CN107226865A - A kind of single-chain antibody and its application in oncotherapy - Google Patents
A kind of single-chain antibody and its application in oncotherapy Download PDFInfo
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- CN107226865A CN107226865A CN201610176685.4A CN201610176685A CN107226865A CN 107226865 A CN107226865 A CN 107226865A CN 201610176685 A CN201610176685 A CN 201610176685A CN 107226865 A CN107226865 A CN 107226865A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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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
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2510/00—Genetically modified cells
Abstract
A kind of application the invention provides single-chain antibody and its in oncotherapy.Specifically, the invention provides a kind of single-chain antibody, the fusion protein containing the single-chain antibody, Chimeric antigen receptor and its related application containing the single-chain antibody.The amino acid sequence of the single-chain antibody such as SEQ ID NO.:Shown in 2, the T cell using the modification of the Chimeric antigen receptor containing the single-chain antibody of the present invention has good fragmentation effect to tumour cell, and toxicity of missing the target is low, can be used for the treatment of entity tumor.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to a kind of single-chain antibody and its in oncotherapy
Application.
Background technology
In the world, a kind of new adoptive cellular immunotherapy technology is a dark horse, here it is application is chimeric
The research of the T cell adoptive ground magnetic target therapy tumour of antigen receptor genetic modification, the research is clinically
Achieve the progress of highly significant.The most representational is using CD19 as the CAR-T cell therapies CD19 targetted
Basis and the clinical research of positive B cell leukemia and lymthoma.June etc. is published in NEJM report
Display：Using CD19CAR-T cell therapies are refractory, Recurrent Acute leukemic lymphoblastoid patient obtains notable
Effect, complete remission rate is up to more than 90%.
At present, global at least 20 Duo Ge research groups are carrying out this clinical treatment research.But, in reality
In terms of the treatment of body tumour, CAR-T cells show limited therapeutic effect.Mainly there is following reason：
(1) target antigen is expressed in the normal tissue, and toxicity of missing the target is the main barrier of CAR-T cell therapy entity tumors
Hinder.(2) because the heterogeneity of tumour makes CAR-T to produce of short duration tumor regression, because last target resists
Former negative tumour cell is by dominance.(3) most of all, by the immunosupress of tumor cell secretion
Cell factor is with inhibitory cells such as regulatory T cells and tumor-associated macrophages near tumor cells shape
Into immunosupress microenvironment significantly suppress the abilities of going back to the nest of CAR-T cells.
Therefore, this area can be applied to the CAR-T cell therapy sides for the treatment of of solid tumor in the urgent need to developing
Method.
The content of the invention
A kind of application it is an object of the invention to provide single-chain antibody and its in oncotherapy.
In the first aspect of the present invention, there is provided a kind of single-chain antibody, the amino acid sequence such as SEQ of the antibody
ID NO.:Shown in 2.
In the second aspect of the present invention there is provided a kind of fusion protein, this hair is contained in described fusion protein
Single-chain antibody described in bright first aspect as fusion protein composed component.
In another preference, described fusion protein is also containing the composed component being selected from the group：
(a) constant region of antibody；
(b) CD28 membrane spaning domain；
(c) CD137 elements (i.e. 4-1BB)；
(d) CD3 ζ intracellular signal domain.
In another preference, the membrane spaning domain that described membrane spaning domain is CD28.
In another preference, described fusion protein has signal peptide element in its N-terminal.
In another preference, described fusion protein antibody constant region and CD28 membrane spaning domain it
Between be additionally provided with the hinge area of antibody.
In another preference, the hinge area of described antibody is IgD antibody hinge region.
In another preference, described fusion protein is Chimeric antigen receptor.
In another preference, the Chimeric antigen receptor is from N-terminal to C-terminal, successively with elements below：
The constant region of single-chain antibody-antibody described in optional signal peptide element-first aspect present invention-
CD28 membrane spaning domain-CD137 element (4-1BB)-CD3 ζ intracellular signal domain.
In the third aspect of the present invention there is provided a kind of Chimeric antigen receptor (CAR), the chimeric antigen by
The ectodomain of body is amino acid sequence such as SEQ ID NO.:Single-chain antibody shown in 2.
In another preference, described Chimeric antigen receptor also contains with the hinge of antibody constant region, antibody
The structure that the intracellular signal domain in area, CD28 transmembrane regions and CD137 and CD3 ζ is in series is signal transduction
Domain.
In another preference, described Chimeric antigen receptor is from N-terminal to C-terminal, successively with elements below：
The constant region of single-chain antibody-antibody described in optional signal peptide element-first aspect present invention-
CD28 membrane spaning domain-CD137 element (4-1BB)-CD3 ζ intracellular signal domain.
In the fourth aspect of the present invention, there is provided a kind of polynucleotides, the described polynucleotide encoding present invention
Single-chain antibody described in first aspect or fusion protein or third aspect present invention containing the single-chain antibody
Described Chimeric antigen receptor.
In another preference, the sequence such as SEQ ID NO. of the polynucleotides:Shown in 1.
In the fifth aspect of the present invention there is provided a kind of carrier, described carrier contains in fourth aspect present invention
Described polynucleotides.
In another preference, the carrier is slow virus carrier.
In the sixth aspect of the present invention there is provided a kind of cell, contain fifth aspect present invention in described cell
Described in carrier or chromosome in be integrated with polynucleotides described in the fourth aspect present invention of external source.
In another preference, described cell is T cell (CAR-T cells).
In the seventh aspect of the present invention there is provided a kind of pharmaceutical composition, the composition contains (a) pharmaceutically
Described in single-chain antibody, second aspect of the present invention described in acceptable carrier and (b) first aspect present invention
Fusion protein, the Chimeric antigen receptor described in third aspect present invention, the core described in fourth aspect present invention
Cell described in carrier, sixth aspect present invention or its combination described in acid molecule, fifth aspect present invention.
In the eighth aspect of the present invention, there is provided the single-chain antibody described in a kind of first aspect present invention, sheet
The Chimeric antigen receptor described in fusion protein, third aspect present invention, the present invention described in invention second aspect
The carrier described in nucleic acid molecules, fifth aspect present invention described in fourth aspect, described in sixth aspect present invention
Cell purposes, for prepare treatment MUC1 positive tumors or suppress MUC1 positive tumor cells medicine.
In another preference, described tumour is selected from the group：Cancer of the esophagus, stomach cancer, breast cancer, oophoroma,
Carcinoma of urinary bladder, adenocarcinoma of seminal vesicle, lung cancer, three cloudy breast cancer or its combination.
In the ninth aspect of the present invention, there is provided a kind of method that external non-therapeutic suppresses tumour cell, bag
Include step：Under conditions of the cell described in sixth aspect present invention is present, tumour cell is cultivated.
In another preference, the tumour cell is MUC1 positive cells.
In another preference, the tumour cell includes T47D cells.
In the tenth aspect of the present invention there is provided a kind of method for treating disease, including to pair for needing to treat
Fusion protein, sheet as described in using the single-chain antibody second aspect of the present invention described in first aspect present invention
The nucleic acid molecules described in Chimeric antigen receptor, fourth aspect present invention described in the invention third aspect, the present invention
Described in carrier, the cell described in sixth aspect present invention or seventh aspect present invention described in 5th aspect
Pharmaceutical composition.
In another preference, described single-chain antibody can be combined with MUC1 positive tumor cells.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and (such as implementation below
Example) in specifically describe each technical characteristic between can be combined with each other, so as to constitute new or preferred skill
Art scheme.As space is limited, no longer tire out one by one herein and state.
Brief description of the drawings
Fig. 1 shows the comparison knot of p-SM3 single-chain antibodies amino acid sequence and SM3 single-chain antibody amino acid sequences
Really.In figure, red amino acid represents the amino acid of mutation.
Fig. 2 shows Chimeric antigen receptor (CAR) structural representation.Wherein, Fig. 2A and Fig. 2 B are shown respectively
Include the structure of MUC1 single-chain antibodies B Chimeric antigen receptor after original MUC1 antibody As and mutation.
Fig. 3 shows Mortaility results of the MUC1-CAR-T cells to MUC1 positive tumor cells T47D.Wherein,
Fig. 3 A show untransfected CAR Mortaility results of the T cell to T47D, and Fig. 3 B show what is transfected with empty carrier
T shows Mortaility results of the A-CAR-T to T47D to T47D fragmentation effect, Fig. 3 C, and Fig. 3 D are shown
Mortaility results of the MUC1-CAR-T cells to T47D.
Embodiment
The present inventor is surprised to find that a kind of single-chain antibody and its swollen first by extensively and in depth studying
Application in knurl treatment.Specifically, the invention provides a kind of single-chain antibody, melting containing the single-chain antibody
Hop protein, Chimeric antigen receptor and its related application containing the single-chain antibody.Experiment shows, using this
The T cell of the modification of the Chimeric antigen receptor containing the single-chain antibody of invention has to tumour cell kills well
Hinder effect, toxicity of missing the target is low, can be used for the treatment of entity tumor.
Term
As used herein, term " single-chain antibody of the invention ", " MUC1 single-chain antibodies of the present invention ", " MUC1
Single-chain antibody B " refers to sequence such as SEQ ID NO.:Single-chain antibody shown in 2.
MUC1 molecules
In order to reduce the toxicity of missing the target of CAR-T cells as far as possible, the selection of tumor targets is particularly important.Grind
It is a good tumor targets to study carefully discovery MUC1 molecules.Mainly there is following reason：First, in up to 80% people
In the tumor tissues of class tumour, MUC1 occurs unconventionality expression, and MUC1 is in the cancer of the esophagus, stomach cancer, breast cancer,
There is different degrees of unconventionality expression in oophoroma, the kinds of tumors tissue such as carcinoma of urinary bladder.2nd, in tumour cell
Upper MUC1 expression is in nonpolar distribution；3rd, because glycosyl transferase activity increases and causes MUC1 to glycosylate
Not exclusively, exposure associated epitope, the novel targets treated as oncobiology.
Chimeric antigen receptor
The invention provides the inosculating antibody for including extracellular domain, membrane spaning domain and intracellular domain
Original receptor (CAR), wherein extracellular domain contain the single-chain antibody of the present invention as target-specific binding member
Part.
Ectodomain includes：Target-specific binding members (also referred to as antigen-binding domains).It is intracellular
Domain includes costimulatory signal conducting region and ζ chain parts.Costimulatory signal conducting region refers to including costimulation point
A part for the intracellular domain of son.Costimulatory molecules is lymphocyte to required for the effective response of antigen
Cell surface molecule, rather than antigen receptor or their part.
Between CAR ectodomain and membrane spaning domain, or the cytoplasmic domain in CAR and transmembrane structure
Between domain, joint may be incorporated into.As used herein, term " joint " is often referred to play membrane spaning domain
It is connected to the ectodomain of polypeptide chain or any oligopeptides or polypeptide of cytoplasmic domain effect.Joint may include
0-300 amino acid, most preferably preferably 2 to 100 amino acid and 3 to 50 amino acid.
In of the invention one preferably embodiment, the invention provides transformed by genetic engineering with table
Up to CAR cell (for example, T cell), it shows significant antitumor property.The CAR of the present invention can be with
Including ectodomain, the ectodomain, which has, is fused to T cell antigen receptor complex ζ chain (examples
Such as, CD3 ζ) Cellular Signaling Transduction Mediated domain antigen-binding domains.The CAR of the present invention is when in NKT
When being expressed in cell, antigen recognizing can be changed based on antigen-binding specificity.
In one embodiment, CAR of the invention includes including signal specific conducting structure domain of the present invention
(CD8 hinge areas and transmembrane region, CD137 and CD3 ζ intracellular signal domain are in series).With other
The CAR of mode is compared, and the signal transduction domain containing single-chain antibody of the present invention significantly increases antitumor work
The internal persistence of property and CART cells.
Hinge region and transmembrane region
For hinge region and transmembrane region (membrane spaning domain), CAR, which can be designed to include, is fused to the extracellular of CAR
The membrane spaning domain of domain.In one embodiment, using naturally with one of the domain in CAR phase
The membrane spaning domain of association.In some instances, membrane spaning domain may be selected, or is entered by amino acid replacement
Row modification, to avoid the transmembrane structure that such domain is bound to identical or different surface membrane protein
Domain, so as to minimize the interaction with other members of receptor complex.
Membrane spaning domain may originate from natural origin or synthesis source.In natural origin, the domain may originate from
Any embrane-associated protein or transmembrane protein.Transmembrane region specifically for the present invention may originate from T-cell receptors
α, β or ζ chain, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,
CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e. at least include it is above-mentioned in transmembrane region
(one or more)).
In the present invention, it can contain between constant region and hinge area and between hinge region and transmembrane region
Or do not contain joint sequence (Linker).In a preference, (light chain can by VH (weight chain variable district) and VL
Become area) between be classified as GGGGSGGGGSGGGGS (SEQ ID NO. in order:5) Linker.
Intracellular domain
The CAR of present invention intracellular domain or other Cellular Signaling Transduction Mediated domain is to cause wherein
The reason for activation at least one normal effect subfunction for placing CAR immunocyte.Term " effector
Function " refers to the proprietary feature of cell.For example, the effector function of T cell can be to include cell factor
The cell lysis activity or auxiliary activity of secretion.Therefore term " Cellular Signaling Transduction Mediated domain " is referred to
Transduction effector function signal and guides the protein part of cell implementation proprietary feature.Although generally can be used whole
Individual Cellular Signaling Transduction Mediated domain, but in many examples, it is not necessary to use whole chain.Just using intracellular
For the truncation part of signal transduction domain, this truncation part can be used for replacing complete chain, as long as it
Transduction effector function signal.Therefore term Cellular Signaling Transduction Mediated domain refers to including being enough effector of transduceing
Any truncation part of the Cellular Signaling Transduction Mediated domain of function signal.
Preferred example for the CAR of present invention Cellular Signaling Transduction Mediated domain includes φt cell receptor
(TCR) the co-receptor of endochylema sequence and corporate action to be transduceed in antigen receptor with reference to rear commencing signal, with
And any derivative or variant and any composition sequence with identical Functional Capability of these sequences.
It is known that complete activating T cell is not enough to by the TCR signals individually produced, and be also required to secondary or common
Stimulus signal.Therefore, T cell activation can be considered as by two inhomogeneous endochylema signal transduction sequence mediations：
By TCR (primary endochylema signal transduction sequence) start antigen-dependence primary activation those and with antigen-
Dependent/non-dependent mode work with provide secondary or costimulatory signal (secondary endochylema signal transduction sequence) that
A bit.
The primary that primary endochylema signal transduction sequence adjusts TCR compounds with stimulation mode or with suppressor mode is living
Change.The primary endochylema signal transduction sequence worked with stimulation mode can include signal transduction motif, known to it
For activation motifs or ITAM based on immunity receptor tyrosine.
The example bag of ITAM comprising the primary endochylema signal transduction sequence in the present invention with particular use
Include and come from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b
With CD66d those.It is particularly preferred that the endochylema signal transduction molecule in the CAR of the present invention includes coming from
CD3 ζ endochylema signal transduction sequence.
In a preferred embodiment, CAR cytoplasmic domain can be designed to include CD3- ζ signals with itself
Conducting structure domain, or can be with any other desired endochylema structure useful in the CAR of present invention content
(one or more) joints in domain.For example, CAR cytoplasmic domain may include CD3 ζ chain parts and costimulation letter
Number conducting region.Costimulatory signal conducting region refers to including a part for the intracellular domain of costimulatory molecules
CAR.Costimulatory molecules be lymphocyte to the cell surface molecule needed for the effective response of antigen, rather than
Antigen receptor or their part.The example of this molecule include CD27, CD28,4-1BB (CD137), OX40,
CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKG2C, B7-H3 and the part etc. specifically bound with CD83.Therefore, although the present invention it is main with
4-1BB as costimulatory signal transport element example, but other costimulation elements also be located at the present invention model
In enclosing.
Endochylema signal transduction sequence in the CAR of present invention endochylema signal transduction part can be at random or to advise
Fixed order is connected with each other.Optionally, short oligopeptides or polypeptide linker, preferred length is in 2 and 10 ammonia
Base acid, can form the connection.Glycine-serine doublet provides specially suitable connector.
Carrier
The present invention includes the DNA construct for including CAR sequences, and the wherein sequence includes being operably coupled to letter
The nucleotide sequence of the antigen-binding domains of the nucleotide sequence in number conducting structure domain.
Coding expects that the nucleotide sequence of molecule is obtained using the recombination method being known in the art, such as example
Such as by screening library from the cell of expressing gene, by being somebody's turn to do from the known carrier including the gene
Gene, or by using the technology of standard, be directly separated from cell and tissue comprising the gene.It is optional
Ground, gene interested can be synthesized production.
Present invention provides the carrier for the DNA for wherein inserting the present invention.Come from such as slow disease of retrovirus
The carrier of poison is the suitable tools for realizing long-term gene transfer, because they allow transgenosis long-term, stabilization
Integrate and it breeds in daughter cell.
Simplified summary, generally by the nucleic acid for the coding CAR polypeptides or part thereof that are operably connected to promoter,
And construct is incorporated to expression vector, realize the expression of coding CAR natural or synthetic nucleic acid.The carrier is fitted
Together in duplication and integration eukaryotic.Typical cloning vector, which is included, can be used for regulation to expect nucleotide sequence expression
Transcription and translation terminator, initiation sequence and promoter.
The nucleic acid can be cloned into the carrier of many types.For example, the nucleic acid can be cloned into such carrier,
It includes but is not limited to plasmid, phasmid, phage-derived thing, animal virus and clay.Specifically feel emerging
Interesting carrier includes expression vector, replicating vector, probe generation vectors and sequencing vector.
Further, expression vector can be supplied to cell in viral vector form.It can be used as the virus of carrier
Including but not limited to retrovirus, adenovirus, adeno-associated virus, herpesviral and slow virus.Generally,
Suitable carrier is included in the replication orgin worked at least one organism, promoter sequence, convenient
Restriction enzyme sites and one or more selectable marks.
Many systems based on virus are developed, for gene transfer to be entered into mammalian cell.For example,
Retrovirus provides the convenient platform for gene delivery system.Using what is be known in the art
Technology is by the gene insertion vector of selection and is packaged into retroviral particle.The recombinant virus can then be divided
From be transferred in vivo or in vitro subject cell.Many retroviral systems are known in the art
's.In some embodiments, using adenovirus vector.Many adenovirus vectors are known in the art
's.In one embodiment, using slow virus carrier.
Extra promoter element, such as enhancer, can adjust the frequency that transcription starts.Normally, this
It is a little to be located in the 30-110bp regions of initiation site upstream, although having shown that many promoters are also included recently
The function element in initiation site downstream.Interval between promoter element is often flexibility, to work as element
When being squeezed relative to another or be mobile, promoter function is kept.In thymidine kinase (tk) promoter,
Interval between promoter element, which can be increased, separates 50bp, and activity is just begun to decline.Depending on promoter,
Showing discrete component can cooperate or independently work, to start transcription.
One example of suitable promoter is instant early stage cytomegalovirus (CMV) promoter sequence.This is opened
Promoter sequences are that can drive any polynucleotide sequence high level expression being operably coupled to thereon
Strong constitutive promoter sequence.Another example of suitable promoter is the elongation growth factor
-1α(EF-1α).It is also possible, however, to use other constitutive promoter sequences, including but not limited to anthropoid cape
Virus-4 0 (SV40) early promoter, mouse mammary cancer viral (MMTV), the long end of human immunodeficiency virus (HIV)
End repeats (LTR) promoter, MoMuLV promoters, avian leukosis virus promoter, Ai Baisitan-Ba Er
(Epstein-Barr) early promoter, Rous sarcoma virus promoter and people's gene are opened virus immediately
Mover, such as, but not limited to actin promoter, Myosin promoter, ferroheme promoter and creatine
Kinase promoter.Further, the present invention should not limited to the application of constitutive promoter.Induction type starts
Son is also contemplated as the part of the present invention.The use of inducible promoter provides molecular switch, and it can
When such expression is desired, the polynucleotide sequence for the inducible promoter that is operably connected is opened
Expression, or the closing expression when expression is undesirable.The example of inducible promoter includes but is not limited to gold
Belong to metallothoinein promoter, Glucocorticoid promoter, progesterone promoter and tetracycline promoter.
In order to assess the expression of CAR polypeptides or part thereof, be introduced into the expression vector of cell can also include it is optional
In the marker gene or reporter selected any one or both, in order to from seeking to be turned by viral vector
Identification and selection expression cell in dye or the cell mass of infection.In other respects, selectable mark can be taken
Band is on independent section of DNA and for cotransfection program.The flank of both selectable mark and reporter
Can all have appropriate regulatory sequence, so as to be expressed in host cell.Useful selectable marker bag
Include such as antibiotics resistance gene, such as neo etc..
Reporter is used for the cell for identifying potential transfection and the feature for evaluating regulatory sequence.Generally
Ground, reporter is following gene：It is not present in recipient organism or tissue or by recipient organism or group
Knit and expressed, and its coded polypeptide, the property such as enzyme that the expression of the polypeptide can be detected easily by some
Activity is clearly showed that.After DNA has been incorporated into recipient cell, the expression of reporter is in the suitable time
Under be measured.Suitable reporter may include the plain enzyme of coding fluorescence, beta galactosidase, chloramphenicol
Transacetylase, SEAP or Green Fluorescent Protein gene gene (for example, Ui-Tei etc.,
2000FEBS Letters479:79-82).Suitable expression system is known and using known technology system
It is standby or commercially obtain.Generally, the reporter expression of display highest level has minimum 5 flanks
The construct in area is accredited as promoter.Such promoter region can be connected to reporter and for evaluating
The ability of reagent regulation promoter-driving transcription.
Gene is introduced into cell and the method that gene expression enters cell is well known in the art.In expression
In the content of carrier, carrier can be easily introduced into host cell by any method in the art, for example,
Mammal, bacterium, yeast or insect cell.For example, expression vector can pass through physics, chemistry or biological
Learn to do section and be transferred to host cell.
The physical method that polynucleotides are introduced into host cell includes calcium phosphate precipitation, lipofection, particle
Bombardment, microinjection, electroporation etc..Production includes the method for the cell of carrier and/or exogenous nucleic acid at this
It is known in field.See (2001, the Molecular Cloning such as Sambrook:A Laboratory
Manual,Cold Spring Harbor Laboratory,New York).Polynucleotides are introduced into host thin
The method for optimizing of born of the same parents is calcium phosphate transfection.
Polynucleotides interested are introduced into the biological method of host cell including the use of DNA and RNA carriers.
Viral vector, particularly retroviral vector, have become it is most widely used by gene insertion lactation move
The method of thing such as people's cell.Other viral vectors may originate from slow virus, poxvirus, herpes simplex virus I,
Adenovirus and adeno-associated virus etc..
The chemical means that polynucleotides are introduced into host cell include dispersion system of colloid, and such as macromolecular is combined
Thing, Nano capsule, microballoon, pearl；With the system based on lipid, including oil in water emulsion, micella, mixing
Micella and liposome.Exemplary colloid system as in vitro and in vivo tool for transmitting (delivery vehicle)
Unite as liposome (for example, artificial membrane vesicle).
In the case of using non-viral delivery system, exemplary tool for transmitting is liposome.Consideration uses fat
Matter preparation, host cell (external, in vitro (ex vivo) or in vivo) is introduced by nucleic acid.On the other hand,
The nucleic acid can be associated with lipid.The nucleic acid associated with lipid can be encapsulated into the aqueous interior of liposome
In, be dispersed in the lipid bilayer of liposome, through with the both associated connections point of liposome and oligonucleotides
Son is attached to liposome, is absorbed in liposome, with lipid bluk recombination, is dispersed in the solution comprising lipid, with
Lipid is mixed, and is combined with lipid, is included in as suspension in lipid, included in micella or multiple with micella
Close, or it is otherwise associated with lipid.Lipid, lipid/DNA or the lipid associated with composition/
Expression vector is not limited to any concrete structure in solution.For example, they may be present in bilayer structure,
As micella or with " (collapsed) of collapse " structure.They also can simply be dispersed in solution
In, it is possible to create size or the inhomogenous aggregation of shape.Lipid is fatty material, and it can be natural generation
Or the lipid of synthesis.For example, lipid includes lipid droplet, it naturally occurs in cytoplasm and includes long-chain
Such change of aliphatic hydrocarbon and their derivative such as aliphatic acid, alcohols, amine, alkamine and aldehydes
In compound.
One in the present invention is preferably carried out in mode, and the carrier is slow virus carrier.The present inventor grinds
Study carefully confirmation, CAR of the present invention is built using the slow virus carrier, it is higher to the transfection efficiency of T cell, and have
The repeatability of height.
One in the present invention is preferably carried out in mode, and signal peptide sequence is also included in the carrier.It is preferred that
Ground, the signal peptide sequence is connected to the upstream of the antigen tuberculosis domain nucleotide sequence.
Therapeutic application
The present invention includes the cell (such as T cell) with slow virus carrier (LV) transduction.
Therefore, present invention provides stimulate T cell-mediation to the target cell of mammal group or tissue
The method of immune response, it comprises the following steps：The T cell that mammal expresses CAR is administered to, to treat
MUC1 positive tumors.
In a preference, tumour of the present invention is selected from the group：Cancer of the esophagus, stomach cancer, breast cancer, ovum
Nest cancer, carcinoma of urinary bladder, adenocarcinoma of seminal vesicle, lung cancer, three cloudy breast cancer or its combination.
The T cell of the CAR- modifications of the present invention can be administered alone or as pharmaceutical composition and diluent and/
Or combined administration with other components such as IL-2 or other cell factors or cell mass.Briefly, it is of the invention
Pharmaceutical composition may include target cell as described herein group, with one or more pharmacy or physiologically may be used
Receive carrier, diluent or excipient to combine.Such composition may include buffer solution such as neutral buffered salt
Water, sulfate buffered saline etc.；Carbohydrate such as glucose, mannose, sucrose or glucan,
Mannitol；Protein；Polypeptide or amino acid such as glycine；Antioxidant；Chelating agent such as EDTA or paddy
The sweet peptide of Guang；Adjuvant (for example, aluminium hydroxide)；And preservative.The composition of the present invention is preferably formulated for quiet
Applied in arteries and veins.
The mode that the pharmaceutical composition of the present invention can be suitable for the disease of (or prevention) to be treated is applied.Using
Quantity and frequency will be determined by such factor, the illness of such as patient and the type of patient disease and serious
Although degree --- appropriate dosage can be determined by clinical test.
When pointing out " effective dose in immunology ", " antitumor effective dose ", " tumour-suppression effective dose "
Or when " therapeutic dose ", the precise volume of the present composition to be administered can be determined by doctor, it considers patient
Age, weight, tumor size, infection or the metastasis degree and the individual difference of illness of (object).Can be usual
Point out：Pharmaceutical composition including T cell described herein can be with 104To 109The agent of individual cell/kg body weight
Amount, preferably 105To 106The dosage (including all integer values in the range of those) of individual cell/kg body weight is applied.
T cell composition can also repeatedly be applied with these dosage.Cell can be by using known in immunotherapy
Injection technique (see such as Rosenberg, NewEng.J.of Med.319:1676,1988) apply.It is right
It can be controlled in the optimal dose and therapeutic scheme of specific patient by monitoring the disease indication of patient and therefore adjusting
Treatment is readily determined by medical domain technical staff.
The administration of object composition thing can be carried out in any convenient manner, including by spray-on process, injection,
Swallow, infuse, be implanted into or transplant.Compositions described herein can by subcutaneous, intracutaneous, knurl, knot in,
In spinal cord, intramuscular, patient is administered to by intravenous (i.v.) injection or intraperitoneal.In an embodiment party
In formula, CAR-T cell compositions of the invention are administered to patient by intracutaneous or hypodermic injection.Another
In individual embodiment, CAR-T cell compositions of the invention are preferably injected by i.v. to be applied.The present invention's
The composition of CAR-T cells can be injected directly into tumour, lymph node or infection position.
In further embodiment, CAR-T cells of the invention can with below in conjunction with using：Chemotherapy,
Radiation, immunodepressant, such as, cyclosporin, imuran, methopterin, mycophenolate and FK506,
Antibody or other immunotherapeutic agents.In further embodiment, cell composition and marrow of the invention
Transplant, combine (example using chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), endoxan
Such as, prior to, concurrently with, or after) and it is administered to patient.For example, in one embodiment, object can be through
The standard care of high dose chemotherapy is gone through, autologous peripheral blood stemcell transplant is carried out afterwards.In some embodiments,
After the transfer, object receives the injection of the immunocyte of the extension of the present invention.In an extra embodiment
In, the cell of extension is applied in surgery operation consent or surgical site infections.
The dosage for the treatment of above of patient is administered to by with the receiving of sanatory exact properties and treatment
Person and change.The practice that people's applied dose ratio can receive according to this area is implemented.Generally, treatment every time
Or each course for the treatment of, can be by 1 × 106It is individual to 1 × 1010Individual T cell of the present invention through modification, is returned for example, by vein
Defeated mode, is applied to patient.
Main advantages of the present invention include：
(a) single-chain antibody of the invention has fabulous recognition capability to MUC1 positive tumor cells.
(b) MUC1-CAR-T cells of the invention have to MUC1 positive tumor cells system significantly kill in vitro
Hinder effect.
(c) toxicity of missing the target of MUC1-CAR-T cells of the invention is than relatively low.
(d) MUC1-CAR-T cells of the invention have significant to tumor focus positive MUC1 in vivo
Lethal effect.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate
The present invention rather than limitation the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally according to normal condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise
Percentage and number are calculated by weight.
Embodiment 1
The mutating experiment of MUC1 single chain antibody sequences
One original MUC1 antibody As, its amino acid sequence such as SEQ ID NO. are provided:Shown in 4, nucleotides
Sequence such as SEQ ID NO.:Shown in 3.Original antibodies A sequence is carried out using molecular biology method
Amino acid mutation, is mutated six amino acid sequences, obtains amino acid sequence such as SEQ ID NO. altogether:Shown in 2
MUC1 single-chain antibodies B.
Fig. 1 shows original antibodies A amino acid sequence and MUC1 single-chain antibodies B amino acid sequence ratio
To result, red amino acid represents the amino acid of mutation.Tri- catastrophe points of wherein A, Q, C are located at CDR region.
Embodiment 2
CAR-T cells in vitro killing experiments
With the MUC1 single-chain antibodies B obtained in embodiment 1 sequence construct MUC1-CAR- slow virus carriers, enter
Row virus packaging, transfecting T cells carry out killing experiments in vitro after preparing MUC1-CAR-T cells.Target cell
For the positive T47D cells of MUC1, effect target ratio is 5:1, the killing time is 12 hours.
Same procedure builds A-CAR-T cells with original antibodies A and carries out body simultaneously with MUC1-CAR-T cells
Outer killing experiments.
The structure of Chimeric antigen receptor (CAR) is as shown in Fig. 2 Fig. 2A and Fig. 2 B are respectively illustrated comprising original anti-
The structure of body A and mutation MUC1 single-chain antibodies B Chimeric antigen receptor.Wherein, A-scFv is original antibodies A
Sequence, B-scFv for mutation MUC1 single chain antibody sequences, Fc is hinge area, CD28 and CD137 (4-1BB)
Molecule is T cell activation costimulatory molecules, and CD3 molecules are the signaling molecule of T cell activation first.
As a result as shown in figure 3, the A-CAR-T cells (fragmentation effects of ＜ 40%) prepared with utilization original antibodies A
Compare, MUC1-CAR-T cells have stronger Cytotoxicity in vitro effect (＞ 50% to MUC1 positive tumor cells T47D
Mortaility results).The T cell transfected with untransfected CAR T cell and empty carrier as a control group (imitate by killing
Rate ＜ 20%).
The above results show have to MUC1 positive tumor cells with the MUC1 single-chain antibodies B CAR-T cells built
Preferably kill ability.
Embodiment 3
MUC1-CAR-T clinical test
One adenocarcinoma of seminal vesicle patient is treated with MUC1-CAR-T, significant therapeutic effect is shown.
Specifically, it is similar to two sizes and biology morphology with A-CAR-T cells with MUC1-CAR-T cells respectively
Tumor focus carried out intratumor injection, other treatment measure is identical.
As a result show, the tumor focus treated with A-CAR-T does not show necrosis phenomena, but uses
There is obvious neoplasm necrosis phenomenon in the tumor focus of MUC1-CAR-T cell therapies, and the above results show, with
Original antibodies A is compared, and MUC1 single-chain antibodies B has more preferable lethal effect to MUC1 positive tumor focuses.
Embodiment 4
MUC1-CAR-T clinical test
With MUC1-CAR-T cells and A-CAR-T the cells essence similar to two sizes and biology morphology respectively
Cystadenocarcinoma focus has carried out intratumor injection, and immunohistochemistry is carried out and other to two tumor focus after treatment
Analysis.
As a result show, the quantity of tumour cell positive the tumor focus MUC1 treated with MUC1-CAR-T is obvious
Reduce, tumor focus has obvious necrosis phenomena, and there are obvious CD4+ and CD8+ lymphocyte infiltrations.
The tumor focus treated with A-CAR-T does not find that MUC1 positive tumor cells have obvious reduction, does not have yet
Obvious lymphocyte infiltration phenomenon.As a result the A-CAR-T cell phases with original antibodies A sequence construct are shown
Than the MUC1-CAR-T cells built with MUC1 single-chain antibodies B have obvious to MUC1 positive tumors focus
Fragmentation effect.
All documents referred in the present invention are all incorporated as reference in this application, just as each document
It is individually recited as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (10)
1. a kind of single-chain antibody, it is characterised in that the amino acid sequence of the antibody such as SEQ ID NO.:2 institutes
Show.
2. a kind of fusion protein, it is characterised in that containing described in claim 1 in described fusion protein
Single-chain antibody as fusion protein composed component.
3. fusion protein as claimed in claim 2, it is characterised in that described fusion protein is inosculating antibody
Original receptor.
4. fusion protein as claimed in claim 3, it is characterised in that the Chimeric antigen receptor from N-terminal to
C-terminal, successively with elements below：
Constant region-the CD28's of single-chain antibody-antibody described in optional signal peptide element-claim 1
Membrane spaning domain-CD137 element-CD3 ζ intracellular signal domain.
5. a kind of Chimeric antigen receptor (CAR), it is characterised in that the extracellular structure of the Chimeric antigen receptor
Domain is amino acid sequence such as SEQ ID NO.:Single-chain antibody shown in 2.
6. a kind of polynucleotides, it is characterised in that the list described in described polynucleotide encoding claim 1
Chimeric antigen receptor described in chain antibody or fusion protein or claim 5 containing the single-chain antibody.
7. a kind of carrier, it is characterised in that described carrier contains the polynucleotides described in claim 6.
8. a kind of cell, it is characterised in that contain the carrier or dye described in claim 7 in described cell
The polynucleotides described in the claim 6 of external source are integrated with colour solid.
9. cell as claimed in claim 8, it is characterised in that described cell is that (CAR-T is thin for T cell
Born of the same parents).
10. a kind of pharmaceutical composition, it is characterised in that the composition contains (a) pharmaceutically acceptable load
The fusion protein described in single-chain antibody, claim 2 described in body and (b) claim 1, claim
The nucleic acid molecules described in Chimeric antigen receptor, claim 6 described in 5, the carrier described in claim 7,
Cell or its combination described in claim 8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109320602A (en) * | 2018-07-04 | 2019-02-12 | 华东师范大学 | A kind of Chimeric antigen receptor T cell and application thereof of Siglec-9 targeting |
| CN110551742A (en) * | 2018-06-01 | 2019-12-10 | 上海恒润达生生物科技有限公司 | MUC 1-targeted chimeric antigen receptors and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103483453A (en) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | Chimeric antigen receptor combining EGFR (epidermal growth factor receptor) family proteins and composition and uses thereof |
| CN103483452A (en) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | Dual-signal independent chimeric antigen receptors (dsCAR) and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103483453A (en) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | Chimeric antigen receptor combining EGFR (epidermal growth factor receptor) family proteins and composition and uses thereof |
| CN103483452A (en) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | Dual-signal independent chimeric antigen receptors (dsCAR) and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110551742A (en) * | 2018-06-01 | 2019-12-10 | 上海恒润达生生物科技有限公司 | MUC 1-targeted chimeric antigen receptors and uses thereof |
| CN109320602A (en) * | 2018-07-04 | 2019-02-12 | 华东师范大学 | A kind of Chimeric antigen receptor T cell and application thereof of Siglec-9 targeting |
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