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CN106519037A - Chimeric receptor capable of being activated - Google Patents

Chimeric receptor capable of being activated Download PDF

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Publication number
CN106519037A
CN106519037A CN201510578444.8A CN201510578444A CN106519037A CN 106519037 A CN106519037 A CN 106519037A CN 201510578444 A CN201510578444 A CN 201510578444A CN 106519037 A CN106519037 A CN 106519037A
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Prior art keywords
chimerical receptor
activable
tissue
chimerical
receptor
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CN106519037B (en
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李宗海
陈骋
蒋华
王华茂
王鹏
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Keji Biomedical Shanghai Co ltd
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Keji Biomedical (shanghai) Co Ltd
Shanghai Cancer Institute
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Priority to PCT/CN2016/098617 priority patent/WO2017041749A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention relates to a chimeric receptor capable of being activated. Only in the presence of protease with specific expression or high expression in pathological tissues, the chimeric receptor can be targeted on corresponding antigens to kill pathological tissues or cells. The chimeric receptor cannot kill pathological tissues or cells in the absence of protease. A feasible solution is provided for the problem of on-target-off-tumor.

Description

Activable Chimerical receptor
Technical field
The invention belongs to immunotherapy of tumors field, more particularly it relates to activable being fitted together to is received Body, its preparation method and its application.
Background technology
Effect of the immune effector cell in tumor immune response is increasingly subject to pay attention to.It is thin based on immunological effect The adoptive immunotherapy of born of the same parents achieves certain effect, and this kind of immunization therapy side in Partial tumors Method can overcome the drawbacks described above of Antybody therapy, but still unsatisfactory in the curative effect of most of tumours [Grupp SA, et al.Adoptive cellular therapy.Curr Top Microbiol Immunol., 2011；344:149-72.].In recent years, according to cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL) t lymphocyte receptor (T Cell Receptor, TCR) is depended on to the identification specificity of target cell Discovery, will be for the CD3 ζ of scFv and the t lymphocyte receptor of the antibody of tumor cell associated antigen Or the intracellular signal such as Fc ε RI γ activation motif is fused into Chimeric antigen receptor (Chimeric antigen Receptor, CAR；Chimerical receptor (Chimeric receptor, CR) is also referred to as now), and passed through such as The mode such as slow-virus infection genetic modification is in T lymphocytic cell surfaces.This CAR T lymphocytes can be with Major Histocompatibility compound (Major Histocompatibility Complex, MHC) is non-limiting T lymphocytes are optionally directed to tumour cell and specifically kill tumour by mode.CAR T drench Bar cell is new Immunotherapy Strategy [Schmitz M, an et al. in immunotherapy of tumors field Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer.J Biomed Biotechnol, 2010, doi:10.1155/2010/956304.].Additionally, CAR modifications NK cells (Klingemann H.Challenges of cancer therapy with natural killer cells. Cytotherapy.2014Dec 18.pii：S 1465-3249 (14) 00791-9) or NKT cells also facing Good antitumor activity (Heczey A1, Liu D1, Tian G2, Courtney is illustrated in research before bed AN1, Wei J1, Marinova E1, Gao X1, Guo L1, Yvon E3, Hicks J2, Liu H4, Dotti G5, Metelitsa LS6.Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy.Blood. 2014；124(18):2824-33).
Chimeric antigen receptor includes extracellular land, transmembrane region and intracellular signal area.Generally extracellular region is included It is capable of identify that the scFv of tumor associated antigen, transmembrane region adopts CD8, the equimolecular transmembrane regions of CD28, Intracellular signal area adopts ITAM (ITAM) CD3 ζ or Fc ε RI γ and costimulation The intracellular signal area of signaling molecule CD28, CD27, CD137, CD134 etc..
Intracellular signal area is first generation CAR T lymphocytes only comprising ITAM, and wherein chimeric antigen is received Body each several part is connected by following form：scFv-TM-ITAM.This kind of CAR T can excite antineoplastic Cytotoxic effect, but cytokine secretion is fewer, and lasting resisting can not be excited in vivo to swell Knurl effect [Zhang T.et al.Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic Pathways, Can Res 2007,67 (22)：11029-11036.].
Subsequently the second generation CAR T lymphocytes of development add CD28 or CD137 (also known as 4-1BB) Intracellular signal area, wherein Chimeric antigen receptor each several part by following form connect：scFv-TM-CD28 - ITAM or scFv-TM-/CD137-ITAM.Intracellular signal area occur B7/CD28 or 4-1BBL/CD137 costimulations effect causes the continuous proliferation of T lymphocytes, and can improve T lymphs Cell secretes the level of the cell factor such as IL-2 and IFN-γ, while improving CAR T survivals in vivo Cycle and antitumous effect [Dotti G.et al.CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients. J Clin Invest, 2011,121 (5)：1822-1826.].
The third generation CAR T lymphocytes for developing in recent years, wherein Chimeric antigen receptor each several part are by as follows Form connects：ScFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, enters One step improves CAR T time to lives in vivo and its antitumous effect [Carpenito C., et al. Control of large established tumor xenografts with genetically retargeted human T cells containing CD28and CD137domains.PNAS, 2009,106 (9)： 3360–3365.]。
Although CAR T lymphocytes have tempting prospect, its high risk in immunotherapy of tumors Also need to consider.Such as, due to some/specificity that can recognize of kind of normal structure low expression CAR resists Original is likely to result in CAR T lymphocytes to expressing the damage of the normal structure of antigen.Such as, for nephrocyte Antigen carbonic anhydrase IX (CAIX) expressed on cancer patient tumors cell is first is used for clinical CAR T lymphocytes are adopted the case for the treatment of, and the case of first miss the target effect of the report containing CAR cells. There is 2-4 level hepatotoxicity wind agitation after repeatedly input CAR T lymphocytes in patient.Analysis reason is on hepatic duct Chrotoplast low expression CAIX, former clinical testing are forced to interrupt while eliminating any of Case treatment effect Evaluate [Stoter G.et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX： First clinical experience.J clin oncol, 2006,24 (13)：e20-e22.；Ngo MC., et al. Ex vivo gene transfer for improved adoptive immunotherapy of cancer.Human Molecular Genetics, 2011, R1-R7].In addition, costimulatory signal excessive in CAR can be reduced Effector cell activation needed for threshold value so that genetic modification T lymphocytes are in low-level antigen or do not have It is likely to be activated under conditions of antigen triggering, cause large amount of cell factor is released so that possible initiation So-called " cytokine storm ".This signal leakage (signal leakage) can cause the cell toxicant that misses the target Property, so as to produce nonspecific tissue damage.For example, using the third generation CAR for Her2 Due in normal lung tissue during one Advanced Colon Cancer patient with liver and Lung metastases of clinical treatment Low expression Her2 and cause so-called " cytokine storm " cause a disease people sudden death [Morgan RA., et al. Report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing Erbb2.Molecular Therapy, 2010,18 (4)：843-851.].
Accordingly, it would be desirable to find suitable method, immune effector cell based on CAR is enable more Tumour is acted on accurately, it is to avoid be applied to the high risk existed during immunotherapy of tumors.
The content of the invention
It is an object of the invention to provide a kind of activable Chimerical receptor, its preparation method and its application.
In a first aspect of the present invention, there is provided a kind of activable Chimerical receptor (Activatable Chimeric Receptor, ACR), which includes：
Chimerical receptor (Chimeric Receptor, CR), which can target (with reference to) pathologic in an active state The antigen of the high expression of tissue；
Closure elements (Blocking element, BE), which can suppress Chimerical receptor with the pathologic tissue The antigen binding of high expression；With
Can shearing elements (Cleavable element, CE), which is located between Chimerical receptor and closure elements.
In a preference, described Chimerical receptor includes what is be linked in sequence：Extracellular antigen binding domain, Transmembrane region and intracellular signal area；Described intracellular signal area is selected from：CD3 ζ, Fc ε RI γ, CD27, CD28, The intracellular signal region sequence of CD137, CD134, CD40 or Myd88, or its combination.
In another preference, described extracellular antigen binding domain is the specific binding pathologic group Knit the antibody of the antigen of high expression.
In another preference, described specifically binds the anti-of the high antigen expressed of the pathologic tissue Body can be：Single-chain antibody (scFV), monoclonal antibody, single domain antibody, Fab fragments, Fd pieces Section, Fv fragments, 2 fragments of F (ab ') and its derivative, or the antibody of other forms.
In another preference, described specifically binds the anti-of the high antigen expressed of the pathologic tissue Body is selected from (but not limited to)：Single-chain antibody or single domain antibody.
In another preference, Chimerical receptor effectively can recognize described in the case of closure elements are non-existent The antigen of the high expression of pathologic tissue, closure elements can disturb or compete Chimerical receptor with the pathologic The combination of the antigen of the high expression of tissue.
In another preference, described activable Chimerical receptor is according to suitable from aminoterminal to c-terminus Sequence, includes successively：Closure elements, can shearing elements, Chimerical receptor.
In another preference, described Chimerical receptor, closure elements, can also wrap between shearing elements Include connection peptide.
In another preference, described activable Chimerical receptor, wherein containing the first connecting peptides (LP1) and the second connecting peptides (LP2)；Wherein activable Chimerical receptor is according to from aminoterminal to c-terminus Order, include successively：Closure elements, the first connecting peptides, can shearing elements, the second connecting peptides, Chimerical receptor.
In another preference, described closure elements are selected from, but not limited to,：Directly receive with described chimeric The polypeptide that body is combined；Or from spatially hindering the polypeptide of described Chimerical receptor and antigen binding.
In another preference, described closure elements be 2～100aa (such as 10aa, 20aa, 30aa, 40aa, 50aa, 60aa, 70aa, 80aa)；The preferably polypeptide of 2～40aa.
In another preference, it is described can shearing elements be can be specific expressed or high by pathologic tissue The element that the protease of expression is sheared, reduces or decomposes, the protease and the pathologic tissue are high The antigen common location of expression is in same pathologic tissue.
In another preference, described pathologic tissue is included but is not limited to：Tumour, LADA Diseased tissue, by the tissue that viral (such as inhibition of HIV) infects.
In another preference, described pathologic tissue is tumour, described pathologic tissue specificity The protease of expression or high expression is included but is not limited to：Plasma urokinase-type plasminogen activator (urokinase-type plasminogen activator, uPA), legumain protease or matriptase (MT-SP1)。
In another preference, described pathologic tissue is specific expressed or protease of high expression is urine Kinases type activator of plasminogen or matriptase, it is described can shearing elements be SEQ ID NO:2 institutes Show the polypeptide of amino acid sequence.
In another preference, the antigen of the high expression of described pathologic tissue is included but is not limited to：GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, Mesothelin, CD19, CD20 or ASGPR1.
In another preference, the antigen of the high expression of described pathologic tissue is GPC3, described envelope Close the Binding peptide that element is GC33 antibody；It is preferred that described closure elements are SEQ ID NO:1 The polypeptide of shown amino acid sequence.
In another preference, when the antigen of the high expression of described pathologic tissue is GPC3, the disease Rationality is organized as tumour, including：Liver cancer, melanoma, clear cell carcinoma of ovary, yolk sac tumor, god Jing blastomas.
In another aspect of this invention, there is provided a kind of polynucleotides, it is activable chimeric described in its coding Acceptor.
In another aspect of this invention, there is provided a kind of expression vector, which includes activable described in coding The nucleic acid of Chimerical receptor.
In another preference, described expression vector derives from slow virus plasmid pWPT.
In another aspect of this invention, there is provided a kind of virus, described virus include the carrier.
In another aspect of this invention, there is provided above arbitrary described activable Chimerical receptor or coding The purposes of its nucleic acid or the expression vector comprising the nucleic acid or virus, for preparing targeting pathologic group The Chimerical receptor immune effector cell knitted.
In another aspect of this invention, there is provided a kind of Chimerical receptor immune effector cell, its transduction has coding The above nucleic acid of arbitrary described activable Chimerical receptor, or foregoing expression vector or described Virus；Or arbitrary described activable Chimerical receptor before its surface expression.
In another preference, described immune effector cell includes：T lymphocytes, NK cells or NKT cells, Treg cells.
In another aspect of this invention, there is provided the purposes of described Chimerical receptor immune effector cell, it is used for The medicine of targeting pathologic tissue is prepared, it is anti-that the pathologic tissue high expression Chimerical receptor can be combined It is former.
In a preference, described pathologic tissue is tumour, described targeting pathologic tissue Medicine is the medicine for suppressing tumour.
In another aspect of this invention, there is provided a kind of pharmaceutical composition, which includes：It is foregoing chimeric Recipient immune effector cell.
The other side of the present invention, due to this disclosure, is aobvious to those skilled in the art And be clear to.
Description of the drawings
The structural representation of Fig. 1, activable Chimerical receptor ACR.
It is followed successively by according to N-terminal → C-terminal：Sp：Secreting signal peptide, BE：Closure elements, LP1：Joint 1；CE1：Can shearing elements；LP2：Joint 2, Ab：Antigen binding domain；LP3：Joint 3；TM： Transmembrane region；SD：Intracellular signal area.
The schematic diagram of Fig. 2, GC33-28BBZ-ACR.
According to the direction of N-terminal → C-terminal, in figure, modules are followed successively by CD8 secretion signals, BE (closings Element), LP1, CE (can shearing elements, the digestion substrate of UP1), LP2, CR (GC33-28BBZ), F2A, eGFP.
The GC33-28BBZ-ACR positive rates of Fig. 3, facs analysis T cell.
By the positive rate of GFP as the GC33-28BBZ-ACR for indicating T cell after slow-virus infection Positive rate.
Fig. 4, in vitro toxicity experiment.Using SMMC-7721 Huh-7 as target cell, effector cell is body The outer culture T cell for expressing GC33-28BBZ-ACR of 11 days, effect target ratio respectively 3:1,1:1 With 1:3, target cell numbers are 10000/hole, according to different effect targets than corresponding effector cell.Respectively plus Enter uPA and MTSP1, a low dose group, a high dose group and blank group are respectively set.Each group is equal If 5 multiple holes, take the mean value of 5 multiple holes.Detection time is 18h.
Specific embodiment
The present inventor discloses one kind based on Chimeric antigen receptor (CAR) technology through extensive research Activable Chimerical receptor, the activable Chimerical receptor are specific expressed or high only in pathologic tissue The protease of expression can target corresponding antigen under conditions of existing, and realize pathologic tissue or cell Lethal effect；And do not play a role under the conditions of protease is non-existent.The present invention is to avoid On-target-off-tumor there is provided feasible solution.
As used herein, described " Chimerical receptor " and " Chimeric antigen receptor " is used interchangeably.
As used in the present invention, described " antigen of the high expression of pathologic tissue " refers to that the chimeric of activation is received The antigen targetted by body, the antigen high expression in pathologic tissue or cell.In the present invention, should " disease Normal structure or cell that the antigen of the high expression of rationality tissue " is likely to beyond pathologic tissue or cell Middle expression.It is preferred that it is tumor associated antigen to be somebody's turn to do " antigen of the high expression of pathologic tissue ", for example, select From (but not limited to)：GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20 or ASGPR1.
As used in the present invention, described " pathologic tissue is specific expressed or the protease of high expression " is Refer to, or the high albumen (water expressed in pathologic tissue or cell Solution) enzyme, which can be hydrolyzed to its specific substrate.
As used in the present invention, described " closure elements " be refer to block described Chimerical receptor and its The polypeptide of corresponding antigen binding, which passes through direct combination with described Chimerical receptor or from spatially hindering The modes such as described Chimerical receptor and antigen binding are playing the work combined with target site by blocking Chimerical receptor With.
As used in the present invention, described " can shearing elements " be one and be located at Chimerical receptor and closure elements Between polypeptide, which is the bottom of " pathologic tissue is specific expressed or the protease of high expression " Thing, when there is described protease, should " can shearing elements " can be sheared, reduce or decompose, from And cause closure elements no longer to block its corresponding antigen of described Chimerical receptor and occur to combine.
As used in the present invention, described " pathologic tissue " is included but is not limited to：Tumour, itself exempts from Epidemic disease diseased tissue, the tissue infected by viral (such as inhibition of HIV) etc..
In the present invention, described pathologic tissue can be various be unfavorable for health harmful groups in body Knit or focus, it is necessary to remove in slave body.Described pathologic target tissue includes tumour.Any ability Tumour known to domain is can be included in the present invention, as long as the tumour can express low expression in normal structure Tumor associated antigen.For example, described tumour is included but is not limited to：Liver cancer, lung cancer, glioma, Breast cancer, cancer of the stomach, prostate cancer, brain tumor, oophoroma, bone tumour, colon cancer, thyroid tumors, Mediastinal tumor, intestinal tumor, kidney neoplasms, adrenal tumor, tumor of bladder, orchioncus, malignant lymphatic Knurl, Huppert's disease, nervous system neoplasm, the cancer of the esophagus, thymus gland celiothelioma, cancer of pancreas, leukaemia, Head and neck neoplasm, cervical carcinoma, cutaneum carcinoma, melanoma, vagina epithelium cancer, carcinoma of gallbladder, malignant fibrous Histocytoma.For example, described tumor associated antigen is included but is not limited to：GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4EGFR, CD19, CD33, IL13R, LMP1, PLAC1, NY-ESO-1, MAGE4, MUC1, MUC16, LeY, CEA, CAIX (carbonic acid Acid anhydride enzyme IX), CD123.
Term " Chimerical receptor epidemic disease effector cell " is it is known in the art which is using genetic modification technology The immune effector cell of expression antigen (such as tumour antigen) specific chimeric acceptor, can targeting performance killing Effect.Described immune effector cell for example includes T cell, NK cells, NKT cells, modulability T cell (Regulatory cell, abbreviation Treg).Conventional preparation " Chimerical receptor immune effector cell " Method be well known by persons skilled in the art, including allowing its expression intracellular costimulation cellular elements intracellular knot Structure domain, such as CD28 (are preferably comprised CD28a, CD28b), CD137, CD27, CD3 ζ (compared with Be CD3 ζ intracellular domains goodly), one kind in CD8, CD19, CD134, CD20, Fc ε RI γ or It is various.Combined with respective ligand by them, activated the secondary signal of immune effector cell, strengthened immunity The secreting function of the multiplication capacity of cell and cell factor, extends the time-to-live of activating immune cell.
In view of the definitely special target spot of tumour is few, therefore the immunity of most CAR modifications is thin The antigen that born of the same parents' (such as CAR T cells) are directed to (such as CD19, CD20, Her2, EGFR, EpCAM etc.) More or less express in the normal tissue, therefore, it is difficult to there is the pair of on-target-off-tumor with avoiding Effect.The effect for how reducing or reducing this On-target-off-tumor becomes extremely important.This A person of good sense widely compares the difference of the microenvironment of tumor tissues and the microenvironment of normal structure, is based on first Some proteolytic enzymes such as uPA, MT-sp1, Legumain protease etc. high expression in tumor tissues And do not express or the characteristics of low expression in normal structure, using the specificity of these hydrolases, transform embedding The immunocyte of acceptor modification is closed, the immunocyte for being obtained only could more after these hydrolysis enzyme effects Anti-tumor function is played effectively, so as to effectively improve the security of immunocyte.
Therefore, the present invention provide a kind of activable Chimerical receptor (Activatable Chimeric Receptor, ACR), the element (Cleavable that it includes Chimerical receptor (Chimeric Receptor, CR), can shear Element, CE) and closure elements (Blocking element, BE) connection, can be by between three Connecting peptides are attached.Wherein CE antigens be sheared, reduce, optical resolution or other modification after, ACRs can show the conformation of activation so that CR can be easier and targeted integration.
As the preferred embodiment of the present invention, a kind of structure of the activable Chimerical receptor ACR of the present invention Schematic diagram is shown in Fig. 1.From left to right arrange, i.e., for rearranging from the N-terminal of protein to C-terminal across Membrane receptor can activate Chimerical receptor ACR schematic diagram.Under unactivated state, BE meetings and Ab Area combines, or spatially hinders the combination of Ab and target antigen；And once CE1 is sheared by protease etc., So Ab just can be combined with target antigen.
Described closure elements can be any by directly being combined or from sky with described Chimerical receptor Between upper hinder the modes such as described Chimerical receptor and antigen binding and tie with target site playing blocking Chimerical receptor The polypeptide of the effect of conjunction.The closure elements can be selected according to the type of the antigen binding domain of Chimerical receptor. For example, described antigen binding domain is a kind of antibody, then the closure elements can be the antibody combination it is many Peptide.In a particular embodiment of the present invention, antibody of the antigen binding domain of Chimerical receptor using resisting GPC 3 GC33, and its Binding peptide (NSQQATPKDNEISTFH) is applied as closure elements.
It is described can shearing elements can be or high expression specific expressed by pathologic tissue protease Shearing, reduction or the substrate for decomposing.Wherein specific table can be selected according to corresponding indication Up to or high expression protease, its substrate can be used as can shearing elements.The expression of described protease is special The opposite sex is higher, then preferred.In a preferred embodiment of the present invention, indication is tumour, tumor group There is different, proteolytic enzyme therein such as uPA from the microenvironment of normal structure in the microenvironment knitted, The high expression in tumor tissues such as MT-sp1, Legumain protease, and do not express in normal structure or Low expression, therefore the present inventor transforms exempting from for Chimerical receptor modification using the specificity of these hydrolases Epidemic disease cell, the immunocyte only could more effectively play anti-tumor function after these hydrolysis enzyme effects, So as to effectively improve the security of the immunocyte of CAR modifications.In a particular embodiment of the present invention, Substrate polypeptide LSGRSDNH using uPA and MT-SP1 protease is used as can shearing elements.
Described Chimerical receptor, closure elements, connection peptide can be may also include between shearing elements.It is described Connection peptide have no particular limits, can be it is any can provide described Chimerical receptor, closure elements, Can be flexibly connected between shearing elements, not affect any polypeptide of each element function of itself.It is preferred that Described connexon includes 2-40 amino acid；Preferably 3-30 amino acid, such as 5,8,10, 15th, 20,25 amino acid.
The present invention also includes the nucleic acid of the coding activable Chimerical receptor.The invention further relates to above-mentioned many The fragment of the variant of nucleotides, its coding and the polypeptide or polypeptide for having identical amino acid sequence of the invention, Analogs and derivatives.
Present invention also offers the carrier of the nucleic acid comprising above-mentioned activable Chimerical receptor.It is concrete at one In embodiment, the carrier that the present invention is used is a kind of slow virus plasmid vector pWPT.It should be understood that its Its expression vector is also available.
Present invention additionally comprises the virus comprising above-mentioned carrier.The present invention virus include pack after with sense The virus of dye power, also include comprising be packaged as having infectious virus institute must composition disease to be packaged Poison.Other known in the art can be used for by foreign gene transduce into immune effector cell virus and its Corresponding plasmid vector can also be used for the present invention.
Present invention also offers the immune effector cell of genetic modification, which is had by transduction, and coding is described to live The nucleic acid of the Chimerical receptor of change is had above-mentioned comprising the recombinant plasmid containing the nucleic acid by transduction, or bag Virus containing the plasmid.The conventional nucleic acid transduction method in this area, including non-viral and viral transduction side Method may be used to the present invention.Electroporation and transposons method are included based on non-viral transduction method.Closely Foreign gene directly can be imported nucleus by the Nucleofector consideration conveys dye instrument of phase Amaxa company's research and development Obtain the high efficiency transduction of genes of interest.In addition, being based on sleeping beauty transposon stand (Sleeping Beauty system) Or the more common electroporation of transduction efficiency of the Transposon System such as PiggyBac transposons improves a lot, will Nucleofector transfects instrument and has been reported [Davies JK., et al. with the system combined application of sleeping beauty transposon stand Combining CD19redirection and alloanergization to generate tumor-specific Human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res, 2010,70 (10):OF1-10.], the method had not only had higher transduction efficiency but also can realize genes of interest Site-directed integration.In one embodiment of the invention, realize Chimerical receptor (for activating in the present invention Chimerical receptor) transduction method of immune effector cell of genetic modification is such as retrovirus based on virus Or the transduction method of slow virus.The method has transduction efficiency high, and foreign gene stably can be expressed, and The advantages of time of in vitro culture immune effector cell arrival clinical number of stages can be shortened.In the transgenosis Immune effector cell surface, the nucleic acid of transduction pass through transcription, accurate translation on its surface.By to various The tumour cell of different cultures carries out cell in vitro poison it is demonstrated experimentally that the immune effector cell tool of the present invention There is the tumor cytotoxicity effect (also known as cytotoxicity) of high degree of specificity.Therefore the encoding chimera of the present invention The nucleic acid of receptor protein, the plasmid comprising the nucleic acid, the virus comprising the plasmid and transduction have above-mentioned nucleic acid, The transgenosis immune effector cell of plasmid or virus can be efficiently used for the immunization therapy of tumour.
Immunocyte of the present invention can also carry the coded sequence of the cell factor of external source；Described Cell factor is included but is not limited to：IL-12, IL-15 or IL-21 etc..These cell factors have immunity Adjust or antineoplastic activity, the function of the NK cells of energy enhancement effect T cell and activation, or directly Antitumor action is waved in sending and receiving.Therefore, it will be understood by those skilled in the art that the utilization of these cell factors Contribute to described immunocyte preferably to play a role.
Immunocyte of the present invention can also be expressed another kind of embedding in addition to above-mentioned Chimerical receptor Acceptor is closed, this receptor does not contain CD3 ζ, but the intracellular signal domain containing CD28, CD137 Intracellular signal domain or combination of the two.
Immunocyte of the present invention can also express chemokine receptors；Described chemokine receptors Including but not limited to CCR2.It will be understood by those skilled in the art that described CCR2 chemotactic factor (CF)s are received Body can cause internal CCR2 competitive bindings therewith, for the transfer of blocking tumour is favourable.
Immunocyte of the present invention can also express the siRNA or blocking that can reduce PD-1 expression The albumen of PD-L1.It will be understood by those skilled in the art that competitive blocking PD-L1 and its acceptor PD-1 Interaction, be conducive to recovering antitumor t cell responses, so as to suppress tumour growth.
Immunocyte of the present invention can also express safety switch；It is preferred that described safety switch Including：ICaspase-9, Truancated EGFR or RQR8.
The Chimerical receptor immune effector cell of the present invention can apply to prepare pharmaceutical composition or diagnosis examination Agent.Described composition can be also included pharmaceutically acceptable except the immunocyte including effective dose Carrier.Term " pharmaceutically acceptable " is referred to and suitably give animal when biomolecule ontology and composition Or during people, they will not produce unfavorable, allergy or other bad reactions.
Can be carbohydrate as the specific example of some materials of pharmaceutically acceptable carrier or its component, such as Lactose, dextrose and saccharose；Starch, such as cornstarch and potato starch；Cellulose and its derivates, Such as sodium carboxymethylcellulose, ethyl cellulose and methylcellulose；Tragacanth powder；Malt；Gelatin； Talcum；Kollag, such as stearic acid and magnesium stearate；Calcium sulfate；Vegetable oil, such as peanut oil, cotton Seed oil, sesame oil, olive oil, corn oil and cupu oil；Polyalcohol, such as propane diols, glycerine, sorb Sugar alcohol, mannitol and polyethylene glycol；Alginic acid；Emulsifying agent, such asWetting agent, the such as moon Osmanthus base sodium sulphate；Colouring agent；Flavor enhancement；Tablet agent, stabilizer；Antioxidant；Preservative；Without heat Raw water；Isotonic salting liquid and phosphate buffer etc..
The composition of the present invention can make various formulations as needed, and can by physician in view patient category, Age, body weight and the substantially factor such as disease condition, administering mode determination dosage beneficial to patient are applied With.Administering mode for example can be using injection or other therapeutic modalities.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for Illustrate the present invention rather than limit the scope of the present invention.The reality of unreceipted actual conditions in the following example Proved recipe method, generally writes according to normal condition such as J. Pehanorm Brookers etc., Molecular Cloning:A Laboratory guide, the Three editions, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.
The structure of embodiment 1, resisting GPC 3 third generation Chimeric antigen receptor
Using anti-phosphatidylinositols proteoglycan -3 (GPC3) third generation Chimeric antigen receptor for building before (Chimeric antigen receptor, CAR), in the CAR, single-chain antibody used is anti-from GC33 Body, the structure of the CAR T referring in Application No. CN201310164725.X, its GPC3-28BBZ It is applied in the present embodiment, also referred to as GC33-28BBZ.
The present inventor is by Binding peptide (NSQQATPKDNEISTFH (the SEQ ID NO of GC33 antibody: 1)) as BE elements, and substrate polypeptide LSGRSDNH (the SEQ ID of uPA and MT-SP1 protease NO:2) as CE elements.Additionally by by from foot and mouth disease virus (food-and-mouth disease Virus, FMDV) ribosomal skip sequence (ribosomal skipping sequence 2A) (abbreviation F2A) EGFP is connected to into the C-terminal of ACR, the coexpression of eGFP and ACR is realized, so as to pass through eGFP Expression carry out the expression of side light ACR.
(1) acquisition of the fragment of Sp+BE+LP1+CE+LP2
The primer of design such as table 1.
Table 1
Wherein the fragment of Sp+BE+LP1+CE+LP2 adopts the primer such as table 1, by Overlap PCR Obtain, amplification condition is：
Denaturation：94 DEG C, 4min；
Following 25 circulations：Denaturation：94 DEG C, 30s,
Annealing：50 DEG C, 30s,
Extend：68 DEG C, 20s；
Then, 68 DEG C re-extend 10min.
Glue reclaim is carried out using conventional method to pcr amplification product subsequently, is obtained The DNA fragmentation that Sp+BE+LP1+CE+LP2 is connected with each other.
(2) acquisition of LP2-GC33-28BBZ fragments
The primer of design such as 2.
Table 2
Take GC33-28BBZ plasmids 1 microlitre of (plasmid is referring to CN201310164725.X patents) (100ng) For template, respectively using GPC3scfv-F, 3z-F2A-R as upstream and downstream primer, 50 microlitres of systems are entered Performing PCR is expanded, and the PCR primer of acquisition carries out cutting glue reclaim, so as to obtain LP2-GC33-28BBZ Fragment.
(3) acquisition of F2A-EGFP
The primer of design such as 3.
Table 3
1 microlitre of GC33-28BBZ plasmids (100ng) are taken for template, respectively by F2A-EGFP-F, Pwpt-EGFP-R enters performing PCR amplification as upstream and downstream primer, and the PCR primer of acquisition carries out cutting glue Reclaim, so as to obtain F2A-EGFP.
(4) acquisition of slow virus plasmid pWPT-GC33-28BBZ-ACR
The three segments that above-mentioned (1)～(3) prepare are carried out into overlap PCR, specifically three sections pieces Section, is mixed according to equimolar ratio, by carrying out overlap PCR, upstream and downstream above-mentioned steps (1) Suo Shu Primer is CD8sp-F and EGFP-R.The PCR primer of acquisition carries out cutting glue reclaim.
Using restriction enzyme to being connected into slow virus plasmid pWPT carriers after MluI and SalI double digestions Corresponding site in.
The recombinant plasmid transformed Escherichia coli Top10 competent cells of acquisition, culture 12h or so are chosen Clone's checking.Selection includes the clone of recombinant plasmid and send sequencing.Correctly clone carries out disease to select sequence Poison is prepared and is packed.
The carrier of acquisition is named as pWPT-GC33-28BBZ-ACR.
The preparation of embodiment 3, CAR-T
1st, the packaging of the slow virus of GC33-28BBZ-ACR
With 6 × 106Density inoculated and cultured to the 6th～10 generation HEK-293T cell (ATCC： CRL-11268) in 10cm culture dishes, 37 DEG C, 5%CO2Overnight incubation prepares for transfecting.Culture Base is the DMEM containing 10% hyclone (purchased from gibco companies) (purchased from gibco companies).
The step of transfection, is as follows：
(1) by 10 μ g genes of interest plasmid pWPT-GC33-28BBZ-ACR, pack with 6.5 μ g respectively Plasmid PAX2：With 3.5 μ g envelope plasmid pMD2.G, the serum-free DMEM cultures of 800 μ L are dissolved in In liquid, mix.
(2) by 60 μ l PEI, (polyethyleneimine, purchased from Polysciences companies, is made into 1 μ g/ μ L concentration Working solution), in adding the serum-free DMEM nutrient solutions of above-mentioned 800 μ L with plasmid, be vortexed mixed It is even, it is stored at room temperature incubation 25min.
(3) 800 μ L of transfection composite are added in HEK-293T cells to be transfected, after 6-8h hours, Liquid is changed with the DMEM training bases of 10%FBS to the 293T cells of transfection.
(4) about 24h after transfection, observation transfection efficiency (i.e. the cell proportion in green fluorescence).In transfection After 72h, virus, ultracentrifugation is collected by filtration using 0.45 μm of filter (purchased from Millipore companies) (Beckman Optima L-100XP ultracentrifuge 28000rpm, 4 DEG C are centrifuged 2 hours), concentration disease Poison.Centrifugation gained precipitation carries out weight with the AIM-V nutrient solutions (purchased from Gibco companies) of 1/30 stoste volume It is outstanding, it is frozen in -80 DEG C with the packing of 100 μ L/ pipes, to infect T lymphocytes.Meanwhile, by what is obtained Concentrating virus are titrated.
2nd, the infection of GC33-28BBZ-ACR
Human peripheral blood single nucleus cell (Shanghai City is obtained by density-gradient centrifugation method by healthy human peripheral blood Blood Center is provided).PMNC is by CD4+/CD8+ cell magnetic beads (purchased from Stem Cell Technologies) negativity method for separating obtains the positive primary human T lymphocytes of CD4+ and CD8+.Point T cell after choosing carries out Flow cytometry its purity, with positive rate >=95% of target cell be advisable into Row next step is operated.With 1 × 106/ mL density adds AIM-V lymphocytes culture medium liquid (purchased from Gibco Company, containing 2% people AB serum) cultivate and with cell：Magnetic bead ratio is 1:1 adds while being coated with The magnetic bead (Invitrogen companies) of AntiCD3 McAb and CD28 antibody and the recombinant human il-2 of final concentration 300U/mL (purchased from Huaxin Advanced Biotechnical Co., Ltd., Shanghai) stimulates culture 24h.Before infection, by CD4+CD8+T Lymphocyte is according to 1:1 ratio mixing, then with MOI ≈ 5-10 by above-mentioned viral concentration liquid with wait to feel Dye T cell mixing, while add the polybrene of final concentration of 6 μ g/mL.Metainfective cell, every It changes liquid, changes without virulent training base.5 × 10 are adopted every other day5The density of/mL is passed on, Add the recombinant human il-2 of final concentration 300U/mL simultaneously in LCF.
Metainfective primary T cells at the 9-11 days (i.e. the first round amplification end cycle), by streaming Cell art detect Chimeric antigen receptor GC33-28BBZ-ACR expression, due to eGFP with GC33-28BBZ-ACR is co-expressed, and detects that the positive cell of eGFP as expresses Chimeric antigen receptor Positive cell, and using the T lymphocytes that are uninfected by as negative control.
As a result such as Fig. 3, the T cell after the positive rate of GFP is as instruction slow-virus infection GC33-28BBZ-ACR positive rates, it is seen that positive rate is 61.0%.
Embodiment 4, external source restructuring uPA promotes the activation of BE-GPC3-CAR T
The material that in vitro toxicity experiment is used is as follows：
Using SMMC-7721 Huh-7 as target cell, effector cell is expressing in vitro culture 11 days The T cell (positive rate 61%) of GC33-28BBZ-ACR, effect target ratio respectively 3:1,1:1 and 1:3, Target cell numbers are 10000/hole, according to different effect targets than corresponding effector cell.It is separately added into uPA With MTSP1, a low dose group (uPA is respectively set：0.08μg/mL；MT-SP1：0.04 μ g/mL), One high dose group (uPA：0.4μg/mL；MT-SP1：0.2 μ g/mL) and blank group (uPA：0μg/mL； MT-SP1：0μg/mL).Each group is all provided with 5 multiple holes, takes the mean value of 5 multiple holes.Detection time is 18h.
Wherein each experimental group and each control group are as follows：
Experimental group (blank group)：Target cell Huh-7+GC33-28BBZ-ACR T cells+variable concentrations Enzyme,
Control group 1：The enzyme of the maximum release LDH+ variable concentrations of target cell,
Control group 2：The enzyme of the spontaneous release LDH+ variable concentrations of target cell,
Control group 3：The enzyme of the spontaneous release LDH+ variable concentrations of effector cell.
Detection method：Using 96 non-radioactive cell toxicity detection kits (Promega companies) of CytoTox Detected.
Cytotoxicity computing formula is：
From fig. 4, it can be seen that GC33-28BBZ-ACR T cells (have infected GC33-28BBZ-ACR T cell) antitumor activity it is relatively low, but can be effective in the presence of uPA or MT-SP1 Ground activation, so as to killing tumor cell.
The above results show that the design of the present invention is rational, that is, true by the effect of closure elements Weaken can the lethal effect of T cell in fact；But if the uPA of q.s in tumor by local environment Or the effect ACR of the corresponding protease such as MT-SP1 can obtain activation killing tumor cell, so as to rise To local antitumoral activity, the damage of normal tissue is reduced.
The all documents referred in the present invention are all incorporated as reference in this application, literary just as each Offer and be individually recited as reference like that.In addition, it is to be understood that in the above-mentioned instruction content for having read the present invention Afterwards, those skilled in the art can be made various changes or modifications to the present invention, and these equivalent form of values are same Fall within the application appended claims limited range.

Claims (22)

1. a kind of activable Chimerical receptor it include：
Chimerical receptor, which can target the antigen of the high expression of pathologic tissue in an active state；
Closure elements, which can suppress the antigen binding of Chimerical receptor and the high expression of the pathologic tissue；With
Can shearing elements, which is located between Chimerical receptor and closure elements.
2. activable Chimerical receptor as claimed in claim 1, it is characterised in that described chimeric to receive Body includes what is be linked in sequence：Extracellular antigen binding domain, transmembrane region and intracellular signal area；Described intracellular Signaling zone is selected from：CD3 ζ, Fc ε RI γ, the intracellular of CD27, CD28, CD137, CD134, CD40 Signal region sequence or Myd88, or its combination.
3. activable Chimerical receptor as claimed in claim 2, it is characterised in that described is extracellular Antigen binding domain is the antibody of the antigen of the high expression of the specific binding pathologic tissue.
4. activable Chimerical receptor as claimed in claim 3, it is characterised in that described specificity It is selected from reference to the antibody of the antigen of the high expression of the pathologic tissue：Single-chain antibody or single domain antibody.
5. activable Chimerical receptor as claimed in claim 1, it is characterised in that described activates Chimerical receptor according to the order from aminoterminal to c-terminus, include successively：Closure elements, can shear unit Part, Chimerical receptor.
6. activable Chimerical receptor as claimed in claim 1, it is characterised in that described chimeric to receive Body, closure elements, can be between shearing elements, also including connection peptide.
7. activable Chimerical receptor as claimed in claim 1, it is characterised in that described closing unit Part is selected from：
The polypeptide for directly being combined with described Chimerical receptor；Or
From the polypeptide for spatially hindering described Chimerical receptor and antigen binding.
8. activable Chimerical receptor as claimed in claim 1, it is characterised in that described shears Element be can the protease of or high expression specific expressed by pathologic tissue shear, reduce or decompose Element, the protease is with the high antigen common location expressed of the pathologic tissue in same pathologic tissue.
9. activable Chimerical receptor as claimed in claim 8, it is characterised in that described pathologic Tissue includes：Tumour, autoimmune disease tissue, the tissue being infected by the virus.
10. activable Chimerical receptor as claimed in claim 9, it is characterised in that described pathology Property tissue be tumour, described pathologic tissue is specific expressed or protease of high expression includes：Urine swashs Enzyme type activator of plasminogen, legumain protease or matriptase.
11. activable Chimerical receptors as claimed in claim 10, it is characterised in that described pathology Property tissue specific expression or high expression protease be plasma urokinase-type plasminogen activator or Matriptase, it is described can shearing elements be SEQ ID NO:The polypeptide of amino acid sequence shown in 2.
12. activable Chimerical receptors as claimed in claim 9, it is characterised in that described pathology Property the high expression of tissue antigen include：GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20 or ASGPR1.
13. activable Chimerical receptors as claimed in claim 12, it is characterised in that described pathology Property the high expression of tissue antigen be GPC3, described closure elements are the Binding peptides of GC33 antibody； It is preferred that described closure elements are SEQ ID NO:The polypeptide of amino acid sequence shown in 1.
A kind of 14. polynucleotides, its arbitrary described activable Chimerical receptor of coding claim 1-13.
A kind of 15. expression vectors, which includes the activable Chimerical receptor described in coding claim 14 Nucleic acid.
16. a kind of viruses, it is characterised in that described virus includes carrier described in claim 15.
The arbitrary described activable Chimerical receptor of 17. claims 1-13 or encode its nucleic acid or The purposes of the expression vector comprising the nucleic acid or virus, for preparing the Chimerical receptor of targeting pathologic tissue Immune effector cell.
A kind of 18. Chimerical receptor immune effector cells, its transduction have coding claim 1-13 arbitrary described Activable Chimerical receptor nucleic acid, or the expression vector described in claim 15 or claim 16 Described virus；Or the arbitrary described activable Chimerical receptor of its surface expression claim 1-13.
19. Chimerical receptor immune effector cells as claimed in claim 18, it is characterised in that described Immune effector cell includes：T lymphocytes, NK cells or NKT cells, Treg cells.
The purposes of the Chimerical receptor immune effector cell described in 20. claims 18 or 19, for preparing The medicine of targeting pathologic tissue, the antigen that the pathologic tissue high expression Chimerical receptor can be combined.
21. purposes as claimed in claim 20, it is characterised in that described pathologic tissue is tumour, The medicine of described targeting pathologic tissue is the medicine for suppressing tumour.
22. a kind of pharmaceutical compositions, it is characterised in which includes：Described in claim 18 or 19 Chimerical receptor immune effector cell.
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