CN106478797A - Stem from the tumor antigen small peptide of SAGE1 - Google Patents
Stem from the tumor antigen small peptide of SAGE1 Download PDFInfo
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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- C07—ORGANIC CHEMISTRY
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- 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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- 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
- C07K16/303—Liver or Pancreas
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- C07—ORGANIC CHEMISTRY
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- 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
- C07K16/3053—Skin, nerves, brain
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- 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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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
Abstract
The present invention relates to the newfound small peptide stemming from tumor antigen SAGE1, the purposes of the complex that this small peptide is formed with MHC molecule and described small peptide and complex.Meanwhile, the invention still further relates to the molecule being combined with above-mentioned small peptide or complex, and the purposes of these molecules.
Description
Technical field
The present invention relates to the newfound small peptide stemming from tumor antigen SAGE1, it is compound that this small peptide and MHC molecule are formed
Thing and the purposes of described small peptide and complex.Meanwhile, the invention still further relates to the molecule being combined with above-mentioned small peptide or complex, with
And the purposes of these molecules.
Background technology
It is known that under many pathological conditions, such as infection, cancer, autoimmune disease etc., all can there is some specific point
Sub is not suitable for expression.Therefore, these molecules have just become " labelling " of pathology or abnormality.These molecules not only can conduct
The label of medical diagnosis on disease, it may also be used for produce diagnostic reagent and/or therapeutic agent.For example, produce spy with the label of cancer
Fixed antibody.In addition, these molecules can also activated cell toxic T lymphocyte (CTL) effectively specific immune response,
Play antitumor efficiency, at the same time it can also the φt cell receptor that can combine with this " labelling " is obtained by the CTL of activation
(TCR) as therapeutic agent.Therefore, these molecules, play very important effect in the diagnosis and treatment of relevant disease.
For tumor, different endogenic tumor antigen molecules delivered by existing a lot of documents.But this is not
The real target spot of relevant disease, because causing CTL immune response and incomplete tumor antigen molecule, and comes from
The specific CTL epitope (Epitope) of antigen.Generally, tumor antigen in the cell pass through proteolysiss by its
Be processed into the polypeptide fragment of 8-16 amino acid length, i.e. CTL epi-position, so with endoplasmic in ajor histocompatibility
Complex (MHC, the MHC of the mankind is commonly referred to HLA gene or HLA complex) molecule combines to form polypeptide-MHC complex
(peptide-MHCcomplex, pMHC), and pMHC submission supplies CD8 to cell surface the most at last+The TCR identification on T cell surface.
Although the autochthonous tumor antigen molecule of correlation has early been delivered, we are simultaneously unaware of the concrete polypeptide fragment being rendered out.
Therefore, either still it is used for producing diagnostic reagent or the therapeutic reagent of relevant disease as vaccine, identifying these is in
It is delivered to the polypeptide fragment of 8-16 amino acid length of cell surface, i.e. CTL epi-position, it is critical that.People in the art
Member is devoted to finding and determine that these are presented to the polypeptide fragment of target cells.
The discovery of this polypeptide fragment by submission is a complicated process with determination, because polypeptide by HLA submission is
The common results of the interaction of the enzymolysis of antigen protein and polypeptide fragment and HLA.This explanation, complete tumor antigen molecule
Any information can not be provided for the discovery of polypeptide fragment with identification.The method using computer simulation delivered by a lot of documents, such as
Public database SYFPEITHI (Rammensee, et al., Immunogenetics.1999 (50):213-219) and BIMAS
(Parker,et al.,J.Immunol.1994.152:163) prediction algorithm, is provided to identify which polypeptide fragment may be by
Submission.But a kind of this simply prediction, has very big uncertainty, because after it is not real intracellular process and translation
Modification.Tumor tissues are after treatment, it is possible to use mass spectrograph Direct Identification goes out what tumor cell surface was gone out by submission
Polypeptide fragment is although this is a complicated process, but the result so obtaining is very reliable.Meanwhile, mass spectrometric spirit
Sensitivity is also sufficient to the modification after differentiating the polypeptide fragment of low concentration and translation, and therefore it is to find and determine tumor surface
The ideal tools of polypeptide fragment.
Research finds, SAGE1 antigen is in melanoma, bladder cancer, hepatocarcinoma, epidermoid carcinoma, nonsmall-cell lung cancer and squamous
All there is expression in the tumor tissues such as cell carcinoma, and do not express in the most normal structures in addition to testis (Martelange V1,
De Smet C,De Plaen E,Lurquin C,Boon T.Cancer Res.2000；60(14):3848-55；
Atanackovic D,etal.,Cancer Biol Ther.2006；5(9):1218-25).Therefore, stem from SAGE1 antigen
Peptide, as the target spot of above-mentioned kinds cancer, not only can be used as the label of above-mentioned medical diagnosis on disease, it may also be used for produce above-mentioned
The prevention reagent of disease and/or therapeutic agent, such as antibody or φt cell receptor.The present invention using spectrometer analysis and identifies swollen
What oncocyte surface was rendered stems from the new polypeptide fragment of tumor antigen SAGE1.
Content of the invention
Object of the present invention is to provide a kind of newfound small peptide stemming from tumor antigen SAGE1, this small peptide with
Complex and the purposes of described small peptide and complex that MHC molecule is formed.
A kind of a first aspect of the present invention, there is provided peptide, described peptide comprises：
() aminoacid sequence VLSTAPPQL (SEQ ID NO:1)；Or
() is in SEQ ID NO:There are in 11,2 or 3 aminoacid replacement, and/or 1,2 or 3 aminoacid
Insertion, and/or the aminoacid sequence of 1,2 or 3 aminoacid deletion；
Wherein, described peptide can form complex with MHC molecule.
In another preference, described peptide is made up of 7-25 aminoacid.
In another preference, described peptide is made up of 8-16 aminoacid.
In another preference, the aminoacid sequence of described peptide is SEQ ID NO:1.
A kind of a second aspect of the present invention, there is provided pMHC complex, described complex comprises first aspect present invention institute
The peptide stated.
In another preference, the aminoacid sequence of the peptide in described pMHC complex is SEQ ID NO:1.
In another preference, the type of MHC molecule is HLA-A*02.
In another preference, the type of MHC molecule is HLA-A*0201.
In another preference, described pMHC complex is polymer.
In another preference, described pMHC complex is solvable.
In another preference, described pMHC complex is biotinylated.
A kind of a third aspect of the present invention, there is provided detached cell, described cell surface presents second aspect present invention
Described pMHC complex.
A kind of a fourth aspect of the present invention, there is provided nucleic acid molecules, described nucleic acid molecules comprise to encode first party of the present invention
The nucleotide sequence of peptide described in face or its complementary series.
A kind of a fifth aspect of the present invention, there is provided carrier, described carrier contains the nucleic acid described in fourth aspect present invention
Molecule.
A kind of a sixth aspect of the present invention, there is provided host cell, contains described in fifth aspect present invention in described cell
Carrier.
A kind of a seventh aspect of the present invention, there is provided molecule, described molecule can be in conjunction with described in first aspect present invention
PMHC complex described in peptide and/or second aspect present invention.
In another preference, described molecule can specifically bind peptide described in first aspect present invention and/or this
PMHC complex described in bright second aspect.
In another preference, described molecule is φt cell receptor.
In another preference, described φt cell receptor is solvable.
In another preference, described molecule is antibody or its binding fragment.
In another preference, described antibody is monoclonal antibody.
A kind of a eighth aspect of the present invention, there is provided detached monoclonal T cell, described T cell combines the present invention second
PMHC complex described in aspect.
In another preference, pMHC complex described in described T cell specific binding second aspect present invention.
A ninth aspect of the present invention, there is provided pMHC described in peptide, second aspect present invention described in first aspect present invention is multiple
The purposes of cell described in compound or third aspect present invention, for activating and/or separating T cell.
A tenth aspect of the present invention, there is provided pMHC described in peptide, second aspect present invention described in first aspect present invention is multiple
The purposes of compound, for screening φt cell receptor or antibody library.
A eleventh aspect of the present invention, there is provided pMHC described in peptide, second aspect present invention described in first aspect present invention
Described in nucleic acid molecules described in cell, fourth aspect present invention, seventh aspect present invention described in complex, third aspect present invention
The purposes of T cell described in molecule or eighth aspect present invention, for the medicine of preparation prevention or treating cancer.
A kind of a twelveth aspect of the present invention, there is provided pharmaceutical composition, described compositionss contain pharmaceutically acceptable
Described in pMHC complex, third aspect present invention described in peptide, second aspect present invention described in carrier and first aspect present invention
Molecule described in cell, seventh aspect present invention or T cell described in eighth aspect present invention.
In another preference, described pharmaceutical composition is vaccine.
A thirteenth aspect of the present invention, there is provided a kind of method of prevention or treatment disease, applies including to the object needing
With described in pMHC complex, third aspect present invention described in peptide, second aspect present invention described in appropriate first aspect present invention
Molecule described in cell, seventh aspect present invention or T cell described in eighth aspect present invention.
A fourteenth aspect of the present invention, there is provided a kind of acquisition is tied with pMHC complex described in right second aspect present invention
The method of the molecule closing, including：
() is by candidate molecules and pMHC complex contacts described in second aspect present invention；
() filters out the molecule being combined with pMHC complex in ().
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, thus constituting new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description
Fig. 1 is the representative mass spectrum identifying small peptide of the present invention.
Fig. 2 is the SDS-PAGE glue figure of solubility pMHC complex of the present invention.Left side band is molecular weight marker, right side bar
Band is respectively light chain and the heavy chain of MHC molecule.
Fig. 3 is the double positive staining result of CD8+ and the tetramer-PE of monoclonal cell.
Fig. 4 is the ELISPOT experimental result picture of monoclonal cell.
The BIAcore collection of illustrative plates that Fig. 5 is combined with pMHC complex of the present invention for sTCR.
Specific embodiment
The present invention, by extensively in-depth study, obtains the peptide stemming from antigen SAGE1, this peptide by MHC molecule is in
It is delivered to tumor cell surface, as tumor marker.Therefore, the invention provides stemming from the peptide of antigen SAGE1, this peptide with
Complex and the purposes of described peptide and complex that MHC molecule is formed.Meanwhile, the invention still further relates to above-mentioned peptide or complex
In conjunction with molecule.It should be understood that in the present invention, the peptide of the present invention is used interchangeably with polypeptide of the present invention or small peptide of the present invention, all
Refer to the peptide stemming from antigen SAGE1 that the present invention provides.
Specifically, a first aspect of the present invention provides a kind of peptide, and described peptide comprises：
() aminoacid sequence VLSTAPPQL (SEQ ID NO:1)；Or
() is in SEQ ID NO:There are in 11,2 or 3 aminoacid replacement, and/or 1,2 or 3 aminoacid
Insertion, and/or the aminoacid sequence of 1,2 or 3 aminoacid deletion；
Wherein, state peptide and can form complex with MHC molecule.
Aminoacid replacement means in same position, and certain amino acid residue is substituted by other amino acid residues.Insertion
Amino acid residue can insert in any position, and the amino acid residue of insertion can also be completely or partially adjacent one another are, or insertion
Aminoacid between not adjacent one another are.Can be from SEQ ID NO：1,2 or 3 aminoacid are deleted in 1 sequence.
It is known to those skilled in the art that peptide of the present invention can one or more positions between aminoacid sequence
Carry out post translational modification.The example of post translational modification can be in the Curr Opin Immunol.2006 such as Engelhard 2 months；
18(1)：Find in 92-7, and include phosphorylation, acetylation and deamidization.
It is preferred that peptide of the present invention and MHC are incorporated into the binding site peptide point of MHC molecule.Generally, the repairing of foregoing description
The aminoacid of decorations will not destroy the binding ability of described peptide and MHC.In one preferred embodiment, described aminoacid is repaiied
Decorations improve the ability that peptide is combined with MHC.For example, mutation is likely to occur in the binding site of peptide and MHC.These binding sites and
On binding site, preferred residue is known in the art, especially (referring to such as the peptide which combines HLA-A*02
Parkhurst etc., J.Immunol.157:2539-2548(1996)).
More specifically, the length of the aminoacid of the peptide of the present invention can be 7-25, preferably 8-16, it is preferred that 9,10,
Or 11.
Peptide of the present invention can be by VLSTAPPQL (SEQ ID NO：1) form, or mainly by VLSTAPPQL (SEQ
ID NO：1) form, it corresponds to the position of the 127-135 residue of SAGE1 full length protein.
Invention also provides SEQ ID NO：Albumen shown in 1 or the analog of peptide.These analog and natural peptide difference
Can be difference on aminoacid sequence or the difference on the modified forms not affecting 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 technology, such as by radiation or sudden and violent
It is exposed to mutagenic agent and produces random mutagenesises, also can pass through site-directed mutagenesis or the biological technology of other known moleculars.Analog
Also include the analog with the residue (as D- aminoacid) different from natural L-amino acids, and have non-naturally occurring or
The analog of the aminoacid (as β, gamma-amino acid) of synthesis.It should be understood that the peptide of the present invention is not limited to the above-mentioned representativeness enumerating
Peptide.
(generally the not changing primary structure) form of modification includes：The chemically derived form such as acetylation of inner or in vitro peptide
Or it is carboxylated.Modify and also include glycosylation, such as those carry out glycosyl in the synthesis and processing of peptide or in further processing step
Change the peptide modified and produce.This modification can carry out (the glycosylation as mammal of glycosylated enzyme by being exposed to peptide
Enzyme or deglycosylating enzyme) and complete.Modified forms are also included with phosphorylated amino acid residue (such as phosphotyrosine, phosphoric acid silk
Propylhomoserin, phosphothreonine) sequence.Also include being modified thus improve its anti-Proteolytic enzyme performance or optimizing solubility property
Peptide.
In the present invention, " SEQ ID NO：Albumen conservative variation's peptide shown in 1 " refers to and SEQ ID NO：1 aminoacid
Sequence is compared, and has at most 3, and more preferably at most 2 aminoacid are replaced by the similar or close aminoacid of property and formed peptide.
These conservative variation's peptides carry out amino acid substitution preferably based on table 1 and produce.
Table 1
| Initial residue | Representational replacement | Preferably 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 be closed with Merrifield synthetic method (be otherwise known as polypeptide solid-state reaction method) is simple
Become.The peptide of GMP rank can use the solid phase synthesis of polypeptide system (Multiple Peptide Systems, San Diego, CA)
Technology is synthesized.Or, described peptide can be re-combined into, if it is desired, can be synthesized with methods known in the art.
Typically such method is related to the use of carrier, and described carrier includes the nucleotide sequence of coded polypeptide, express polypeptide in vivo；Example
As expressed in antibacterial, yeast, insecticide or mammalian cell.Or, table can also be carried out using external cell-free system
Reach.Such system is known in the art, and can obtain from commercial channels.Described peptide can be detached and/or with base
Pure form in basis provides.For example, they can be provided in the form of other peptides or albumen with a kind of there is no.
Tumor antigen is processed into the polypeptide piece for 8-16 amino acid length by proteolysiss in the cell
Section, i.e. CTL epi-position, and then combine to form polypeptide-MHC complex (peptide-MHC with the MHC molecule in endoplasmic
Complex, pMHC), submission is to cell surface together.Therefore, a second aspect of the present invention provides a kind of pMHC complex, institute
State the peptide comprising in complex described in first aspect present invention.It is preferred that described polypeptide is incorporated into the peptide-binding groove of MHC molecule
On.Described MHC molecule can be MHC I quasi-molecule or MHC class Ⅱmolecule it is preferable that described MHC molecule is MHC I class divides
Son.In one preferred embodiment, described MHC molecule is HLA-A*02, it is highly preferred that described MHC molecule is HLA-A*
0201.
PMHC complex of the present invention can be existed with multimeric forms, for example, dimer or the tetramer or five
Aggressiveness or six aggressiveness or eight aggressiveness or bigger.Produce the polymeric proper method of pMHC and may be referred to pertinent literature, such as
(Greten et al., Clin.Diagnostic Lab.Immunol.2002：216-220).
Generally, can be produced with being combined by fluorescent labeling Streptavidin with the pMHC complex with biotin residue
PMHC polymer.Or, described pMHC polymer can also be used as molecular scaffold by immunoglobulin and be formed.At this it is
In system, the extracellular region of MHC molecule and the constant region of heavy chain immunoglobulin are passed through a short catenation sequence (linker) and are combined
Together.In addition, forming pMHC polymer can also utilize carrier molecule, such as dextran (WO02072631).PMHC poly
Body is favorably improved the detection of part in connection, such as φt cell receptor.Or, improve pMHC complex in related application
Effect, such as activation T cell.
PMHC complex of the present invention can be provided with soluble form.For obtaining the pMHC complex of soluble form,
Preferably, in described pMHC complex, MHC molecule does not contain transmembrane region.Specifically, in pMHC complex, mhc class i molecule is permissible
It is made up of the ectodomain of its light chain and all or part of heavy chain.Or, MHC molecule is the piece only comprising its functional domain
Section.
The method producing solubility pMHC complex of the present invention is well known by persons skilled in the art, includes, but not limited to
Method described in the embodiment of the present invention 2.MHC molecule in solubility pMHC complex of the present invention can also utilize synthetic method
Produce, the then peptide refolding with the present invention.By determine peptide and MHC molecule whether can refolding it may be determined that the present invention
Peptide can form complex with which class MHC molecule.
The solubility pMHC complex of the present invention can be used for screening or detecting molecule in connection, such as TCR or antibody.
Methods described includes contacting described pMHC complex with bound fraction to be measured, and measure bound fraction to be measured whether with complex
In conjunction with.The assay method of the combination of pMHC complex is well known in the art.Preferably method includes but is not limited to, surface etc. from
Daughter is resonated, or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy.Or, this
Outward, described combination can be detected by carrying out functional examination to the biological response combining generation, such as release of cytokines or thin
Born of the same parents' apoptosis.
Similarly, the solubility pMHC complex of the present invention can be also used for screening TCR or antibody library.Using phage
Display technique come to build antibody library be well known in the art, such as list of references Aitken, Antibody phage display:
Described in Methods and Protocols (2009, Humana, New York).In one preferred embodiment, this
Bright pMHC complex is used for screening the multiformity TCR library being showed in phage particle surface.The TCR of described display library
Non-natural mutation may be contained.
Therefore, the solubility pMHC complex of the present invention can be fixed on suitable solid phase carrier by junctional complex.Gu
The example of phase carrier includes, but not limited to pearl, film, agarose gel, magnetic bead, substrate, pipe, post.PMHC complex is permissible
It is fixed on ELISA Sptting plate, magnetic bead or surface plasma resonance biosensor chip.PMHC complex is fixed to
The method of solid phase carrier is well known by persons skilled in the art, and includes, for example, using affine combination pair, such as biotin
And Streptavidin, or antibody and antigen.In one preferred embodiment, pMHC complex biotin labeling, and
It is fixed on the coated surface of Streptavidin.
Peptide of the present invention can together with MHC complex submission to cell surface.Therefore, present invention also offers one
Plant cell, described cell is capable of the pMHC complex of the submission present invention to its surface.Such cell can be mammalian cell,
It is preferred that immune system cell, and it is preferably special antigen-presenting cell, such as dendritic cell or B cell.Other are preferred
Cell include T2 cell (Hosken, et al., Science.1990.248:367-70).Present peptide of the present invention or
The cell of pMHC complex can be detached, it is preferred that in the form of cell colony, or provide in a substantially pure form.
Described cell can not be natural submission complex of the present invention, or described cell delivery is in the level of complex than natural
Height under state.Such cell can enter horizontal pulse process with peptide of the present invention and obtain.Pulse processes and is related to described
Peptide incubated cell a few houres are it is preferable that the concentration of peptide used is 10-5-10-12M.Additionally, described cell can also use HLA-A*02
Molecule is transduceed, the submission of further induction peptide.The cell of submission pMHC of the present invention complex can be used for separating T
Cell and φt cell receptor, described T cell is sorted out by described cell-stimulating and further, and then is also obtained in that expression
φt cell receptor on described T cell surface.
In one preferred embodiment, the method obtaining above-mentioned T cell is included using above-mentioned submission pMHC of the present invention
The fresh blood that the cytositimulation of complex obtains at healthy volunteer.Can be through the stimulation of several wheels, such as 3-4 wheel.Activation
The identification of T cell the release (ratio of cytokine by the presence of the T2 cell of the peptide pulse of the present invention, can be measured
As IFN-γ ELISpot tests).Using traget antibody, active cell can be sorted by flow cytometer (FACS),
The cell of sorting can be verified with amplification culture with further, for example, is detected by ELISpot and/or the cell for target cell
Toxicity and/or the dyeing of pMHC polymer are verified.TCR chain from the T cell clone of empirical tests can pass through cDNA end
Rapid amplifying (RACE) is exaggerated, and is sequenced.
Present invention also offers a kind of nucleic acid molecules, described nucleic acid molecules include encoding the nucleotide sequence of the peptide of the present invention.
Described nucleic acid can be cDNA.Described nucleic acid molecules can be mainly made up of the nucleotide sequence 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 nucleotide sequence can encode identical aminoacid sequence.
Present invention also offers a kind of carrier, described carrier includes nucleotide sequence of the present invention.Suitable carrier
It is known to vector construction field, including selection and other controlling elements, such as enhancer element of promoter.Of the present invention
Carrier include the sequence that is adapted for introduction into cell.Such as, described carrier can be expression vector, in this carrier, described polypeptide
Coded sequence controlled by its own cis-acting regulatory element, the design of carrier be easy to 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, such as plasmid, bites
Thalline, virus or other carriers, it contains one or more heterologous or restructuring nucleotide sequence.Suitable phage and virus
Carrier includes, but are not limited to：Lambda phage, EMBL phage, simian viruss, cattle wart virus, Epstein-Barr virus, adenopathy
Poison, herpesviruss, mouse sarcoma viruses, murine mammary tumor virus, slow viruss etc..
Present invention also offers a kind of binding molecule, described molecule can be used to as immunotherapeutic agent or diagnostic reagent.
This binding molecule can only combine with peptide, or the complex being formed with peptide and MHC molecule is combined.In latter event, described knot
Close molecule can partly be attached on MHC molecule, meanwhile, it is also combined with the peptide of the present invention.The bound fraction of the present invention is permissible
It is detached and/or solvable and/or non-naturally occurring, do not have equivalent in the Nature, and/or pure, and/or people
Work synthesis.
In a preference of the present invention, described binding molecule is φt cell receptor (TCR).Can be using international immunity
Genetics information system (IMGT) is describing TCR.Natural α β heterodimeric TCR has α chain and β chain.In a broad sense, each chain comprises
Variable region, bonding pad and constant region, β chain generally contains short variable region also between variable region and bonding pad, but this variable region
Often it is regarded as a part for bonding pad.
The TCR of the present invention can be any form known in the art.For example, described TCR can be heterodimer, or with
Presented in single-stranded.Described TCR can be soluble form (i.e. no cross-film or cytoplasmic domain), and specifically, described TCR can comprise
All or part of TCR ectodomain.Described TCR can also be the total length chain comprising its transmembrane region.Described TCR can be provided
To cell surface, such as T cell.
The TCR of solubility can be obtained in conjunction with the prior art in this area, for example, in the perseverance of the α and β chain of α β TCR
Introduce between localization and introduce artificial disulfide bond between artificial disulfide bond, or the α chain variable region in α β TCR and β chain constant region.
The TCR of the present invention can be used for for cytotoxic agent or immunostimulant being delivered to target cell, or it is thin to be transformed into T
Born of the same parents, enable the T cell of this TCR of expression to destroy tumor cell, to give in the therapeutic process be referred to as adoptive immunotherapy
Give patient.In addition, mutation can also be contained it is preferable that the TCR after mutation is to pMHC complex of the present invention in the TCR of the present invention
Affinity increase.The TCR of the present invention can be used alone, and also can be combined with covalent or other modes with conjugate, excellent
Choosing is combined with covalent manner.Described conjugate includes detectable, and (for diagnostic purpose, wherein said TCR for detection is in
Pass the presence of the cell of pMHC complex of the present invention), therapeutic agent, PK (protein kinase) modify part or any the above material
Combination combine or be coupled.The TCR of the present invention preferably can also be combined with covalent manner with the antibodies of anti-CD3, with weight
Orientation T cell, thus kill target cell.
In another preference, the binding molecule of the present invention is antibody.As used herein, term " antibody " refers to immune globulin
White molecule and the immunoactive portions of immunoglobulin molecules, i.e. the molecule containing specific binding site, its can with All Pure Nature,
Or part synthetic or whole synthetic.Term " antibody " include antibody fragment, its derivant, functional equivalents with
And homologous antibody, humanized antibody, described antibody fragment includes immunoglobulin land, and described land is antigen-binding site
Or with antigen-binding site homology.It can be with All Pure Nature or part synthetic or whole synthetic.Humanized antibody is permissible
It is the antibody modified, it contains the constant region of the variable region of non-human antibody (for example, mice) and human antibody.
The example of antibody can be immunoglobulin isotypes (such as IgG, IgE, IgM, IgD and IgA) and they with
The subclass of type；Fragment includes antigen binding domain, such as Fab, scFv, Fv, dAb, Fd；And double-chain antibody.Antibody can be many
Resist or monoclonal antibody, preferably monoclonal antibody.
Above-mentioned TCR is well known by persons skilled in the art with the preparation method of antibody, including but not limited to, from escherichia coli
Express in cell or insect cell, and be purified.
On the other hand, invention further provides the peptide of the present invention, pMHC complex, nucleic acid molecules, carrier, cell
And purposes in terms of pharmacy for the binding molecule.Described peptide, pMHC complex, nucleic acid, carrier, cell or binding molecule can be by
For treatment or prophylaxis of cancer, preferably melanoma, bladder cancer, hepatocarcinoma, epidermoid carcinoma, nonsmall-cell lung cancer and squamous cell carcinoma
Deng.
Present invention also offers a kind of pharmaceutical composition, it comprises the peptide of the present invention, pMHC complex, the nucleic acid of the present invention
The binding molecule of molecule, the cell of the present invention or the present invention, and pharmaceutically acceptable carrier.Described pharmaceutical composition is permissible
It is any suitable form, (medication needing depending on patient).It can be provided in the form of unit dosage forms, generally puts
In sealing container, and can be used as the part offer of test kit.Such test kit generally (but being not required) comprises to make
With explanation.It can comprise multiple described unit dosage forms.
Described pharmaceutical composition is applied to any suitable route of administration, and such as injection (includes subcutaneous, muscle, abdominal cavity or quiet
Arteries and veins is injected), suck or the oral or approach such as per nasal or per anum.Described compositionss can be by any known to pharmaceutical field
Prepared by method, such as aseptically, by mixing active component with carrier or excipient.
According to for treatment disease or disease (such as cancer, viral infection or autoimmune disease), patient
Body age and situation etc., the dosage of invention formulation can change in 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 complex together with MHC molecule or passs
In the cell of pMHC complex, T cell or B cell can be activated so as to play a role.
Therefore, the cell of the peptide of the present invention, pMHC complex or submission pMHC complex can be with the shape of vaccine combination
Formula provides.Described vaccine combination can be used for treatment or prophylaxis of cancer.This based composition all of is included in the present invention.
It should be understood that described vaccine can be various ways (Schlom J.J Natl Cancer Inst.2012104 (8)：599-
613).For example, the peptide of the present invention is used directly for immune patients (Salgaller ML.Cancer Res.1996.56 (20)：
4749-57and Marchand M.Int J Cancer.1999.80(2)：219-230).Described vaccine combination can comprise
Extra peptide is so that the peptide of the present invention is one of peptide mixer.Described vaccine combination can add adjuvant, is exempted from strengthening
Epidemic disease is reacted.Or, described vaccine combination can be the form of the antigen presenting cell presenting peptide of the present invention and MHC complex.
Preferably, described antigen-presenting cell is immunocyte, more preferably dendritic cell.Described peptide pulse can also arrive cell
Surface (Thurner BI.et al., J.Exp.Med.1999.190:, or can be by the code nucleic acid of peptide of the present invention 1669)
It is incorporated in dendritic cell, for example, using electroporation (Van Tendeloo, VF.etal., Blood 2001.98:49).
Following specific embodiment, is expanded on further the present invention.It should be understood that these embodiments be merely to illustrate the present invention and
It is not used in restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally 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 condition proposed by manufacturer.Unless
In addition illustrate, otherwise percentage ratio and number are calculated by weight.
Embodiment 1 stems from the polypeptide of SAGE1 antigen by Mass Spectrometric Identification
By the tumor cell line HCCC9810 (people's intrahepatic cholangiocarcinoma cell) purchased from ATCC, train at the standard conditions
Support.
Carry out purification HLA-A*02 complex using commercialization anti-HLA-A*02 antibody BB7.2.Specifically, with containing non-from
The buffer cell lysis of subtype surfactant Triton X-100 (1%v/v), add 1ml lysate by 2*10^7 cell,
Roll incubation 1h at 4 DEG C.Centrifugation removes cell debriss, supernatant elder generation and antibody incubation, adds rProtein A-Sepharose
Capture " antibody-HLA-A*02 complex ".Cross post, " rProtein A-Sepharose- antibody-HLA-A*02 is combined for collection
Thing ".Wash pillar with less salt and high-salt buffer, be finally hung on HLA complex on immune affinity column with 10% acetic acid eluting,
Heat through 95 DEG C again, and 10KDa (AmiconR Ultr Centrifugal Filters, MILLIPORE) ultrafilter membrane mistake
Filter macromole, finally give mixtures of polypeptides.
Mixtures of polypeptides is through Agilent 1260 high performance liquid chromatography fractional distillation：ZORBAX 300SB-C18；1.0*150mm,
3.5um；Mobile phase A is 98% water, 2% acetonitrile, 0.1% trifluoroacetic acid, and Mobile phase B is 98% acetonitrile, 2% water, 0.1% trifluoro
Acetic acid, eluent gradient is that in 10 minutes, Mobile phase B is raised to 70% by 5%.Collect a fraction within each minute.Total run time is
30 minutes.
After the HPLC fraction of polypeptide is concentrated, sample introduction nanoLC-MSMS systematic analysiss：
Eksigent nanoLC-AB Sciex Triple TOF 5600 system：Mass spectrum adopts IDA analysis method.Liquid phase
Chromatograph adopts：Pre-column：(Eksigent) column.5 μm C18.100 μm * 2.5cm, 910-00050 of NanoLC Trap, analysis
Post：(Eksigent)C18-CL-120,3μm,0.075 × 150mm, 805-00120.
Dionex Ultimate3000-Thermo QE Plus system：Mass spectrum adopts ddms2 analysis method. liquid chromatograph
Using：Pre-column：(Thermo)Acclaim100um × 2cm, nanoViper, C18,5um, 100A,
164564, analytical column：(Thermo)Acclaim75um × 15cm, nanoViper, C18,3um, 100A,
164568.
The mobile phase A of flow chromatography received of above-mentioned two system is 98% water, 2% acetonitrile, 0.1% formic acid, and Mobile phase B is
98% acetonitrile, 2% water, 0.1% formic acid, eluent gradient is that in 74 minutes, Mobile phase B is raised to 50% by 5%.Total run time
For 90 minutes.
Mass spectrometry results, by searching library software ProteinPilot and Peaks, search for the Uniprot number of human protein
According to storehouse.The result being given according to software, as shown in figure 1, finally draw the antigen short peptide sequence of the present invention.
The preparation of embodiment 2 solubility pMHC complex
The heavy chain of I type HLA-A*0201 molecule and light chain (β 2m) respectively in the form of inclusion body in escherichia coli
(E.coli) express.It should be noted that for the pMHC complex obtaining solubility, HLA-A*0201 molecule used in the present embodiment
Heavy chain does not comprise its transmembrane region and cytoplasmic region.In addition, carrying out biotinylation for convenience of the follow-up pMHC complex to solubility, can
Biotinylation tag is added with the C-terminal in heavy chain.The detailed process preparing solubility pMHC complex of the present invention is as follows：
A. purification
Collect the E.coli bacterium solution of 100ml abduction delivering heavy chain or light chain, after 4 DEG C of 8000g are centrifuged 10min, use 10ml
PBS washing thalline once, uses 5ml BugBuster Master Mix Extraction Reagents (Merck) acutely afterwards
Shake resuspended thalline, and in room temperature rotate incubation 20min, after 4 DEG C, 6000g be centrifuged 15min, supernatant discarded, collection is forgiven
Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation incubation 5min；Plus 30ml
The BugBuster of 10 times of dilution, mixes, and 4 DEG C of 6000g are centrifuged 15min；Supernatant discarded, plus the BugBuster of 10 times of 30ml dilution
Resuspended inclusion body, mixes, and 4 DEG C of 6000g are centrifuged 15min, are repeated twice, plus the resuspended bag of 30ml 20mM Tris-HCl pH 8.0
Contain body, mix, 4 DEG C of 6000g are centrifuged 15min, finally use 20mM Tris-HCl 8M carbamide to dissolve inclusion body, SDS-PAGE detects
Inclusion body purity, BCA test kit surveys concentration.
B. renaturation
The peptide VLSTAPPQL (synthesis of Beijing SBS Genetech gene technology company limited) of the present invention is dissolved in DMSO extremely
The concentration of 20mg/ml.The inclusion body of light chain and heavy chain is dissolved with 8M carbamide, 20mM Tris pH 8.0,10mM DTT, renaturation
Front addition 3M guanidine hydrochloride, 10mM Sodium Acetate Trihydrate, the further degeneration of 10mM EDTA.VLSTAPPQL peptide is added with 25mg/L (final concentration)
Enter renaturation buffer (0.4M L-Arginine, 100mM Tris pH 8.3,2mM EDTA, 0.5mM GSSG, 5mM
Reduced glutathion, 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 adds in three times, 8h/ time), renaturation carries out at least 3 days to completing at 4 DEG C, and can SDS-PAGE detection renaturation
Success.
C. purification after renaturation
Change renaturation buffer as dialysis with the 20mM Tris pH 8.0 of 10 volumes, at least change buffer twice
Fully 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).Using Akta purification instrument (the general electricity of GE
Gas company), the 0-400mMNaCl linear gradient liquid wash-out protein that 20mM Tris pH 8.0 prepares, pMHC is about in 250mM
Eluting at NaCl, collects all peaks component, and SDS-PAGE detects purity.The glue figure of the solubility pMHC complex of the present invention obtaining is such as
Shown in Fig. 2.
D. biotinylation
With Millipore super filter tube by the pMHC molecular concentration of purification, buffer exchange is 20mM Tris pH simultaneously
8.0, it is subsequently adding biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D-
Biotin, 100 μ g/ml BirA enzyme (GST-BirA), overnight, whether SDS-PAGE detection biotinylation for incubated at room mixture
Completely.
E. the complex after purifying biological elementization
With Millipore super filter tube by the pMHC molecular concentration after biotinylation labelling to 1ml, using gel permeation chromatography
The pMHC of purifying biological elementization, using Akta purification instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibration HiPrepTM
16/60S200HR post (GE General Electric Co. Limited), loads the concentrated biotinylation pMHC molecule of 1ml, then uses PBS with 1ml/
Min flow velocity eluting.
Embodiment 3 combines the TCR of pMHC complex of the present invention
Present embodiments provide the illustration obtaining monoclonal T cell and TCR using pMHC complex of the present invention.
Those skilled in the art know the multiple methods obtaining TCR, including but not limited to, by TCR α from T cell clone
Separate with the sequence of β chain, described T cell clone is by the cytositimulation of submission pMHC of the present invention complex.Obtain
TCR sequence can be cloned into above suitable carrier, then expresses in escherichia coli such as E.coli, or expression is in phage
Surface.
The T cell clone being obtained using the small peptide of the present invention is as shown in Figure 3.Tested by ELISPOT and detect this T further
The function of cell clone and specificity.Those skilled in the art know the method using ELISPOT experiment detection cell function.This
In embodiment IFN-γ ELISPOT experiment, effector lymphocyte used is the T cell clone obtaining in the present invention, and target cell is load
The T2 cell of small peptide of the present invention, matched group is to have loaded the T2 cell of other small peptides and the T2 cell of unsupported any small peptide.
Prepare ELISPOT flat board first, ELISPOT experimental procedure is as follows：In the following order each component of test is added
Enter ELISPOT flat board：40 μ l T2 cells 5 × 105Individual cells/ml (i.e. 20,000 T2 cells/well), 40 μ l effector lymphocytes
After (2000 T cell clone/holes), experimental group adds 20 μ l specificity small peptides, and matched group adds 20 μ l non-specificity small peptides, empty
White group adds 20 μ l culture medium (test medium), and arranges 2 multiple holes.Then be incubated overnight (37 DEG C, 5%CO2).Subsequently wash
Flat board simultaneously carries out secondary detection and colour developing, flat board is dried 1 hour, recycles immunodotting plate reader (ELISPOT
READER system；AID company) speckle being formed is counted on film.Experimental result is as shown in figure 4, the specific antigen obtaining is special
Property T cell clone to load small peptide of the present invention T2 cell have specific reaction.
Use Quick-RNATMMiniPrep (ZYMO research) extracts the total serum IgE of above-mentioned T cell clone, and obtains
TCR sequence.TCR for verifying acquisition further can be in conjunction with the pMHC complex of the present invention, the present embodiment table in E.coli
Reach solvable TCR albumen, and detect the combination of itself and pMHC complex by BIAcore.It should be noted that can be according to existing
Technology obtaining the TCR of solubility, including but not limited to, described in patent documentation PCT/CN2015/093806.
Detect the binding activity of soluble TCR protein and pMHC complex using BIAcore T200 real-time analyzer.
The antibody (GenScript) of anti-Streptavidin is added coupling buffer (10mM sodium-acetate buffer, pH 4.77), then
Antibody is flow through the CM5 chip being activated with EDC and NHS in advance, makes antibody be fixed on chip surface, finally use the salt of ethanolamine
Acid solution closes unreacted activating surface, completes coupling process, coupling level is about 15,000RU.Make the strepto- parent of low concentration
Flow through the chip surface of coated antibody with element, then logical by crossing detection by the pMHC logistics that mode described in embodiment 2 is obtained
Road, another passage is as reference channel, then the biotin of 0.05mM is flow through chip 2min, sealed joint with the flow velocity of 10 μ L/min
The remaining binding site of mould Avidin.
Using BIAcore Evaluation computed in software kinetic parameter, obtain the TCR molecule of solubility of the present invention with
The kinetic profile that pMHC complex of the present invention combines is as shown in figure 5, collection of illustrative plates shows the combination of the two.Meanwhile, also utilize
The combination of TCR molecule and other several irrelevant antigen small peptides and HLA complex that said method have detected solubility of the present invention is lived
Property, result shows that TCR molecule of the present invention is all no combined with other irrelevant antigen.
The all documents referring in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
1. a kind of peptide is it is characterised in that described peptide comprises：
() aminoacid sequence VLSTAPPQL (SEQ ID NO:1)；Or
() is in SEQ ID NO:There are in 11,2 or 3 aminoacid replacement, and/or 1,2 or 3 aminoacid are inserted
Enter, and/or the aminoacid sequence of 1,2 or 3 aminoacid deletion；
Wherein, described peptide can form complex with MHC molecule.
2. a kind of pMHC complex is it is characterised in that described complex comprises the peptide described in claim 1.
3. a kind of detached cell is it is characterised in that described cell surface presents pMHC complex described in claim 2.
4. a kind of nucleic acid molecules are it is characterised in that described nucleic acid molecules comprise to encode the nucleotide sequence of peptide described in claim 1
Or its complementary series.
5. a kind of carrier is it is characterised in that described carrier contains the nucleic acid molecules described in claim 4.
6. a kind of host cell is it is characterised in that containing the carrier described in claim 5 in described cell.
7. a kind of molecule is it is characterised in that described molecule can be in conjunction with institute in peptide described in claim 1 and/or claim 2
State pMHC complex.
8. a kind of detached monoclonal T cell is it is characterised in that described cell combines pMHC complex described in claim 2.
9. the cell described in the peptide described in claim 1, the pMHC complex described in claim 2, claim 3, right are wanted
Seek the purposes of the nucleic acid molecules described in 4, the molecule described in claim 7 or the T cell described in present claims 8, for preparing
Prevention or the medicine for the treatment of cancer.
10. a kind of pharmaceutical composition, described compositionss contain peptide described in pharmaceutically acceptable carrier and claim 1,
Cell described in pMHC complex described in claim 2, claim 3, the molecule described in claim 7 or claim 8
Described T cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107936109A (en) * | 2016-10-12 | 2018-04-20 | 广东香雪精准医疗技术有限公司 | Tumour antigen small peptide derived from SAGE1 |
| CN108997481A (en) * | 2017-06-07 | 2018-12-14 | 中国科学院广州生物医药与健康研究院 | Antigen small peptide from LMP1 |
| CN110272483A (en) * | 2018-03-14 | 2019-09-24 | 中国科学院广州生物医药与健康研究院 | Identify the T cell receptor of SAGE1 antigen small peptide |
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| WO2010006343A2 (en) * | 2008-07-11 | 2010-01-14 | Sloan-Kettering Institute For Cancer Research | Glycopeptide constructs and uses thereof |
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| WO2010006343A2 (en) * | 2008-07-11 | 2010-01-14 | Sloan-Kettering Institute For Cancer Research | Glycopeptide constructs and uses thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107936109A (en) * | 2016-10-12 | 2018-04-20 | 广东香雪精准医疗技术有限公司 | Tumour antigen small peptide derived from SAGE1 |
| CN107936109B (en) * | 2016-10-12 | 2022-02-08 | 香雪生命科学技术（广东）有限公司 | Tumor antigen short peptide derived from SAGE1 |
| CN108997481A (en) * | 2017-06-07 | 2018-12-14 | 中国科学院广州生物医药与健康研究院 | Antigen small peptide from LMP1 |
| CN110272483A (en) * | 2018-03-14 | 2019-09-24 | 中国科学院广州生物医药与健康研究院 | Identify the T cell receptor of SAGE1 antigen small peptide |
| CN110272483B (en) * | 2018-03-14 | 2022-08-19 | 中国科学院广州生物医药与健康研究院 | T cell receptor for recognizing SAGE1 antigen short peptide |
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