs C contains SEQ ID NO:Nucleotide sequence shown in 2.

This paper nucleic acid constructs C can be kind of a recombinant expression carrier (recombinant expression carrier C), for express the CAR, ScFv and the optional brake molecule.Preferably, expression vector is transposon vector.In certain embodiments, carrier For the one or more in following transposon vector：Piggybac, sleeping beauty, frog prince, Tn5 and Ty.In addition to the nucleotide sequence contained by nucleic acid constructs C, generally also containing the generally contained other elements of carrier in expression vector, Such as multiple cloning sites, resistant gene, replication origin etc..

In certain embodiments, the recombinant expression carrier uses pUC18, pUC19, pMD18-T, pMD19-T, pGM- Carrier T, pUC57, pMAX or pDC315 serial carrier are as skeleton.In other embodiments, the recombinant expression carrier is adopted With pCDNA3 serial carriers, pCDNA4 serial carriers, pCDNA5 serial carriers, pCDNA6 serial carriers, pRL serial carriers, PUC57 carriers, pMAX carriers or pDC315 serial carriers are as skeleton.In certain embodiments, the present invention uses CN The pSN carriers of 201510638974.7 structures, its carrier structure is as shown in this application Fig. 1.

The nucleic acid constructs C/ recombinant expression carriers C of the present invention can be transferred in cell interested.The method being transferred to is The conventional method in this area, includes but is not limited to：Viral transduction, microinjection, particle bombardment, via Particle Bombardment Transformation and electricity turn etc.. In certain embodiments, turned using electricity by the nucleic acid constructs or recombinant expression carrier.

Cell interested can be various T cells well known in the art, including but not limited to periphery blood T lymphocyte, Cell toxicant killer T cell (CTL), helper cell, suppression/regulatory T cells, gamma delta T cells and cytokine induction kill Hinder the T cell of the mixed cell populations such as cell (CIK), tumor infiltrating lymphocyte (TIL).

When nucleic acid constructs C/ recombinant expression carriers C is transfected into T cell interested, because it contains needed for swivel base ITR elements and transposase, therefore the nucleic acid constructs or the inverted terminal repeat of transposons 5 ' contained by recombinant expression carrier Nucleotide sequence (including 5 ' and 3 ' inverted terminal repeats are in itself) between the inverted terminal repeat of transposons 3 ' is whole In the genome for closing cells of interest.When nucleic acid constructs C/ recombinant expression carriers C contains the nucleic acid sequence of coding brake molecule During row, cell will further express brake molecule.

Therefore, a kind of transgenic T cells are also provided herein, stable integration includes coding in the genome of the T cell The expression cassette of the nucleotide sequence of the activated form antibody of CAR and co-stimulators or its acceptor (single-chain antibody such as interested). Further, the inverted terminal repeat of transposons 5 ' (5 ' that is sequentially connected of stable integration in the genome of the T cell ITR), control encodes the activated form antibody of brake molecule, Chimeric antigen receptor (CAR), co-stimulators or its acceptor (such as Single-chain antibody interested), the promoter of the nucleotide sequence expression of the hinge area of the targeted antigen of antibody and transmembrane region, coding stops Car molecule, Chimeric antigen receptor (CAR), the activated form antibody of co-stimulators or its acceptor are (such as interested single-stranded anti- Body), the nucleotide sequence of the targeted extracellular hinge area of antigen of antibody and transmembrane region, polyA tailing signal sequences, and transposons 3 ' inverted terminal repeats (3 ' ITR).

In certain embodiments, the transgenic T cells expression activated form antibody.The antibody can resist for secreting type Body or film anchor type, preferably film anchor type.The antibody can be the co-stimulators activated form for acting on T cell itself Antibody.After the transgenic T cells express the antibody, constellation effect can be strengthened.

In certain embodiments, this paper T cell stably expresses CAR interested and scFv interested.Some In embodiment, this paper T cell has been transferred to nucleic acid constructs C/ recombinant expression carriers C as described herein.

According to the biological function of expressed antibody, this paper transgenic T cells can have different biological activities, Including but not limited to suppress tumour, suppress virus, suppress bacterium etc..Therefore, this paper CAR-T cells can be used for suppression tumour thin The growth of born of the same parents, the growth for suppressing virus, treatment tumour, treatment disease of viral infection, treatment bacterial infection disease, Yi Jizhi Treat autoimmune disease.Tumour includes but is not limited to liver cancer, lung cancer, colon cancer, cancer of pancreas, stomach cancer, breast cancer, nasopharyngeal carcinoma, leaching Bar knurl, oophoroma, carcinoma of urinary bladder, prostate cancer, head and neck neoplasm.

Therefore, a kind of pharmaceutical composition is also provided herein, the pharmaceutical composition contain transgenic T cells as described herein and Pharmaceutically acceptable carrier or excipient.Transgenic T cells as described herein are also provided herein to prepare for suppressing tumour The medicine of cell growth, prepare for suppress viral growth medicine, prepare for treat tumour medicine, prepare for treating The medicine of disease of viral infection, prepare the medicine for treating bacterial infection disease or prepare for treating autoimmunity Purposes in the medicine of disease.

In this paper one or more embodiments, the transgenic T cells are CAR-T cells.

Therefore, present invention also offers a kind of kit, the kit to contain recombinant expression carrier as described herein.Examination Agent box, which also contains, is applied to the various reagents that are transfected into the recombinant expression carrier cell, and optionally instruct ability The recombinant expression carrier is transfected into the specification of cell by field technique personnel.

Therefore, in certain embodiments, the present invention also relates to a kind of composition of recombinant expression carrier, said composition sheet Recombinant expression carrier described in text.Corresponding solvent or carrier can also be contained in composition.

The present inventor passes through substantial amounts of preliminary experiment and performing creative labour, successfully constructs a kind of high efficiency stable expression activation The multipotency CAR-T cells of type antibody, by Transposon System stable integration, activated form resists in multipotency CAR-T cellular genomes Body (the especially activated form antibody of costimulatory molecules and its acceptor) expression cassette, so as to keep its original CAR-T cell killing On the premise of activity, possess the activity of stability and high efficiency expression activated form antibody, the cell of feedback is quickly increased in tumor locus Grow.

Instant invention overcomes current conventional Gene transfer vector system is low to T cell transfection efficiency, mediate antibody expression The defects of low, immunologic cytotoxicity cell high level is stably expressed Chimeric antigen receptor and activated form antibody, overcome in entity The problem of CAR-T cells are difficult to reach effective quantity in knurl CAR-T cell therapy procedures.To avoid activated form antibody-secreting to carefully After extracellular, uncontrollable activating immune system, immune radical response is caused, may be selected by antibody anchorage on CAR-T cell membranes, Pass through direct contact activation adjacent cells.Meanwhile the cell and can is quickly removed by listing monoclonal antibody (such as Mabthera), is improved The security for the treatment of.Transgenic T cells produced by the present invention can be used in Several Kinds of Malignancy and the treatment of virus.

Embodiment involved in the present invention is described in detail below in conjunction with case study on implementation.Those skilled in the art It will be understood that case study on implementation below is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.In case study on implementation Unreceipted particular technique or condition person, according to the technology described by document in the art or condition (such as with reference to J. Pehanorm cloth Luke etc. writes, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or according to product description Carry out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.

Embodiment 1：Recombinant plasmid pNB328-herinCAR-CD28 structure

By SEQ ID NO:HerinCAR coded sequences (the targeting EGFR family of the molecule of Mabthera containing CD20- brake shown in 1 CAR, referring to CN 201510812654.9) with SEQ ID NO:(the targeting EGFR men of herinCAR-CD28 coded sequences shown in 2 Race and CAR and CD28 comprising the brake of CD20- Mabtheras molecule it is single-stranded-film combination type antibody, centre is connected with 2A), in commission Hai Jierui biotech firms synthesize, and introduce EcoRI and SalI restriction enzyme sites respectively in its upstream and downstream, load pNB328 carriers, point PNB328-herinCAR, pNB328-herinCAR-CD28 are not named as it.

The herinCAR coded sequences of the molecule of Mabthera containing CD20- brake：

GCCACCATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAG TGT GGTGGAGGTGGAGGTGGAGGTGGAGGTGGTACCCACTCACTGC CCCCGAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTTCCTCAGA CCCTCTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCCCTATATCCCC TGTCAGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAAGGCGAGCCCAAAT CTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAGTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACC ACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTG CCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGG CCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAGTAAGAGGAGCAGGCTC CTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACC ACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGA ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCT GAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGA GGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGATAA(SEQ ID NO:1) wherein double underline represents restriction enzyme site, the CD20 epitope coding sequences for anti-Rituximab antibodies identification of wave underline.

HerinCAR-CD28 coded sequences：

GCCACCATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAG TGT GGTGGAGGTGGAGGTGGAGGTGGAGGTGGTACCCACTCACTG CCCCCGAGGCCAGCTGCAGTTCCTGTCCCTCTGCGCATGCAGCCTGGCCCAGCCCACCCTGTCCTATCCTT CCTCAGACCCTCTTGGGACCTAGTCTCTGCCTTCTACTCTCTACCCCTGGCCCCCCTCAGCCCTACAAGTGTCC CTATATCCCCTGTCAGTGTGGGGAGGGGCCCGGACCCTGATGCTCATGTGGCTGTTGACCTGTCCCGGTATGAA GGCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAGTTCGTGCCGGTCTTCCT GCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCC TGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTAC ATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAG TAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACC AGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCG TACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCGTAAAAG GCGAGCTCCTGTTAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTCGAGTCCAACCCTGGGCCCA TGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGT ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGG AAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGTGATAA (SEQ ID NO:2), wherein double underline represents restriction enzyme site, and single underscore represents Furin 2A coded sequence, under dotted line The coded sequence of the CD28 single-chain antibodies of line.

The ideograph of pNB328-herinCAR-CD28 carriers is shown in Fig. 1.

Embodiment 2：The structure of herinCAR-CD28 cells

Prepare 1 × 10⁷PMNC (the Peripheral blood mononuclear that fresh separated obtains Cell, PBMC), by Lonza 2b-Nucleofector instruments, by 6 μ g pNB328-herinCAR-CD28, plasmid transfection Into nucleus, 37 DEG C, 5%CO are put₂Incubator culture；Anti-cd 3 antibodies containing 30ng/mL, 3000IU/mL are transferred to after 6 hours In IL-2 (being purchased from Novoprotein companies) 6 orifice plates, 37 DEG C, 5%CO are put₂Incubator culture.After cell covers with, by 1:5 Ratio Secondary Culture.Produce at the same express targeting EGFR family and the molecule of Mabthera containing CD20- brake CAR, CD28 it is single-stranded anti- The genetic modification T cell of body, abbreviation herinCAR-CD28 cells.Meanwhile identical source PBMC transfections pNB328-herinCAR Plasmid, obtain herinCAR-T cells.

Embodiment 3：The detection of herinCAR-CD28 cell surface CD28 antibody molecules

Collect suspend embodiment 2 build herinCAR-CD28 cells and control herinCAR-T cells, after counting with 1×10⁶Individual cell/pipe is separately added into 2 1.5ml EP pipes, twice of PBS, 1200rpm centrifugation 5min, abandons supernatant；Point 2 μ l anti-human igg Fab2 ' antibody (being purchased from Jackson ImmunoResearch companies) are not added, and flicking precipitation makes its mixing equal Even, room temperature lucifuge is incubated 30min, and PBS one time, 1200rpm centrifugation 5min, the physiological saline for abandoning 400 μ l of supernatant addition will Cell is transferred in streaming pipe, upper machine testing.Experimental result is found, relative to control cell, herinCAR-CD28 cell surfaces It is specific as shown in Figure 2 with CD28 antibody molecules.

Embodiment 4：The detection of herinCAR-CD28 cell surface CD28 molecules

Collect suspend embodiment 2 build herinCAR-CD28 cells and control herinCAR-T cells, after counting with 1×10⁶Individual cell/pipe is separately added into 2 1.5ml EP pipes, twice of PBS, 1200rpm centrifugation 5min, abandons supernatant；Point 2 μ l isotype control Ab IgG1-PE, anti-CD28-PE antibody (being purchased from BD companies) is not added, and flicking precipitation makes it mixed Uniform, room temperature lucifuge incubation 30min, PBS one time are closed, 1200rpm centrifuges 5min, abandons the physiology salt that supernatant adds 400 μ l Cell is transferred in streaming pipe by water, upper machine testing.Experimental result is found, relative to herinCAR control cells, herinCAR- CD28 cells CD28 positive rate significantly improves, and shows that herinCAR-CD28 cells, can be effective by its film surface C D28 antibody The CD28 signals of the neighbouring T cell of activation, it is specific as shown in Figure 3.

Embodiment 5：The propagation detection of herinCAR-CD28 cells

The herinCAR-CD28 cells and herinCAR cells that embodiment 2 is built are by 4 × 10⁴/ hole is layered on 96 orifice plates In, every kind of cell spreads 3 multiple holes, and cumulative volume is 200 μ l, is placed in 37 DEG C, 5%CO₂Incubator culture, 24h is being cultivated respectively, 20 μ l CCK8 reagents are added after 48h, 72h, 96h, 37 DEG C of lucifuges are incubated 6h, and 450nm surveys OD values on ELIASA.As a result find, The growth rate of herinCAR-CD28 cells illustrates herinCAR-CD28 cell membrane tables apparently higher than control herinCAR cells The CD28 antibody in face can promote the propagation of T cell, specific as shown in Figure 4.

Embodiment 6：The cytotoxicity detection of herinCAR-CD28 cells

Spread on RTCA cells propagation plate (being purchased from ACEA Biosciences companies of the U.S.) in the ratio in 10000/hole Stomach cancer cell line BGC-27, Ovarian Cancer Cells SK-OV3 (being purchased from Unite States Standard biology product collecting center, ATCC), are placed in On the multi-functional real-time n cell analyzers of xCELLigence RTCA DP, the growing state of cell is recorded in real time (to measure Cell index reflect that numerical value is higher shows that cell state is better).After 24 hours, respectively by 8:1、4:1、2:1、0:1 effect target Than (E:T), herinCAR cells and herinCAR-CD28 cells that embodiment 2 is built are added, is replaced in xCELLigence RTCA DP are multi-functional to carry out cell growth status detection in real time on n cell analyzer, after 48 hours, according to what is measured Cell index and 0 under the conditions of each effect target ratio：The ratio for the corresponding cell index that (is not added with effector cell) under the conditions of 1 effect target ratio, it is determined that Tumor cell lysis rate.

As a result show, relative to the herinCAR-T cells for not expressing CD28 antibody, the herinCAR- of expression CD28 antibody T cell, i.e. herinCAR-CD28 cells significantly improve (Fig. 5) to the killing activity of BGC-27 and SK-OV3 cells.Result above Show, after co-expressing CD28 antibody, the killing activity of CAR-T cells against tumor cells can be strengthened.

Embodiment 7：Killing activity is identified inside herinCAR-CD28 cells

5 × 10 are subcutaneously injected in NOD-SCID mouse (being purchased from Shanghai Slac Experimental Animal Co., Ltd.)⁶BGC- 27 pernicious stomach cancer cells, inject herinCAR cells, the herinCAR-CD28 of the structure of embodiment 2 after 10 days respectively through tail vein Cell (injection dosage 2 × 10⁵) or PBS, determine the upgrowth situation of transplantable tumor.As a result show, relative to control group, HerinCAR-CD28 cells have significant difference (Fig. 6) to the inhibitory action of liver cancer, show to co-express CD28 antibody HerinCAR-T cells have good antitumor action in vivo.

Embodiment 8：Propagation detection of the herinCAR-CD28 cells in transplantable tumor

The 35th day after the treatment of embodiment 7, tumor-bearing mice is put to death, takes transplanting tumor tissue, extraction genomic DNA (is pressed The tissue DNA extraction agent box of Sigma-Aldrich companies is operated).Moved using RT-PCR detection herinCAR genes Plant Relative copy number (the primer such as SEQ ID NO in tumor tissue:3 and SEQ ID NO:Shown in 4, reaction system and program are daily RealMaster Mix (SYBR Green) detection kit operation of root biology).As a result show, it is thin relative to herinCAR-T Born of the same parents, copy number of the herinCAR-CD28 cells in transplantable tumor significantly improve (Fig. 7), show that expressing CD28 antibody can strengthen Time-to-live of the CAR-T cells in transplantable tumor.

F：CAGTGAGATTGGGATGAAAGG(SEQ ID NO:3)；

R：GAAGGGCGTCGTAGGTGTC(SEQ ID NO:4).

Embodiment 9：Clearance test (checking of molecule brake function) inside herinCAR-CD28 cells

It is thin in BABL/c nude mices (being purchased from Shanghai Slac Experimental Animal Co., Ltd.) tail vein injection herinCAR-CD28 Born of the same parents' (injection dosage 5 × 10⁶), 100 μ g anti-Rituximab antibodies or human IgG control antibodies are injected intravenously after 3 days.After 12 hours, Blood and bone marrow specimens are gathered, (CD20 and CD3 is double positive thin with the ratio of flow cytomery herinCAR-CD28 cells Born of the same parents).

As a result show, relative to the control group of injection human IgG antibody, after injection anti-Rituximab antibodies (Rituxan), infusion Ratio of the herinCAR-CD28 cells in blood and marrow that builds of embodiment 2 significantly reduce (Fig. 8).It can be seen that CD20 points Son brake can effectively play a role in vivo, can be by ADCC and CDC effects by the herinCAR-CD28 containing CD20 epitopes Cell clearance.

Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, those details can be carried out with various modifications and replacement, these change in the guarantor of the present invention Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.

Claims (11)

1. A kind of 1. transgenic T cells, it is characterised in that in the T cell genome stable integration containing encoding chimeric antigen by Body, and the expression cassette of the nucleotide sequence of the activated form antibody of co-stimulators or its acceptor, and the both ends of expression cassette include The inverted terminal repeat of transposons.
2. 2. transgenic T cells as claimed in claim 1, it is characterised in that the transposons is selected from：piggybac、 Sleeping beauty, frog prince, Tn5 and Ty；Preferably, the transposons is piggybac.
3. 3. transgenic T cells as claimed in claim 1, it is characterised in that the activated form antibody is selected from：Antibody full length sequence Or its functional fragment；Preferably, it is selected from：Fab, Fab ', F (ab ') 2, Fv, scFv and scFv-Fc；It is further preferred that institute It is scFv to state antibody.
4. 4. transgenic T cells as claimed in claim 1, it is characterised in that the activated form antibody of the T cell expression is secretion Type or film anchor type, it is preferable that the antibody is film anchor type.
5. 5. transgenic T cells as claimed in claim 1, it is characterised in that the Chimeric antigen receptor is directed in following antigen One or more：CD19, CD20, CEA, GD2 (also known as B4GALNT1, β Isosorbide-5-Nitraes-acetyl group-galactosaminyl transferase 1), FR (Flavin reductases), PSMA (PSMA), PMEL premelanosomes albumen), CA9 (carbonic anhydrases IX), CD171/L1-CAM, IL-13R α 2, MART-1 (also known as mucoprotein-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/ Testis antigen 1B), MAGE (melanoma associated antigen E1) family protein, BAGE (B melanoma antigens family) family protein, GAGE (somatotropin releasing factor) family protein, AFP (α-fetoprotein), MUC1 (mucin 1, cell surface related), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R α, EGP-2, EGP-40, FBP, GD3 are (also known as ST8SIA1, ST8 α-N- acetyl group-ceramide α -2,8- sialyltransferase 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, the related peptase 3 of kallikrein), HMGA2, fetal type acetylcholinergic receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 are (also known as MUC16, mucin 16, cell surface are related), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (again Claim SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25- dihydrovitamin D3) by Body), β 2-MG (beta-2-microglobulin) and PROGRP (GRP gastrin releasing peptides)；Preferably, the Chimeric antigen receptor is pin To the Chimeric antigen receptor of EGFR families.
6. 6. transgenic T cells as claimed in claim 1, it is characterised in that the transgenic T cells are also comprising brake molecule； Preferably, the brake molecule is the membranous antigen that can have been listed antibody drug identification；Preferably, the membranous antigen be selected from CD11a, CD15、CD19、CD20、CD25、CD44、CD47、CD52、EGFR、ERBB2、ERBB3、ERBB4、VEGFR1、VEGFR2、 The integrin of EpCAM, MSLN, GPIIb/IIIa, α 4 and the integrins of 4 β of α 7；Preferably, the membranous antigen is CD20.
7. 7. transgenic T cells as claimed in claim 1, it is characterised in that the activated form antibody is directed in following antigen It is one or more：CD28、CD137、CD134、CD40、CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、 DR3、SLAM、CD226、CD80、CD86；Preferably, the activated form antibody is the scFv of anti-CD28 antibody.
8. 8. such as the transgenic T cells any one of claim 1-7, it is characterised in that the lethal cell of transgenosis It has been transferred to following nucleic acid constructs C：

   Nucleic acid constructs C：Containing the inverted terminal repeat of transposons 5 ' (5 ' ITR), optional brake molecule, inosculating antibody are encoded The nucleotide sequence of the activated form antibody of original receptor (CAR) and co-stimulators or its acceptor and control the nucleotide sequence express Promoter, polyA tailing signal sequences, the inverted terminal repeat of transposons 3 ' (3 ' ITR), transposase coding sequence and control The promoter of transposase coding sequence expression processed；

   Optionally, one or more methods in being turned using viral transduction, microinjection, particle bombardment, via Particle Bombardment Transformation and electricity The nucleic acid constructs is transferred in the cell, turned preferably by electricity.
9. 9. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition contains to be turned any one of claim 1-8 Transgenic T cells and pharmaceutically acceptable auxiliary material.
10. 10. the purposes of the pharmaceutical composition described in transgenic T cells or claim 9 any one of claim 1-8, Characterized in that, the purposes is selected from：

    Prepare for suppress growth of tumour cell medicine, prepare for suppress viral growth medicine, prepare for treat it is swollen The medicine of knurl, prepare for treat disease of viral infection medicine, prepare medicine for treating bacterial infection disease and Prepare the medicine for treating autoimmune disease；

    Wherein, the tumour is selected from：Liver cancer, lung cancer, colon cancer, cancer of pancreas, stomach cancer, breast cancer, nasopharyngeal carcinoma, lymthoma, ovary Cancer, carcinoma of urinary bladder, prostate cancer and head and neck neoplasm.
11. 11. a kind of nucleic acid constructs, it is characterised in that the nucleic acid constructs contains the inverted terminal repeat of transposons 5 ' (5 ' ITR), encode optional brake molecule, Chimeric antigen receptor (CAR) and the activated form of co-stimulators or its acceptor The nucleotide sequence of antibody and the promoter for controlling the nucleotide sequence to express, polyA tailing signal sequences, the opposing end of transposons 3 ' The promoter of repetitive sequence (3 ' ITR), transposase coding sequence and control transposase coding sequence expression；

    Preferably, the activated form antibody is single-chain antibody；

    Preferably, the transposase is the transposase from piggybac transposon systems, the 5 ' inverted terminal repeat (5 ' ITR) and 3 ' inverted terminal repeats are that 5 ' inverted terminal repeats of piggybac transposons and 3 ' opposing ends repeat Sequence；

    Preferably, the brake molecule is CD20；

    Preferably, the Chimerical receptor antigen is the Chimeric antigen receptor for EGFR families；

    Preferably, the activated form antibody is directed to the one or more in following antigen：CD28、CD137、CD134、CD40、 CD40L、ICOS、HVEM、CD2、CD27、CD30、GITR、LIGHT、DR3、SLAM、CD226、CD80、CD86；Preferably, it is described Activated form antibody is the scFv of anti-CD28 antibody；

    Preferably, the nucleic acid constructs contains SEQ ID NO:Nucleotide sequence shown in 2.