CN106519037B - Activable Chimerical receptor - Google Patents
Activable Chimerical receptor Download PDFInfo
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- CN106519037B CN106519037B CN201510578444.8A CN201510578444A CN106519037B CN 106519037 B CN106519037 B CN 106519037B CN 201510578444 A CN201510578444 A CN 201510578444A CN 106519037 B CN106519037 B CN 106519037B
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- chimerical receptor
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Classifications
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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
Abstract
The present invention relates to activable Chimerical receptors.Activable Chimerical receptor of the invention only pathologic tissue is specific expressed or highly expressed protease existing under the conditions of can target corresponding antigen, realize the lethal effect of pathologic tissue or cell；And do not play a role under conditions of protease is not present, feasible solution is provided for the problems such as deduction and exemption on-target-off-tumor.
Description
Technical field
The invention belongs to immunotherapy of tumors fields, more particularly it relates to activable Chimerical receptor, preparation
Method and its application.
Background technique
Effect of the immune effector cell in tumor immune response is paid more and more attention.Adopting based on immune effector cell
Property immunization therapy achieves certain effect in Partial tumors, and this kind of immunotherapy method can overcome Antybody therapy
Drawbacks described above, but in still unsatisfactory [Grupp SA, the et al.Adoptive cellular of the curative effect of most of tumours
Therapy.Curr Top Microbiol Immunol., 2011；344:149-72.].In recent years, it is drenched according to cytotoxic T
Bar cell (cytotoxic lymphocyte, CTL) depends on t lymphocyte receptor (T to the identification specificity of target cell
Cell Receptor, TCR) discovery, will be for the scFv and t lymphocyte receptor of the antibody of tumor cell associated antigen
The intracellular signals such as CD3 ζ or Fc ε RI γ activation motif be fused into Chimeric antigen receptor (Chimeric antigen receptor,
CAR；Now also referred to as Chimerical receptor (Chimeric receptor, CR)), and it is passed through into the modes gene such as such as slow-virus infection
Modification is on T lymphocyte surface.This CAR T lymphocyte can be with Major Histocompatibility compound (Major
Histocompatibility Complex, MHC) T lymphocyte is selectively directed to tumour cell by non-limiting way
And specifically kill tumour.CAR T lymphocyte is a new Immunotherapy Strategy in immunotherapy of tumors field
[Schmitz M, et al.Chimeric antigen receptor-engineered T cells for
Immunotherapy of Cancer.J Biomed Biotechnol, 2010, doi:10.1155/2010/956304.].This
Outside, NK cell (the Klingemann H.Challenges of cancer therapy with natural of CAR modification
Killer cells.Cytotherapy.2014Dec 18.pii:S1465-3249 (14) 00791-9) or NKT cell also exist
Good anti-tumor activity (Heczey A1, Liu D1, Tian G2, Courtney AN1, Wei are illustrated in preclinical study
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 combined area, transmembrane region and intracellular signal area.Usual extracellular region includes that can identify
The scFv of tumor associated antigen, transmembrane region use CD8, the equimolecular transmembrane region of CD28, and intracellular signal area uses immunity receptor junket
Propylhomoserin activation motifs (ITAM) CD3 ζ or Fc ε RI γ and costimulatory signal molecule CD28, CD27, CD137, CD134's etc. is intracellular
Signaling zone.
Intracellular signal area only includes that ITAM is first generation CAR T lymphocyte, and wherein Chimeric antigen receptor each section is pressed
Following form connection: scFv-TM-ITAM.This kind of CAR T can excite antitumoral cytotoxic effect, but cell factor
It secretes fewer, and lasting anti-tumor effect [Zhang T.et al.Chimeric NKG2D- cannot be excited in vivo
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.]。
The second generation CAR T lymphocyte then developed joined the intracellular signal of CD28 or CD137 (also known as 4-1BB)
Area, wherein Chimeric antigen receptor each section is connected by following form: scFv-TM-CD28-ITAM or scFv-TM-/CD137-
ITAM.B7/CD28 the or 4-1BBL/CD137 costimulation effect that intracellular signal area occurs causes the continuous proliferation of T lymphocyte,
And it can be improved the level of the T lymphocyte secretion cell factors such as IL-2 and IFN-γ, while improving the survival of CAR T in vivo
Period and antitumous effect [Dotti G.et al.CD28costimulation 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 lymphocyte developed in recent years, wherein Chimeric antigen receptor each section is connected by following form:
ScFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM further improves CAR T depositing in vivo
Period living 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 lymphocyte has tempting prospect in immunotherapy of tumors, high risk also needs to examine
Consider.For example, since certain/kind of normal tissue low expression CAR specific antigen that can be identified may cause CAR T lymphocyte
Damage to the normal tissue of expression antigen.Such as, for the antigen carbonic anhydrase IX expressed on renal cell carcinoma patients tumour cell
(CAIX) it is first to adopt the case and first report cell containing CAR for the treatment of for clinical CAR T lymphocyte
The case of undershooting-effect.There are 2-4 grades of hepatotoxicity wind agitation after repeatedly input CAR T lymphocyte in patient.Analysis reason is hepatic duct
Epithelial cell low expression CAIX, former clinical test are forced to interrupt while eliminating any evaluation [Stoter of Case treatment effect
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 reduce the required threshold value of effector cell's activation,
So that the T lymphocyte of gene modification causes in low-level antigen or without that may also can be activated under conditions of antigen triggering
Being released so that for large amount of cell factor may cause so-called " cytokine storm ".This signal leakage (signal
Leakage it) will lead to cytotoxicity of missing the target, to generate nonspecific tissue damage.For example, using for Her2
Due to table low in normal lung tissue during the three generations CAR clinical treatment one Advanced Colon Cancer patient with liver and Lung metastases
Cause so-called " cytokine storm " pathogenic people's sudden death [Morgan RA., et al.Report of a up to Her2
serious adverse event following the administration of T cells transduced with
A chimeric antigen receptor recognizing Erbb2.Molecular Therapy, 2010,18 (4):
843-851.]。
Therefore, it is necessary to find suitable method, to enable the immune effector cell based on CAR more accurately to act on
Tumour avoids being applied to existing high risk when immunotherapy of tumors.
Summary of the invention
The purpose of the present invention is to provide a kind of activable Chimerical receptor, preparation method and its application.
In the first aspect of the present invention, a kind of activable Chimerical receptor (Activatable Chimeric is provided
Receptor, ACR) comprising:
Chimerical receptor (Chimeric Receptor, CR) can target (in conjunction with) pathologic tissue height in an active state
The antigen of expression；
Closure elements (Blocking element, BE) can inhibit Chimerical receptor and the pathologic tissue height to express
Antigen binding；With
Can shearing elements (Cleavable element, CE), between Chimerical receptor and closure elements.
In a preferred embodiment, the Chimerical receptor includes to be linked in sequence: extracellular antigen binding domain, transmembrane region and
Intracellular signal area；The intracellular signal area is selected from: CD3 ζ, Fc ε RI γ's, CD27, CD28, CD137, CD134, CD40 is intracellular
Signal region sequence or Myd88, or combinations thereof.
In another preferred example, the extracellular antigen binding domain is the specific binding pathologic tissue height expression
Antigen antibody.
In another preferred example, the antibody of the described specific binding highly expressed antigen of pathologic tissue can be with
Be: single-chain antibody (scFV), monoclonal antibody, single domain antibody, Fab segment, Fd segment, Fv segment, 2 segment of F (ab ') and
The antibody of its derivative or other forms.
In another preferred example, the antibody of the specific binding highly expressed antigen of pathologic tissue is selected from
(but being not limited to): single-chain antibody or single domain antibody.
In another preferred example, Chimerical receptor can effectively identify the pathologic group in the case where closure elements are not present
Highly expressed antigen is knitted, closure elements can interfere or compete the knot of Chimerical receptor and the highly expressed antigen of the pathologic tissue
It closes.
In another preferred example, the activable Chimerical receptor is according to the sequence from aminoterminal to c-terminus, successively
Include: closure elements, can shearing elements, Chimerical receptor.
In another preferred example, the Chimerical receptor, closure elements, can further include link peptide between shearing elements.
In another preferred example, the activable Chimerical receptor, wherein containing the first connecting peptides (LP1) and second
Connecting peptides (LP2)；Wherein activable Chimerical receptor successively includes: closing member according to the sequence from aminoterminal to c-terminus
Part, the first connecting peptides, can shearing elements, the second connecting peptides, Chimerical receptor.
In another preferred example, the closure elements are selected from but not limited to: directly in conjunction with the Chimerical receptor
Polypeptide；Or from the polypeptide for spatially hindering the Chimerical receptor and antigen binding.
In another preferred example, the closure elements be 2~100aa (such as 10aa, 20aa, 30aa, 40aa, 50aa,
60aa, 70aa, 80aa)；The preferably polypeptide of 2~40aa.
In another preferred example, it is described can shearing elements be can by pathologic tissue specific expressed or highly expressed egg
The element that white enzyme is sheared, restores or decomposed, the protease is with the highly expressed antigen common location of the pathologic tissue in same
One pathologic tissue.
In another preferred example, the pathologic tissue includes but is not limited to: tumour, autoimmune disease tissue,
The tissue infected by viral (such as inhibition of HIV).
In another preferred example, the pathologic tissue is tumour, and the pathologic tissue is specific expressed or high
The protease of expression includes but is not limited to: plasma urokinase-type plasminogen activator (urokinase-type plasminogen
Activator, uPA), legumain protease or matriptase (MT-SP1).
In another preferred example, the pathologic tissue is specific expressed or highly expressed protease is urokinase type fibre
Plasminogen activator or matriptase, it is described can shearing elements be amino acid sequence shown in SEQ ID NO:2 polypeptide.
In another preferred example, the highly expressed antigen of the pathologic tissue includes but is not limited to: GPC3, EGFR,
HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20 or
ASGPR1。
In another preferred example, the highly expressed antigen of the pathologic tissue is GPC3, and the closure elements are
The combination polypeptide of GC33 antibody；Preferably, the closure elements are the polypeptides of amino acid sequence shown in SEQ ID NO:1.
In another preferred example, when the highly expressed antigen of the pathologic tissue is GPC3, which is
Tumour, comprising: liver cancer, melanoma, clear cell carcinoma of ovary, yolk sac tumor, neuroblastoma.
In another aspect of this invention, a kind of polynucleotides, the coding activable Chimerical receptor are provided.
In another aspect of this invention, a kind of expression vector is provided, it includes the coding activable Chimerical receptors
Nucleic acid.
In another preferred example, the expression vector derives from slow virus plasmid pWPT.
In another aspect of this invention, a kind of virus is provided, the virus includes the carrier.
In another aspect of this invention, provide any activable Chimerical receptor in front or encode its nucleic acid,
Or the purposes of the expression vector or virus comprising the nucleic acid, the Chimerical receptor immunological effect for being used to prepare targeting pathologic tissue are thin
Born of the same parents.
In another aspect of this invention, a kind of Chimerical receptor immune effector cell is provided, transduction has coding front any
The nucleic acid or mentioned-above expression vector of the activable Chimerical receptor or the virus；Or before its surface expression
Any activable Chimerical receptor in face.
In another preferred example, the immune effector cell includes: T lymphocyte, NK cell or NKT cell, Treg
Cell.
In another aspect of this invention, the purposes of the Chimerical receptor immune effector cell is provided, targeting is used to prepare
The drug of pathologic tissue, the pathologic tissue height express the antigen that the Chimerical receptor can combine.
In a preferred embodiment, the pathologic tissue is tumour, and the drug of the targeting pathologic tissue is suppression
The drug of tumour processed.
In another aspect of this invention, a kind of pharmaceutical composition is provided comprising: the immune effect of mentioned-above Chimerical receptor
Answer cell.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
The structural schematic diagram of Fig. 1, activable Chimerical receptor ACR.
Successively according to N-terminal → C-terminal are as follows: Sp: secreting signal peptide, BE: closure elements, LP1: connector 1；CE1: member can be sheared
Part；LP2: connector 2, Ab: antigen binding domain；LP3: connector 3；TM: transmembrane region；SD: intracellular signal area.
The schematic diagram of Fig. 2, GC33-28BBZ-ACR.
According to N-terminal → C-terminal direction, modules are followed successively by CD8 secretion signal, BE (closure elements), LP1, CE in figure
(can shearing elements, the digestion substrate of UP1), LP2, CR (GC33-28BBZ), F2A, eGFP.
The GC33-28BBZ-ACR positive rate of Fig. 3, facs analysis T cell.
GC33-28BBZ-ACR positive rate by the positive rate of GFP as T cell after instruction slow-virus infection.
Fig. 4, in vitro toxicity experiment.Using liver cancer cell lines Huh-7 as target cell, effector cell is in vitro culture 11 days
The T cell of GC33-28BBZ-ACR is expressed, for effect target than being 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 and MTSP1, a low dose group, a high dose group are respectively set
And blank group.Each group is all provided with 5 multiple holes, takes the average value of 5 multiple holes.Detection time is 18h.
Specific embodiment
The present inventor discloses a kind of activable based on Chimeric antigen receptor (CAR) technology by extensive research
Chimerical receptor, the activable Chimerical receptor only pathologic tissue is specific expressed or highly expressed protease existing for item
Corresponding antigen can be targeted under part, realize the lethal effect of pathologic tissue or cell；And the item being not present in protease
It does not play a role under part.The present invention is to avoid on-target-off-tumor's from providing feasible solution.
As used herein, " Chimerical receptor " and " Chimeric antigen receptor " is used interchangeably.
As used in the present invention, " the highly expressed antigen of pathologic tissue " refers to what the Chimerical receptor of activation was targeted
Antigen, the antigen high expression in pathologic tissue or cell.In the present invention, it is somebody's turn to do " the highly expressed antigen of pathologic tissue "
May other than pathologic tissue or cell normal tissue or cell in express.Preferably, being somebody's turn to do, " pathologic tissue is highly expressed
Antigen " is tumor associated antigen, such as selected from (but being 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 specific expressed or highly expressed protease " refers to only in pathology
It is expressed in property tissue or cell, or highly expressed albumen (hydrolysis) enzyme in pathologic tissue or cell, it can be special to its
Property substrate is hydrolyzed.
As used in the present invention, " closure elements " are to refer to block its corresponding antigen of the Chimerical receptor
In conjunction with polypeptide, by directly in conjunction with the Chimerical receptor or from spatially hindering the Chimerical receptor and antigen knot
It the modes such as closes and blocks effect of the Chimerical receptor in conjunction with target site to play.
As used in the present invention, described " can shearing elements " be one more between Chimerical receptor and closure elements
Peptide is the substrate of " pathologic tissue specific expressed or highly expressed protease ", when there are the protease
When, should " can shearing elements " can be sheared, restore or decompose so that closure elements no longer block the Chimerical receptor with
Its corresponding antigen combines.
As used in the present invention, " pathologic tissue " includes but is not limited to: tumour, autoimmune disease group
It knits, the tissue infected by viral (such as inhibition of HIV).
In the present invention, the pathologic tissue can be the intracorporal various adverse tissues for being unfavorable for health of machine or disease
Stove, it is necessary to removal in slave body.The pathologic target tissue includes tumour.Any tumour known in the art may include
In the present invention, as long as the tumour can express the tumor associated antigen of low expression in normal tissue.For example, the tumour packet
It includes (but being not limited to): liver cancer, lung cancer, glioma, breast cancer, gastric cancer, prostate cancer, brain tumor, oophoroma, bone tumour, colon
Cancer, thyroid tumors, mediastinal tumor, intestinal tumor, kidney neoplasms, adrenal tumor, tumor of bladder, orchioncus, malignant lymphoma,
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, gallbladder cancer, malignant fibrous histiocytoma.For example, the tumor associated antigen
Including but not limited to: GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2,
EpCAM, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4 EGFR, CD19, CD33, IL13R, LMP1,
PLAC1, NY-ESO-1, MAGE4, MUC1, MUC16, LeY, CEA, CAIX (carbonic anhydrase IX), CD123.
Term " Chimerical receptor epidemic disease effector cell " is it is known in the art that it is to utilize genetic modification technological expression antigen
The immune effector cell of (such as tumour antigen) specific chimeric receptor, can targeting performance lethal effect.The immunological effect
Cell is for example including T cell, NK cell, NKT cell, regulatory T cells (Regulatory cell, abbreviation Treg).Conventional
The method of preparation " Chimerical receptor immune effector cell " is known to the skilled in the art, including it is allowed to express costimulation intracellular
Cellular elements intracellular domain, such as CD28 (being preferably comprised CD28a, CD28b), CD137, CD27, CD3 ζ is (preferably
CD3 ζ intracellular domain), CD8, CD19, CD134, CD20, one of Fc ε RI γ or a variety of.Pass through they and respective ligand knot
It closes, activates the second signal of immune effector cell, enhance the proliferative capacity of immunocyte and the secreting function of cell factor, extend
The time-to-live of activating immune cell.
It is fewer and fewer in view of the absolutely special target spot of tumour, therefore immunocyte (such as CAR T of most of CAR modification
Cell) antigen (such as CD19, CD20, Her2, EGFR, EpCAM etc.) that is directed to more or less expresses in the normal tissue, therefore
There is the side effect of on-target-off-tumor with being difficult to avoid that.How this On-target-off- is mitigated or reduced
The effect of tumor becomes extremely important.The present inventor widely compares the microenvironment of tumor tissues and the micro-loop of normal tissue
The difference in border is high in tumor tissues based on some proteolytic enzymes such as uPA, MT-sp1, Legumain protease for the first time
Expression and do not expressed in normal tissue or the characteristics of low expression, using the specificity of these hydrolases, Chimerical receptor be transformed and has repaired
The immunocyte of decorations, immunocyte obtained only could more effectively play antitumor function after these hydrolyze enzyme effect
Can, so as to effectively improve the safety of immunocyte.
Therefore, the present invention provide a kind of activable Chimerical receptor (Activatable Chimeric Receptor,
ACR), it includes Chimerical receptor (Chimeric Receptor, CR), the element (Cleavable element, CE) that can be sheared
And closure elements (Blocking element, BE) connect, and can be attached by connecting peptides between three.Wherein CE
Antigen is sheared, restores, after optical resolution or other modification, and ACRs can show the conformation of activation, and CR is more held
Easily and targeted integration.
As preferred embodiment of the invention, a kind of structural schematic diagram of activable Chimerical receptor ACR of the invention is shown in figure
1.It from left to right arranges, i.e., rearrange from the N-terminal of protein to C-terminal a transmembrane receptor can activate to obtain Chimerical receptor
The schematic diagram of ACR.Under unactivated state, the area Hui Yu Ab BE is combined, or spatially hinders the combination of Ab and target antigen；And one
Denier CE1 is sheared by protease etc., then Ab can be in conjunction with target antigen.
The closure elements can be it is any can be by directly in conjunction with the Chimerical receptor or from spatially hindering
The modes such as the Chimerical receptor and antigen binding block the polypeptide of effect of the Chimerical receptor in conjunction with target site to play.It can be with
The closure elements are selected according to the type of the antigen binding domain of Chimerical receptor.For example, the antigen binding domain is a kind of anti-
Body, then the closure elements can be the combination polypeptide of the antibody.In a specific embodiment of the present invention, the antigen knot of Chimerical receptor
The antibody GC33 that area uses resisting GPC 3 is closed, and applies it that polypeptide (NSQQATPKDNEISTFH) is combined to be used as closure elements.
It is described can shearing elements be can by pathologic tissue specific expressed or highly expressed proteolytic cleavage, reduction
Or the substrate decomposed.It can be according to corresponding indication, to select wherein specific expressed or highly expressed protease, bottom
Object can be used as can shearing elements.The expression specificity of the protease is higher, then preferred.Of the invention preferred
In mode, indication is tumour, and there are different, proteolysis therein from the microenvironment of normal tissue for the microenvironment of tumor tissues
Enzyme such as uPA, MT-sp1, Legumain protease etc. the high expression in tumor tissues, and do not expressed or low table in normal tissue
It reaches, therefore the present inventor utilizes the specificity of these hydrolases, the immunocyte of Chimerical receptor modification, the immunocyte is transformed
Anti-tumor function could be more effectively only played after these hydrolyze enzyme effect, to effectively improve the immune of CAR modification
The safety of cell.In a specific embodiment of the present invention, made using the substrate polypeptide LSGRSDNH of uPA and MT-SP1 protease
For can shearing elements.
The Chimerical receptor, can may also include link peptide between shearing elements at closure elements.The link peptide does not have
Have special limitation, can be it is any be capable of providing the Chimerical receptor, closure elements, can between shearing elements it is flexible even
It connects, does not influence any polypeptide of the function of each element itself.Preferably, the connexon includes 2-40 amino acid；Preferably
Ground is 3-30 amino acid, such as 5,8,10,15,20,25 amino acid.
The present invention also includes the nucleic acid for encoding the activable Chimerical receptor.The invention further relates to above-mentioned polynucleotides
Variant, coding have the polypeptide of identical amino acid sequence or the segment of polypeptide, analogs and derivatives with the present invention.
The present invention also provides the carriers of the nucleic acid comprising above-mentioned activable Chimerical receptor.In a specific embodiment
In, the carrier that the present invention uses is a kind of slow virus plasmid vector pWPT.It should be understood that other expression vectors are also available.
The invention also includes the viruses comprising above-mentioned carrier.Virus of the invention includes the infectious disease of tool after packaging
Poison also includes the virus to be packaged for being packaged as having infectious viral institute essential component.Known in the art its
Its viral and its corresponding plasmid vector into immune effector cell that can be used for transduceing foreign gene can also be used for the present invention.
The present invention also provides the immune effector cell of gene modification, being transduceed, there have to be activable chimeric described in coding
The nucleic acid of receptor is had above-mentioned comprising the recombinant plasmid containing the nucleic acid, or the virus comprising the plasmid by transduction.Ability
The nucleic acid transduction method of domain routine may be used to the present invention including non-viral and viral transduction method.Based on non-viral
Transduction method includes electroporation and transposons method.The Nucleofector nuclear transfection instrument of recent Amaxa company research and development can be straight
It connects and foreign gene is imported into the high efficiency transduction that nucleus obtains target gene.In addition, being based on sleeping beauty transposon stand (Sleeping
Beauty system) or the more common electroporation of transduction efficiency of the Transposon Systems such as PiggyBac transposons improve a lot, will
[Davies JK., et has been reported in nucleofector transfection instrument and the system combined application of sleeping beauty transposon stand
al.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.], this method not only transduction efficiency with higher but also can be realized the site-directed integration of target gene.
In one embodiment of the invention, realize that Chimerical receptor (being activable Chimerical receptor in the present invention) gene modification is exempted from
The transduction method of epidemic disease effector cell is based on virus such as retrovirus or the transduction method of slow virus.This method has transduction effect
Rate is high, and foreign gene can stablize expression, and can shorten the time that in vitro culture immune effector cell reaches clinical number of stages
The advantages that.On the transgenosis immune effector cell surface, the nucleic acid of transduction is by transcription, accurate translation on its surface.By right
The tumour cell of a variety of different cultures carries out cell in vitro poison it is demonstrated experimentally that immune effector cell of the invention has height special
Anisotropic tumor cytotoxicity effect (also known as cytotoxicity).Therefore the nucleic acid of encoding chimera receptor protein of the invention, include
The plasmid of the nucleic acid, virus and transduction comprising the plasmid have above-mentioned nucleic acid, the transgenosis immune effector cell of plasmid or virus
It 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；The cell factor
Including but not limited to: IL-12, IL-15 or IL-21 etc..These cell factors have immunological regulation or antitumor activity, can increase
The function of strong effector T cell and the NK cell of activation, or directly play antitumor action.Therefore, those skilled in the art can be with
Understand, the utilization of these cell factors facilitates the immunocyte and preferably plays a role.
Immunocyte of the present invention can also express another Chimerical receptor other than above-mentioned Chimerical receptor, should
Receptor does not contain CD3 ζ, but the intracellular signal structural domain containing CD28, the intracellular signal structural domain of CD137 or the group of the two
It closes.
Immunocyte of the present invention can also express chemokine receptors；The chemokine receptors includes but not
It is limited to CCR2.It will be understood by those skilled in the art that the CCR2 chemokine receptors can make intracorporal CCR2 therewith
Competitive binding, for blocking the transfer of tumour to be advantageous.
Immunocyte of the present invention can also express the siRNA that can reduce PD-1 expression or the egg for blocking PD-L1
It is white.It will be understood by those skilled in the art that the competitive interaction for blocking PD-L1 and its receptor PD-1, is conducive to restore anti-
Tumor T cells reaction, to inhibit tumour growth.
Immunocyte of the present invention can also express safety switch；Preferably, the safety switch includes:
ICaspase-9, Truancated EGFR or RQR8.
Chimerical receptor immune effector cell of the invention can be applied to preparation pharmaceutical composition or diagnostic reagent.Described
Composition also may include pharmaceutically acceptable carrier in addition to including a effective amount of immunocyte.Term " can pharmaceutically connect
Receive " refer to when biomolecule ontology and composition suitably give animal or people, they will not be generated it is unfavorable, allergy or its
Its adverse reaction.
The specific example that can be used as pharmaceutically acceptable carrier or some substances of its component is carbohydrate, such as lactose, Portugal
Grape sugar and sucrose；Starch, such as cornstarch and potato starch；Cellulose and its derivates, as sodium carboxymethylcellulose, ethyl are fine
Dimension element and methylcellulose；Tragacanth powder；Malt；Gelatin；Talcum；Solid lubricant, such as stearic acid and magnesium stearate；Sulphur
Sour calcium；Vegetable oil, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil；Polyalcohol, such as propylene glycol, sweet
Oil, D-sorbite, mannitol and polyethylene glycol；Alginic acid；Emulsifier, such asWetting agent, such as lauryl sulfate
Sodium；Colorant；Flavoring agent；Tablet agent, stabilizer；Antioxidant；Preservative；Apirogen water；Isotonic salting liquid and phosphate are slow
Fliud flushing etc..
Various dosage forms can be made in composition of the invention as needed, and can be by doctor according to patient category, age, weight
Substantially the factors such as disease condition, administration mode determine that the dosage beneficial to patient is administered.Administration mode can for example adopt
With injection or other therapeutic modalities.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
The building of embodiment 1, resisting GPC 3 third generation Chimeric antigen receptor
Utilize anti-phosphatidylinositols proteoglycan -3 (GPC3) third generation Chimeric antigen receptor (Chimeric constructed before
Antigen receptor, CAR), single-chain antibody used in the CAR is originated from GC33 antibody, and the building of the CAR T is referring to application
Number in CN201310164725.X, GPC3-28BBZ is applied in the present embodiment, also referred to as GC33-28BBZ.Wherein institute
The amino acid sequence for the single-chain antibody stated (VL as shown in SEQ ID NO:13GPC3-(G4S)3-VHGPC3)。
The present inventor is by the combination polypeptide (NSQQATPKDNEISTFH (SEQ ID NO:1)) of GC33 antibody as BE member
Part, and the substrate polypeptide LSGRSDNH (SEQ ID NO:2) of uPA and MT-SP1 protease is used as CE element.Additionally by origin
From the ribosomal skip sequence (ribosomal of foot and mouth disease virus (food-and-mouth disease virus, FMDV)
Skipping sequence 2A) eGFP is connected to the C-terminal of ACR by (abbreviation F2A), realize the coexpression of eGFP and ACR, thus
By the expression of eGFP come the expression of side light ACR.
(1) acquisition of the segment of Sp+BE+LP1+CE+LP2
The primer of design such as table 1.
Table 1
Wherein the segment of Sp+BE+LP1+CE+LP2 uses the primer such as table 1, is obtained by Overlap PCR, expands item
Part are as follows:
Initial denaturation: 94 DEG C, 4min；
Following 25 circulations: denaturation: 94 DEG C, 30s,
Annealing: 50 DEG C, 30s,
Extend: 68 DEG C, 20s；
Then, 10min is re-extended for 68 DEG C.
Glue recycling is then carried out to pcr amplification product using conventional method, Sp+BE+LP1+CE+LP2 is obtained and is connected with each other
DNA fragmentation.
(2) acquisition of LP2-GC33-28BBZ segment
The primer of design such as 2.
Table 2
Taking GC33-28BBZ plasmid 1 microlitre of (plasmid is referring to CN201310164725.X patent) (100ng) is template, point
Not using GPC3scfv-F, 3z-F2A-R as upstream and downstream primer, 50 microlitres of systems carry out PCR amplification, the PCR product of acquisition into
Row gel extraction, to obtain LP2-GC33-28BBZ segment.
(3) acquisition of F2A-EGFP
The primer of design such as 3.
Table 3
1 microlitre of GC33-28BBZ plasmid (100ng) is taken for template, respectively using F2A-EGFP-F, pwpt-EGFP-R as upper
Downstream primer carries out PCR amplification, and the PCR product of acquisition carries out gel extraction, to obtain F2A-EGFP.
(4) acquisition of slow virus plasmid pWPT-GC33-28BBZ-ACR
The three segments that above-mentioned (1)~(3) are prepared carry out overlap PCR, specifically three segments, according to
Equimolar ratio is mixed, and by above-mentioned steps (1) the progress overlap PCR, upstream and downstream primer is CD8sp-F and EGFP-
R.The PCR product of acquisition carries out gel extraction.
Using restriction enzyme to the corresponding site for being connected into slow virus plasmid pWPT carrier after MluI and SalI double digestion
It is interior.
The recombinant plasmid transformed Escherichia coli Top10 competent cell of acquisition, culture 12h or so carry out choosing clone's verifying.
Selection includes that the clone of recombinant plasmid send sequencing.Selection sequence correctly clones the viral preparation of progress and packaging.
The carrier of acquisition is named as pWPT-GC33-28BBZ-ACR.
The preparation of embodiment 3, CAR-T
1, 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
In culture dish, 37 DEG C, 5%CO2Overnight incubation prepares for transfecting.Culture medium is (public purchased from gibco containing 10% fetal calf serum
Department) DMEM (be purchased from gibco company).
The step of transfection, is as follows:
(1) by 10 μ g target gene plasmid pWPT-GC33-28BBZ-ACR, respectively with 6.5 μ g packaging plasmid PAX2: and
3.5 μ g envelope plasmid pMD2.G, dissolve in the serum-free DMEM culture solution of 800 μ L, mix.
(2) by 60 μ l PEI, (polyethyleneimine is purchased from Polysciences company, is made into the work of 1 μ g/ μ L concentration
Liquid), it is added in the serum-free DMEM culture solution of the above-mentioned 800 μ L with plasmid, is vortexed and mixes, be stored at room temperature and be incubated for 25min.
(3) 800 μ L of transfection composite is added in HEK-293T cell to be transfected, after 6-8h hours, with 10%FBS's
DMEM training base changes liquid to the 293T cell of transfection.
(4) after transfection about for 24 hours, transfection efficiency (being in the cell proportion of green fluorescence) is observed.After transfecting 72h, make
Virus is collected by filtration with 0.45 μm of filter (being purchased from Millipore company), (Beckman Optima L-100XP is super for ultracentrifugation
Fast centrifuge 28000rpm, 4 DEG C are centrifuged 2 hours), concentrating virus.The AIM-V culture of centrifugation gained 1/30 stoste volume of precipitating
Liquid (being purchased from Gibco company) is resuspended, and is frozen with the packing of 100 μ L/ pipes in -80 DEG C, to infect T lymphocyte.Meanwhile it will
The concentrating virus of acquisition is titrated.
2, the infection of GC33-28BBZ-ACR
Human peripheral blood single nucleus cell (Shanghai City Blood Center is obtained by density-gradient centrifugation method by healthy human peripheral blood
It provides).Peripheral blood mononuclear cells passes through CD4+/CD8+ cell magnetic bead (being purchased from Stem Cell Technologies) negativity point
The primary human T lymphocyte of choosing method acquisition CD4+ and the CD8+ positive.T cell after sorting carries out Flow cytometry, and its is pure
Degree, is advisable with positive rate >=95% of target cell and carries out next step operation.With 1 × 106It is thin that AIM-V lymph is added in/mL density
Born of the same parents' liquid medium (being purchased from Gibco company, contain 2% people AB serum) is cultivated and with cell: magnetic bead ratio is that 1:1 is added simultaneously
The magnetic bead (Invitrogen company) of AntiCD3 McAb and CD28 antibody and the recombinant human il-2 of final concentration 300U/mL are coated with (purchased from upper
Hai Hua neoformation Hitek Ltd) stimulation culture is for 24 hours.Before infection, by CD4+CD8+T lymphocyte according to the ratio of 1:1
Mixing, is then mixed above-mentioned viral concentration liquid with T cell to be infected with MOI ≈ 5-10, while final concentration of 6 μ g/mL is added
Polybrene.Metainfective cell, changes liquid every other day, changes without virulent training base.Every other day use 5 × 105/ mL's
Density is passed on, while the recombinant human il-2 of final concentration 300U/mL is added in lymphocyte culture medium.
Metainfective primary T cells are examined at the 9-11 days (i.e. the first round expands end cycle) by flow cytometry
The expression for surveying Chimeric antigen receptor GC33-28BBZ-ACR detects eGFP's since eGFP and GC33-28BBZ-ACR is co-expressed
Positive cell is the positive cell for expressing Chimeric antigen receptor, and the T lymphocyte to be uninfected by is as negative control.
As a result positive as the GC33-28BBZ-ACR of T cell after instruction slow-virus infection by the positive rate of GFP such as Fig. 3
Property rate, it is seen that positive rate 61.0%.
Embodiment 4, external source recombination uPA promote the activation of BE-GPC3-CAR T
The material that in vitro toxicity experiment uses is as follows:
Using liver cancer cell lines Huh-7 as target cell, effector cell is to express GC33-28BBZ- in vitro culture 11 days
The T cell (positive rate 61%) of ACR, effect target is than being respectively 3:1,1:1 and 1:3, and target cell numbers are 10000/hole, according to not
With effect target than corresponding effector cell.It is separately added into uPA and MTSP1, a low dose group (uPA:0.08 μ g/mL is respectively set；MT-
SP1:0.04 μ g/mL), a high dose group (uPA:0.4 μ g/mL；MT-SP1:0.2 μ g/mL) and blank group (uPA:0 μ g/mL；
MT-SP1:0 μ g/mL).Each group is all provided with 5 multiple holes, takes the average 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 cell+various concentration enzyme,
Control group 1: target cell maximum discharges the enzyme of LDH+ various concentration,
Control group 2: the enzyme of the spontaneous release LDH+ various concentration of target cell,
Control group 3: the enzyme of the spontaneous release LDH+ various concentration of effector cell.
Detection method: it is examined using 96 non-radioactive cell toxicity detection kit (Promega company) of CytoTox
It surveys.
Cytotoxicity calculation formula are as follows:
From fig. 4, it can be seen that GC33-28BBZ-ACR T cell (T cell for having infected GC33-28BBZ-ACR) is anti-swollen
Tumor activity is lower, but can effectively activate under the action of uPA MT-SP1, thus killing tumor cell.
The above results show that design of the invention is reasonable, that is, T can be made thin really by the effect of closure elements
The lethal effect of born of the same parents weakens；But if there is the corresponding protease such as the uPA of sufficient amount or MT-SP1 in tumor by local environment
Effect ACR can obtain activation and killing tumor cell to play local antitumoral activity reduces the damage of normal tissue
Wound.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (17)
1. a kind of activable Chimerical receptor comprising:
Chimerical receptor targets the highly expressed antigen of pathologic tissue in an active state；The Chimerical receptor includes extracellular
Antigen binding domain, be single-chain antibody；The highly expressed antigen of the pathologic tissue is GPC3；The single-chain antibody
Amino acid sequence is as shown in SEQ ID NO:13；
Closure elements inhibit Chimerical receptor and the highly expressed antigen binding of the pathologic tissue；The closure elements are
The combination polypeptide of GC33 antibody is the polypeptide of amino acid sequence shown in SEQ ID NO:1；With
Can shearing elements, between Chimerical receptor and closure elements.
2. activable Chimerical receptor as described in claim 1, which is characterized in that the Chimerical receptor includes to be linked in sequence
: extracellular antigen binding domain, transmembrane region and intracellular signal area；The intracellular signal area is selected from: CD3 ζ, Fc ε RI γ,
The intracellular signal region sequence or Myd88 of CD27, CD28, CD137, CD134, CD40, or combinations thereof.
3. activable Chimerical receptor as described in claim 1, which is characterized in that the activable Chimerical receptor according to
Sequence from aminoterminal to c-terminus successively includes: closure elements, can shearing elements, Chimerical receptor.
4. activable Chimerical receptor as described in claim 1, which is characterized in that the Chimerical receptor, closure elements, can
It further include link peptide between shearing elements.
5. activable Chimerical receptor as described in claim 1, which is characterized in that it is described can shearing elements be by pathologic
The element that tissue specific expression or highly expressed protease are sheared, restore or decomposed, the protease and the pathologic
Organize highly expressed antigen common location in same pathologic tissue.
6. activable Chimerical receptor as claimed in claim 5, which is characterized in that the pathologic tissue includes: tumour.
7. activable Chimerical receptor as claimed in claim 6, which is characterized in that the pathologic tissue is specific expressed
Or highly expressed protease includes: plasma urokinase-type plasminogen activator, legumain protease or matriptase.
8. activable Chimerical receptor as claimed in claim 7, which is characterized in that the pathologic tissue is specific expressed
Or highly expressed protease is plasma urokinase-type plasminogen activator or matriptase, it is described can shearing elements be SEQ ID
The polypeptide of amino acid sequence shown in NO:2.
9. a kind of polynucleotides, any activable Chimerical receptor of coding claim 1-8.
10. a kind of expression vector, it includes polynucleotides as claimed in claim 9.
11. a kind of virus, which is characterized in that the virus includes expression vector described in any one of claim 10.
12. any activable Chimerical receptor of claim 1-8 encodes its nucleic acid or the table comprising the nucleic acid
Up to carrier or the purposes of virus, it is used to prepare the Chimerical receptor immune effector cell of targeting pathologic tissue.
13. a kind of Chimerical receptor immune effector cell, transduction has coding claim 1-8 any described activable chimeric
Virus described in the nucleic acid of receptor or expression vector described in any one of claim 10 or claim 11；Or its surface expression right
It is required that any activable Chimerical receptor of 1-8.
14. Chimerical receptor immune effector cell as claimed in claim 13, which is characterized in that the immune effector cell packet
It includes: T lymphocyte, NK cell or NKT cell, Treg cell.
15. the purposes of Chimerical receptor immune effector cell described in claim 13 or 14, is used to prepare targeting pathologic tissue
Drug, which expresses the antigen that the Chimerical receptor can combine, and the antigen is GPC3.
16. purposes as claimed in claim 15, which is characterized in that the pathologic tissue is tumour, the targeting disease
The drug of rationality tissue is the drug for inhibiting tumour.
17. a kind of pharmaceutical composition, characterized in that it comprises: Chimerical receptor immunological effect described in claim 13 or 14
Cell.
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| BR122023022856A2 (en) * | 2014-01-31 | 2023-12-12 | Cytomx Therapeutics, Inc. | ISOLATED POLYPEPTIDE COMPRISING MATRIPTASE AND PLASMINOGEN ACTIVATOR U SUBSTRATES AND OTHER CLEAABLE MOTIONS, PHARMACEUTICAL COMPOSITION COMPRISING SAID POLYPEPTIDE, AS WELL AS METHODS FOR PRODUCING AND MANUFACTURING AN ISOLATED POLYPEPTIDE COMPRISING A CLEAABLE MOTION AND USE AND THERAPEUTICLY EFFECTIVE AMOUNT OF SAID PHARMACEUTICAL COMPOSITION |
| CA2986604A1 (en) * | 2015-06-26 | 2016-12-29 | University Of Southern California | Masking chimeric antigen receptor t cells for tumor-specific activation |
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| CN106519037A (en) | 2017-03-22 |
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