CN108218976A - Tumour antigen small peptide derived from LMP1 - Google Patents
Tumour antigen small peptide derived from LMP1 Download PDFInfo
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- CN108218976A CN108218976A CN201611130630.6A CN201611130630A CN108218976A CN 108218976 A CN108218976 A CN 108218976A CN 201611130630 A CN201611130630 A CN 201611130630A CN 108218976 A CN108218976 A CN 108218976A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—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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- 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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- Peptides Or Proteins (AREA)
Abstract
The compound and the purposes of the small peptide and compound formed the present invention relates to the tumour antigen small peptide derived from LMP1, the small peptide with MHC molecule.Meanwhile the invention further relates to the molecules and the purposes of these molecules combined with above-mentioned small peptide or compound.
Description
Technical field
The present invention relates to the small peptide of newfound derivative self tumor antigen LMP1, which forms compound with MHC molecule
Object and the purposes of the small peptide and compound.Meanwhile the invention further relates to the molecule combined with above-mentioned small peptide or compound, with
And the purposes of these molecules.
Background technology
It is well known that under many pathological conditions, such as infection, cancer, autoimmune disease, can all there be some specific point
Son is not suitable for expressing.Therefore, these molecules are just into pathology or " label " of abnormality.These molecules not only can conduct
The marker of medical diagnosis on disease, it may also be used for generate diagnostic reagent and/or therapeutic agent.For example, generate 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) as therapeutic agent.Therefore, these molecules play very important effect in the diagnose and treat of relevant disease.
For tumour, have many documents and delivered different endogenic tumour antigen molecules.But this is not
The real target spot of relevant disease because causing the not complete tumour antigen molecule of CTL immune responses, and comes from
The specific CTL epitope (Epitope) of antigen.Under normal circumstances, tumour antigen in the cell by proteolysis by its
Be processed into the polypeptide fragment of 8-16 amino acid length, i.e. CTL epitopes, so with the ajor histocompatibility in endoplasmic
Complex (MHC, the MHC of the mankind is commonly referred to as HLA genes or HLA complexs) molecule combines and forms polypeptide-MHC compounds
(peptide-MHC complex, pMHC), and most pMHC submissions supply CD8 to cell surface at last+The TCR on T cell surface knows
Not.It is delivered although relevant autochthonous tumor antigen molecule is early existing, we are simultaneously unaware of the specific polypeptide piece being rendered out
Section.Therefore, these quilts are identified still for generating the diagnostic reagent or therapeutic reagent of relevant disease either as vaccine
The polypeptide fragment of 8-16 amino acid length of cell surface, i.e. CTL epitopes are presented to, is vital.Art technology
Personnel are dedicated to finding and determine these polypeptide fragments for being presented to target cell surface.
The discovery of this polypeptide fragment by submission is with determining it is a complicated process, because polypeptide is by HLA submissions
The common results of the interaction of the enzymolysis and polypeptide fragment and HLA of antigen protein.This explanation, complete tumour antigen molecule
For the discovery of polypeptide fragment any information can not be provided with identification.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) prediction algorithm, is provided 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 real intracellular processing and translates
Modification.Tumor tissues after treatment, can go out what tumor cell surface was gone out by submission using mass spectrograph Direct Identification
Polypeptide fragment obtaining the result is that very reliable although this is a complicated process in this way.Meanwhile mass spectrometric spirit
Sensitivity be also sufficient to differentiate low concentration polypeptide fragment and translation after modification, therefore it be find and determining tumor surface
The ideal tools of polypeptide fragment.
EBV is a kind of nerpes vinrus hominis of global range generally existing.In adult human body of the studies have shown that more than 95%
Containing the antibody for this virus, this also means that they were infected in a certain stage by this virus.It is most of to be felt
The internal all one's life of the people contaminated all generally can seldom go wrong there are EBV.However, in some cases, EBV and some cancers
The generation of disease is related, including Burkitt lymphoma (Burkitt ' s lymphoma), Hodgkin lymphoma (Hodgkin
Lymphoma), lymphocytic hyperplasia disease (PTLD) or nasopharyngeal carcinoma etc. after EBV positive grafts.For example, LMP1 and LMP2 belong to EBV
Incubation period memebrane protein can express (Raab-Traub N.Epstein-Barr virus in the by most nasopharyngeal carcinoma cells
pathogenesis of NPC[J].Semin Cancer Biol,2002,12(6):431-441.).Therefore, derived from LMP1
Peptide, not only can be as the marker of above-mentioned medical diagnosis on disease as the target spot of above-mentioned kinds cancer, it may also be used for generate it is above-mentioned
The prevention reagent and/or therapeutic agent of disease, such as antibody or T cell receptor.The present invention is identified using spectrometer analysis, is found for the first time
The polypeptide fragment of derivative self tumor antigen LMP1 that tumor cell surface is rendered.
Invention content
The object of the present invention is to provide a kind of small peptide of newfound derivative self tumor antigen LMP1, the small peptide with
The compound and the purposes of the small peptide and compound that MHC molecule is formed.
The first aspect of the present invention, provides a kind of peptide, and the peptide includes：
(I) amino acid sequence MLWRLGATI (SEQ ID NO:1)；Or
(II) is in SEQ ID NO:There is 1,2 or 3 amino acid substitution and/or 1,2 or 3 amino acid in 1
Insertion and/or the amino acid sequence of 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 compounds, 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 compounds 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 compounds are polymer.
In another preferred example, the pMHC compounds are soluble.
In another preferred example, the pMHC compounds are biotinylated.
The third aspect of the present invention, provides a kind of cell of separation, and the cell surface presents second aspect of the present invention
The pMHC compounds.
The fourth aspect of the present invention provides a kind of nucleic acid molecules, and the nucleic acid molecules, which include, encodes 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 the 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 with reference to described in first aspect present invention
PMHC compounds 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 compounds 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 monoclonal T cell of separation, and the T cell combines the present invention second
The aspect pMHC compounds.
In another preferred example, pMHC compounds described in the T cell specific binding second aspect of the present invention.
The ninth aspect of the present invention provides peptide described in first aspect present invention, pMHC described in second aspect of the present invention is answered
The purposes of cell described in object or third aspect present invention is closed, for activating and/or detaching T cell.
The tenth aspect of the present invention provides peptide described in first aspect present invention, pMHC described in second aspect of the present invention is answered
The purposes of object is closed, for screening T cell receptor or antibody library.
The eleventh aspect of the present invention provides peptide described in first aspect present invention, pMHC described in second aspect of the present invention
Cell described in compound, third aspect present invention, the nucleic acid molecules described in fourth aspect present invention, described in seventh 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, pMHC compounds described in second aspect of the present invention, described in third aspect 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, and the object including giving needs is applied
Described in suitable first aspect present invention peptide, pMHC compounds described in second aspect of the present invention, described in third aspect 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 acquisition and pMHC compound knots described in right second aspect of the present invention
The method of the molecule of conjunction, including：
(I) is by pMHC complex contacts described in candidate molecules and second aspect of the present invention；
The molecule that (II) is filtered out with pMHC compounds are combined in (I).
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to form new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Fig. 1 is the representative mass spectrogram for identifying small peptide of the present invention.
Fig. 2 is the SDS-PAGE glue figures of solubility pMHC compounds of the invention.Left-hand bar band is molecular weight marker
(Marker), right side band is respectively the light chain and heavy chain of MHC molecule.
Fig. 3 is the double positive staining results of CD8+ and the tetramer-PE of monoclonal cell.
Fig. 4 is the contractile studies figure of monoclonal cell obtained using small peptide of the present invention.
Fig. 5 is the BIAcore collection of illustrative plates that sTCR is combined with pMHC compounds of the present invention.
Specific embodiment
The present invention and in-depth study, obtains the peptide derived from antigen LMP1, which is presented by MHC molecule by extensive
To tumor cell surface, as tumor marker.Therefore, the present invention provides the peptide derived from antigen LMP1, which divides with MHC
The compound and the purposes of the peptide and compound that son is formed.Meanwhile the invention further relates to combined with above-mentioned peptide or compound
Molecule.It should be understood that in the present invention, peptide of the invention is used interchangeably with polypeptide of the present invention or small peptide of the present invention, all refer to this
The peptide derived from antigen LMP1 provided is provided.
Specifically, the first aspect of the present invention provides a kind of peptide, and the peptide includes：
(I) amino acid sequence MLWRLGATI (SEQ ID NO:1)；Or
(II) is in SEQ ID NO:There is 1,2 or 3 amino acid substitution and/or 1,2 or 3 amino acid in 1
Insertion and/or the amino acid sequence of 1,2 or 3 amino acid deletions；
Wherein, compound can be formed with MHC molecule by stating peptide.
Amino acid substitution means that in same position some amino acid residue is substituted by other amino acid residues.It is inserted into
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 be inserted into
Amino acid between it is not adjacent to each other.It can be from SEQ ID NO：1,2 or 3 amino acid is deleted in 1 sequence.
It is known to those skilled in the art that peptide of the present invention can be between amino acid sequence one or more positions
Carry out posttranslational modification.The example of posttranslational modification can be 2 months in the Curr such as Engelhard OpinImmunol.2006；18
(1)：It is found in 92-7, and including phosphorylation, acetylation and deamidization.
Preferably, peptide of the present invention is incorporated into the binding site peptide point of MHC molecule with MHC.In general, foregoing description is repaiied
The amino acid of decorations will not destroy the peptide and the binding ability of MHC.In one preferred embodiment, the amino acid is repaiied
Decorations improve the ability that peptide is combined 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 which combines HLA-A*02 (referring to, such as
Parkhurst etc., J.Immunol.157:2539-2548(1996)).
More specifically, the length of the amino acid of the peptide of the present invention can be 7-25, preferably 8-16, preferably, 9,10,
Or 11.
Peptide of the present invention can be by MLWRLGATI (SEQ ID NO：1) it forms or mainly by MLWRLGATI (SEQ
ID NO：1) it forms, corresponds to the position of the 129-137 residues of LMP1 full length proteins.
Invention also provides SEQ ID NO：The analog of albumen or peptide shown in 1.These analogs and natural peptide difference
It can be the difference on amino acid sequence or 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 analog with the residue (such as D- amino acid) different from natural L-amino acids and with non-naturally occurring or
The analog of the amino acid (such as β, gamma-amino acid) of synthesis.It should be understood that the peptide of the present invention is not limited to enumerated representativeness
Peptide.
Modification (not changing primary structure usually) form includes：Such as acetylation of the chemical derivative form of in vivo or in vitro peptide
Or carboxylated.Modification further includes glycosylation, and glycosyl is carried out in the synthesis and processing of peptide or in further processing step such as those
The peptide changed modification and generated.This modification can carry out the glycosylated enzyme (glycosylation of such as mammal by the way that peptide is exposed to
Enzyme or deglycosylating enzyme) and complete.Modified forms are further included with phosphorylated amino acid residue (such as phosphotyrosine, phosphoric acid silk
Propylhomoserin, phosphothreonine) sequence.It further includes and is modified to improve its anti-proteolytic properties or optimize solubility property
Peptide.
In the present invention, " SEQ ID NO：Albumen conservative variation peptide shown in 1 " refers to and SEQ ID NO：1 amino acid
Sequence is compared, and has at most 3, and 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 | Preferred substitution |
| 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 methods (be otherwise known as polypeptide solid-state reaction method)
Into.The peptide of GMP ranks 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 of coding 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 detached 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 is processed into the polypeptide piece for 8-16 amino acid length by proteolysis in the cell
Section, i.e. CTL epitopes, and then combined to form polypeptide-MHC compounds (peptide-MHC 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 compounds, institute
It states in compound comprising the 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 classes molecules or MHC class Ⅱmolecules, it is preferable that the MHC molecule is MHC I class molecules.
In one preferred embodiment, the MHC molecule is HLA-A*02, it is highly preferred that the MHC molecule is HLA-A*
0201。
PMHC compounds of the present invention can exist with multimeric forms, for example, dimer or the tetramer or five
Aggressiveness or six aggressiveness or eight aggressiveness or bigger.Pertinent literature can be referred to by generating the proper method of pMHC polymers, such as
(Greten et al., Clin.Diagnostic Lab.Immunol.2002：216-220).
In general, with the pMHC compounds with biotin residue and the compound generation of fluorescent marker Streptavidin can be passed through
PMHC polymers.Alternatively, the pMHC polymers can also be used as molecular scaffold by immunoglobulin to be formed.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, carrier molecule can also be utilized by forming pMHC polymers, such as dextran (WO02072631).PMHC polies
Body helps to improve the detection of part in connection, such as T cell receptor.Alternatively, pMHC compounds are improved in related application
Effect such as activates T cell.
PMHC compounds of the present invention can be provided with soluble form.To obtain the pMHC compounds of soluble form,
Preferably, MHC molecule is free of transmembrane region in the pMHC compounds.Specifically, in pMHC compounds, 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 compounds 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 compounds of the invention can also utilize synthetic method
Generate, then with the present invention peptide refolding.By determining whether peptide and MHC molecule being capable of refoldings, it may be determined that the present invention
Peptide can form compound with which class MHC molecule.
The soluble pMHC compounds of the present invention can be used for screening or detect molecule in connection, such as TCR or antibody.
The method includes the pMHC compounds are contacted and measured with bound fraction to be measured bound fraction to be measured whether with compound
With reference to.The assay method of the combination of pMHC compounds is well known in the art.Preferred method includes but not limited to, surface etc. from
Daughter is resonated 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 with reference to generation, such as cytokine release or carefully
Born of the same parents' apoptosis.
Similarly, soluble pMHC compounds of the invention can be also used for screening TCR or antibody library.Utilize bacteriophage
Display technique is come to build antibody library be 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 compounds are used for the diversity TCR libraries that screening is showed in phage particle surface.The TCR of the display library
Non-natural mutation may be contained.
Therefore, soluble pMHC compounds of the invention can be fixed to by attachment on appropriate solid phase carrier.Gu
The example of phase carrier includes, but are not limited to pearl, film, Ago-Gel, magnetic bead, substrate, pipe, column.PMHC compounds can be with
It is fixed on ELISA reaction plates, magnetic bead or surface plasma resonance biosensor chip.PMHC compounds are 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 compounds biotin labeling, and
It is fixed on the coated surface of Streptavidin.
Peptide of the present invention can together with MHC compounds submission to cell surface.Therefore, the present invention also provides one
Kind of cell, the cell are capable of the pMHC compounds of the submission present 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.Other are preferably
Cell include T2 cells (Hosken, et al., Science.1990.248:367-70).Present peptide of the present invention or
The cell of pMHC compounds can be separation, preferably, providing in the form of cell colony or in a substantially pure form.
The cell can not be natural submission compound of the present invention or described cell delivery in the horizontal than natural of compound
Height under state.Such cell can carry out pulse processing with peptide of the present invention and obtain.Pulse processing is related to described
Peptide incubated cell a few houres, it is preferable that a concentration of the 10 of peptide used-5-10-12M.In addition, the cell can also use HLA-A*02
Molecule is transduceed, the submission of further induction peptide.The cell of submission pMHC compounds of the present invention can be used to detach T
Cell and T cell receptor, the T cell is by the cell-stimulating and is further sorted out, and then can also be expressed
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 at healthy volunteer.The stimulation of several wheels can be passed through, as 3-4 takes turns.Activation
T cell identification can by the present invention peptide pulse T2 cells in the presence of, come measure the release of cell factor (ratio
Such as, IFN-γ ELISpot is tested).Using labelled antibody, active cell can be sorted by flow cytometer (FACS),
The cell of sorting can expand culture and further verification, for example, passing through ELISpot detections and/or the cell for target cell
Toxicity and/or the dyeing of pMHC polymers are verified.The TCR chains of T cell clone from empirical tests can pass through cDNA ends
Rapid amplifying (RACE) is amplified, and be sequenced.
The present invention also provides a kind of nucleic acid molecules, the nucleic acid molecules include the nucleic acid sequence of the peptide of the coding present invention.
The nucleic acid can be cDNA.The nucleic acid molecules can mainly be 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.
The present invention also provides a kind of carrier, the carrier includes nucleic acid sequence of the present invention.Suitable carrier
It is selection and other controlling elements including promoter known to vector construction field, such as enhancer element.It is of the present invention
Carrier include being adapted for introduction into the sequence of cell.For example, the carrier can be expression vector, and in the carrier, the polypeptide
Coded sequence controlled by own cis-acting regulatory element, carrier design 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, bites
Thalline, 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 bacteriophages, simian virus, cattle wart virus, Epstein-Barr viruses, 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 compound that the binding molecule can only be combined with peptide or be formed with peptide and MHC molecule is combined.In latter situation, the knot
Closing molecule can partly be attached on MHC molecule, meanwhile, it is also combined with peptide of the invention.The bound fraction of the present 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 the preference of the present invention, the binding molecule is T cell receptor (TCR).It may be used international immune
Genetics information system (IMGT) describes TCR.Natural α β heterodimerics TCR has α chains and β chains.In a broad sense, each chain includes
Variable region, bonding pad and constant region, β chains contain short variable region, but the variable region usually also between variable region and bonding pad
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, 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 can be included
All or part of TCR extracellular domains.The TCR can also be the overall length chain for including its transmembrane region.The TCR can be provided
To cell surface, such as T cell.
Soluble TCR can be obtained with reference to 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 chains variable region of α β TCR and β chain constant regions.
The TCR of the present 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 of the expression TCR to destroy tumour cell, to be given in the therapeutic process for being referred to as adoptive immunotherapy
Give patient.In addition, mutation can also be contained in the TCR of the present invention, it is preferable that the TCR after mutation is to pMHC compounds of the present invention
Affinity increase.The TCR of the present invention can be used alone, and can also be combined with conjugate with covalent or other modes, 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 compounds of the present invention), therapeutic agent, PK (protein kinase) modified parts or any the above substance
Combination combine or coupling.The TCR of the present invention can also be combined with the antibody of anti-CD3, preferably be combined with covalent manner, with weight
T cell is oriented, so as 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, the i.e. molecule containing specific binding site, can it is all natural,
Or part is artificial synthesized or all artificial synthesized.Term " antibody " including 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
It is or 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
Be modification antibody, 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 the peptide of the present invention, pMHC compounds, nucleic acid molecules, carrier, cells
And purposes of the binding molecule in terms of pharmacy.The peptide, pMHC compounds, nucleic acid, carrier, cell or binding molecule can be by
For treating or preventing cancer, preferably melanoma, carcinoma of urinary bladder, 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 the peptide of the present invention, pMHC compounds, nucleic acid of the invention
The binding molecule and pharmaceutically acceptable carrier of molecule, the cell of the present invention or the present invention.Described pharmaceutical composition can be with
It is any suitable form, (depending on the medication that patient needs).It can be provided in the form of unit dosage forms, usually be put
In sealing container, and it can be provided as a part for kit.Such kit usually (but be not required) is comprising making
With explanation.It can include multiple unit dosage forms.
Described pharmaceutical composition is suitable for any appropriate administration route, such as inject (including subcutaneous, muscle, abdominal cavity or quiet
Arteries and veins is injected), sucking or the oral or approach such as intranasal or per anum.The composition can be by any known to pharmaceutical field
Prepared by method, such as aseptically, by the way that active constituent is mixed with carrier or excipient.
According to the disease for treatment or illness (such as cancer, viral infection or autoimmune disease), of patient
Body age and situation etc., the dosage of invention formulation can change in a wider scope.Appropriate dosage will be by
Doctor finally determines.
According to the state of the art, the peptide of cell surface, pMHC compounds are presented to together with MHC molecule or is passed
In the cell of pMHC compounds, T cell or B cell can be activated, it is made to play a role.
Therefore, the cell of peptide of the invention, pMHC compounds or submission pMHC compounds 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 CancerInst.2012 104 (8)：599-
613).For example, the peptide of the present invention is used directly for immune patients (SalgallerML.Cancer Res.1996.56 (20)：
4749-57and Marchand M.Int J Cancer.1999.80(2)：219-230).The vaccine composition can include
Additional peptide so that peptide of the invention is one in peptide mixer.The vaccine composition can add in adjuvant, be exempted from enhancing
Epidemic disease is reacted.Alternatively, the vaccine composition can be the form for the antigen presenting cell for presenting peptide and MHC compounds of the present invention.
Preferably, the antigen presenting cell is immunocyte, more preferably dendritic cells.The peptide can also pulse arrive cell
Surface (Thurner BI.et al., J.Exp.Med.1999.190:It 1669) or can be by the code nucleic acid of peptide of the present invention
It is introduced into dendritic cells, for example, utilizing 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.Test method without specific conditions in the following example, 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 houses) 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 otherwise instructed, material therefor is commercial product in the embodiment of the present invention.
Embodiment 1 is derived from the polypeptide of LMP1 antigens by Mass Spectrometric Identification
Before Mass Spectrometric Identification is carried out, the present invention further demonstrates LMP1 antigens table in kinds of tumor cells
It reaches.Specifically, it is detected (nanostring) using digital unimolecule multiple gene expression spectrum analysis system.As a result it shows
Show, LMP1 antigens have expression in the tumor tissues such as nasopharyngeal carcinoma, gastric cancer, Burkitt lymphoma and Hodgkin lymphoma.
HLA- small peptide compounds are purified using commercial antibodies BB7.2.Specifically, with containing non-ionic surfactant
The buffer solution cracking tumour cell of agent Triton X-100 (1%v/v), adds in 1ml lysates, in 4 DEG C of rollings by 2*10^7 cells
It is dynamic to be incubated 1h.Centrifugation removal cell fragment, supernatant elder generation and antibody incubation, it is " anti-to add rProtein A-Sepharose captures
Body-HLA- small peptides compound ".Column is crossed, is collected " rProtein A-Sepharose- antibody-HLA- small peptides compound ".Use less salt
Pillar is washed with high-salt buffer, is finally hung on HLA- small peptides compound on immune affinity column, then pass through with 10% acetic acid elution
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 be 98% water, 2% acetonitrile, 0.1% trifluoroacetic acid, Mobile phase B be 98% acetonitrile, 2% water, 0.1% trifluoro
Acetic acid, eluent gradient are raised to 70% for Mobile phase B in 10 minutes by 5%.Collect a fraction within each minute.Total run time is
30 minutes.
After the HPLC fractions of polypeptide are concentrated, sample introduction nanoLC-MSMS network analyses：
5600 systems of Eksigent nanoLC-AB Sciex Triple TOF：Mass spectrum uses IDA analysis methods.Liquid phase
Chromatography uses：Pre-column：(Eksigent) NanoLC Trap column.5 μm C18.100 μm * 2.5cm, 910-00050, analysis
Column：(Eksigent)C18-CL-120,0.075 × 150mm, 805-00120.
Dionex Ultimate3000-Thermo QE Plus systems：Mass spectrum uses ddms2 analysis method liquid chromatograies
Using：Pre-column：(Thermo)Acclaim100,100um × 2cm, nanoViper, C18,5um, 100A,
164564, analytical column：(Thermo)Acclaim100,75um × 15cm, nanoViper, C18,3um, 100A,
164568。
Above-mentioned two system receive flow chromatography mobile phase A for 98% water, 2% acetonitrile, 0.1% formic acid, Mobile phase B is
98% acetonitrile, 2% water, 0.1% formic acid, eluent gradient are raised to 50% for Mobile phase B in 74 minutes by 5%.Total run time
It is 90 minutes.
Mass spectrometry results by library software ProteinPilot and Peaks is searched, search for the Uniprot numbers of human protein
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 compounds of embodiment
The heavy chain and light chain (β 2m) of I type HLA-A*0201 molecules are respectively in the form of inclusion body in Escherichia coli
(E.coli) it expresses.It should be noted that obtain soluble pMHC compounds, HLA-A*0201 molecules used in the present embodiment
Heavy chain does not include its transmembrane region and cytoplasmic region.In addition, for convenience of subsequently biotinylation is carried out to soluble pMHC compounds, it can
To add in biotinylation tag in the C-terminal of heavy chain.The detailed process for preparing solubility pMHC compounds of the invention is as follows：
A. it purifies
The E.coli bacterium solutions of 100ml induced expressions heavy chain or light chain are collected, 10ml is used after 4 DEG C of 8000g centrifuge 10min
PBS washing thallines are primary, violent with 5ml BugBuster Master Mix ExtractionReagents (Merck) later
Thalline is resuspended in concussion, and is rotated in room temperature and be incubated 20min, after 4 DEG C, 6000g centrifugation 15min are discarded supernatant, collection is forgiven
Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Add 30ml
The BugBuster of 10 times of dilution, mixing, 4 DEG C of 6000g centrifuge 15min；It discards supernatant, the BugBuster that 30ml is added to dilute 10 times
Inclusion body, mixing is resuspended, 4 DEG C of 6000g centrifuge 15min, are repeated twice, and add 30ml 20mM Tris-HCl pH 8.0 that packet is resuspended
Contain body, mixing, 4 DEG C of 6000g centrifuge 15min, finally dissolve inclusion body, SDS-PAGE detections with 20mM Tris-HCl 8M urea
Inclusion body purity, BCA kits survey concentration.
B. renaturation
The peptide MLWRLGATI (synthesis of Beijing SBS Genetech gene technology Co., Ltd) of the present 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 hydrochlorides, 10mM sodium acetates, 10mM EDTA are further denaturalized.MLWRLGATI peptides are added with 25mg/L (final concentration)
Enter renaturation buffer (0.4M L-arginines, 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 add in three times, 8h/ times), renaturation carry out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detections 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 replace buffer solution and come 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 volumes).Instrument (the general electricity of GE is purified using Akta
Gas company), the 0-400mM NaCl linear gradients liquid elution albumen that 20mM Tris pH 8.0 are prepared, pMHC is about in 250mM
It is eluted at NaCl, collects all peak components, SDS-PAGE detection purity.The obtained glue figure of solubility pMHC compounds of the invention is such as
Shown in Fig. 2.
D. biotinylation
With Millipore super filter tubes by the pMHC molecular concentrations of purifying, while it is 20mM Tris pH by buffer exchange
8.0, then add in biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D-
Biotin, 100 μ g/ml BirA enzymes (GST-BirA), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detections biotinylation
Completely.
E. the compound after purifying biological element
PMHC molecular concentrations after biotinylation is marked with Millipore super filter tubes are to 1ml, using gel permeation chromatography
Using Akta purifying instrument (GE General Electric Co. Limited), HiPrepTM is pre-equilibrated with filtered PBS by the pMHC of purifying biological element
16/60S200HR columns (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated loading 1ml, then with PBS with 1ml/
Min flow velocitys elute.
Embodiment 3 is cloned using the T cell that small peptide of the present invention obtains
It present embodiments provides and obtains the illustration of monoclonal T cell using pMHC compounds of the present invention.
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 chains, the T cell clone is by the cytositimulation of submission pMHC compounds of the present invention.It obtains
TCR sequences can be cloned into above suitable carrier, are then expressed or are expressed in bacteriophage in Escherichia coli such as E.coli
Surface.
Using the peripheral blood lymphocytes of the small peptide stimulation healthy volunteer of the present invention, limiting dilution assay is then used in sorting
Colony Culture is carried out to obtain T cell clone, the results are shown in Figure 3 for CD8+ and the double positive stainings of the tetramer-PE.
The function and specificity of T cell clone is further detected by ELISPOT experiments.Those skilled in the art are known
The method that detection cell function is tested using ELISPOT.The present embodiment IFN-γ ELISPOT experiment used in effector cell be
The T cell clone obtained in the present invention, target cell are the T2 cells for having loaded small peptide of the present invention, and control group is short to have loaded other
The T2 cells of the T2 cells of peptide and unsupported any small peptide.
Prepare ELISPOT tablets first, ELISPOT experimental procedures are as follows：Each component of experiment is added in the following order
Enter ELISPOT tablets：40 5 × 105 cells/mls of μ l T2 cells (i.e. 20,000 T2 cells/wells), 40 μ l effector cells
After (2000 T cell clone/holes), experimental group adds in 20 μ l specificity small peptides, and control group adds in 20 μ l non-specificity small peptides, empty
White group adds in 20 μ l culture mediums (test medium), and sets 2 multiple holes.Then it is incubated overnight (37 DEG C, 5%CO2).Then washing
Tablet simultaneously carries out secondary detection and colour developing, and dry tablet 1 hour recycles immunodotting plate reader (ELISPOT
READER system；AID companies) count the spot formed on film.Experimental result is as shown in figure 4, obtained specific antigen is special
Property T cell clone have specific reaction to the T2 cells for loading small peptide of the present invention, and other unrelated peptides are not reacted substantially.
Embodiment 4 combines the TCR of pMHC compounds of the present invention
Use Quick-RNATMThe total serum IgE of T cell clone described in MiniPrep (ZYMO research) extracting embodiments 3,
And obtain TCR sequences.TCR further to verify acquisition can combine the pMHC compounds of the present invention, and the present embodiment exists
Soluble TCR albumen has been given expression in E.coli, and has passed through the combination that BIAcore detects itself and pMHC compounds.It should be noted that can
To obtain soluble TCR according to the prior art, including but not limited to, described in patent document PCT/CN2015/093806.
The combination activity of soluble TCR protein and pMHC compounds is detected using BIAcore T200 real-time analyzers.
The antibody (GenScript) of anti-Streptavidin is added in into coupling buffer (10mM sodium-acetate buffers, pH 4.77), then
Antibody is flowed through to the CM5 chips activated in advance with EDC and NHS, antibody is made to be fixed on chip surface, finally with 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
The chip surface of coated antibody is flowed through with element, it is logical to cross detection for the then pMHC logistics made from mode as described in embodiment 2
Road, another channel flow through chip 2min, sealed joint as reference channel, then by the biotin of 0.05mM with the flow velocity of 10 μ L/min
The mould remaining binding site of Avidin.
Using BIAcore Evaluation software computational dynamics parameters, obtain the TCR molecules of solubility of the invention with
The kinetic profile that pMHC compounds of the present invention combine is as shown in figure 5, collection of illustrative plates shows the combination of the two.Meanwhile it also utilizes
The combination of TCR molecules and other several irrelevant antigen small peptides and HLA compounds that the above method has detected solubility of the invention is lived
Property, as a result show TCR molecules of the present invention with other irrelevant antigens without combination.
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 be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited
It encloses.
Sequence table
<110>Guangzhou Xiangxue Pharmaceutical Co
<120>Tumour antigen small peptide derived from LMP1
<130> P2016-2059
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213>Artificial sequence
<400> 1
Met 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 MLWRLGATI (SEQ ID NO:1)；Or
(II) is in SEQ ID NO:In 1 there is 1,2 or 3 amino acid substitution and/or 1,2 or 3 amino acid to insert
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 compounds, which is characterized in that the compound includes the peptide described in claim 1.
3. a kind of cell of separation, which is characterized in that the cell surface presents pMHC compounds 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 the 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 combine institute in peptide described in claim 1 and/or claim 2
State pMHC compounds.
A kind of 8. monoclonal T cell of separation, which is characterized in that pMHC compounds described in the cell combination claim 2.
9. the cell described in pMHC compounds, claim 3, right described in peptide described in claim 1, claim 2 will
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 is sought, feature exists
In being 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
The pMHC compounds described in peptide, claim 2 described in 1, the cell described in claim 3, the molecule described in claim 7 or
T cell according to any one of claims 8.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102812043A (en) * | 2009-11-19 | 2012-12-05 | 新加坡国立大学 | Method for preparing T cell receptor-like monoclonal antibody and use thereof |
| CN102695717B (en) * | 2009-09-29 | 2016-08-10 | Ucl商务股份有限公司 | T cell receptor |
-
2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102695717B (en) * | 2009-09-29 | 2016-08-10 | Ucl商务股份有限公司 | T cell receptor |
| CN102812043A (en) * | 2009-11-19 | 2012-12-05 | 新加坡国立大学 | Method for preparing T cell receptor-like monoclonal antibody and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| YOSHIZAKI, T: "A Novel Immune Evasion Mechanism of LMP-1, an EBV-Primary Oncogene, in Nasopharyngeal Carcinoma", 《ADV OTORHINOLARYNGOL》 * |
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