CN107266552A - Tumour antigen small peptide from PRAME - Google Patents
Tumour antigen small peptide from PRAME Download PDFInfo
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- CN107266552A CN107266552A CN201710169806.7A CN201710169806A CN107266552A CN 107266552 A CN107266552 A CN 107266552A CN 201710169806 A CN201710169806 A CN 201710169806A CN 107266552 A CN107266552 A CN 107266552A
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the newfound small peptide from tumour antigen PRAME, the small peptide and the compound of MHC molecule formation and the purposes of the small peptide and compound.Meanwhile, the invention further relates to the purposes of the molecule combined with above-mentioned small peptide or compound, and these molecules.
Description
Technical field
The present invention relates to the newfound small peptide from tumour antigen PRAME, the small peptide is compound with MHC molecule formation
The purposes of thing and 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 have some specific point
Sub is not suitable for expression.Therefore, these molecules are just into pathology or " mark " of 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, producing spy with the label 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 φt cell receptor that can be combined with " mark " 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 tumour, existing many documents delivered different endogenic tumour antigen molecules.But this is not
The real target spot of relevant disease, because causing CTL immune responses and incomplete tumour antigen molecule, and comes from
The specific CTL epitope (Epitope) of antigen.Generally, 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 to form 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.Although related autochthonous tumor antigen molecule has early been delivered, we are simultaneously unaware of the specific polypeptide piece that is rendered out
Section.Therefore, either still it is used to as vaccine produce the diagnostic reagent or therapeutic reagent of relevant disease, identifies these quilts
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 directed to finding and determine these polypeptide fragments for being presented to target cells.
The discovery of this polypeptide fragment by submission and determination are a complicated processes, 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) identify which polypeptide fragment may be by there is provided prediction algorithm
Submission.But this is a kind of prediction, with very big uncertainty, because after it is not real intracellular processing and translated
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, although this is a complicated process, but the result so obtained is very reliable.Meanwhile, mass spectrometric spirit
Sensitivity is also sufficient to differentiate the modification after the polypeptide fragment of low concentration and translation, therefore it is discovery and determines tumor surface
The ideal tools of polypeptide fragment.
PRAME is melanoma specific antigen (preferentially expressed antigen of
Melanoma, PRAME), 88% it is initial and 95% transfer melanoma in have expression (Ikeda H, et
al.Immunity,1997,6(2):199-208), normal skin tissue and benign melanocytic tumor cell are not expressed then.Except black
Plain knurl, PRAME is expressed also in kinds of tumor cells, including squamous cell lung carcinoma, breast cancer, clear-cell carcinoma, head and neck neoplasm,
(van't Veer LJ, the et al.Nature, 2002,415 such as Huo Jiejin lymphomas, sarcoma and medulloblastoma
(6871):530-536；Boon K,et al.Oncogene,2003,22(48):7687-7694).Therefore, from PRAME
The peptide of antigen, as the target spot of above-mentioned kinds cancer, not only can as above-mentioned medical diagnosis on disease label, it may also be used for produce
The prevention reagent and/or therapeutic agent of above-mentioned disease, such as antibody or φt cell receptor.The present invention is using spectrometer analysis and identifies
The new polypeptide fragment from tumour antigen PRAME that tumor cell surface is rendered.
The content of the invention
Object of the present invention is to provide a kind of newfound small peptide from tumour antigen PRAME, the small peptide with
The compound of MHC molecule formation and the purposes of the small peptide and compound.
The first aspect of the present invention is included there is provided a kind of peptide, the peptide：
(I) amino acid sequence AAFDGRHSQTLK (SEQ ID NO:1)；Or
(II) is in SEQ ID NO:There is 1,2 or 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and/or 1,2 or 3 amino acid in 1
Insertion, and/or 1,2 or 3 amino acid deletions amino acid sequence；
Wherein, the peptide can be with MHC molecule formation compound.
In another preference, the peptide is made up of 7-25 amino acid.
In another preference, the peptide is made up of 8-16 amino acid.
In another preference, the amino acid sequence of the peptide is SEQ ID NO:1.
The second aspect of the present invention includes first aspect present invention institute there is provided a kind of pMHC compounds, the compound
The peptide stated.
In another preference, the amino acid sequence of the peptide in the pMHC compounds is SEQ ID NO:1.
In another preference, the type of MHC molecule is HLA-A*11.
In another preference, the type of MHC molecule is HLA-A*1101.
In another preference, the pMHC compounds are polymer.
In another preference, the pMHC compounds are solvable.
In another preference, the pMHC compounds are biotinylated.
The third aspect of the present invention presents second aspect of the present invention there is provided a kind of cell of separation, the cell surface
The pMHC compounds.
The fourth aspect of the present invention includes coding first party of the present invention there is provided a kind of nucleic acid molecules, the nucleic acid molecules
The nucleotide sequence of peptide described in face or its complementary series.
The fifth aspect of the present invention contains the nucleic acid described in fourth aspect present invention there is provided a kind of carrier, the carrier
Molecule.
The sixth aspect of the present invention is there is provided a kind of host cell, containing described in fifth aspect present invention in the cell
Carrier.
The seventh aspect of the present invention is there is provided 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 preference, 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 preference, the molecule is φt cell receptor.
In another preference, the φt cell receptor is solvable.
In another preference, the molecule is antibody or its binding fragment.
In another preference, the antibody is monoclonal antibody.
The eighth aspect of the present invention combines the present invention second there is provided a kind of monoclonal T cell of separation, the T cell
The aspect pMHC compounds.
In another preference, pMHC compounds described in the T cell specific binding second aspect of the present invention.
The ninth aspect of the present invention is answered there is provided pMHC described in peptide described in first aspect present invention, second aspect of the present invention
The purposes of cell described in compound or third aspect present invention, for activating and/or separating T cell.
The tenth aspect of the present invention is answered there is provided pMHC described in peptide described in first aspect present invention, second aspect of the present invention
The purposes of compound, for screening φt cell receptor or antibody library.
There is provided pMHC described in peptide described in first aspect present invention, second aspect of the present invention for the eleventh 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, the medicine for preparing prevention or treating cancer.
The twelveth aspect of the present invention contains pharmaceutically acceptable there is provided a kind of pharmaceutical composition, the composition
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 preference, described pharmaceutical composition is vaccine.
The thirteenth aspect of the present invention is applied there is provided a kind of method prevented or treat disease, including to the object of needs
With described in pMHC compounds described in peptide described in appropriate first aspect present invention, second aspect of the present invention, 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 obtains what is combined with pMHC compounds described in second aspect of the present invention there is provided a kind of
The method of molecule, including：
(I) is by candidate molecules and pMHC complex contacts described in 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 constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 is the representative mass spectrogram for identifying small peptide of the present invention.
Fig. 2 is the glue figure of solubility pMHC compounds of the invention.Right side band is molecular weight marker, and left-hand bar 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 the 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.
Embodiment
Present invention in-depth study by extensive, obtains the peptide from antigen PRAME, the peptide is in by MHC molecule
Tumor cell surface is delivered to, tumor marker is used as.Therefore, the invention provides the peptide from antigen PRAME, the peptide with
The compound of MHC molecule formation and the purposes of the peptide and compound.Meanwhile, the invention further relates to above-mentioned peptide or compound
With reference to 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 the peptide from antigen PRAME that the present invention is provided.
Specifically, the first aspect of the present invention provides a kind of peptide, and the peptide is included：
(I) amino acid sequence AAFDGRHSQTLK (SEQ ID NO:1)；Or
(II) is in SEQ ID NO:There is 1,2 or 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and/or 1,2 or 3 amino acid in 1
Insertion, and/or 1,2 or 3 amino acid deletions amino acid sequence；
Wherein, the peptide can be with MHC molecule formation compound.
49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor means that in same position some amino acid residue is substituted by other amino acid residues.Insertion
Amino acid residue can be inserted in any position, and the amino acid residue of insertion can also be completely or partially adjacent to each other, or insertion
Amino acid between it is not adjacent to each other.Can be from SEQ ID NO：Deletion 1,2 or 3 amino acid 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 Opin such as Engelhard Immunol.2006；
18(1)：Found in 92-7, and including phosphorylation, acetylation and deamidization.
It is preferred that peptide of the present invention is incorporated into the binding site peptide point of MHC molecule with MHC.Generally, foregoing description is repaiied
The amino acid of decorations will not destroy the peptide and MHC binding ability.In one preferred embodiment, described amino acid is repaiied
Decorations improve the ability that peptide is combined with MHC.For example, mutation is likely to occur in peptide and MHC binding site.These binding sites and
Preferred residue is known in the art on binding site, especially (referring to such as the peptide which combines HLA-A*11
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, it is preferred that 9,10,
Or 11.
Peptide of the present invention can be by AAFDGRHSQTLK (SEQ ID NO：1) constitute, or it is main by
AAFDGRHSQTLK(SEQ ID NO：1) constitute, it corresponds to the position of the 409-418 residues of PRAME 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 difference on amino acid sequence or do 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 pass through radiation or sudden and violent
It is exposed to mutagens and produces random mutagenesis, can also be by the technology of site-directed mutagenesis or other known molecular biology.Analog
Also include the 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 the above-mentioned representativeness enumerated
Peptide.
Modification (not changing primary structure generally) form includes：The chemically derived form such as acetylation of inner or in vitro peptide
Or carboxylated.Modification also includes glycosylation, and such as those carry out glycosyl in the synthesis and processing of peptide or in further processing step
The peptide changed modification and produced.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 also include having phosphorylated amino acid residue (such as phosphotyrosine, phosphoric acid silk
Propylhomoserin, phosphothreonine) sequence.Also include being modified improving its anti-proteolysis performance or optimizing 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 the similar or close amino acid of property and form peptide.
These conservative variation's peptides carry out amino acid substitution preferably based on table 1 and produced.
Table 1
| Initial residue | Representational substitution | It is preferred that 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.Or, 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 nucleotide sequence of coded polypeptide, polypeptide is expressed in vivo；Example
Such as, expressed in bacterium, yeast, insect or mammalian cell.Or, external cell-free system carry out table can also be used
Reach.Such system is known in the art, and can be obtained from commercial channels.The peptide can be separated and/or with base
Pure form is provided in sheet.For example, they can there is no offer in the form of other peptide or proteins with a kind of.
Tumour antigen is processed into as the polypeptide piece of 8-16 amino acid length by proteolysis in the cell
Section, i.e. CTL epitopes, and then polypeptide-MHC compounds (peptide-MHC is combined to form with the MHC molecule in endoplasmic
Complex, pMHC), submission is to cell surface together.Therefore, the second aspect of the present invention provides a kind of pMHC compounds, institute
State in compound comprising the peptide described in first aspect present invention.It is preferred that the polypeptide is incorporated into the peptide-binding groove of MHC molecule
On.The MHC molecule can be MHC I quasi-molecules or MHC class Ⅱmolecules, it is preferable that the MHC molecule is MHC I quasi-molecules.
In one preferred embodiment, the MHC molecule is HLA-A*11, it is highly preferred that the MHC molecule is HLA-A*
1101。
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.The proper method for producing pMHC polymers may be referred to pertinent literature, such as
(Greten et al., Clin.Diagnostic Lab.Immunol.2002：216-220).
Generally, the pMHC compounds with biotin residue can be used with being produced by the way that fluorescence labeling Streptavidin is compound
PMHC polymers.Or, 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 catenation sequence (linker) one short
Together.In addition, carrier molecule, such as dextran (WO02072631) can also be utilized by forming pMHC polymers.PMHC polies
Body is favorably improved the detection of part in connection, such as φt cell receptor.Or, pMHC compounds are improved in related application
Effect, such as activation 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 up of the ectodomain of its light chain and all or part of heavy chain.Or, MHC molecule is the piece for only including its functional domain
Section.
The method for producing solubility pMHC compounds of the invention is well known by persons skilled in the art, is included, but 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
Produce, then with the peptide refolding of the present invention.By determining whether peptide and MHC molecule being capable of refoldings, it may be determined that the present invention
Peptide can be with which class MHC molecule formation compound.
The soluble pMHC compounds of the present invention can be used for screening or detect molecule in connection, such as TCR or antibody.
Methods described includes contacting the pMHC compounds with bound fraction to be measured, and measure 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.It is preferred that method include but is not limited to, surface etc. from
Daughter is resonated, or any other biosensor technique, ELISA, flow cytometry, chromatography, microexamination.Or, this
Outside, the combination can be by detecting to combining the biological response produced progress functional examination, such as cytokine release or thin
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 come build antibody library be it is well known in the art, such as bibliography Aitken, Antibody phage display:
Described in Methods and Protocols (2009, Humana, New York).In one preferred embodiment, this hair
Bright pMHC compounds be used to screen the diversity TCR libraries for being 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 on appropriate solid phase carrier by attachment.Gu
The example of phase carrier includes, but not limited to pearl, film, Ago-Gel, magnetic bead, substrate, pipe, post.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, present invention also offers one
Cell is planted, the cell is capable of submission pMHC compounds of the invention to its surface.Such cell can be mammalian cell,
It is preferred that immune system cell, and preferably special antigen presenting cell, such as dendritic cells or B cell.Other are preferred
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, it is preferred that being provided 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 the cell delivery compares natural in the level of compound
Height under state.Such cell can enter horizontal pulse processing and obtain with peptide of the present invention.Pulse processing is related to described
Peptide incubated cell a few houres, it is preferable that the concentration of peptide used is 10-5-10-12M.In addition, the cell can also use HLA-A*11
Molecule is transduceed, the submission of further induction peptide.The cell of submission pMHC compounds of the present invention can be used to separate T
Cell and φt cell receptor, the T cell is by the cell-stimulating and is further sorted out, and then also results in expression
φt cell receptor on the T cell surface.
In one preferred embodiment, the method for above-mentioned T cell is obtained using above-mentioned submission pMHC of the present invention
The new blood that the cytositimulation of compound is obtained at healthy volunteer.The stimulation of several wheels, such as 3-4 wheels can be passed through.Activation
T cell identification can by the present invention peptide pulse T2 cells in the presence of, come determine cell factor release (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 checking, for example, being detected by ELISpot and/or for the cell of 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 exaggerated, and is sequenced.
Present invention also offers a kind of nucleic acid molecules, the nucleic acid molecules include the nucleotide sequence of the peptide of the coding present invention.
The nucleic acid can be cDNA.The nucleic acid molecules mainly can be made up of the nucleotide 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 nucleotide sequences can encode identical amino acid sequence.
Present invention also offers a kind of carrier, the carrier includes nucleotide sequence of the present invention.Suitable carrier
It is known to vector construction field, to include selection and other controlling elements, such as enhancer element of promoter.It is of the present invention
Carrier include being adapted for introduction into the sequence of cell.Such as, the carrier can be expression vector, in the carrier, the 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 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..
Present invention also offers a kind of binding molecule, the molecule can be used as immunotherapeutic agent or diagnostic reagent.
The binding molecule can be combined only with peptide, or be combined with the compound of peptide and MHC molecule formation.In latter event, the knot
Closing molecule partly can 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
It is separation and/or solvable and/or non-naturally occurring, i.e., does not have equivalent in the Nature, and/or it is pure, and/or people
Work synthesis.
In the preference of the present invention, the binding molecule is φt cell receptor (TCR).Can be using 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 is included
Variable region, bonding pad and constant region, β chains generally contain short variable region, but the variable region 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 is present.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 ectodomains.The TCR can also be the total length chain for including its transmembrane region.The TCR can be provided
To cell surface, such as T cell.
The prior art in this area can be combined to obtain the TCR of solubility, for example, the perseverance of α and the β chain in α β TCR
Artificial disulfide bond is introduced between localization, or artificial disulfide bond is introduced between α β TCR α chains variable region 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 expression TCR T cell to destroy tumour cell, to be given in the therapeutic process for be 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.It (is diagnostic purpose, wherein the TCR is in for detection that the conjugate, which includes detectable,
Pass the presence of the cell of pMHC compounds of the present invention), therapeutic agent, PK (protein kinase) modified parts or any the above material
Combination combine or be coupled.The present invention TCR can also be with anti-CD3 antibody binding, preferably combined with covalent manner, with weight
T cell is oriented, so as to kill target cell.
In another preference, 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, its can with All Pure Nature,
Or part is artificial synthesized or all artificial synthesized.Term " antibody " include antibody fragment, its derivative, functional equivalents with
And homologous antibody, humanized antibody, the antibody fragment include immunoglobulin land, the land is antigen-binding site
Or it is homologous with antigen-binding site.It can be artificial synthesized with All Pure Nature or part or all artificial synthesized.Humanized antibody can be with
It is the antibody of modification, its variable region (for example, mouse) for containing non-human antibody and the constant region of 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；Fragment includes antigen binding domain, such as Fab, scFv, Fv, dAb, Fd；And double-chain antibody.Antibody can be many
Anti- or monoclonal antibody, preferably monoclonal antibody.
Above-mentioned TCR and antibody preparation method are well known by persons skilled in the art, are included but is not limited to, from Escherichia coli
Express, and be 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, cell
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.
Present invention also offers a kind of pharmaceutical composition, its peptide comprising the present invention, pMHC compounds, 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 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, generally be put
In sealing container, and it can be provided as a part for kit.Such kit generally (but being not required), which is included, to be made
With explanation.It can include multiple described unit dosage forms.
Described pharmaceutical composition is applied to any appropriate method of administration, such as inject (including subcutaneous, muscle, abdominal cavity or quiet
Arteries and veins is injected), the approach such as suction or oral or 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 component 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 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 played a role.
Therefore, the cell of peptide of the invention, pMHC compounds or submission pMHC compounds can be with the shape of vaccine combination
Formula is provided.The vaccine combination can be used for treating or preventing cancer.This all based compositions are included in the present invention.
It should be understood that the vaccine can be diversified forms (Schlom J.J Natl Cancer Inst.2012 104 (8)：599-
613).For example, 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).The vaccine combination can be included
Extra peptide so that peptide of the invention is one in peptide mixer.The vaccine combination can add adjuvant, be exempted from strengthening
Epidemic disease is reacted.Or, the vaccine combination can be the form for the antigen presenting cell for presenting peptide of the present invention and MHC compounds.
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:, or can be by the code nucleic acid of peptide of the present invention 1669)
It is incorporated into dendritic cells, for example, utilizing 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 limitation the scope of the present invention.The experimental method 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 houses) described in condition, or according to the condition proposed by manufacturer.Unless
Illustrate in addition, otherwise percentage and number are calculated by weight.
Unless otherwise instructed, material used in the present invention and reagent are commercially available prod.
Embodiment 1 is derived from the polypeptide of DAGE by Mass Spectrometric Identification
Before Mass Spectrometric Identification is carried out, the present invention further demonstrates DAGE table in kinds of tumor cells
Reach.Specifically, detected (nanostring) using digital unimolecule multiple gene expression spectral analysis system.As a result show
Show, DAGE is in melanoma, gland cancer, non-small cell lung cancer, hepatocellular carcinoma, myeloma, osteosarcoma, clear-cell carcinoma, lymph
There is expression in the tumour cells such as knurl.
HLA- small peptide compounds are purified using commercial antibodies W6/32.Specifically, with containing non-ionic surfactant
Agent Triton X-100 (1%v/v) buffer solution cracking tumour cell, adds 1ml lysates, in 4 DEG C of rollings by 2*10^7 cells
It is dynamic to be incubated 1h.Centrifugation removes cell fragment, supernatant elder generation and antibody incubation, adds rProtein A-Sepharose captures " anti-
Body-HLA- small peptides compound ".Post is crossed, is collected " rProtein A-Sepharose- antibody-HLA- small peptides compound ".Use less salt
Pillar is washed with high-salt buffer, finally HLA- small peptides compound on immune affinity column is hung on 10% acetic acid elution, then pass through
95 DEG C of heating are crossed, and 10KDa (AmiconR Ultr Centrifugal Filters, MILLIPORE) ultrafiltration membrance filter falls
Macromolecular, finally gives mixtures of polypeptides.
Mixtures of polypeptides is fractionated through the high performance liquid chromatography of Agilent 1260：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 is that Mobile phase B is raised to 70% by 5% in 10 minutes.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：
The systems of Eksigent nanoLC-AB Sciex Triple TOF 5600：Mass spectrum uses IDA analysis methods.Liquid phase
Chromatogram is used：Pre-column：(Eksigent) NanoLC Trap column.5 μm C18.100 μm * 2.5cm, 910-00050, analysis
Post：(Eksigent)C18-CL-120,3μm,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。
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 Mobile phase B is raised to 50% by 5% in 74 minutes.Total run time
For 90 minutes.
Mass spectrometry results, by library software ProteinPilot and Peaks is searched, search for the Uniprot numbers of human protein
According to storehouse.The result provided according to software, as shown in figure 1, the final antigen short peptide sequence for drawing the present invention.
The preparation of the solubility pMHC compounds of embodiment 2
The heavy chain and light chain (β 2m) of I type HLA-A*1101 molecules are respectively in the form of inclusion body in Escherichia coli
(E.coli) express.It should be noted that to obtain HLA-A*1101 molecules used in soluble pMHC compounds, the present embodiment
Heavy chain does not include its transmembrane region and cytoplasmic region.In addition, biotinylation is carried out to soluble pMHC compounds for convenience of follow-up, can
To add biotinylation tag in the C-terminal of heavy chain.The detailed process for preparing solubility pMHC compounds of the invention is as follows：
A. purify
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 once, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) afterwards
Thalline is resuspended in concussion, and is incubated 20min in room temperature rotation, after 4 DEG C, 6000g centrifugation 15min, supernatant discarding, collection is forgiven
Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Plus 30ml
The BugBuster of 10 times of dilution, is mixed, and 4 DEG C of 6000g centrifuge 15min；Supernatant discarding, plus 30ml dilute 10 times of BugBuster
Inclusion body is resuspended, mixes, 4 DEG C of 6000g centrifuge 15min, are repeated twice, plus bag is resuspended in 30ml 20mM Tris-HCl pH 8.0
Contain body, mix, 4 DEG C of 6000g centrifuge 15min, finally with 20mM Tris-HCl 8M urea dissolving inclusion body, SDS-PAGE detections
Inclusion body purity, BCA kits survey concentration.
B. renaturation
The peptide AAFDGRHSQTLK (synthesis of Beijing SBS Genetech gene technology Co., Ltd) of the present invention is dissolved in DMSO extremely
20mg/ml concentration.The inclusion body of light chain and heavy chain is dissolved with 8M urea, 20mM Tris pH 8.0,10mM DTT, renaturation
Preceding addition 3M guanidine hydrochlorides, 10mM sodium acetates, 10mM EDTA are further denatured.AAFDGRHSQTLK peptides is (dense eventually with 25mg/L
Degree) add renaturation buffer (0.4ML- arginine, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidisability gluathiones
Peptide, 5mM reduced glutathiones, 0.2mM PMSF, be cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L
Heavy chain (final concentration, heavy chain is added in three times, 8h/ times), renaturation carries out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detections
Renaturation success.
C. purified after renaturation
Make dialysis to change renaturation buffer with the 20mM Tris pH 8.0 of 10 volumes, at least change 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
Eluted at NaCl, collect all peak components, SDS-PAGE detection purity.The glue figure of obtained 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 being 20mM Tris pH by buffer exchange
8.0, then add 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 elementization
PMHC molecular concentrations after biotinylation is marked with Millipore super filter tubes are to 1ml, using gel permeation chromatography
The pMHC of purifying biological elementization, purifies instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibrations HiPrepTM using Akta
16/60S200HR posts (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated loading 1ml, then with PBS with 1ml/
Min flow velocitys are eluted.
The T cell clone that embodiment 3 is obtained using small peptide of the present invention
Present embodiments provide using pMHC compounds of the present invention to obtain the illustration of monoclonal T cell.
Those skilled in the art know a variety of methods for obtaining TCR, include but is not limited to, by TCR α from T cell clone
Separated with the sequence of β chains, the T cell clone is by the cytositimulation of submission pMHC compounds of the present invention.Obtain
TCR sequences can be cloned into above suitable carrier, then expressed in Escherichia coli such as E.coli, or are expressed in bacteriophage
Surface.
The PBLC of healthy volunteer is stimulated using the small peptide of the present invention, limiting dilution assay is then used in sorting
Carry out Colony Culture to obtain T cell clone, its CD8+ and the double positive staining results of the tetramer-PE are as shown in Figure 3.
The function and specificity that further detect that the T cell is cloned are tested by ELISPOT.Those skilled in the art know
The method that detection cell function is tested using ELISPOT.Effector cell used is in the present embodiment IFN-γ ELISPOT experiments
The T cell clone obtained in the present invention, target cell is 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 flat boards first, ELISPOT experimental procedures are as follows：Each component of experiment is added in the following order
Enter ELISPOT flat boards：40 μ l T2 cells 5 × 105Individual cells/ml (i.e. 20,000 T2 cells/wells), 40 μ l effector cells
After (2000 T cell clone/holes), experimental group adds the specific small peptides of 20 μ l, and control group adds the non-specific small peptides of 20 μ l, empty
White group adds 20 μ l culture mediums (test medium), and sets 2 multiple holes.Then it is incubated overnight (37 DEG C, 5%CO2).Then washing
Flat board simultaneously carries out secondary detection and colour developing, dries flat board 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, it is and very poor to other unrelated reactive polypeptides.
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.The pMHC compounds of the present invention can be combined for the TCR that further checking is obtained, the present embodiment exists
Solvable TCR albumen has been given expression in E.coli, and has detected by BIAcore the combination of itself and pMHC compounds.It should be noted that can
To obtain the TCR of solubility according to prior art, include but is not limited to, described in patent document PCT/CN2015/093806.
The binding activity of soluble TCR protein and pMHC compounds is detected using BIAcore T200 real-time analyzers.
The antibody (GenScript) of anti-Streptavidin is added 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 fixed on chip surface, finally with the salt of monoethanolamine
Acid solution closes unreacted activating surface, completes coupling process, coupling level is about 15,000RU.Make the strepto- parent of low concentration
The chip surface of coated antibody is flowed through with element, the then pMHC logistics made from mode as described in embodiment 2 is crossed detection and led to
Road, another passage with 10 μ L/min flow velocity flows through chip 2min, sealed joint as reference channel, then by 0.05mM biotin
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 are combined is as shown in figure 5, collection of illustrative plates shows the combination of the two.Meanwhile, also utilize
The combination of TCR molecules and other several irrelevant antigen small peptides and HLA compounds that the above method have 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 documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made 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.
Sequence table
<110>Guangzhou Xiangxue Pharmaceutical Co
<120>Tumour antigen small peptide from PRAME
<130> P2017-0414
<150> CN 201610192759.3
<151> 2016-03-30
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 12
<212> PRT
<213>Artificial sequence
<400> 1
Ala Ala Phe Asp Gly Arg His Ser Gln Thr Leu Lys
1 5 10
Claims (10)
1. a kind of peptide, it is characterised in that the peptide is included：
(I) amino acid sequence AAFDGRHSQTLK (SEQ ID NO:1)；Or
(II) is in SEQ ID NO:There is 1,2 or 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in 1, and/or 1,2 or 3 amino acid is inserted
Enter, and/or 1,2 or 3 amino acid deletions amino acid sequence；
Wherein, the peptide can be with MHC molecule formation compound.
2. a kind of pMHC compounds, it is characterised in that the compound includes the peptide described in claim 1.
3. a kind of cell of separation, it is characterised in that the cell surface presents pMHC compounds described in claim 2.
4. a kind of nucleic acid molecules, it is characterised in that the nucleic acid molecules include the nucleotide sequence of peptide described in coding claim 1
Or its complementary series.
5. a kind of carrier, it is characterised in that the carrier contains the nucleic acid molecules described in claim 4.
6. a kind of host cell, it is characterised in that contain the carrier described in claim 5 in the cell.
7. a kind of molecule, it is characterised in that the molecule can combine institute in peptide described in claim 1 and/or claim 2
State pMHC compounds.
8. a kind of monoclonal T cell of separation, it is characterised in that pMHC compounds described in the cell combination claim 2.
9. the pMHC compounds described in peptide, claim 2 described in claim 1, the cell described in claim 3, right will
The purposes of the T cell described in the nucleic acid molecules described in 4, the molecule described in claim 7 or present claims 8 is sought, for preparing
Prevention or the medicine for the treatment of cancer.
10. a kind of pharmaceutical composition, the composition contain pharmaceutically acceptable carrier and peptide described in claim 1,
Cell, the molecule described in claim 7 or the claim 8 described in pMHC compounds, claim 3 described in claim 2
Described T cell.
Applications Claiming Priority (2)
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| CN201610192759 | 2016-03-30 |
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| CN118221811A (en) * | 2023-08-25 | 2024-06-21 | 立凌生物制药(苏州)有限公司 | Single domain antibody targeting PRAME polypeptide and its use |
| WO2025040598A3 (en) * | 2023-08-18 | 2025-05-01 | Immatics Biotechnologies Gmbh | Peptides displayed by mhc for use in immunotherapy against different types of cancer |
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