CN110272483A - Identify the T cell receptor of SAGE1 antigen small peptide - Google Patents

Identify the T cell receptor of SAGE1 antigen small peptide Download PDF

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CN110272483A
CN110272483A CN201810210760.3A CN201810210760A CN110272483A CN 110272483 A CN110272483 A CN 110272483A CN 201810210760 A CN201810210760 A CN 201810210760A CN 110272483 A CN110272483 A CN 110272483A
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tcr
cell
ser
chain
leu
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CN110272483B (en
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李懿
陈安安
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Guangzhou Institute of Biomedicine and Health of CAS
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Guangzhou Institute of Biomedicine and Health of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The present invention provides the T cell receptors (TCR) that one kind can specifically bind the small peptide ATIIHNLREEK derived from SAGE1 antigen, specifically, the antigen small peptide ATIIHNLREEK can form compound with HLA A1101 and be presented to cell surface together, and the present invention provides one kind being capable of the specifically T cell receptor in conjunction with ATIIHNLREEK-HLA-A1101 compound (TCR).Carrier the present invention also provides the sequence of nucleic acid molecules for encoding the TCR and comprising the sequence of nucleic acid molecules.In addition, the present invention also provides the cells for the TCR of the present invention that transduces.

Description

Identify the T cell receptor of SAGE1 antigen small peptide
Technical field
The present invention relates to the T cell receptors of identification SAGE1 antigen small peptide, and the invention further relates to transduce above-mentioned TCR to obtain SAGE1 specificity T cell and they prevent and treat SAGE1 antigen protein related disease in purposes.
Background technique
SAGE1 can be degraded to micromolecule polypeptide after expressing in the cell as a kind of autochthonous tumor antigen protein, and Cell surface is presented to after forming compound in conjunction with MHC (main histocompatibility complex) molecule.Studies have shown that ATIIHNLREEK is the small peptide derived from SAGE1 antigen protein.SAGE1 antigen is in melanoma, bladder cancer, liver cancer, epiderm-like There is expression in the tumor tissues such as cancer, non-small cell lung cancer and squamous cell carcinoma, and in most normal tissues in addition to testis Do not express (Martelange V1, De Smet C, De Plaen E, Lurquin C, Boon T.Cancer Res.2000；60 (14):3848-55；Atanackovic D,etal.,Cancer BiolTher.2006；5(9):1218-25).For above-mentioned The treatment of disease can use the methods of chemotherapy and radiation treatment, but can all damage to the normal cell of itself.
T cell adoptive immunotherapy is that will there is the reaction-ive T cell of specificity to be transferred in patient body target cell antigen, It is set to play a role for target cell.T cell receptor (TCR) is a kind of memebrane protein on T cell surface, can be identified corresponding The antigen small peptide of target cell surface.In immune system, pass through the TCR and the main histocompatbility of small peptide-of antigen small peptide specificity The combination of complex (pMHC compound) causes T cell and antigen presenting cell (APC) is directly physically contacted, then T cell And other cell membrane surface molecules of both APC just interact, and cause a series of subsequent cell signal transmitting and its His physiological reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, this field skill Art personnel are dedicated to isolating the TCR for having specificity to SAGE1 antigen small peptide, so that it is played a role, or the TCR is transduceed T cell has specific T cell to SAGE1 antigen small peptide to obtain, so that them be made to play work in cellular immunotherapy With.
Summary of the invention
The purpose of the present invention is to provide a kind of T cell receptors for identifying SAGE1 antigen small peptide.
First aspect present invention provides a kind of T cell receptor (TCR), and the TCR can be with ATIIHNLREEK-HLA- A1101 compound combines.
In another preferred example, the TCR includes TCR α chain variable domain and TCR β chain variable domain, the TCR α chain variable domain CDR3 amino acid sequence be AESPPDNYGQNFV (SEQ ID NO:13)；And/or the CDR3 of the TCR β chain variable domain Amino acid sequence is ASSPVAGELF (SEQ ID NO:10).
In another preferred example, 3 complementary determining regions (CDR) of the TCR α chain variable domain are as follows:
αCDR1-DSSSTY(SEQ ID NO:6)
αCDR2-IFSNMDM(SEQ ID NO:7)
αCDR3-AESPPDNYGQNFV(SEQ ID NO:13)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-KGHSH(SEQ ID NO:5)
βCDR2-LQKENI(SEQ ID NO:8)
βCDR3-ASSPVAGELF(SEQ ID NO:10)。
In another preferred example, the TCR includes TCR α chain variable domain and TCR β chain variable domain, the TCR α chain variable domain To have the amino acid sequence of at least 90% sequence identity with SEQ ID NO:1；And/or the TCR β chain variable domain be with SEQ ID NO:2 has the amino acid sequence of at least 90% sequence identity.
In another preferred example, the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.
In another preferred example, the TCR includes β chain variable domain amino acid sequence SEQ ID NO:2.
In another preferred example, the TCR is α β heterodimer, and it includes TCR α chain constant region TRAC*01 and TCR β Chain constant region TRBC1*01 or TRBC2*01.
In another preferred example, the α chain amino acid sequence of the TCR is the β of SEQ ID NO:3,15 and/or the TCR Chain amino acid sequence is SEQ ID NO:4,16.
In another preferred example, the α chain nucleotides sequence of the TCR is classified as SEQ ID's NO.:12, the 23 and/or TCR β chain nucleotides sequence is classified as SEQ ID NO.:14,24.
In another preferred example, the TCR is soluble.
In another preferred example, the TCR includes (a) all or part of TCR α chain in addition to transmembrane domain；And (b) all or part of TCR β chain in addition to transmembrane domain；
And (a) and (b) respectively contains functional variable domain, or includes functional variable domain and the TCR At least part of chain constant domain.
In another preferred example, the TCR is single-stranded.
In another preferred example, TCR is formed by connecting with β chain variable domain by connecting peptide sequence by α chain variable domain.
In another preferred example, cysteine residues form artificial disulfide bond between α the and β chain constant domain of the TCR.
In another preferred example, the cysteine residues of artificial disulfide bond are formed in the TCR instead of selected from following One or more groups of sites:
The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；With
The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1.
In another preferred example, the α chain amino acid sequence of the TCR is the β of SEQ ID NO:3,15 and/or the TCR Chain amino acid sequence is SEQ ID NO:4,16.
In another preferred example, the amino acid sequence of the single-stranded TCR α chain is SEQ ID NO.:25 and/or described single-stranded The amino acid sequence of TCR β chain is SEQ ID NO.:27.
In another preferred example, the nucleotides sequence of the single-stranded TCR α chain is classified as SEQ ID NO.:26 and/or described single-stranded The nucleotides sequence of TCR β chain is classified as SEQ ID NO.:28.
In another preferred example, artificial interchain disulfide bond is contained between the α chain variable region of the TCR and β chain constant region.
In another preferred example, the TCR is comprising α chain variable domain and β chain variable domain and in addition to transmembrane domain All or part of β chain constant domain, but it does not contain α chain constant domain, the α chain variable domain and β chain of the TCR forms heterogeneous dimerization Body.
In another preferred example, the α chain of the TCR and/or the end C- or N- of β chain are combined with conjugate.
In another preferred example, the conjugate in conjunction with the T cell receptor is detectable marker, therapeutic agent, PK are repaired The combination of decorations part or any of these substances.
In another preferred example, the therapeutic agent is anti-CD 3 antibodies.
Second aspect of the present invention provides a kind of multivalent TCR complex, and it includes at least two TCR molecules, and wherein At least one TCR molecule be first aspect present invention described in TCR.
Third aspect present invention provides a kind of nucleic acid molecules, and the nucleic acid molecules include coding first aspect present invention institute The nucleic acid sequence or its complementary series for the TCR molecule stated.
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chain variable domain: 9。
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID of coding TCR β chain variable domain NO:11.
In another preferred example, the nucleic acid molecules include nucleotide sequence SEQ ID NO:12 of coding TCR α chain, 23 And/or nucleotide sequence SEQ ID NO:14 comprising encoding TCR β chain, 24.
Fourth aspect present invention provides a kind of carrier, and the carrier contains nucleic acid described in third aspect present invention point Son；Preferably, the carrier is viral vectors；It is highly preferred that the carrier is slow virus carrier.
Fifth aspect present invention provides a kind of isolated host cell, contains the present invention the 4th in the host cell Nucleic acid molecules described in the third aspect present invention of external source are integrated in carrier described in aspect or genome.
Sixth aspect present invention provides a kind of cell, nucleic acid molecules described in the cell transduction third aspect present invention Or carrier described in fourth aspect present invention；Preferably, the cell is T cell or stem cell.
Seventh aspect present invention provides a kind of pharmaceutical composition, the composition contain pharmaceutically acceptable carrier with And TCR described in first aspect present invention, described in TCR compound, third aspect present invention described in second aspect of the present invention Cell described in carrier described in nucleic acid molecules, fourth aspect present invention or sixth aspect present invention.
Eighth aspect present invention provides T cell receptor described in first aspect present invention or second aspect of the present invention institute Nucleic acid molecules described in the TCR compound stated, third aspect present invention, carrier or the present invention described in fourth aspect present invention The purposes of cell described in 6th aspect, is used to prepare the drug for the treatment of tumour or autoimmune disease.
Ninth aspect present invention provides a kind of method for treating disease, including suitable to object in need for the treatment of application TCR compound, third aspect present invention described in T cell receptor described in first aspect present invention or second aspect of the present invention Cell described in carrier described in the nucleic acid molecules, fourth aspect present invention or sixth aspect present invention or the present invention the Pharmaceutical composition described in seven aspects；
In another preferred example, the disease is tumour.
In another preferred example, the tumour is selected from the group: melanoma, gastric cancer, lung cancer (e.g., squamous cell lung carcinoma), Cancer of the esophagus, bladder cancer, head and neck neoplasm (e.g., head and neck squamous cell carcinoma), prostate cancer, breast cancer, colon cancer, oophoroma, Clear-cell carcinoma, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukaemia, or combinations thereof.
In another preferred example, the tumour is selected from the group: melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small thin Born of the same parents' lung cancer, squamous cell carcinoma, or combinations thereof.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively that TCR α chain variable domain amino acid sequence, TCR α chain are variable Domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, the TCR α chain amino acid sequence with leader sequence And the TCR α chain nucleotide sequence with leader sequence.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively that TCR β chain variable domain amino acid sequence, TCR β chain are variable Domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, the TCR β chain amino acid sequence with leader sequence And the TCR β chain nucleotide sequence with leader sequence.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chain.
Fig. 5 a and Fig. 5 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chain.
Fig. 6 is the glue figure of the sTCR obtained after purification.Leftmost side swimming lane is to go back virgin rubber, and intermediate swimming lane is molecular weight It marks (marker), rightmost side swimming lane is non-reduced glue.
Fig. 7 is Biacore dynamics of the sTCR of the present invention in conjunction with ATIIHNLREEK-HLA A1101 compound Map.
Fig. 8 is the thin testing result of primary T of tetramer staining TCR transduction.
Fig. 9 is ELISPOT testing inspection result.
Figure 10 is lethal effect result of the T cell to specificity target cell of TCR of the present invention transduction.
Specific embodiment
After extensive and in-depth study, having found can be with SAGE1 antigen small peptide ATIIHNLREEK (SEQ by the present inventor ID NO:29) specific binding TCR, the antigen small peptide ATIIHNLREEK can form compound and together with HLA-A1101 It is presented to cell surface.The present invention also provides the sequence of nucleic acid molecules for encoding the TCR and include the nucleic acid molecules The carrier of sequence.In addition, the present invention also provides the cells for the TCR of the present invention that transduces.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, for The presentation of antigen has specificity, and different individuals has different MHC, can present small peptide different in a kind of proteantigen to respectively From APC cell surface.The MHC of the mankind is commonly referred to as HLA gene or HLA complex.
T cell receptor (TCR) is the unique of specific antigen peptide of the presentation on main histocompatibility complex (MHC) Receptor.In immune system, T cell is caused by the combination of the TCR and pMHC compound of antigentic specificity and antigen presentation is thin Born of the same parents (APC) are directly physically contacted, and then other cell membrane surface molecules of both T cell and APC just interact, this A series of subsequent cell signal transmitting and other physiological reactions are just caused, so that the T cell of different antigentic specificities Immunological effect is played to its target cell.
TCR be as α chain/β chain or γ chain/δ chain in the form of heterodimer existing for cell membrane surface glycoprotein.? TCR heterodimer is made of α and β chain in 95% T cell, and 5% T cell has the TCR being made of γ and δ chain.It The right heterogeneous dimerization TCR of α β has α chain and β chain, and α chain and β chain constitute the subunit of α β heterodimeric TCR.In a broad sense, α and β are each Chain includes variable region, bonding pad and constant region, and β chain usually contains short variable region also between variable region and bonding pad, but should Variable region is often regarded as a part of bonding pad.Each variable region includes 3 be entrenched in frame structure (framework regions) A CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compound, wherein CDR3 by Variable region and bonding pad recombinate, referred to as hypervariable region.α the and β chain of TCR generally regards that each there are two " structural domains " can be changed as Domain and constant domain, variable domain are made of the variable region connected and bonding pad.The sequence of TCR constant domain can be in international immune genetic It learns and is found in the public database of information system (IMGT), if the constant domain sequence of TCR molecule alpha chain is " TRAC*01 ", TCR divides The constant domain sequence of sub- β chain is " TRBC1*01 " or " TRBC2*01 ".In addition, α the and β chain of TCR also includes transmembrane region and cytoplasm Area, cytoplasmic region are very short.
In the present invention, term " polypeptide of the present invention ", " TCR of the invention ", " T cell receptor of the invention " is interchangeable makes With.
Native interchain disulfide bond and artificial interchain disulfide bond
Natural TCR membrane-proximal region C α and C β interchain exist one group of disulfide bond, the present invention in referred to as " two sulphur of native interchain Key ".In the present invention, by what is be artificially introduced, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond claim For " artificial interchain disulfide bond ".
For convenience of the position of description disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequence in the present invention Position Number by from N-terminal to C-terminal sequence successively carry out Position Number, in TRBC1*01 or TRBC2*01, by from N-terminal to The 60th amino acid of the sequence of C-terminal successively is P (proline), then can describe it as TRBC1*01 or TRBC2*01 in the present invention The Pro60 of exons 1 can also be stated that the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1, for another example It is Q (glutamine) by the 61st amino acid of the sequence from N-terminal to C-terminal successively in TRBC1*01 or TRBC2*01, then it is of the invention In can describe it as the Gln61 of TRBC1*01 or TRBC2*01 exons 1, can also be stated that TRBC1*01 or TRBC2* 61st amino acids of 01 exons 1, other and so on.In the present invention, the amino acid sequence of variable region TRAV and TRBV Position Number, according to the Position Number listed in IMGT.Such as some amino acid in TRAV, the Position Number listed in IMGT is 46, then the 46th amino acids of TRAV, other and so on are described it as in the present invention.In the present invention, the sequence of other amino acid Column position number has specified otherwise, then presses specified otherwise.
Detailed description of the invention
As used herein, term " SAGE1TCR-T " refers to can react for SAGE1 Antigenic Peptide of the invention, but to it His Antigenic Peptide is responseless cell.
As used herein, term " T cell of non-SAGE1TCR transduction ", " Non-SAGE1TCR-T " are used interchangeably, Refer to the T cell for expressing other TCR, it is understood that it is to be for SAGE1 Antigenic Peptide of the invention, but the TCR that function is bad, this A little T cells are added together with SAGE1 Antigenic Peptide of the invention can not cause to react.
TCR molecule
In antigen processing pathways, antigen is degraded in the cell, is then carried by MHC molecule to cell surface.T is thin Born of the same parents' receptor can identify the peptide-MHC compound of Antigen Presenting Cell surface.Therefore, the first aspect of the present invention provides one kind It can be in conjunction with the TCR molecule of ATIIHNLREEK-HLA-A1101 compound.Preferably, the TCR molecule is separation or purifying 's.α the and β chain of the TCR respectively has 3 complementary determining regions (CDR).
It is preferably carried out in mode at of the invention one, the α chain of the TCR includes with following amino acid sequence CDR:
αCDR1-DSSSTY(SEQ ID NO:6)
αCDR2-IFSNMDM(SEQ ID NO:7)
αCDR3-AESPPDNYGQNFV(SEQ ID NO:13)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-KGHSH(SEQ ID NO:5)
βCDR2-LQKENI(SEQ ID NO:8)
βCDR3-ASSPVAGELF(SEQ ID NO:10)。
The CDR region amino acid sequence of aforementioned present invention can be embedded into chimeric to prepare in any suitable frame structure TCR.As long as frame structure is compatible with the CDR region of TCR of the invention, those skilled in the art's disclosed CDR region according to the present invention It can design or synthesize the TCR molecule with corresponding function.Therefore, TCR molecule of the present invention refers to comprising above-mentioned α and/or β The TCR molecule of chain CDR region sequence and any suitable frame structure.TCR α chain variable domain of the present invention is to have with SEQ ID NO:1 There are at least 90%, preferably 95%, the more preferably amino acid sequence of 98% sequence identity；And/or TCR β chain of the present invention can Variable domain is to have at least 90%, preferably 95% with SEQ ID NO:2, the amino acid sequence of more preferably 98% sequence identity Column.
In a preference of the invention, TCR molecule of the invention is the heterodimer being made of α and β chain.Specifically Ground, on the one hand the α chain of the heterogeneous dimerization TCR molecule includes variable domain and constant domain, the α chain variable domain amino acid sequence packet CDR1 (SEQ ID NO:6), CDR2 (SEQ ID NO:7) and CDR3 (SEQ ID NO:13) containing above-mentioned α chain.Preferably, institute Stating TCR molecule includes α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domain ammonia of the TCR molecule Base acid sequence is SEQ ID NO:1.On the other hand, the β chain of the heterogeneous dimerization TCR molecule includes variable domain and constant domain, institute State CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:8) and the CDR3 that β chain variable domain amino acid sequence includes above-mentioned β chain (SEQ ID NO:10).Preferably, the TCR molecule includes β chain variable domain amino acid sequence SEQ ID NO:2.It is highly preferred that The β chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO:2.
In a preference of the invention, TCR molecule of the invention is the portion by some or all of α chain and/or β chain The single chain TCR molecules for dividing or all forming.Description in relation to single chain TCR molecules can be with bibliography Chung et al (1994) Proc.Natl.Acad.Sci.USA 91,12654-12658.According to document, those skilled in the art can be easily Building includes the single chain TCR molecules in the area CDRs of the present invention.Specifically, the single chain TCR molecules include V α, V β and C β, preferably According to the sequential connection from N-terminal to C-terminal.
CDR1 (SEQ ID NO:6) of the α chain variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned α chain, CDR2 (SEQ ID NO:7) and CDR3 (SEQ ID NO:13).Preferably, the single chain TCR molecules include α chain variable domain amino Acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:1. The β chain variable domain amino acid sequence of the single chain TCR molecules includes CDR1 (SEQ ID NO:5), the CDR2 (SEQ of above-mentioned β chain ID NO:8) and CDR3 (SEQ ID NO:10).Preferably, the single chain TCR molecules include β chain variable domain amino acid sequence SEQ ID NO:2.It is highly preferred that the β chain variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:2.
In a preference of the invention, the constant domain of TCR molecule of the invention is the constant domain of people.Art technology Personnel know or can be obtained by consulting the public database of pertinent texts or IMGT (international immunogenetics information system) Obtain the constant domain amino acid sequence of people.For example, the constant domain sequence of TCR molecule alpha chain of the present invention can be " TRAC*01 ", TCR divides The constant domain sequence of sub- β chain can be " TRBC1*01 " or " TRBC2*01 ".The amino acid sequence provided in the TRAC*01 of IMGT The 53rd be Arg, indicate herein are as follows: the Arg53 of TRAC*01 exons 1, other and so on.Preferably, TCR of the present invention The amino acid sequence of molecule alpha chain is SEQ ID NO:3,15 and/or the amino acid sequence of β chain is SEQ ID NO:4,16.
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.As immunoglobulin (antibody) is made The same for antigen recognition molecule, at this moment TCR can also need to obtain soluble TCR points by development and application in diagnosing and treating Son.Soluble TCR molecule does not include its transmembrane region.STCR has very extensive purposes, it cannot be only used for research TCR With the interaction of pMHC, it is also possible to make the diagnostic tool of detection infection or the marker as autoimmunity disease.Similarly, may be used Dissolubility TCR can be used to for therapeutic agent (such as cytotoxin compounds or immunostimulating compound) to be transported to presentation specificity The cell of antigen, in addition, sTCR can also with other molecules (e.g., anti-CD 3 antibodies) in conjunction with redirecting T cell, from And make the cell of its targeting presentation specific antigen.The present invention also obtains the solubility for having specificity to SAGE1 antigen small peptide TCR。
To obtain sTCR, on the one hand, TCR of the present invention can be to be introduced between the residue of itself α and β chain constant domain The TCR of artificial disulfide bond.Cysteine residues form artificial interchain disulfide bond between α the and β chain constant domain of the TCR.Half Guang Histidine residue can be substituted in other amino acid residues of appropriate site in natural TCR to form artificial interchain disulfide bond.For example, Replace the Thr48 of TRAC*01 exons 1 and replaces the cysteine residues of the Ser57 of TRBC1*01 or TRBC2*01 exons 1 To form disulfide bond.It introduces cysteine residues and may also is that TRAC*01 exons 1 with other sites for forming disulfide bond The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1；The Tyr10 and TRBC1*01 of TRAC*01 exons 1 or The Ser17 of TRBC2*01 exons 1；Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1 Asp59；The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；TRAC*01 exons 1 Arg53 and TRBC1*01 or TRBC2*01 exons 1 Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1 or The Ala19 of TRBC2*01 exons 1；Or Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1 Glu20.I.e. cysteine residues are instead of any group of site in above-mentioned α and β chain constant domain.It can be in TCR constant domain of the present invention One or more C-terminals truncate most 50 or most 30 or most 15 or most 10 or most 8 or less Amino acid can also be by the way that day will be formed so that it does not include cysteine residues to achieve the purpose that lack natural disulphide bonds The cysteine residues of right disulfide bond sport another amino acid to reach above-mentioned purpose.
As described above, TCR of the invention may be embodied in the artificial disulfide bond introduced between the residue of itself α and β chain constant domain. It should be noted that the artificial disulfide bond with or without introducing described above between constant domain, TCR of the invention can be constant containing TRAC Domain sequence and TRBC1 or TRBC2 constant domain sequence.The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequence of TCR can It is connected by the natural disulphide bonds being present in TCR.
To obtain sTCR, on the other hand, TCR of the present invention further includes the TCR to mutate in its hydrophobic core region, The mutation of these hydrophobic core regions is preferably capable making the stability-enhanced mutation of sTCR of the present invention, such as in publication number Described in patent document for WO2014/206304.Such TCR can mutate in its following hydrophobic core position of variable domain: (α and/or β chain) variable region amino acid the 11st, 13,19,21,53,76,89,91,94 and/or α chain J gene (TRAJ) small peptide Amino acid position the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptide amino acid position reciprocal is 2nd, 4,6 reciprocal, wherein ammonia The Position Number of base acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).Those skilled in the art Member knows above-mentioned international immunogenetics information system, and the amino acid residue that different TCR can be obtained according to the database exists Position Number in IMGT.
The TCR that hydrophobic core region mutates in the present invention can be by α and the β chain of a flexible peptide chain link TCR can Variable domain and the solvable single-stranded TCR of stability constituted.It should be noted that in the present invention flexible peptide chain can be any suitable connection TCR α and The peptide chain of β chain variable domain.
In addition, patent document PCT/CN2016/077680 is also disclosed in the α chain variable region of TCR for stability Introducing artificial interchain disulfide bond between β chain constant region can be such that the stability of TCR significantly improves.Therefore, height parent of the invention Artificial interchain disulfide bond can also be contained between the α chain variable region and β chain constant region of power TCR.Specifically, in the α of the TCR The cysteine residues of artificial interchain disulfide bond are formed between chain variable region and β chain constant region instead of the 46th ammonia of TRAV 60th amino acids of base acid and TRBC1*01 or TRBC2*01 exons 1；The 47th amino acids and TRBC1*01 of TRAV or 61 amino acids of TRBC2*01 exons 1；The of the 46th amino acids of TRAV and TRBC1*01 or TRBC2*01 exons 1 61 amino acids；Or TRAV the 47th amino acids and TRBC1*01 or TRBC2*01 exons 1 the 60th amino acids.It is preferred that Ground, such TCR may include all or part of TCR α chain of (I) in addition to its transmembrane domain, and (II) removes its cross-film knot All or part of TCR β chain other than structure domain, wherein (I) and (II) variable domain comprising TCR chain and at least part is constant Domain, α chain and β chain form heterodimer.It is highly preferred that such TCR may include α chain variable domain and β chain variable domain and All or part of β chain constant domain in addition to transmembrane domain, but it does not contain α chain constant domain, the α chain variable domain of the TCR Heterodimer is formed with β chain.
TCR of the invention can also be provided in the form of multivalence complex.Multivalent TCR complex of the invention include two, Three, four or more TCR of the present invention are combined and the polymer that is formed, can such as be generated with four dimerization domains of p53 The compound that the tetramer or multiple TCR of the present invention are formed in conjunction with another molecule.TCR compound of the invention can be used for body Outer or tracking in vivo or targeting present the cell of specific antigen, it can also be used to which generating has other multivalence TCR of such application multiple Close the intermediate of object.
TCR of the invention can be used alone, can also with conjugate with covalent or other modes in conjunction with, preferably with covalently side Formula combines.The conjugate includes that detectable marker (for diagnostic purpose, presents wherein the TCR is used to detect The presence of the cell of ATIIHNLREEK-HLA-A1101 compound), therapeutic agent, PK (protein kinase) modified part or it is any with The combination of these upper substances combines or coupling.
Detectable marker for diagnostic purposes includes but is not limited to: fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.
Can in conjunction with TCR of the present invention or coupling therapeutic agent include but is not limited to: 1. radionuclides (Koppe etc., 2005, (Cancer metastasis reviews) 24,539 is commented in metastasis of cancer)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc Segment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；5. antibody ScFv segment (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold medal (Lapotko etc., 2005, cancer communicates (Cancer letters) 239,36 to nano particle/nanometer rods；Huang etc., 2006, beauty Chemical Society, state magazine (Journal of the American Chemical Society) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,11631)；9. magnetic nanosphere；10. pro-drug activation enzymes are (for example, DT- diaphorase (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
In addition, TCR of the invention can also be comprising derived from the heterozygosis TCR more than a kind of species sequence.For example, grinding Studying carefully display Muridae TCR can more effectively express in human T-cell than people TCR.Therefore, TCR of the present invention may include people's variable domain With the constant domain of mouse.The defect of this method is possible to cause immune response.Therefore, when it is used for the treatment of adoptive T cell There should be regulation scheme to carry out immunosupress, to allow to express the implantation of the T cell of Muridae.
It should be understood that amino acid name herein is indicated using international single English alphabet or three English alphabets, amino The corresponding relationship of the single English alphabet and three English alphabets of sour title is as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser (S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
Nucleic acid molecules
The second aspect of the present invention provides the nucleic acid molecules of coding first aspect present invention TCR molecule or part thereof, institute Stating part can be one or more CDR, the variable domain and α chain and/or β chain of α and/or β chain.
The nucleotide sequence for encoding first aspect present invention TCR molecule alpha chain CDR region is as follows:
αCDR1-gacagctcctccacctac(SEQ ID NO:17)
αCDR2-attttttcaaatatggacatg(SEQ ID NO:18)
αCDR3-gcagagagtccgcctgataactatggtcagaattttgtc(SEQ ID NO:19)。
The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR region is as follows:
βCDR1-aaaggacacagtcat(SEQ ID NO:20)
βCDR2-ctccagaaagaaaatatc(SEQ ID NO:21)
βCDR3-gccagctcaccggttgccggggagctgttt(SEQ ID NO:22)。
Therefore, the nucleotide sequence for encoding the nucleic acid molecules of the present invention of TCR α chain of the present invention includes SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, and/or the nucleotide sequence of nucleic acid molecules of the present invention of coding TCR β chain of the present invention includes SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22.
The nucleotide sequence of nucleic acid molecules of the present invention can be it is single-stranded or double-stranded, the nucleic acid molecules can be RNA or DNA, and may include or not include introne.Preferably, the nucleotide sequence of nucleic acid molecules of the present invention does not include introne But polypeptide of the present invention can be encoded, such as encodes the nucleotide sequence packet of the nucleic acid molecules of the present invention of TCR α chain variable domain of the present invention The nucleotide sequence for including the nucleic acid molecules of the present invention of SEQ ID NO:9 and/or coding TCR β chain variable domain of the present invention includes SEQ ID NO:11.It is highly preferred that the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:12 and/or SEQ ID NO: 14。
It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.Therefore, it compiles The nucleic acid sequence that code book invents TCR can variant identical as present invention nucleic acid sequence shown in the drawings or degeneracy.With One of example in the present invention illustrates that " variant of degeneracy " refer to that coding has the protein sequence of SEQ ID NO:1, But the differentiated nucleic acid sequence of sequence with SEQ ID NO:9.
Nucleotide sequence can be through codon optimization.Different cells is above different in the utilization of specific codon , the codon in sequence can be changed to increase expression quantity according to the type of cell.Mammalian cell and various other The codon usage table of biology is well known to those skilled in the art.
Nucleic acid molecules full length sequence or its segment of the invention usually can with but be not limited to PCR amplification method, recombination method or Artificial synthesized method obtains.At present, it is already possible to obtain encoding completely by chemical synthesis TCR of the present invention (or its segment, Or derivatives thereof) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or Such as carrier) and cell in.DNA can be coding strand or noncoding strand.
Carrier
It, can in vivo or body the invention further relates to the carrier comprising nucleic acid molecules of the invention, including expression vector The construct of outer expression.Common carrier includes bacterial plasmid, bacteriophage and animals and plants virus.
Viral delivery systems include but is not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, Retroviral vector, slow virus carrier, baculovirus vector.
Preferably, nucleotide of the invention can be transferred in cell by carrier, such as in T cell, so that the cell table Up to the TCR of SAGE1 antigentic specificity.Ideally, which can should express to continual high levels in T cell.
Cell
The invention further relates to the genetically engineered host cell of carrier or coded sequence of the invention.The host Contain in carrier or chromosome of the invention in cell and is integrated with nucleic acid molecules of the invention.Host cell is selected from: prokaryotic cell And eukaryocyte, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..
In addition, the invention also includes the isolated cell for expressing TCR of the invention, especially T cell.The T cell can spread out It is born from the T cell separated from subject, or can be the mixed cellularity group separated from subject, such as periphery hemolymph is thin A part of born of the same parents (PBL) group.Such as, which can be isolated from peripheral blood mononuclear cells (PBMC), can be CD4+T helper cell Or CD8+Cytotoxic T cell.The cell can be in CD4+T helper cell/CD8+In the mixing group of cytotoxic T cell.Generally Ground, the cell can be activated with antibody (e.g., the antibody of anti-CD3 or anti-CD28), to allow them to more easily receive to turn Dye, such as transfected with the carrier of the nucleotide sequence comprising encoding TCR molecule of the present invention.
Alternatively, cell of the invention can also be or derived from stem cell, such as candidate stem cell (HSC).Gene is turned Moving to HSC not will lead in cell surface expression TCR, because stem cell surface does not express CD3 molecule.However, when stem cell point It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecule will start in thymocyte The surface expression introducing TCR molecule.
There are many method be suitable for being carried out with the DNA or RNA of coding TCR of the present invention T cell transfection (e.g., the such as Robbins, (2008)J.Immunol.180:6116-6131).The T cell for expressing TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat Rev for carrying out adoptive treatment Cancer8 (4): 299-308).
SAGE1 antigen-related disease
The invention further relates to the method with SAGE1 antigen protein related disease, packet are treated and/or prevented in subject The step of adoptive transfer SAGE1 specific T-cells are included to the subject.The SAGE1 specific T-cells can recognize ATIIHNLREEK-HLA-A1101 compound.
The T cell of SAGE1 specificity of the invention can be used for treating any presentation SAGE1 antigen small peptide ATIIHNLREEK- The SAGE1 antigen protein related disease of HLA-A1101 compound, including but not limited to tumour, such as melanoma and other realities Body tumour such as gastric cancer, lung cancer, cancer of the esophagus, bladder cancer, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, ovary Cancer etc..
Treatment method
Can by separation with the patient of SAGE1 antigen-related disease or the T cell of volunteer, and will be of the invention TCR is imported in above-mentioned T cell, is then fed back to the cell that these genetic engineerings are modified in patient body to treat.Therefore, The present invention provides a kind of method for treating SAGE1 related disease, the T cell including the expression TCR of the present invention that will be separated, preferably Ground, the T cell derive from patient itself, are input in patient body.Generally, the T cell including (1) separation patient, (2) are with originally Invention nucleic acid molecules or the nucleic acid molecules ex vivo transduction T cell that can encode TCR molecule of the present invention, (3) modify genetic engineering T cell be input in patient body.The quantity of separation, transfection and the cell fed back can be determined by doctor.
Main advantages of the present invention include:
(1) TCR of the invention can be with SAGE1 antigen small peptide compound ATIIHNLREEK-HLA-A1101 specificity knot It closes, while the cell for the TCR of the present invention that transduceed can have very strong specific killing by specific activation and to target cell Effect.
Following specific embodiment, the present invention is further explained.It should be understood that these embodiments be merely to illustrate the present invention and It is not used in and limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, Such as (Sambrook and Russell et al., molecular cloning: laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing house) described in condition, or according to the normal condition proposed by manufacturer.Unless In addition illustrate, otherwise percentage and number are calculated by weight.Unless otherwise stated, otherwise percentage and number are calculated by weight. Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
Embodiment 1 clones SAGE1 antigen small peptide specific T-cells
It is stimulated using synthesis small peptide SAGE1 388-398ATIIHNLREEK (Nanjing Jin Sikang Biotechnology Co., Ltd) From the peripheral blood lymphocytes (PBL) for the healthy volunteer that genotype is HLA-A1101.By SAGE1 388- 398ATIIHNLREEK small peptide and the HLA-A*1101 renaturation for having biotin labeling, prepare pHLA monoploid.These monoploid It is combined into the tetramer of PE label with the Streptavidin (BD company) marked with PE, sorts the tetramer and anti-CD8-APC is bis- Positive cell.The cell of sorting is expanded, and carries out secondary sorting according to the above method, then carries out monoclonal training with limiting dilution assay It supports.Monoclonal cell tetramer staining, the double positive colonies screened are as shown in Figure 3.
Embodiment 2 obtains the building of the tcr gene and carrier of SAGE1 antigen small peptide specific T-cell clones
Use Quick-RNATMThe SAGE1 388-398 screened in MiniPrep (ZYMO research) extracting embodiment 1 The total serum IgE of the restrictive T cell clone of specificity, HLA-A1101.The synthesis of cDNA uses the SMART RACE of clontech CDNA amplification kit, the primer of use are the C-terminal conserved regions designed in mankind's tcr gene.Sequence is cloned into carrier T (TAKARA) it is sequenced on.Through being sequenced, the α chain and β chain-ordering structure of the TCR of double positive colonies expression respectively such as Fig. 1 and Shown in Fig. 2, Fig. 1 a, Fig. 1 b, Fig. 1 c and Fig. 1 d are respectively TCR α chain variable domain amino acid sequence, TCR α chain variable domain nucleotides sequence Column, TCR α chain amino acid sequence and TCR α chain nucleotide sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c and Fig. 2 d are respectively TCR β chain variable domain Amino acid sequence, TCR β chain variable domain nucleotide sequence, TCR β chain amino acid sequence and TCR β chain nucleotide sequence.
Identified, α chain includes the CDR with following amino acid sequence:
αCDR1-DSSSTY(SEQ ID NO:6)
αCDR2-IFSNMDM(SEQ ID NO:7)
αCDR3-AESPPDNYGQNFV(SEQ ID NO:13)
β chain includes the CDR with following amino acid sequence:
βCDR1-KGHSH(SEQ ID NO:5)
βCDR2-LQKENI(SEQ ID NO:8)
βCDR3-ASSPVAGELF(SEQ ID NO:10)。
The full-length gene of TCR α chain and β chain is cloned into Lentiviral respectively by overlapping (overlap) PCR pLenti(addgene).Specifically: it is attached the full-length gene of TCR α chain and TCR β chain to obtain TCR with overlap PCR α -2A-TCR β segment.It connects Lentiviral and TCR α -2A-TCR β digestion to obtain pLenti-SAGE1TRA-2A- TRB-IRES-NGFR plasmid.It is used as control, while the also slow virus carrier pLenti-eGFP of building expression eGFP.Later again Pseudovirus is packed with 293T/17.
Expression, refolding and the purifying of the solvable TCR of the antigen small peptide of the present invention specificity of embodiment 3
To obtain soluble TCR molecule, α the and β chain of TCR molecule of the invention can only include its variable domain and portion respectively Point constant domain, and a cysteine residues are introduced in the constant domain of α and β chain respectively to form artificial interchain disulfide bond, The position of introducing cysteine residues is respectively the Ser57 of the Thr48 and TRBC2*01 exons 1 of TRAC*01 exons 1；Its α The amino acid sequence and nucleotide sequence of chain are distinguished as shown in figures 4 a and 4b, the amino acid sequence and nucleotide sequence of β chain Respectively as shown in figure 5 a and 5b, the cysteine residues of introducing with overstriking and underline alphabetical indicate.Pass through " molecular cloning Laboratory manual " (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) Described in standard method the objective gene sequence of above-mentioned TCR α and β chain is inserted respectively into expression vector pET28a after synthesizing + (Novagene), the cloning site of upstream and downstream are NcoI and NotI respectively.Insert Fragment is errorless by sequencing confirmation.
The expression vector of TCR α and β chain is converted by chemical transformation respectively and enters expression bacterium BL21 (DE3), bacterium It is grown with LB culture solution, is induced when OD600=0.6 with final concentration 0.5mM IPTG, the packet formed after α the and β chain expression of TCR Contain body to extract by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, forgives Body is finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT) (DTT), 10mM ethylenediamine tetra-acetic acid (EDTA), 20mMTris (pH 8.1) in.
Dissolved TCR α and β chain are quickly mixed in 5M urea, 0.4M arginine, 20mM Tris with the mass ratio of 1:1 (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Solution is placed in dialysis (4 DEG C) in the deionized water of 10 times of volumes afterwards, changes deionized water into buffer after 12 hours (20mMTris, pH 8.0) continues at 4 DEG C and dialyses 12 hours.Solution after the completion of dialysis leads to after 0.45 μM of membrane filtration Cross anion-exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains renaturation successful α and β bis- The TCR of aggressiveness is confirmed by SDS-PAGE glue.TCR then passes through gel permeation chromatography (HiPrep 16/60, Sephacryl S- 100HR, GE Healthcare) it is further purified.TCR purity after purification by SDS-PAGE measurement be greater than 90%, concentration by The measurement of BCA method.The SDS-PAGE glue figure for the sTCR that the present invention obtains is as shown in Figure 6.
Embodiment 4 combines characterization
BIAcore analysis
This example demonstrated soluble TCR molecules of the present invention can be with ATIIHNLREEK-HLA A1101 compound Specific binding.
Use TCR molecule and ATIIHNLREEK- obtained in BIAcore T200 real-time analyzer detection embodiment 3 The combination activity of HLA A1101 compound.Coupling buffer (10mM is added in the antibody (GenScript) of anti-Streptavidin Sodium-acetate buffer, pH 4.77), then antibody is flowed through to the CM5 chip activated in advance with EDC and NHS, fix antibody In chip surface, unreacted activating surface finally is closed with the hydrochloric acid solution of ethanol amine, completes coupling process, coupling is horizontal about It is 15,000RU.
The Streptavidin of low concentration is set to flow through the chip surface of coated antibody, then by ATIIHNLREEK-HLA A1101 compound flows through sense channel, and another channel is as reference channel, then by the biotin of 0.05mM with the stream of 10 μ L/min Speed flows through chip 2min, closes the remaining binding site of Streptavidin.
The preparation process of above-mentioned ATIIHNLREEK-HLA A1101 compound is as follows:
A. it purifies
The E.coli bacterium solution for collecting 100ml inducing expression heavy chain or light chain uses 10ml after 4 DEG C of 8000g are centrifuged 10min PBS washing thalline is primary, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) later Thallus is resuspended in concussion, and rotates in room temperature and be incubated for 20min, and later in 4 DEG C, 6000g is centrifuged 15min, discards supernatant, collection is forgiven Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated for 5min；Add 30ml The BugBuster of 10 times of dilution is mixed, and 4 DEG C of 6000g are centrifuged 15min；It discards supernatant, 30ml is added to dilute 10 times of BugBuster Inclusion body is resuspended, mixes, 4 DEG C of 6000g are centrifuged 15min, are repeated twice, and the resuspension of 30ml 20mMTris-HCl pH 8.0 is added to forgive Body mixes, and 4 DEG C of 6000g are centrifuged 15min, finally dissolves inclusion body, SDS-PAGE detection packet with 20mMTris-HCl 8M urea Contain body purity, BCA kit surveys concentration.
B. renaturation
The small peptide ATIIHNLREEK (Nanjing Jin Sikang Biotechnology Co., Ltd) of synthesis is dissolved in DMSO to 20mg/ The concentration of ml.The inclusion body of light chain and heavy chain is dissolved with 8M urea, 20mMTris pH 8.0,10mM DTT, is added before renaturation 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA are further denaturalized.ATIIHNLREEK peptide is added again with 25mg/L (final concentration) Property buffer (0.4M L-arginine, 100mMTris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduction Type glutathione, 0.2mM PMSF, are cooled to 4 DEG C), the heavy chain of the light chain and 90mg/L that then sequentially add 20mg/L is (dense eventually Degree, heavy chain are added in three times, and 8h/ times), renaturation carries out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detection renaturation success.
C. it is purified after renaturation
Make dialysis with the 20mMTris pH 8.0 of 10 volumes to replace renaturation buffer, at least replacement buffer comes twice Sufficiently reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volume).Instrument (the general electricity of GE is purified using Akta Gas company), the 0-400mMNaCl linear gradient liquid that 20mMTris pH 8.0 is prepared elutes albumen, and pMHC is about in 250mMNaCl Place's elution, collects all peak components, and SDS-PAGE detects purity.
D. biotinylation
It with Millipore super filter tube by the pMHC molecular concentration of purifying, while being 20mMTris pH by buffer exchange 8.0, then be added biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mMMgOAc, 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 detects biotinylation complete.
E. the compound after purifying biological element
PMHC molecular concentration after being marked biotinylation with Millipore super filter tube is to 1ml, using gel permeation chromatography The pMHC of purifying biological element, purifies instrument (GE General Electric Co. Limited) using Akta, pre-equilibrates HiPrepTM with filtered PBS 16/60S200HR column (GE General Electric Co. Limited), load 1ml concentrated biotinylation pMHC molecule, then with PBS with 1ml/ The elution of min flow velocity.Biotinylated pMHC molecule occurs in about 55ml as unimodal elution.Merge the group containing protein Point, it is concentrated with Millipore super filter tube, BCA method (Thermo) measures protein concentration, and protease inhibitors cocktail is added (Roche) packing of biotinylated pMHC molecule is stored in -80 DEG C.Power is calculated using BIAcore Evaluation software Parameter is learned, it is as shown in Figure 7 to obtain kinetic profile of the TCR molecule of solubility of the invention in conjunction with compound.
Embodiment 5SAGE1 specific t-cell receptor slow virus packaging and primary T cells transduction SAGE1TCR
(a) (Express-In-mediated transient is transiently transfected by the quick mediation of 293T/17 cell Transfection slow virus) is prepared
Utilize the slow virus of gene of the third generation slow virus packaging system packaging containing TCR needed for encoding.It is situated between using quick Lead transient transfection (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)) with 4 kinds of plasmids, (one kind containing pLenti-SAGE1TRA-2A-TRB-IRES-NGFR described in embodiment 2 is slow Viral vectors, and 3 kinds of plasmids containing other components necessary to building infectiousness but non-replicating lentiviral particle) transfection 293T/17 cell.
To be transfected, the 0th day kind cell, in 15 cm dishes, kind upper 1.7 × 107A 293T/17 cell makes thin Born of the same parents are evenly distributed on culture dish, and convergence degree is slightly above 50%.1st day transfected plasmids pack pLenti-SAGE1TRA-2A- TRB-IRES-NGFR and pLenti-eGFP pseudovirus, by the above expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV It is mixed with pMD.2G, the dosage of a 15 cm diameter plates is as follows: 15 micrograms: 5 micrograms: 5 micrograms: 5 micrograms.Transfection reagent The ratio of PEI-MAX and plasmid is 2:1, and the usage amount of each plate is 60 micrograms.Concrete operations are as follows: expression plasmid and packaging Plasmid be added 1800 microlitres of OPTI-MEM (be uniformly mixed in (Ji Bu can company (Gibco), catalog number (Cat.No.) 31985-070) culture medium, Being stored at room temperature 5 minutes becomes DNA mixed liquor；Corresponding amount PEI is taken to be uniformly mixed with 1800 microlitres of OPTI-MEM culture mediums, room temperature is quiet Setting 5 minutes becomes PEI mixed liquor.DNA mixed liquor and PEI mixed liquor are mixed and be stored at room temperature 30 minutes, then is added Add 3150 microlitres of OPTI-MEM culture mediums, is added to the 293T/17 for being converted into 11.25 milliliters of OPTI-MEM after mixing In cell, culture dish is shaked gently, is uniformly mixed culture medium, 37 DEG C/5%CO2Lower culture.Transfection 5-7 hours, removal transfection Culture medium changes the DMEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt)) containing 10% fetal calf serum into completely Culture medium, 37 DEG C/5%CO2Lower culture.3rd and the 4th day culture medium supernatant of the collection containing wrapped slow virus.For harvest packet Collected culture supernatant 3000g is centrifuged 15 minutes removal cell fragments by the slow virus of dress, then through 0.22 micron filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB) filtering, it is finally (silent with the concentration tube of 50KD interception Ke Milibo (Merck Millipore), catalog number (Cat.No.) UFC905096) it is concentrated, most of supernatant is removed, is finally concentrated To 1 milliliter, frozen for -80 DEG C after equal part packing.Pseudovirus sample is taken to carry out virus titer measurement, step is referring to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit specification.It is used as control, while also packet turns the pseudovirus of pLenti-eGFP.
(b) with the lentiviruses transduction primary T cells containing SAGE1 specific t-cell receptor gene
CD8 is separated to from the blood of healthy volunteer+T cell, then with the lentiviruses transduction packed.It is thin to count these Born of the same parents, in 48 orifice plates, containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10%FBS (Ji Bu can company (Gibco), Catalog number (Cat.No.) C10010500BT) 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture mediums in 1 × 106 A cells/ml (0.5 milliliter/hole) and AntiCD3 McAb/CD28 antibody-coating globule (T cell amplified matter, the life pre-washed Technologies, catalog number (Cat.No.) 11452D) be incubated overnight stimulation altogether, cell: pearl=2:1.
After stimulation overnight, according to the virus titer that p24ELISA kit is measured, it is added in the ratio of MOI=10 dense The slow virus of the SAGE1 specific t-cell receptor gene of contracting, 32 DEG C, 900g centrifugation infection 1 hour.It is removed after infection slow Cell is resuspended with 1640 culture mediums containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10%FBS in virus infection liquid, and 37 DEG C/5%CO2Lower culture 3 days.Transduction counted cell, diluting cells to 0.5 × 10 after 3 days6A cells/ml.It counts within every two days Cell, replacement or is added the fresh culture containing 50IU/ml IL-2 and 10ng/ml IL-7, maintain cell 0.5 × 106-1×106A cells/ml.Flow cytometry cell was begun through from the 3rd day, is tried since the 5th day for function Test (for example, ELISPOT and non-radioactive cell toxicity detection of IFN-γ release).Slow down point since the 10th day or in cell It splits when becoming smaller with size, stored frozen etc. divides cell, at least 4 × 106A cell/pipe (1 × 107A cells/ml, 90% FBS/10%DMSO).
(c) primary T cells of tetramer staining TCR transduction
SAGE1 388-398ATIIHNLREEK small peptide and the HLA-A*1101 renaturation for having biotin labeling, prepare pHLA Monoploid.The Streptavidin (BD) that these monoploid are marked with PE is combined into the tetramer of PE label, referred to as SAGE1- tetramer-PE.The T cell for expressing SAGE1 specific t-cell receptor gene can be labeled as positive cell by this tetramer. T cell sample in (b) through transfecting is incubated for 30 minutes on ice with SAGE1-tetramer-PE, anti-mouse is then added β chain-APC (biolegend) antibody continues to be incubated for 15 minutes on ice.Sample uses BD after cleaning 2 times with the PBS containing 2%FBS Calibur or BD Arial detection or sorting express the SAGE1-tetramer-PE of SAGE1 specific t-cell receptor gene With the T cell of the bis- positives of CD8, data analysis using CellQuest software (BD) or FlowJo software (Tree Star Inc, Ashland, OR) analysis.
Through testing and analyzing, as a result as shown in figure 8, with after SAGE1-tetramer-PE and the dyeing of anti-mouse β chain antibody, not The seldom SAGE1-tetramer-PE of blank control group T cell and anti-mouse β chain-APC through TCR slow-virus infection are double positive thin Born of the same parents, and there are the double sun of SAGE1-tetramer-PE and anti-mouse β chain-APC of expression TCR through the T cell of TCR slow-virus infection Property cell, it is only a small amount of nonspecific double positive when with other tetramer-PE of non-SAGE1-tetramer-PE dyeing Cell.
Embodiment 6 verifies SAGE1 specificity TCR function
4.1ELISPOT scheme
Following tests is carried out to prove the activation that target cell specificity reacts of T cell of TCR- transduction.It utilizes Readout of the IFN-γ yield of ELISPOT testing inspection as t cell activation.
Reagent
Test medium: 10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), (Ji Bu can by RPMI1640 Company (Gibco), catalog number (Cat.No.) C11875500bt)
Washing buffer: 0.01M PBS/0.05% polysorbas20
PBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT)
96 orifice plate of PVDF ELISPOT (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) MSIPS4510)
People's IFN-γ ELISPOT PVDF- enzyme reagent kit (BD) (captures equipped with required every other reagent and detection is anti- Body, Streptavidin-alkaline phosphatase and BCIP/NBT solution)
Method
Target cell preparation
The target cell of the present embodiment is the B-lymphoblastoid cell lines (LCL) of Epstein-Barr viral (EBV) conversion. B95-8 cell induces culture medium supernatant of the production containing EBV, 4 DEG C/600g centrifugation 10 through myristoyl acetic acid phorbol ester (TPA) Minute removal impurity, then crosses 0.22 micron filter, and equal part dispenses -70 DEG C of preservations.It is HLA-A11/A02/A24 from genotype The peripheral blood lymphocytes (PBL) of the healthy volunteer of (including homozygote and heterozygote), taking 10 milliliters of concentration is 2 × 107/ milli The PBL suspension risen is added after cyclosporin in 25 square centimeters of culture bottle in 37 DEG C/CO2It is small that 1 is incubated in incubator When, quick-thawing portion EBV, by 1/10 dilution be added in above-mentioned cell, gently shake up and culture bottle is uprightly placed in 37 DEG C/ CO2It is cultivated in incubator.10 milliliters of culture mediums of addition continue to cultivate after culture 12 days, and further expansion culture is gone forward side by side after about 30 days Row flow cytometer detection, wherein CD19+CD23hiCD58+For B-lymphoblastoid cell lines (LCL).This ELISPOT is tested with HLA- A11/02 is target cell.
Effector cell's preparation
Effector cell's (T cell) of this test is to express SAGE1 specificity TCR through flow cytometry in embodiment 3 CD8+T cell, and with the CD8 of same volunteer+T is as negative control effector cell.Pearl (T cell is coated with AntiCD3 McAb/CD28 Amplified matter, life technologies) stimulation T cell, with the lentiviruses transduction (foundation for carrying SAGE1 specificity TCR gene Embodiment 3), in the 1640 culture mediums amplification containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 until transduction 9-12 days afterwards, then these cells are placed in test medium, 300g room temperature is centrifuged 10 minutes and is washed.Then by cell With 2 × required final concentration is resuspended in test medium.Same processing negative control effector cell.
ELISPOT
According to the specification that manufacturer provides, prepare orifice plate as described below: with 10 milliliters of sterile PBS of every block of plate by 1:200 It dilutes anti-human IFN-γ and captures antibody, 100 microlitres of dilution is then captured into antibody etc. point, each hole is added.Orifice plate is incubated at 4 DEG C Overnight.After incubation, orifice plate is washed to remove extra capture antibody.The RPMI 1640 of 100 microlitres/Kong Hanyou 10%FBS is added Culture medium simultaneously incubates orifice plate 2 hours at room temperature to close orifice plate.Then culture medium is washed away from orifice plate, by flicking on paper The washing buffer of any remnants is removed with ELISPOT orifice plate is patted.
The T cell that SAGE1TCR-T cell (T cell of SAGE1TCR transduction, effector cell), non-SAGE1TCR transduce (Non-SAGE1TCR-T expresses the effector cell of other TCR) and LCLs-A11/02 (target cell) make according to described in embodiment 3 It is standby, and corresponding small peptide is added in corresponding experimental group, wherein PSAGE1For SAGE1 388-398ATIIHNLREEK small peptide, PA02And PA11 For non-SAGE1TCR specific bond small peptide.
Then all components of test are added by ELISPOT orifice plate using following sequence:
130 microlitres of target cells, 154000 cells/mls (obtain about 20000 target cell/holes in total).
50 microlitres of effector cells's (2000 SAGE1TCR-T cells).
20 microlitre 10-4The SAGE1PX251 388-398ATIIHNLREEK/P of mol/LA02/PA11Small peptide solution is (dense eventually Degree is 10-6Mol/L).
All holes prepare addition in triplicate.
Then orifice plate (37 DEG C/5%CO2) second day overnight is incubated, culture medium is abandoned, is washed orifice plate 2 times with distilled water, then use Washing buffer is washed 3 times, is patted on paper handkerchief to remove remaining washing buffer.Then dilute with the PBS containing 10%FBS Detection primary antibody is released, each hole is added by 100 microlitres/hole.It incubates orifice plate 2 hours, then is washed 3 times with washing buffer at room temperature, Orifice plate is patted on paper handkerchief to remove excessive washing buffer.
Streptavidin-alkaline phosphatase is diluted by 1:10000 with the PBS containing 10%FBS, it is diluted by 100 microlitres Streptavidin-alkaline phosphatase is added each hole and incubates orifice plate 1 hour at room temperature.Then it is washed 3 times with washing buffer PBS is washed 2 times, pats orifice plate on paper handkerchief to remove excessive washing buffer and PBS.Kit is added after washing to mention Develop in the 100 microlitres/hole of BCIP/NBT solution of confession.It is protected from light during development with masking foil covering orifice plate, stands 5-15 points Clock.The spot of conventional detection development orifice plate during this period, determines the Best Times for terminating reaction.Removal BCIP/NBT solution is used in combination Distilled water rinses orifice plate to stop developing reaction, then drying removes orifice plate bottom, is dried at room temperature for orifice plate until each Hole is completely dried, and recycles immunodotting plate count meter (CTL, Celltech Ltd. (Cellular Technology Limited the)) spot that counterdie is formed in counting orifice.
As a result
The T cell (as described above) for examining SAGE1TCR transduction is tested to load SAGE1388- by ELISPOT The IFN-γ release that the target cell of 398ATIIHNLREEK small peptide and the target cell of non-specific small peptide react.It utilizes Graphpad prism6 draws the ELSPOT amount of speckle observed in each hole.
Experimental result is as shown in figure 9, individually SAGE1TCR-T cell (effector cell) or LCL cell (target cell) addition Corresponding small peptide is discharged without IFN-γ.
SAGE1TCR-T cell (effector cell) can be with load PSAGE1LCLs-A11/02 cell react release it is more IFN-γ。
SAGE1TCR-T cell (effector cell) is to load PA11Or PA02LCLs-A11/02 cell IFN-γ release very It is few.
The T cell (Non-SAGE1TCR-T expresses the effector cell of other TCR) of non-SAGE1TCR transduction is to load PSAGE1 LCLs-A11/02 cell IFN-γ release seldom, i.e., non-SAGE1TCR to SAGE1388-398ATIIHNLREEK small peptide without Recognition reaction.
4.2 non-radioactive cell toxicity test schemes
The test is51Cr discharges the colorimetric alternate test of cell toxicity test, quantitative determines the cream discharged after cell cracking Acidohydrogenase (LDH).The LDH of release in the medium is detected using the enzyme reaction of coupling in 30 minutes, LDH can in enzyme reaction A kind of tetrazolium salts (INT) are made to be converted into red formazan (formazan).The amount of the red product of generation and the cell number of cracking It is directly proportional.Collection 490nm visible light extinction Value Data can be collected with 96 hole read plates of standard.
Material
CytoToxNon-radioactive cell toxicity detection (Pu Luomaige company, G1780) contains substrate mixture, examination Test buffer, cracked solution and stop buffer.
Test medium: 10%FBS (it is heat-inactivated, Ji Bu can company, catalog number (Cat.No.) 16000-044), containing phenol red 90% RPMI 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt), 1% penicillin/streptomycin (Ji Bu can company, mesh Record 15070-063).
Micropore round bottom tissue culturing plates with 96 hole (Nucor Corporation (Nunc), catalog number (Cat.No.) 163320)
96 hole immuno plate Maxisorb (Nucor Corporation (Nunc), catalog number (Cat.No.) 442404)
Method
Target cell preparation
Target cell LCLs used in the test is as the target cell preparation method in aforementioned ELISPOT scheme.It is testing Target cell is prepared in culture medium: target cell concentration is adjusted to 3 × 105A/milliliter, every hole take 50 microlitres to obtain 1.5 × 104It is a thin Born of the same parents/hole.
Effector cell's preparation
Effector cell's (T cell) of this test is to express SAGE1 specificity TCR through flow cytometry in embodiment 3 CD8+T cell (SAGE1TCR-T).To express other special TCR of non-SAGE1 small peptide as control group.
Effector cell and target cell ratio use 1:1 and 2:1, and wherein target cell is quantitative (3 × 105A/milliliter, every hole 50 microlitres are taken to obtain 1.5 × 104A cells/well), effector cell is according to effect target than variation.
The preparation of small peptide solution
SAGE1 388-398ATIIHNLREEK (or PX1And PX2Two kinds of non-specific small peptides) RPMI of the small peptide containing 5%FBS 1640 culture mediums are diluted to 10 by 10 times of dilution methods-6The working solution of M, ultimate density is 10 after so that it is added to experimental group-6M。
(a) effector cell is detected by the small peptide that target cell loads various concentration and kills ability
Test prepares
All components of test are added by micropore round bottom tissue culturing plates with 96 hole using following sequence:
Each hole is added in -80ul target cell (preparation as described above)
Each hole is added in -100ul effector cell (preparation as described above)
Each hole (do not load small peptide experimental group and directly mend 20ul culture medium) is added in -20ul small peptide solution.
Prepare control group as described below:
Small peptide experimental group is not loaded: containing 100ul effector cell and 100ul target cell.
The spontaneous release of effector cell: only 100ul effector cell.
The spontaneous release of target cell: only 100ul target cell.
Target cell maximum release: only 100ul target cell.
Reagent culture medium control: only 200ul culture medium.
All holes prepare in triplicate, and final volume is 200ul (inadequate is supplied with culture medium).
37 DEG C incubate 24 hours.Before collecting all hole supernatants, by target cell maximum release control wells in -70 DEG C of placement cells About 30 minutes, then melt 15 minutes at 37 DEG C, so that target cell all cracks.
It is centrifuged plate 4 minutes in 250g.The 50ul supernatant in each hole of test panel is transferred to 96 hole immuno plates The corresponding aperture of Maxisorb plate.Using test buffer (12ml) reconstituted substrate mixture, then plus 50ul is to each hole of plate.It is flat Plate close the lid after at shady place incubation at room temperature 30 minutes.Each hole of plate is added to terminate reaction in 50ul stop bath.It is added The absorbance of record 490nm is counted after stop bath in 1 hour.
Calculated result
Culture medium is deducted from all absorbance values of release group from experimental group, the spontaneous release group of target cell and effector cell The absorbance value of background.
It brings the corrected value obtained among the above into following formula, calculates each effect target than generated cytotoxicity Percentage.
Cytotoxicity=100 % × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximum-target cell from Hair)
As a result
By non-radioactive cell toxicity detection inspection SAGE1TCR-T cell (as described above) to load SAGE1 388- The LDH release that the target cell of 398ATIIHNLREEK small peptide and non-specific small peptide reacts.It is drawn using Graphpad prism6 Make 490nm visible light light absorption value in each hole.
Experimental data statistical result is as shown in Figure 10, load 10-6Under the small peptide concentration of M, SAGE1TCR-T cell is thin to target The lethal effect of born of the same parents LCLs-A11/02 is gradually increased with the raising of effector cell and target ration；To do not load small peptide or Load PX1/PX2The target cell LCLs-A11/02 of non-specific small peptide is without lethal effect.
The T cell (Non-SAGE1TCR-T expresses the effector cell of other TCR) of the non-SAGE1TCR transduction of control group is to negative Carry PSAGE1LCLs-A11/02 cell without dissolution, it is seen that non-SAGE1TCR cell is to SAGE1 388- 398ATIIHNLREEK small peptide is without recognition reaction.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>T cell receptor of SAGE1 antigen small peptide is identified
<130> P2017-2276
<160> 29
<170> PatentIn version 3.5
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Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp
1 5 10 15
Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu
20 25 30
Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr
35 40 45
Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu
50 55 60
Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln
65 70 75 80
Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ser Pro Pro Asp Asn
85 90 95
Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu Ser Val Leu
100 105 110
Pro Tyr
<210> 2
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Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg Gly
1 5 10 15
Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His Val
20 25 30
Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val Tyr
35 40 45
Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu Arg
50 55 60
Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile Gln
65 70 75 80
Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser Pro
85 90 95
Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
<210> 3
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Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp
1 5 10 15
Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu
20 25 30
Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr
35 40 45
Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu
50 55 60
Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln
65 70 75 80
Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ser Pro Pro Asp Asn
85 90 95
Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu Ser Val Leu
100 105 110
Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser
115 120 125
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln
130 135 140
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys
145 150 155 160
Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val
165 170 175
Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn
180 185 190
Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys
195 200 205
Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val
225 230 235 240
Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 4
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<213>artificial sequence (artificial sequence)
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Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg Gly
1 5 10 15
Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His Val
20 25 30
Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val Tyr
35 40 45
Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu Arg
50 55 60
Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile Gln
65 70 75 80
Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser Pro
85 90 95
Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu
100 105 110
Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
115 120 125
Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu
130 135 140
Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
145 150 155 160
Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys
165 170 175
Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu
180 185 190
Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
195 200 205
Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
210 215 220
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
225 230 235 240
Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser
245 250 255
Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala
260 265 270
Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp
275 280 285
Ser Arg Gly
290
<210> 5
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<213>artificial sequence (artificial sequence)
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Lys Gly His Ser His
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<210> 6
<211> 6
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 6
Asp Ser Ser Ser Thr Tyr
1 5
<210> 7
<211> 7
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 7
Ile Phe Ser Asn Met Asp Met
1 5
<210> 8
<211> 6
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<213>artificial sequence (artificial sequence)
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Leu Gln Lys Glu Asn Ile
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<210> 9
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<212> DNA
<213>artificial sequence (artificial sequence)
<400> 9
ggagaggatg tggagcagag tcttttcctg agtgtccgag agggagacag ctccgttata 60
aactgcactt acacagacag ctcctccacc tacttatact ggtataagca agaacctgga 120
gcaggtctcc agttgctgac gtatattttt tcaaatatgg acatgaaaca agaccaaaga 180
ctcactgttc tattgaataa aaaggataaa catctgtctc tgcgcattgc agacacccag 240
actggggact cagctatcta cttctgtgca gagagtccgc ctgataacta tggtcagaat 300
tttgtctttg gtcccggaac cagattgtcc gtgctgccct at 342
<210> 10
<211> 10
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 10
Ala Ser Ser Pro Val Ala Gly Glu Leu Phe
1 5 10
<210> 11
<211> 336
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 11
aatgccggcg tcatgcagaa cccaagacac ctggtcagga ggaggggaca ggaggcaaga 60
ctgagatgca gcccaatgaa aggacacagt catgtttact ggtatcggca gctcccagag 120
gaaggtctga aattcatggt ttatctccag aaagaaaata tcatagatga gtcaggaatg 180
ccaaaggaac gattttctgc tgaatttccc aaagagggcc ccagcatcct gaggatccag 240
caggtagtgc gaggagattc ggcagcttat ttctgtgcca gctcaccggt tgccggggag 300
ctgttttttg gagaaggctc taggctgacc gtactg 336
<210> 12
<211> 762
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 12
ggagaggatg tggagcagag tcttttcctg agtgtccgag agggagacag ctccgttata 60
aactgcactt acacagacag ctcctccacc tacttatact ggtataagca agaacctgga 120
gcaggtctcc agttgctgac gtatattttt tcaaatatgg acatgaaaca agaccaaaga 180
ctcactgttc tattgaataa aaaggataaa catctgtctc tgcgcattgc agacacccag 240
actggggact cagctatcta cttctgtgca gagagtccgc ctgataacta tggtcagaat 300
tttgtctttg gtcccggaac cagattgtcc gtgctgccct atatccagaa ccctgaccct 360
gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 420
tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480
actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540
aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600
ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 660
acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 720
gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 762
<210> 13
<211> 13
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 13
Ala Glu Ser Pro Pro Asp Asn Tyr Gly Gln Asn Phe Val
1 5 10
<210> 14
<211> 873
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 14
aatgccggcg tcatgcagaa cccaagacac ctggtcagga ggaggggaca ggaggcaaga 60
ctgagatgca gcccaatgaa aggacacagt catgtttact ggtatcggca gctcccagag 120
gaaggtctga aattcatggt ttatctccag aaagaaaata tcatagatga gtcaggaatg 180
ccaaaggaac gattttctgc tgaatttccc aaagagggcc ccagcatcct gaggatccag 240
caggtagtgc gaggagattc ggcagcttat ttctgtgcca gctcaccggt tgccggggag 300
ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaaaaa cgtgttccca 360
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420
ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480
gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 540
ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600
aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660
tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 720
gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 780
tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 840
atggccatgg tcaagagaaa ggattccaga ggc 873
<210> 15
<211> 275
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 15
Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu
1 5 10 15
Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser
20 25 30
Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser
35 40 45
Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu
50 55 60
Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln
65 70 75 80
Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg
85 90 95
Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu
100 105 110
Ser Pro Pro Asp Asn Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr
115 120 125
Arg Leu Ser Val Leu Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr
130 135 140
Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr
145 150 155 160
Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val
165 170 175
Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys
180 185 190
Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala
195 200 205
Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser
210 215 220
Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr
225 230 235 240
Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile
245 250 255
Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu
260 265 270
Trp Ser Ser
275
<210> 16
<211> 310
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 16
Met Asp Thr Arg Leu Leu Cys Cys Ala Val Ile Cys Leu Leu Gly Ala
1 5 10 15
Gly Leu Ser Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg
20 25 30
Arg Arg Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His
35 40 45
Ser His Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe
50 55 60
Met Val Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro
65 70 75 80
Lys Glu Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu
85 90 95
Arg Ile Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala
100 105 110
Ser Ser Pro Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu
115 120 125
Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
130 135 140
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
145 150 155 160
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
165 170 175
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
180 185 190
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
195 200 205
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
210 215 220
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
225 230 235 240
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
245 250 255
Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly
260 265 270
Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
275 280 285
Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
290 295 300
Arg Lys Asp Ser Arg Gly
305 310
<210> 17
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 17
gacagctcct ccacctac 18
<210> 18
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 18
attttttcaa atatggacat g 21
<210> 19
<211> 39
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 19
gcagagagtc cgcctgataa ctatggtcag aattttgtc 39
<210> 20
<211> 15
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 20
aaaggacaca gtcat 15
<210> 21
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 21
ctccagaaag aaaatatc 18
<210> 22
<211> 30
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 22
gccagctcac cggttgccgg ggagctgttt 30
<210> 23
<211> 825
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 23
atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60
agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120
ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180
ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240
agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300
cagactgggg actcagctat ctacttctgt gcagagagtc cgcctgataa ctatggtcag 360
aattttgtct ttggtcccgg aaccagattg tccgtgctgc cctatatcca gaaccctgac 420
cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540
aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660
ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720
gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780
gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825
<210> 24
<211> 930
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 24
atggacacca gactactctg ctgtgcggtc atctgtcttc tgggggcagg tctctcaaat 60
gccggcgtca tgcagaaccc aagacacctg gtcaggagga ggggacagga ggcaagactg 120
agatgcagcc caatgaaagg acacagtcat gtttactggt atcggcagct cccagaggaa 180
ggtctgaaat tcatggttta tctccagaaa gaaaatatca tagatgagtc aggaatgcca 240
aaggaacgat tttctgctga atttcccaaa gagggcccca gcatcctgag gatccagcag 300
gtagtgcgag gagattcggc agcttatttc tgtgccagct caccggttgc cggggagctg 360
ttttttggag aaggctctag gctgaccgta ctggaggacc tgaaaaacgt gttcccaccc 420
gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480
gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540
aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600
aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660
ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720
acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780
gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840
gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900
gccatggtca agagaaagga ttccagaggc 930
<210> 25
<211> 208
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 25
Met Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly
1 5 10 15
Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr
20 25 30
Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr
35 40 45
Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val
50 55 60
Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr
65 70 75 80
Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ser Pro Pro Asp
85 90 95
Asn Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu Ser Val
100 105 110
Leu Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp
115 120 125
Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser
130 135 140
Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp
145 150 155 160
Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
165 170 175
Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn
180 185 190
Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
<210> 26
<211> 624
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 26
atgggtgaag atgttgaaca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 60
ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 120
ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 180
agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 240
cagactgggg actcagctat ctacttctgt gcagagagtc cgcctgataa ctatggtcag 300
aattttgtct ttggtcccgg aaccagattg tccgtgctgc cctatatcca gaaccctgac 360
cctgccgtgt accagctgag agactctaag tcgagtgaca agtctgtctg cctattcacc 420
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480
aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600
ttcttcccca gcccagaaag ttcc 624
<210> 27
<211> 243
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 27
Met Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg
1 5 10 15
Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His
20 25 30
Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val
35 40 45
Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu
50 55 60
Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile
65 70 75 80
Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser
85 90 95
Pro Val Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val
100 105 110
Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp
<210> 28
<211> 729
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 28
atgaatgcag gtgttatgca gaatccaaga cacctggtca ggaggagggg acaggaggca 60
agactgagat gcagcccaat gaaaggacac agtcatgttt actggtatcg gcagctccca 120
gaggaaggtc tgaaattcat ggtttatctc cagaaagaaa atatcataga tgagtcagga 180
atgccaaagg aacgattttc tgctgaattt cccaaagagg gccccagcat cctgaggatc 240
cagcaggtag tgcgaggaga ttcggcagct tatttctgtg ccagctcacc ggttgccggg 300
gagctgtttt ttggagaagg ctctaggctg accgtactgg aggacctgaa aaacgtgttc 360
ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 420
acactggtgt gcctggccac cggtttctac cccgaccacg tggagctgag ctggtgggtg 480
aatgggaagg aggtgcacag tggggtctgc acagacccgc agcccctcaa ggagcagccc 540
gccctcaatg actccagata cgctctgagc agccgcctga gggtctcggc caccttctgg 600
caggaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 660
gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 720
agagcagac 729
<210> 29
<211> 11
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 29
Ala Thr Ile Ile His Asn Leu Arg Glu Glu Lys
1 5 10

Claims (10)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be with ATIIHNLREEK-HLA-A1101 compound In conjunction with.
2. T cell receptor as described in claim 1, which is characterized in that the TCR includes TCR α chain variable domain and TCR β chain can Variable domain, the amino acid sequence of the CDR3 of the TCR α chain variable domain are AESPPDNYGQNFV (SEQ ID NO:13)；And/or institute The amino acid sequence for stating the CDR3 of TCR β chain variable domain is ASSPVAGELF (SEQ ID NO:10).
3. T cell receptor as claimed in claim 2, which is characterized in that 3 complementary determining regions of the TCR α chain variable domain (CDR) are as follows:
αCDR1-DSSSTY (SEQ ID NO:6)
αCDR2-IFSNMDM (SEQ ID NO:7)
αCDR3-AESPPDNYGQNFV (SEQ ID NO:13)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-KGHSH (SEQ ID NO:5)
βCDR2-LQKENI (SEQ ID NO:8)
βCDR3-ASSPVAGELF (SEQ ID NO:10)。
4. a kind of multivalent TCR complex, which is characterized in that it includes at least two TCR molecules, and it is therein at least one TCR molecule is TCR described in claim 1.
5. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules include the core for encoding TCR molecule described in claim 1 Acid sequence or its complementary series.
6. a kind of carrier, which is characterized in that the carrier contains nucleic acid molecules described in claim 5；Preferably, described Carrier is viral vectors；It is highly preferred that the carrier is slow virus carrier.
7. a kind of isolated host cell, which is characterized in that in the host cell containing carrier as claimed in claim 6 or Nucleic acid molecules described in the claim 5 of external source are integrated in genome.
8. a kind of cell, which is characterized in that described in nucleic acid molecules described in the cell transduction claim 5 or claim 6 Carrier；Preferably, the cell is T cell or stem cell.
9. a kind of pharmaceutical composition, which is characterized in that the composition contains pharmaceutically acceptable carrier and claim 1 The TCR, TCR compound as claimed in claim 4, nucleic acid molecules, load as claimed in claim 6 described in claim 5 Body or cell according to any one of claims 8.
10. core described in T cell receptor described in claim 1 or TCR compound as claimed in claim 4, claim 5 The purposes of acid molecule, carrier as claimed in claim 6 or cell according to any one of claims 8, which is characterized in that be used to prepare and control Treat the drug of tumour or autoimmune disease.
CN201810210760.3A 2018-03-14 2018-03-14 T cell receptor for recognizing SAGE1 antigen short peptide Active CN110272483B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478797A (en) * 2015-12-10 2017-03-08 广州市香雪制药股份有限公司 Stem from the tumor antigen small peptide of SAGE1
WO2017203526A1 (en) * 2016-05-23 2017-11-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Methods of diagnosing cancer using cancer testis antigens

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106478797A (en) * 2015-12-10 2017-03-08 广州市香雪制药股份有限公司 Stem from the tumor antigen small peptide of SAGE1
WO2017203526A1 (en) * 2016-05-23 2017-11-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Methods of diagnosing cancer using cancer testis antigens

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMEAGA MAHESWARAN 等: "Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer", 《PLOS ONE》 *
YAO-TSENG CHEN 等: "Cancer-testis antigen expression in digestive tract carcinomas: frequent expression in esophageal squamous cell carcinoma and its precursor lesions", 《CANCER IMMUNOL RES》 *

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