CN108997481A - Antigen small peptide from LMP1 - Google Patents
Antigen small peptide from LMP1 Download PDFInfo
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- CN108997481A CN108997481A CN201710424928.6A CN201710424928A CN108997481A CN 108997481 A CN108997481 A CN 108997481A CN 201710424928 A CN201710424928 A CN 201710424928A CN 108997481 A CN108997481 A CN 108997481A
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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
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
- C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
- C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention relates to the small peptide of the newfound tumour antigen LMP1 from EBV virus, the purposes of compound and the small peptide and compound that the small peptide and MHC molecule are formed.Meanwhile the invention further relates to the purposes of molecule and these molecules in conjunction with above-mentioned small peptide or compound.
Description
Technical field
The present invention relates to the small peptide of the newfound tumour antigen LMP1 from EBV virus, the small peptide and MHC molecule shape
At compound and the small peptide and compound purposes.Meanwhile the invention further relates in conjunction with above-mentioned small peptide or compound
Molecule and these molecules purposes.
Background technique
It is well known that under many pathological conditions, such as infection, cancer, autoimmune disease, can all there are some specific points
Son is not suitable for expressing.Therefore, these molecules are just at pathology or " label " of abnormality.These molecules not only can be used as
The marker of medical diagnosis on disease, it may also be used for generate diagnostic reagent and/or therapeutic agent.For example, generating spy with the marker of cancer
Fixed antibody.In addition, these molecules can also effectively activated cell toxic T lymphocyte (CTL) specific immune response,
Antitumor efficiency is played, at the same time it can also obtain the T cell receptor that can be combined with " label " by the CTL of activation
(TCR) it is used as therapeutic agent.Therefore, these molecules play a very important role in the diagnosing and treating of related disease.
For tumour, has many documents and delivered different endogenic tumour antigen molecules.But this is not
The real target spot of related disease because causing the not complete tumour antigen molecule of CTL immune response, and comes from
The specific CTL epitope (Epitope) of antigen.Under normal circumstances, tumour antigen pass through in the cell proteolysis by its
Be processed into the polypeptide fragment of 8-16 amino acid length, i.e. CTL epitope, so with the ajor histocompatibility in endoplasmic
Complex (MHC, the MHC of the mankind are commonly referred to as HLA gene or HLA complex) molecule combines and forms polypeptide-MHC compound
(peptide-MHC complex, pMHC), and finally by pMHC submission to cell surface for CD8+The TCR on T cell surface knows
Not.It is delivered although relevant autochthonous tumor antigen molecule early has, we are simultaneously unaware of the specific polypeptide piece being rendered out
Section.Therefore, it either still is used to generate the diagnostic reagent or therapeutic reagent of related disease as vaccine, identifies these quilts
It is presented to the polypeptide fragment of 8-16 amino acid length of cell surface, i.e. CTL epitope, is vital.Art technology
Personnel are dedicated to finding and determining that these are presented to the polypeptide fragment of target cell surface.
The discovery and determination of this polypeptide fragment by submission are a complicated processes, because polypeptide is by HLA submission
The common results of the interaction of the enzymatic hydrolysis and polypeptide fragment and HLA of antigen protein.This explanation, complete tumour antigen molecule
Any information can not be provided for the discovery and identification of polypeptide fragment.Many documents have delivered the method using computer simulation, such as
Public database SYFPEITHI (Rammensee, et al., Immunogenetics.1999 (50): 213-219) and BIMAS
(Parker, et al., J.Immunol.1994.152:163) provides prediction algorithm to identify which polypeptide fragment may be by
Submission.But this is a kind of prediction, has very big uncertainty, because after it is not really processing intracellular and translates
Modification.Tumor tissues after treatment, can use mass spectrograph Direct Identification and go out what tumor cell surface was gone out by submission
Polypeptide fragment obtaining the result is that very reliable although this is a complicated process in this way.Meanwhile mass spectrometric spirit
Sensitivity is also sufficient to the polypeptide fragment for identifying low concentration and the modification after translation, therefore it is discovery and determining tumor surface
The ideal tools of polypeptide fragment.
The study found that Epstein-Barr virus (EBV) can infect lymphocyte and epithelial cell, and malignant tumour phase is developed into it
It closes.EBV can be detected in all cells of most of pernicious nasopharyngeal carcinoma, and the frequent table of oncoprotein LMP1 therein
It reaches.(Raab-Traub N.2007.EBV-induced oncogenesis,p 986–1006.In Arvin A,
Campadelli-Fiume G,Mocarski E,Moore PS,Roizman B,Whitley R,Yamanishi K(ed),
Human herpesviruses:biology,therapy,and immunoprophylaxis.Cambridge
University Press,Cambridge,United Kingdom；Raab-Traub N.2002.Epstein-Barr
virus in the pathogenesis of NPC.Semin Cancer Biol 12:431–441.).Therefore, it is derived from
The peptide of LMP1 antigen not only can be used as the marker of above-mentioned medical diagnosis on disease as the target spot of above-mentioned cancer, it may also be used for generate
The prevention reagent and/or therapeutic agent of above-mentioned disease, such as antibody or T cell receptor.The present invention is using spectrometer analysis and identifies
The new polypeptide fragment from tumour antigen LMP1 that tumor cell surface is rendered.
Summary of the invention
The object of the present invention is to provide the small peptide of newfound tumour antigen LMP1 from EBV virus a kind of,
The purposes of compound and the small peptide and compound that the small peptide and MHC molecule are formed.
The first aspect of the present invention provides a kind of peptide, and the peptide includes:
(I) amino acid sequence ILWRLGATI (SEQ ID NO:1)；Or
(II) has 1,2 or 3 amino acid substitution and/or 1,2 or 3 amino acid in SEQ ID NO:1
The amino acid sequence of insertion and/or 1,2 or 3 amino acid deletions；
Wherein, the peptide can form compound with MHC molecule.
In another preferred example, the peptide is made of 7-25 amino acid.
In another preferred example, the peptide is made of 8-16 amino acid.
In another preferred example, the amino acid sequence of the peptide is SEQ ID NO:1.
The second aspect of the present invention, provides a kind of pMHC compound, and the compound includes first aspect present invention institute
The peptide stated.
In another preferred example, the amino acid sequence of the peptide in the pMHC compound is SEQ ID NO:1.
In another preferred example, the type of MHC molecule is HLA-A*02.
In another preferred example, the type of MHC molecule is HLA-A*0201.
In another preferred example, the pMHC compound is polymer.
In another preferred example, the pMHC compound is soluble.
In another preferred example, the pMHC compound is biotinylated.
The third aspect of the present invention, provides a kind of isolated cell, and the cell surface presents second aspect of the present invention
The pMHC compound.
The fourth aspect of the present invention, provides a kind of nucleic acid molecules, and the nucleic acid molecules include to encode first party of the present invention
The nucleic acid sequence of peptide described in face or its complementary series.
The fifth aspect of the present invention, provides a kind of carrier, and the carrier contains nucleic acid described in fourth aspect present invention
Molecule.
The sixth aspect of the present invention provides a kind of host cell, contains described in fifth aspect present invention in the cell
Carrier.
The seventh aspect of the present invention, provides a kind of molecule, and the molecule can be in conjunction with described in first aspect present invention
PMHC compound described in peptide and/or second aspect of the present invention.
In another preferred example, the molecule can specifically bind peptide and/or this hair described in first aspect present invention
PMHC compound described in bright second aspect.
In another preferred example, the molecule is T cell receptor.
In another preferred example, the T cell receptor is soluble.
In another preferred example, the molecule is antibody or its binding fragment.
In another preferred example, the antibody is monoclonal antibody.
The eighth aspect of the present invention, provides a kind of isolated monoclonal T cell, and the T cell combines the present invention second
The aspect pMHC compound.
In another preferred example, pMHC compound described in the T cell specific binding second aspect of the present invention.
It is multiple to provide pMHC described in peptide, second aspect of the present invention described in first aspect present invention for the ninth aspect of the present invention
The purposes for closing cell described in object or third aspect present invention, for activating and/or separating T cell.
It is multiple to provide pMHC described in peptide, second aspect of the present invention described in first aspect present invention for the tenth aspect of the present invention
The purposes for closing object, for screening T cell receptor or antibody library.
The eleventh aspect of the present invention provides pMHC described in peptide, second aspect of the present invention described in first aspect present invention
Cell described in compound, third aspect present invention, described in nucleic acid molecules, seventh aspect present invention described in fourth aspect present invention
The purposes of T cell described in molecule or eighth aspect present invention is used to prepare the drug of prevention or treating cancer.
The twelveth aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains pharmaceutically acceptable
Peptide described in carrier and first aspect present invention, described in pMHC compound, third aspect present invention described in second aspect of the present invention
T cell described in molecule described in cell, seventh aspect present invention or eighth aspect present invention.
In another preferred example, described pharmaceutical composition is vaccine.
The thirteenth aspect of the present invention provides a kind of method prevented or treat disease, including applying to the object needed
The peptide described in suitable first aspect present invention, described in pMHC compound, third aspect present invention described in second aspect of the present invention
T cell described in molecule described in cell, seventh aspect present invention or eighth aspect present invention.
The fourteenth aspect of the present invention provides a kind of obtain and pMHC compound knot described in right second aspect of the present invention
The method of the molecule of conjunction, comprising:
(I) is by pMHC complex contacts described in candidate molecules and second aspect of the present invention；
(II) filters out the molecule in conjunction with pMHC compound in (I).
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 is the representative mass spectrogram for identifying small peptide of the present invention.
Fig. 2 is the SDS-PAGE glue figure of solubility pMHC compound of the invention.Left-hand bar band is molecular weight marker, right side item
The light chain and heavy chain of band respectively MHC molecule.
Fig. 3 is the double positive staining results of CD8+ and the tetramer-PE of monoclonal cell.
Fig. 4 is double positive staining results of monoclonal cell TCR transduction primary T cells
Fig. 5 is the ELISPOT experimental result of monoclonal cell TCR transduction primary T cells.
Fig. 6 is BIAcore map of the sTCR in conjunction with pMHC compound of the present invention.
Specific embodiment
The present invention obtains the peptide of the tumour antigen LMP1 from EBV virus, the peptide by extensive and in-depth research
Tumor cell surface is presented to by MHC molecule, as tumor marker.Therefore, the present invention provides be derived from the swollen of EBV virus
The purposes of compound and the peptide and compound that the peptide of tumor antigen LMP1, the peptide and MHC molecule are formed.Meanwhile the present invention
Further relate to the molecule in conjunction with above-mentioned peptide or compound.It should be understood that in the present invention, peptide of the invention is with polypeptide of the present invention or originally
Invention small peptide is used interchangeably, and all refers to the peptide of the tumour antigen LMP1 provided by the invention from EBV virus.
Specifically, the first aspect of the present invention provides a kind of peptide, and the peptide includes:
(I) amino acid sequence ILWRLGATI (SEQ ID NO:1)；Or
(II) has 1,2 or 3 amino acid substitution and/or 1,2 or 3 amino acid in SEQ ID NO:1
The amino acid sequence of insertion and/or 1,2 or 3 amino acid deletions；
Wherein, the peptide can form compound with MHC molecule.
Amino acid substitution means that in same position, some amino acid residue is substituted by other amino acid residues.Insertion
Amino acid residue can be inserted into any position, and the amino acid residue of insertion can also be completely or partially adjacent to each other, or insertion
Amino acid between it is not adjacent to each other.1,2 or 3 amino acid can be deleted from the sequence of SEQ ID NO:1.
It is known to those skilled in the art that peptide of the present invention can one or more positions between amino acid sequence
Carry out posttranslational modification.The example of posttranslational modification can be Immunol.2006 2 months in the Curr Opin such as Engelhard；
18 (1): finding in 92-7, and including phosphorylation, acetylation and deamidization.
Preferably, peptide of the present invention and MHC are incorporated into the binding site peptide point of MHC molecule.In general, foregoing description is repaired
The amino acid of decorations will not destroy the peptide and the binding ability of MHC.In one preferred embodiment, the amino acid is repaired
Decorations improve ability of the peptide in conjunction with MHC.For example, mutation is likely to occur in peptide and the binding site of MHC.These binding sites and
Preferred residue is known in the art on binding site, especially for the peptide to which in conjunction with HLA-A*02 (referring to, such as
Parkhurst etc., J.Immunol.157:2539-2548 (1996)).
More specifically, the length of the amino acid of peptide of the invention can be 7-25, preferably 8-16, preferably, 9,10,
Or 11.
Peptide of the present invention can be made of ILWRLGATI (SEQ ID NO:1), or mainly by ILWRLGATI (SEQ
ID NO:1) composition, correspond to the position of the 129-137 residue of LMP1 full length protein.
Invention also provides the analog of albumen shown in SEQ ID NO:1 or peptide.These analogs and natural peptide difference
It can be the difference on amino acid sequence, be also possible to not influence the difference on the modified forms of sequence, or have both at the same time.This
A little peptides include natural or induction genetic variant.Induction variant can be obtained by various technologies, such as by radiation or cruelly
It is exposed to mutagens and generates random mutagenesis, can also pass through the technology of site-directed mutagenesis or other known molecular biology.Analog
Further include with different from natural L-amino acids residue (such as D- amino acid) analog, and have it is non-naturally occurring or
The analog of the amino acid (such as β, gamma-amino acid) of synthesis.It should be understood that peptide of the invention is not limited to enumerated representativeness
Peptide.
Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetylation of internal or external peptide
Or carboxylated.Modification further includes glycosylation, carries out glycosyl in the synthesis and processing of peptide or in further processing step such as those
The peptide changing modification and generating.This modification can carry out the glycosylated enzyme (glycosylation of such as mammal by the way that peptide to be exposed to
Enzyme or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphotyrosine, phosphoric acid silk
Propylhomoserin, phosphothreonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimize solubility property
Peptide.
In the present invention, " albumen conservative variation peptide shown in SEQ ID NO:1 " refers to the amino acid with SEQ ID NO:1
Sequence is compared, and has at most 3, more preferably at most 2 amino acid are replaced by amino acid with similar or analogous properties and form peptide.
These conservative variation's peptides carry out amino acid substitution preferably based on table 1 and generate.
Table 1
| Initial residue | Representative substitution | It is preferred to replace |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Peptide of the present invention can simply be closed with Merrifield synthetic method (be otherwise known as polypeptide solid-state reaction method)
At.The peptide of GMP rank can use the synthesis in solid state of polypeptide system (Multiple Peptide Systems, San Diego, CA)
Technology is synthesized.Alternatively, the peptide can be re-combined into, if it is desired, can be synthesized with methods known in the art.
Typical such method is related to the use of carrier, and the carrier includes the nucleic acid sequence for encoding polypeptide, expresses polypeptide in vivo；Example
Such as, it is expressed in bacterium, yeast, insect or mammalian cell.Alternatively, external cell-free system can also be used to carry out table
It reaches.Such system is known in the art, and can be obtained from commercial channels.The peptide can be separation and/or with base
Pure form provides in sheet.For example, they can by it is a kind of there is no other peptide or proteins in the form of provide.
Tumour antigen passes through proteolysis in the cell and is processed into as the polypeptide piece of 8-16 amino acid length
Section, i.e. CTL epitope, and then polypeptide-MHC compound (peptide-MHC is formed in conjunction with the MHC molecule in endoplasmic
Complex, pMHC), submission to cell surface together.Therefore, the second aspect of the present invention provides a kind of pMHC compound, institute
It states in compound comprising peptide described in first aspect present invention.Preferably, the polypeptide is incorporated into the peptide-binding groove of MHC molecule
On.The MHC molecule can be MHC I class molecule or MHC class Ⅱmolecule, it is preferable that the MHC molecule is MHC I class molecule.
In one preferred embodiment, the MHC molecule is HLA-A*02, it is highly preferred that the MHC molecule is HLA-A*
0201。
PMHC compound of the present invention can exist with multimeric forms, for example, dimer or the tetramer or five
Aggressiveness or six aggressiveness or eight aggressiveness are bigger.The proper method for generating pMHC polymer can refer to pertinent literature, such as
(Greten et al., Clin.Diagnostic Lab.Immunol.2002:216-220).
In general, with the pMHC compound with biotin residue and the compound generation of fluorescent marker Streptavidin can be passed through
PMHC polymer.Alternatively, the pMHC polymer can also be formed by immunoglobulin as molecular scaffold.It is at this
In system, the extracellular region of MHC molecule and the constant region of heavy chain immunoglobulin are combined by a short catenation sequence (linker)
Together.In addition, forming pMHC polymer also can use carrier molecule, such as dextran (WO02072631).PMHC poly
Body helps to improve the detection of part in connection, such as T cell receptor.Alternatively, improving pMHC compound in related application
Effect such as activates T cell.
PMHC compound of the present invention can be provided with soluble form.For obtain soluble form pMHC compound,
Preferably, MHC molecule is free of transmembrane region in the pMHC compound.Specifically, in pMHC compound, mhc class i molecule can be with
It is made of the extracellular domain of its light chain and all or part of heavy chain.Alternatively, MHC molecule is the piece for only including its functional domain
Section.
The method for generating solubility pMHC compound of the invention is known to the skilled in the art, and is included, but are not limited to
Method described in the embodiment of the present invention 2.MHC molecule in solubility pMHC compound of the invention also can use synthetic method
Generate, then with peptide refolding of the invention.By determine peptide and MHC molecule whether can refolding, can determine the present invention
Peptide can form compound with which class MHC molecule.
Soluble pMHC compound of the invention can be used for screening or detecting molecule in connection, such as TCR or antibody.
The method includes the pMHC compound is contacted with bound fraction to be measured, and measurement bound fraction to be measured whether with compound
In conjunction with.The measuring method of the combination of pMHC compound is well known in the art.Preferred method includes but is not limited to, surface etc. from
Daughter resonance or any other biosensor technique, ELISA, flow cytometry, chromatography, microexamination.Alternatively, this
Outside, the combination can be detected by carrying out functional examination to the biological response in conjunction with generation, such as cytokine release or carefully
Born of the same parents' apoptosis.
Similarly, soluble pMHC compound of the invention can be also used for screening TCR or antibody library.Utilize bacteriophage
Display technique come construct antibody library be it is well known in the art, such as bibliography Aitken, Antibody phage display:
Described in Methods and Protocols (2009, Humana, New York).In one preferred embodiment, this hair
Bright pMHC compound be used to screen the library diversity TCR for being showed in phage particle surface.The TCR of the display library
Non-natural mutation may be contained.
Therefore, soluble pMHC compound of the invention can be fixed on solid phase carrier appropriate by attachment.Gu
The example of phase carrier includes, but are not limited to pearl, film, Ago-Gel, magnetic bead, substrate, pipe, column.PMHC compound can be with
It is fixed on ELISA reaction plate, magnetic bead or surface plasma resonance biosensor chip.PMHC compound is fixed to
The method of solid phase carrier is well known by persons skilled in the art, and including, for example, using affine combination pair, such as biotin
And Streptavidin or antibody and antigen.In one preferred embodiment, pMHC compound biotin labeling, and
It is fixed on the coated surface of Streptavidin.
Peptide of the present invention can together with MHC compound submission to cell surface.Therefore, the present invention also provides one
Kind of cell, the cell can submission pMHC compound of the invention to its surface.Such cell can be mammalian cell,
Preferably, immune system cell, and preferably special antigen presenting cell, such as dendritic cells or B cell (immortalize leaching
Bar mother cell system --- LCL).The cell for presenting peptide or pMHC compound of the present invention can be separation, preferably, with
The form of cell colony, or provide in a substantially pure form.It is of the present invention multiple that the cell can not be natural submission
Conjunction object the or described cell delivery is in the level of compound than high under native state.Such cell can be with of the present invention
Peptide carries out pulse processing and obtains.Pulse processing was related to described peptide incubated cell a few houres, it is preferable that the concentration of peptide used is
10-5-10-12M.In addition, the cell can also be transduceed with HLA-A*02 molecule, the submission of further induction peptide.Submission sheet
The cell for inventing the pMHC compound can be used to separate T cell and T cell receptor, and the T cell is swashed by the cell
It lives and is further sorted out, and then can also obtain expression in the T cell receptor on the T cell surface.
In one preferred embodiment, the method for above-mentioned T cell is obtained including the use of above-mentioned submission pMHC of the present invention
The new blood that the cytositimulation of compound obtains from healthy volunteer.The stimulation that several wheels can be passed through is swashed if 3-4 takes turns
The meeting of T cell living is proliferated, and using labelled antibody, the cell that is activated can be sorted by flow cytometer (FACS), point
The cell of choosing can expand culture and further verifying, for example, by ELISpot detection and/or for the cell toxicant of target cell
Property and/or pMHC polymer dyeing verified.The TCR chain cloned from verified T cell can be fast by the end cDNA
Speed amplification (RACE) is amplified, and is sequenced.
The present invention also provides a kind of nucleic acid molecules, the nucleic acid molecules include the nucleic acid sequence of coding peptide of the invention.
The nucleic acid can be cDNA.The nucleic acid molecules can be mainly made of the nucleic acid sequence for encoding peptide of the present invention, or can
Only to encode peptide of the present invention.Such nucleic acid molecules can be synthesized with methods known in the art.Due to genetic code
Degeneracy, it will be understood by those skilled in the art that the nucleic acid molecules of different nucleic acid sequences can encode identical amino acid sequence.
It include nucleic acid sequence of the present invention in the carrier the present invention also provides a kind of carrier.Suitable carrier
It is the selection known to vector construction field, including promoter and other controlling elements, such as enhancer element.It is of the present invention
Carrier include the sequence for being adapted for introduction into cell.For example, the carrier can be expression vector, and in the carrier, the polypeptide
Control of the coded sequence by own cis-acting regulatory element, the design of carrier convenient for host cell gene integration or
Gene replacement etc..
It will be recognized by one of ordinary skill in the art that in the present invention, term " carrier " includes DNA molecular, for example plasmid, is bitten
Thallus, virus or other carriers, it contains one or more heterologous or recombination nucleic acid sequences.Suitable bacteriophage and virus
Carrier includes, but are not limited to: lambda phage, EMBL bacteriophage, simian virus, cattle wart virus, Epstein-Barr virus, adenopathy
Poison, herpesviral, mouse sarcoma virus, murine mammary tumor virus, slow virus etc..
The present invention also provides a kind of binding molecule, the molecule can be used as immunotherapeutic agent or diagnostic reagent.
The binding molecule can be combined only with peptide, or in conjunction with the compound that peptide and MHC molecule are formed.In latter situation, the knot
Closing molecule can partially be integrated on MHC molecule, meanwhile, it is also in conjunction with peptide of the invention.Bound fraction of the invention can be with
Be separation and/or it is soluble and/or non-naturally occurring, i.e., there is no equivalent and/or pure and/or people in the Nature
Work synthesis.
In a preference of the invention, the binding molecule is T cell receptor (TCR).It can be using international immune
Genetics information system (IMGT) describes TCR.Natural α β heterodimeric TCR has α chain and β chain.In a broad sense, each chain includes
Variable region, bonding pad and constant region, β chain usually contain short variable region, but the variable region also between variable region and bonding pad
Often it is regarded as a part of bonding pad.
TCR of the invention can be any form known in the art.For example, the TCR can be heterodimer, or with
Single-stranded form exists.The TCR can be soluble form (i.e. without cross-film or cytoplasmic domain), and specifically, the TCR may include
All or part of TCR extracellular domain.The TCR is also possible to the overall length chain comprising its transmembrane region.The TCR can be provided
To cell surface, such as T cell.
Soluble TCR can be obtained in conjunction with the prior art in this field, for example, the perseverance of α and the β chain in α β TCR
Artificial disulfide bond is introduced between localization, or introduces artificial disulfide bond between the α chain variable region and β chain constant region of α β TCR.
TCR of the invention can be used for cytotoxic agent or immunostimulant being delivered to target cell, or to be transformed into T thin
Born of the same parents enable the T cell for expressing the TCR to destroy tumour cell, to give in the therapeutic process of referred to as adoptive immunotherapy
Give patient.In addition, mutation can also be contained in TCR of the invention, it is preferable that the TCR after mutation is to pMHC compound of the present invention
Affinity increase.TCR of the invention can be used alone, can also with conjugate with covalent or other modes in conjunction with, it is excellent
Choosing is combined with covalent manner.The conjugate includes detectable marker (for diagnostic purpose, wherein the TCR is in for detecting
Pass the presence of the cell of pMHC compound of the present invention), therapeutic agent, PK (protein kinase) modified part or any the above substance
Combination combine or coupling.TCR of the invention can also preferably be combined in conjunction with the antibody of anti-CD3 with covalent manner, with weight
T cell is oriented, to kill target cell.
In another preferred example, binding molecule of the invention is antibody.As used herein, term " antibody " refers to immune globulin
The immunoactive portions of white molecule and immunoglobulin molecules, i.e., containing the molecule of specific binding site, can it is all natural,
Or part is artificial synthesized or all artificial synthesized.Term " antibody " include antibody fragment, its derivative, functional equivalents with
And homologous antibody, humanized antibody, the antibody fragment include immunoglobulin combined area, the combined area is antigen-binding site
Or it is homologous with antigen-binding site.It can all natural or part it is artificial synthesized or all it is artificial synthesized.Humanized antibody can be with
It is the antibody of modification, the constant region of variable region (for example, mouse) and human antibody containing non-human antibody.
The example of antibody can be immunoglobulin isotypes (such as IgG, IgE, IgM, IgD and IgA) and they are same
The subclass of type；Segment includes antigen binding domain, such as Fab, scFv, Fv, dAb, Fd；And double-chain antibody.Antibody can be more
Anti- or monoclonal antibody, preferably monoclonal antibody.
The preparation method of above-mentioned TCR and antibody is known to the skilled in the art, including but not limited to, from Escherichia coli
It expresses, and is purified in cell or insect cell.
On the other hand, invention further provides peptide of the invention, pMHC compound, nucleic acid molecules, carriers, cell
And purposes of the binding molecule in terms of pharmacy.The peptide, pMHC compound, nucleic acid, carrier, cell or binding molecule can be by
For treating or preventing cancer, preferably melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer and squamous cell carcinoma
Deng.
The present invention also provides a kind of pharmaceutical compositions, and it includes peptide of the invention, pMHC compound, nucleic acid of the invention
Molecule, cell of the invention or binding molecule of the invention and pharmaceutically acceptable carrier.Described pharmaceutical composition can be with
It is any suitable form, (medication needed depending on patient).It can be provided in the form of unit dosage forms, usually be set
In sealing container, and a part that can be used as kit provides.Such kit usually (but being not required) is comprising making
With explanation.It may include multiple unit dosage forms.
Described pharmaceutical composition is suitable for any administration route appropriate, such as inject (including subcutaneous, muscle, abdominal cavity or quiet
Arteries and veins injection), sucking or the oral or approach such as intranasal or per anum.The composition can be by any known to pharmaceutical field
Method preparation, such as aseptically, by the way that active constituent is mixed with carrier or excipient.
According to of disease or illness (such as cancer, viral infection or autoimmune disease), patient for treatment
The dosage at body age and situation etc., invention formulation can change in a wider scope.Appropriate dosage will be by
Doctor finally determines.
According to the state of the art, it is presented to the peptide of cell surface, pMHC compound together with MHC molecule or passs
In the cell of pMHC compound, T cell or B cell can be activated, it is made to play a role.
Therefore, the cell of peptide of the invention, pMHC compound or submission pMHC compound can be with the shape of vaccine composition
Formula provides.The vaccine composition can be used for treating or preventing cancer.All this kind of compositions are included in the present invention.
It should be understood that the vaccine can be diversified forms (Schlom J.J Natl Cancer Inst.2012 104 (8): 599-
613).For example, peptide of the invention be used directly for immune patients (Salgaller ML.Cancer Res.1996.56 (20):
4749-57 and Marchand M.Int J Cancer.1999.80 (2): 219-230).The vaccine composition can wrap
Containing additional peptide, so that peptide of the invention is one in peptide mixer.Adjuvant can be added in the vaccine composition, with enhancing
Immune response.Alternatively, the vaccine composition can be the shape for presenting the antigen presenting cell of peptide and MHC compound of the present invention
Formula.Preferably, the antigen presenting cell is immunocyte, more preferably dendritic cells.The peptide can also be with pulse to carefully
The surface (Thurner BI.et al., J.Exp.Med.1999.190:1669) of born of the same parents, or can be by the coding of peptide of the present invention
Nucleic acid is introduced into dendritic cells, for example, using electroporation (Van Tendeloo, VF.etal., Blood 2001.98:
49)。
Following specific embodiment, the present invention is further explained.It should be understood that these embodiments be merely to illustrate the present invention and
It is not used in and limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition,
Such as (Sambrook and Russell et al., molecular cloning: laboratory manual (Molecular Cloning-A Laboratory
Manual) (third edition) (2001) CSHL publishing house) described in condition, or according to the normal condition proposed by manufacturer.Unless
In addition illustrate, otherwise percentage and number are calculated by weight.
Unless stated otherwise, otherwise material used in the embodiment of the present invention and reagent are commercial product.
Embodiment 1 is derived from the polypeptide of Epstein-Barr virus LMP1 antigen by Mass Spectrometric Identification
Before carrying out Mass Spectrometric Identification, the present invention further demonstrates LMP1 antigen table in kinds of tumor cells
It reaches.Specifically, (nanostring) is detected using digital unimolecule multiple gene expression spectrum analysis system.As a result it shows
Show, LMP1 antigen has expression in the tumor tissues such as nasopharyngeal carcinoma, gastric cancer, Burkitt lymphoma and Hodgkin lymphoma.
HLA- small peptide compound is purified using commercial antibodies BB7.2.Specifically, with containing non-ionic surfactant
The buffer of agent Triton X-100 (1%v/v) cracks tumour cell, and 1ml lysate is added by 2*10^7 cell, rolls at 4 DEG C
It is dynamic to be incubated for 1h.Centrifugation removal cell fragment, supernatant elder generation and antibody incubation, it is " anti-to add rProtein A-Sepharose capture
Body-HLA- small peptide compound ".Column is crossed, is collected " rProtein A-Sepharose- antibody-HLA- small peptide compound ".Use less salt
Pillar is washed with high-salt buffer, is finally hung on HLA- small peptide compound on immune affinity column with 10% acetic acid elution, then pass through
It crosses 95 DEG C of heating and 10KDa (AmiconR Ultr Centrifugal Filters, MILLIPORE) ultrafiltration membrance filter falls
Macromolecular finally obtains mixtures of polypeptides.
Mixtures of polypeptides is fractionated through 1260 high performance liquid chromatography of Agilent: ZORBAX 300SB-C18；1.0*150mm,
3.5um；Mobile phase A is 98% water, and 2% acetonitrile, 0.1% trifluoroacetic acid, Mobile phase B is 98% acetonitrile, 2% water, 0.1% trifluoro
Acetic acid, eluent gradient are that Mobile phase B by 5% is raised to 70% in 10 minutes.Each minute one fraction of collection.Total run time is
30 minutes.
After the HPLC fraction of polypeptide is concentrated, sample introduction nanoLC-MSMS network analysis:
5600 system of Eksigent nanoLC-AB Sciex Triple TOF: mass spectrum uses IDA analysis method.Liquid phase
Chromatography uses: pre-column: (Eksigent) NanoLC Trap column.5 μm C18.100 μm * 2.5cm, 910-00050, analysis
Column: C18-CL-120,3 μm of (Eksigent),0.075 × 150mm, 805-00120.
Dionex Ultimate3000-Thermo QE Plus system: mass spectrum uses ddms2 analysis method liquid chromatogram
Using: pre-column: (Thermo) Acclaim100,100um × 2cm, nanoViper, C18,5um, 100A,
164564, analytical column: (Thermo) Acclaim100,75um × 15cm, nanoViper, C18,3um, 100A,
164568。
The mobile phase A of flow chromatography of above-mentioned two system received is 98% water, and 2% acetonitrile, 0.1% formic acid, Mobile phase B is
98% acetonitrile, 2% water, 0.1% formic acid, eluent gradient are that Mobile phase B by 5% is raised to 50% in 74 minutes.Total run time
It is 90 minutes.
Mass spectrometry results search for the Uniprot number of human protein by library software ProteinPilot and Peaks is searched
According to library.According to software as a result, comprehensive analysis, finally obtains antigen short peptide sequence of the invention, as shown in Figure 1.
The preparation of 2 solubility pMHC compound of embodiment
The heavy chain and light chain (β 2m) of I type HLA-A*0201 molecule are respectively in the form of inclusion body in Escherichia coli
(E.coli) it expresses.It should be noted that obtain soluble pMHC compound, HLA-A*0201 molecule used in the present embodiment
Heavy chain does not include its transmembrane region and cytoplasmic region.In addition, biotinylation is carried out to soluble pMHC compound for convenience of subsequent, it can
Biotinylation tag is added in the C-terminal of heavy chain.Preparing present invention solubility pMHC compound, detailed process is as follows:
A. it purifies
The E.coli bacterium solution for collecting 100ml inducing expression heavy chain or light chain uses 10ml after 4 DEG C of 8000g are centrifuged 10min
PBS washing thalline is primary, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) later
Thallus is resuspended in concussion, and rotates in room temperature and be incubated for 20min, and later in 4 DEG C, 6000g is centrifuged 15min, discards supernatant, collection is forgiven
Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated for 5min；Add 30ml
The BugBuster of 10 times of dilution is mixed, and 4 DEG C of 6000g are centrifuged 15min；It discards supernatant, 30ml is added to dilute 10 times of BugBuster
Inclusion body is resuspended, mixes, 4 DEG C of 6000g are centrifuged 15min, are repeated twice, and add 30ml 20mM Tris-HCl pH 8.0 that packet is resuspended
Contain body, mix, 4 DEG C of 6000g are centrifuged 15min, finally dissolve inclusion body, SDS-PAGE detection with 20mM Tris-HCl 8M urea
Inclusion body purity, BCA kit survey concentration.
B. renaturation
Peptide ILWRLGATI (synthesis of Nanjing Jin Sikang Biotechnology Co., Ltd) of the invention is dissolved in DMSO extremely
The concentration of 20mg/ml.The inclusion body of light chain and heavy chain 8M urea, 20mM Tris pH 8.0,10mM DTT dissolve, renaturation
Preceding addition 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA are further denaturalized.ILWRLGATI peptide is added with 25mg/L (final concentration)
Enter renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM
Reduced glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add the light chain of 20mg/L and the heavy chain of 90mg/L
(final concentration, heavy chain are added in three times, 8h/ times), renaturation carry out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detection renaturation
Success.
C. it is purified after renaturation
Make dialysis with the 20mM Tris pH 8.0 of 10 volumes to replace renaturation buffer, at least replacement buffer comes twice
Sufficiently reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into
On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volume).Instrument (the general electricity of GE is purified using Akta
Gas company), the 0-400mMNaCl linear gradient liquid that 20mM Tris pH 8.0 is prepared elutes albumen, and pMHC is about in 250mM
It is eluted at NaCl, collects all peak components, SDS-PAGE detects purity.The glue figure of obtained solubility pMHC compound of the invention is such as
Shown in Fig. 2.
D. biotinylation
It with Millipore super filter tube by the pMHC molecular concentration of purifying, while being 20mM Tris pH by buffer exchange
8.0, biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D- is then added
Biotin, 100 μ g/ml BirA enzymes (GST-BirA), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detects biotinylation
Completely.
E. the compound after purifying biological element
PMHC molecular concentration after being marked biotinylation with Millipore super filter tube is to 1ml, using gel permeation chromatography
The pMHC of purifying biological element, purifies instrument (GE General Electric Co. Limited) using Akta, pre-equilibrates HiPrepTM with filtered PBS
16/60 S200 HR column (GE General Electric Co. Limited), the concentrated biotinylation pMHC molecule of load 1ml, then with PBS with
The elution of 1ml/min flow velocity.
Embodiment 3 combines the TCR of pMHC compound of the present invention
It present embodiments provides using pMHC compound of the present invention and obtains the illustration of monoclonal T cell and TCR.
The known a variety of methods for obtaining TCR of those skilled in the art, including but not limited to, by TCR α from T cell clone
It is separated with the sequence of β chain, the T cell clone is by the cytositimulation of submission pMHC compound of the present invention.It obtains
TCR sequence can be cloned into above suitable carrier, then be expressed in Escherichia coli such as E.coli, or expression is in bacteriophage
Surface.
The T cell obtained using small peptide of the invention is cloned as shown in figure 3, using Quick-RNATMMiniPrep(ZYMO
Research the total serum IgE of above-mentioned T cell clone) is extracted, and obtains TCR sequence.The TCR α chain of this TCR and the area V of TCR β chain
Gene carries out splicing using lentiviruses transduction primary T cells with the conserved region area C of mouse TCR α chain and TCR β chain respectively, and streaming is thin
Born of the same parents' instrument detection expression is as shown in figure 4, further detect the TCR function and specificity of T cell clone by ELISPOT experiment.
The method they those skilled in the art known lentiviruses transduction primary T cells and detect cell function using ELISPOT experiment.This reality
Applying effector cell used in an IFN-γ ELISPOT experiment is the TCR transduction that the T cell clone and separate obtained in the present invention arrives
Primary T cells, target cell is the LCL-A02 cell for having loaded small peptide of the present invention, and control group is to have loaded other small peptides
The LCL-A02 cell of LCL-A02 cell and unsupported any small peptide.
Prepare ELISPOT plate first, ELISPOT experimental procedure is as follows: in the following order adding each component of test
Enter ELISPOT plate: 130 microlitres of target cells, 77000 cells/mls (obtaining about 10000 target cell/holes in total), 50 micro-
After rising effector cell (1000 LMP1TCR positive T cells), 20 μ l specificity small peptides are added in experimental group, and 20 μ l are added in control group
Non-specific small peptide, 20 μ l culture mediums (test medium) are added in blank group, and 3 multiple holes are arranged.Then be incubated overnight (37 DEG C,
5%CO2).Subsequent washing flat board simultaneously carries out secondary detection and colour developing, dry plate 1 hour, recycles immunodotting plate reading
Count (ELISPOT READER system；AID company) count the spot formed on film.Experimental result is as shown in figure 5, obtain
Specific antigen specific T-cell clones TCR has specific reaction to the LCL-A02 cell for loading small peptide of the present invention.
It can be in conjunction with pMHC compound of the invention, the present embodiment table in E.coli further to verify the TCR of acquisition
Up to having gone out soluble TCR albumen, and the combination of itself and pMHC compound is detected by BIAcore.It should be noted that can be according to existing
Technology obtains soluble TCR, including but not limited to, described in patent document PCT/CN2015/093806.
The combination activity of soluble TCR protein and pMHC compound is detected using BIAcore T200 real-time analyzer.
Coupling buffer (10mM sodium-acetate buffer, pH 4.77) is added in the antibody (GenScript) of anti-Streptavidin, then
Antibody is flowed through to the CM5 chip activated in advance with EDC and NHS, antibody is made to be fixed on chip surface, finally uses the salt of ethanol amine
Acid solution closes unreacted activating surface, completes coupling process, coupling horizontal about 15,000RU.Make the strepto- parent of low concentration
Then it is logical will to be crossed detection by pMHC logistics made from mode described in embodiment 2 for the chip surface that coated antibody is flowed through with element
Chip 2min, sealed joint are flowed through as reference channel, then by the biotin of 0.05mM with the flow velocity of 10 μ L/min in road, another channel
The mould remaining binding site of Avidin.
Using BIAcore Evaluation software computational dynamics parameter, obtain the TCR molecule of solubility of the invention with
The kinetic profile that pMHC compound of the present invention combines is as shown in fig. 6, map shows the combination of the two.
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.
Sequence table
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>it is derived from the antigen small peptide of LMP1
<130> P2017-1187
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213>artificial sequence
<400> 1
Ile Leu Trp Arg Leu Gly Ala Thr Ile
1 5
Claims (10)
1. a kind of peptide, which is characterized in that the peptide includes:
(I) amino acid sequence ILWRLGATI (SEQ ID NO:1)；Or
(II) there is 1,2 or 3 amino acid substitution and/or 1,2 or 3 amino acid to insert in SEQ ID NO:1
Enter and/or the amino acid sequence of 1,2 or 3 amino acid deletions；
Wherein, the peptide can form compound with MHC molecule.
2. a kind of pMHC compound, which is characterized in that the compound includes peptide described in claim 1.
3. a kind of isolated cell, which is characterized in that the cell surface presents pMHC compound described in claim 2.
4. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules include the nucleic acid sequence of peptide described in coding claim 1
Or its complementary series.
5. a kind of carrier, which is characterized in that the carrier contains nucleic acid molecules described in claim 4.
6. a kind of host cell, which is characterized in that contain the carrier described in claim 5 in the cell.
7. a kind of molecule, which is characterized in that the molecule can be in conjunction with institute in peptide described in claim 1 and/or claim 2
State pMHC compound.
8. a kind of isolated monoclonal T cell, which is characterized in that pMHC compound described in the cell combination claim 2.
9. peptide described in claim 1, pMHC compound as claimed in claim 2, cell as claimed in claim 3, right are wanted
The purposes of nucleic acid molecules described in asking 4, molecule as claimed in claim 7 or monoclonal T cell according to any one of claims 8, it is special
Sign is, is used to prepare the drug of prevention or treating cancer.
10. a kind of pharmaceutical composition, which is characterized in that the composition contains pharmaceutically acceptable carrier and claim
Peptide described in 1, pMHC compound as claimed in claim 2, cell as claimed in claim 3, molecule as claimed in claim 7 or
Monoclonal T cell according to any one of claims 8.
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| US20090305324A1 (en) * | 2005-10-28 | 2009-12-10 | Medical And Biological Laboratories Co., Ltd | Cytotoxic t-cell epitope peptides that specifically attack epstein-barr virus-infected cells and uses thereof |
| WO2016203577A1 (en) * | 2015-06-17 | 2016-12-22 | 株式会社医学生物学研究所 | Cytotoxic t-cell epitope peptide and use thereof |
| US20170056473A1 (en) * | 2015-09-01 | 2017-03-02 | The Catholic University Of Korea Industry-Academic Cooperation Foundation | Ebv antigen specific t-cell receptor and use thereof |
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| CN107001444A (en) * | 2014-12-17 | 2017-08-01 | 中国科学院广州生物医药与健康研究院 | Recognize the φt cell receptor of Epstein-Barr virus small peptide |
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| US20090305324A1 (en) * | 2005-10-28 | 2009-12-10 | Medical And Biological Laboratories Co., Ltd | Cytotoxic t-cell epitope peptides that specifically attack epstein-barr virus-infected cells and uses thereof |
| CN107001444A (en) * | 2014-12-17 | 2017-08-01 | 中国科学院广州生物医药与健康研究院 | Recognize the φt cell receptor of Epstein-Barr virus small peptide |
| WO2016203577A1 (en) * | 2015-06-17 | 2016-12-22 | 株式会社医学生物学研究所 | Cytotoxic t-cell epitope peptide and use thereof |
| US20170056473A1 (en) * | 2015-09-01 | 2017-03-02 | The Catholic University Of Korea Industry-Academic Cooperation Foundation | Ebv antigen specific t-cell receptor and use thereof |
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