CN110272482A - Identify the T cell receptor of PRAME antigen small peptide - Google Patents

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

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CN110272482A
CN110272482A CN201810210194.6A CN201810210194A CN110272482A CN 110272482 A CN110272482 A CN 110272482A CN 201810210194 A CN201810210194 A CN 201810210194A CN 110272482 A CN110272482 A CN 110272482A
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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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Abstract

The present invention provides the T cell receptors (TCR) of small peptide EVLVDLFLK of the energy specific recognition derived from PRAME antigen a kind of, specifically, the present invention provides a kind of T cell receptor, the TCR can be in conjunction with the EVLVDLFLK-HLA-A*1101 compound for being presented to cell surface.Carrier the present invention also provides the sequence of nucleic acid molecules for encoding the TCR and comprising the sequence of nucleic acid molecules.

Description

Identify the T cell receptor of PRAME antigen small peptide
Technical field
The present invention relates to the TCR of energy specific recognition PRAME antigen small peptide, and the invention further relates to transduce above-mentioned TCR to obtain PRAME specificity T cell and they prevention and treatment PRAME related disease in purposes.
Background technique
RPAME is degraded to micromolecule polypeptide, and and MHC after generating in the cell as a kind of autochthonous tumor antigen (main histocompatibility complex) molecule combines and forms compound, is presented to cell surface.Studies have shown that EVLVDLFLK is Small peptide derived from PRAME.PRAME antigen is removed in melanoma, clear-cell carcinoma, lung cancer, breast cancer, medulloblastoma etc. Solid tumor expression is outer, also expresses in the Malignancy of part, as acute myeloid leukemia, chronic myelogenous leukemia, (Kessler JH, et al.J Exp Med, 2001,193 (1): 73-88.Chang such as acute lymphoblastic cancer, myeloma AY, et al.J Clin Invest.2017,127 (7): 2705-2718).Treatment for above-mentioned disease can use chemotherapy The methods of with radiation treatment, but the normal cell of itself can all be damaged.Therefore, to be derived from the short of PRAME antigen Target spot of the peptide as above-mentioned kinds cancer, not only can be used as the marker of above-mentioned medical diagnosis on disease, it may also be used for generate above-mentioned disease The prevention reagent and/or therapeutic agent of disease, such as antibody or T cell receptor.
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 specific recognition capability to PRAME antigen small peptide, it is made to play a role, or will The TCR transduces T cell to obtain the T cell for having specific recognition capability to PRAME antigen small peptide, to make them in cell It plays a role in immunization therapy.
Summary of the invention
The purpose of the present invention is to provide a kind of T cell receptors of energy specific recognition PRAME antigen small peptide.
First aspect present invention provides a kind of T cell receptor (TCR), and the TCR can be with EVLVDLFLK-HLA-A* 1101 compounds combine.
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 AATSGGSYIPT (SEQ ID NO:12)；And/or the ammonia of the CDR3 of the TCR β chain variable domain Base acid sequence is ASSLSGRLGEQF (SEQ ID NO:15).
In another preferred example, 3 complementary determining regions (CDR) of the TCR α chain variable domain are as follows:
α CDR1-DSAIYN (SEQ ID NO:10)
α CDR2-IQSSQRE (SEQ ID NO:11)
α CDR3-AATSGGSYIPT (SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
β CDR1-MNHNY (SEQ ID NO:13)
β CDR2-SVGAGI (SEQ ID NO:14)
βCDR3-ASSLSGRLGEQF (SEQ ID NO:15)。
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 SEQ ID NO:3,5,25 and/or the TCR β chain amino acid sequence is SEQ ID NO:4,6,27.
In another preferred example, the TCR is soluble.
In another preferred example, the TCR is single-stranded.
In another preferred example, the TCR is formed by connecting with β chain variable domain by peptide catenation sequence by α chain variable domain.
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, 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 SEQ ID NO:3,5,25 and/or the TCR β chain amino acid sequence is SEQ ID NO:4,6,27.
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, which is characterized in that the cysteine residues of artificial interchain disulfide bond are formed in the TCR Instead of selected from following one or more groups of sites:
The 46th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1；
The 47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1；
The 46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1；Or
The 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1.
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.Preferably, 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: 7。
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID of coding TCR β chain variable domain NO:8.
In another preferred example, the nucleic acid molecules include coding TCR α chain nucleotide sequence SEQ ID NO:9,16, 26 and/or comprising encode TCR β chain nucleotide sequence SEQ ID NO:17,18,28.
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；
Preferably, the disease is tumour, and the preferably described tumour is selected from the group: melanoma, clear-cell carcinoma, lung Cancer, breast cancer, medulloblastoma, Malignancy, 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 respectively illustrate TCR α chain variable domain amino acid sequence, TCR α chain Variable domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, the TCR α chain amino acid with leader sequence Sequence and 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 respectively illustrate TCR β chain variable domain amino acid sequence, TCR β chain Variable domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, the TCR β chain amino acid with leader sequence Sequence and TCR β chain nucleotide sequence with leader sequence.
Fig. 3 shows the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b respectively illustrate the amino acid sequence and nucleotide sequence of sTCR α chain.
Fig. 5 a and Fig. 5 b respectively illustrate the amino acid sequence and nucleotide sequence of sTCR β chain.
Fig. 6 shows the glue figure of the sTCR obtained after purification.Leftmost side swimming lane is to go back virgin rubber, and intermediate swimming lane is non- Also virgin rubber, rightmost side swimming lane are molecular weight marker (marker).
Fig. 7 shows Biacore power of the sTCR of the present invention in conjunction with EVLVDLFLK-HLA-A*1101 compound Learn map.
Fig. 8 shows the thin testing result of primary T of tetramer staining TCR transduction.
Fig. 9 shows ELISPOT testing inspection result.
Figure 10 shows lethal effect of the T cell to specificity target cell of TCR transduction of the present invention.
Specific embodiment
The present inventor after extensive and in-depth study, has found and PRAME antigen small peptide EVLVDLFLK (SEQ ID NO:29) the TCR that can be specifically bound, the antigen small peptide EVLVDLFLK can form compound and together with HLA-A*1101 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 " PRAME TCR-T " refers to can react for PRAME antigen peptide of the invention, but right Other Antigenic Peptides are responseless cells.
As used herein, term " T cell of non-PRAME TCR transduction ", " Non-PRAME TCR-T " are used interchangeably, Refer both to express the T cell of other TCR, it is understood that it is to be for PRAME antigen peptide of the invention, but the TCR that function is bad, These T cells are added together with PRAME antigen 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 EVLVDLFLK-HLA-A*1101 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:
α CDR1-DSAIYN (SEQ ID NO:10)
α CDR2-IQSSQRE (SEQ ID NO:11)
α CDR3-AATSGGSYIPT (SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
β CDR1-MNHNY (SEQ ID NO:13)
β CDR2-SVGAGI (SEQ ID NO:14)
β CDR3-ASSLSGRLGEQF (SEQ ID NO:15)。
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:10), CDR2 (SEQ ID NO:11) and CDR3 (SEQ ID NO:12) containing above-mentioned α chain.Preferably, The TCR molecule includes α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domain of the TCR molecule Amino 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, The β chain variable domain amino acid sequence include above-mentioned β chain CDR1 (SEQ ID NO:13), CDR2 (SEQ ID NO:14) and CDR3(SEQ ID NO:15).Preferably, the TCR molecule includes β chain variable domain amino acid sequence SEQ ID NO:2.It is more excellent Selection of land, the β chain variable domain amino acid sequence of the TCR molecule are 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:10) of the α chain variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned α chain, CDR2 (SEQ ID NO:11) and CDR3 (SEQ ID NO:12).Preferably, the single chain TCR molecules include α chain variable domain ammonia Base 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:13), the CDR2 of above-mentioned β chain (SEQ ID NO:14) and CDR3 (SEQ ID NO:15).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 that the amino acid sequence of SEQ ID NO:3,5,25 and/or β chain is SEQ ID NO:4,6,27.
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 PRAME antigen small peptide TCR.In a preferred embodiment, the amino acid sequence of sTCR α chain of the invention is as shown in SEQ ID NO.:25, core Nucleotide sequence is as shown in SEQ ID NO.:26；The amino acid sequence of TCR β chain of the invention is as shown in SEQ ID NO.:27, core Nucleotide sequence such as SEQ ID NO.:28.
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 EVLVDLFLK-HLA-A*1101 compound), therapeutic agent, PK (protein kinase) modified part or it is any more than The combination of these 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:
α CDR1-gatagcgctatttacaac (SEQ ID NO:19)
α CDR2-attcagtcaagtcagagagag (SEQ ID NO:20)
α CDR3-gctgctacatcaggaggaagctacatacctaca (SEQ ID NO:21)。
The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR region is as follows:
β CDR1-atgaaccataactac (SEQ ID NO:22)
β CDR2-tcagttggtgctggtatc (SEQ ID NO:23)
β CDR3-gccagcagccttagcgggaggttgggtgagcagttc (SEQ ID NO:24)。
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:19, SEQ ID NO:20 and SEQ ID NO:21, 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:22, SEQ ID NO:23 and SEQ ID NO:24.
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:7 and/or coding TCR β chain variable domain of the present invention includes SEQ ID NO:8.It is highly preferred that the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:9,16 and/or SEQ ID NO: 17、18。
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:7.
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 PRAME antigen 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).
PRAME antigen related disease
The invention further relates to the method with PRAME related disease is treated and/or prevented in subject comprising adoptive Shift the step of PRAME specific T-cells are to the subject.The PRAME specific T-cells can recognize EVLVDLFLK and HLA- A*1101 compound.
The T cell of PRAME specificity of the invention can be used for treating any presentation PRAME antigen small peptide EVLVDLFLK- The PRAME related disease of HLA-A*1101 compound.Including but not limited to, melanoma, clear-cell carcinoma, lung cancer, breast cancer, at The solid tumors such as Medulloblastoma and Malignancy, such as acute myeloid leukemia, chronic myelogenous leukemia, acute Lymphocyte cancer, myeloma etc..
Treatment method
Can by separation with the patient of PRAME 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 PRAME 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 tied specifically with PRAME antigen small peptide compound EVLVDLFLK-HLA-A*1101 It closes, while the cell for the TCR of the present invention that transduceed can have apparent killing to make by specific activation and to specific target cell With.
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 PRAME antigen small peptide specific T-cells
Using synthesis small peptide PRAME 176-184EVLVDLFLK (Beijing SBS Genetech gene technology Co., Ltd) stimulation come From in the peripheral blood lymphocytes (PBL) for the healthy volunteer that genotype is HLA-A11.By PRAME176-184EVLVDLFLK Small peptide and the HLA-A*1101 renaturation for having biotin labeling, prepare the mono- aggressiveness of pHLA.These single aggressiveness and the chain marked with PE Mould Avidin (BD company) is combined into the tetramer of PE label, sorts the tetramer and anti-CD8-APC double positive cells.Amplification point The cell of choosing, and secondary sorting is carried out according to the above method, then Colony Culture is carried out with limiting dilution assay.Monoclonal cell is used The tetramer and the dyeing of anti-CD8 antibody, the double positive colonies screened are as shown in Figure 3.
Embodiment 2 obtains the building of the tcr gene and carrier of PRAME antigen small peptide specific T-cell clones of the present invention
Use Quick-RNATMThe PRAME 176- screened in MiniPrep (ZYMO research) extracting embodiment 1 The total serum IgE of the restrictive T cell clone of 184EVLVDLFLK specificity, HLA-A11.The synthesis of cDNA is using clontech's SMART RACE cDNA amplification kit, the primer of use are the C-terminal conserved regions designed in mankind's tcr gene.By sequence gram It is grand to being sequenced on carrier T (TAKARA).Through being sequenced, the α chain and β chain-ordering structure point of the TCR of double positive colony expression Not as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c and Fig. 1 d 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 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 nucleotides sequence Column.
Identified, α chain includes the CDR with following amino acid sequence:
α CDR1-DSAIYN (SEQ ID NO:10)
α CDR2-IQSSQRE (SEQ ID NO:11)
α CDR3-AATSGGSYIPT (SEQ ID NO:12)
β chain includes the CDR with following amino acid sequence:
β CDR1-MNHNY (SEQ ID NO:13)
β CDR2-SVGAGI (SEQ ID NO:14)
β CDR3-ASSLSGRLGEQF (SEQ ID NO:15)。
By overlapping (overlap) PCR respectively by the variable domain of TCR α chain and β chain respectively with mouse TCR α chain and β chain Conservative domain is spliced into full-length gene and is connected to Lentiviral pLenti (addgene).Specifically: use overlap The full-length gene of TCR α chain and TCR β chain is attached to obtain TCR α -2A-TCR β segment by PCR.By Lentiviral and TCR α -2A-TCR β digestion connects to obtain pLenti-PRAME TRA-2A-TRB-IRES-NGFR plasmid.It is used as control, simultaneously Also the slow virus carrier pLenti-eGFP of building expression eGFP.Pseudovirus is packed with 293T/17 again later.
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, 20mMTris (pH with the mass ratio of 1:1 8.1) in, 3.7mMcystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.It will after mixing Solution is placed in the deionized water of 10 times of volumes dialysis (4 DEG C), after 12 hours by deionized water change into buffer (20mMTris, PH 8.0) continue at 4 DEG C of dialysis 12 hours.Solution after the completion of dialysis is handed over after 0.45 μM of membrane filtration by anion Change column (HiTrap Q HP, 5ml, GE Healthcare) purifying.The TCR that eluting peak contains the successful α and β dimer of renaturation is logical Cross the confirmation of 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 is greater than 90% by SDS-PAGE measurement, and concentration is surveyed by BCA method It is fixed.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 molecule of the present invention can specifically with EVLVDLFLK-HLA-A*1101 Compound combines.
Use TCR molecule and EVLVDLFLK- obtained in BIAcore T200 real-time analyzer detection embodiment 3 The combination activity of HLA-A*1101 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 EVLVDLFLK-HLA-A* 1101 compounds flow 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.Above-mentioned EVLVDLFLK-HLA-A*1101 compound Preparation process is as follows:
A. it purifies
The E.coli bacterium solution for collecting 100ml inducing expression heavy chain or light chain uses 10ml after 4 DEG C of 8000g are centrifuged 10min PBS washing thalline is primary, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) later Thallus is resuspended in concussion, and rotates in room temperature and be incubated for 20min, and later in 4 DEG C, 6000g is centrifuged 15min, discards supernatant, collection is forgiven Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated for 5min；Add 30ml The BugBuster of 10 times of dilution is mixed, and 4 DEG C of 6000g are centrifuged 15min；It discards supernatant, 30ml is added to dilute 10 times of BugBuster Inclusion body is resuspended, mixes, 4 DEG C of 6000g are centrifuged 15min, are repeated twice, and 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 EVLVDLFLK (Beijing SBS Genetech gene technology Co., Ltd) of synthesis is dissolved in DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain is dissolved with 8M urea, 20mMTris pH 8.0,10mM DTT, and 3M is added before renaturation Guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA are further denaturalized.Renaturation is added with 25mg/L (final concentration) in LVLGTLEEV peptide to delay Fliud flushing (0.4M L-arginine, 100mMTris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced form paddy The sweet peptide of Guang, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add the light chain of 20mg/L and heavy chain (final concentration, the weight of 90mg/L Chain is 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 0-400mM NaCl linear gradient liquid that 20mM Tris pH 8.0 is prepared elutes albumen, and pMHC is about in 250mM It is eluted at NaCl, collects all peak components, SDS-PAGE detects purity.
D. biotinylation
It with Millipore super filter tube by the pMHC molecular concentration of purifying, while being 20mM Tris pH by buffer exchange 8.0, biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D- is then added Biotin, 100 μ g/ml BirA enzymes (GST-BirA), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detects biotinylation Completely.
E. the compound after purifying biological element
PMHC molecular concentration after being marked biotinylation with Millipore super filter tube is to 1ml, using gel permeation chromatography The pMHC of purifying biological element, purifies instrument (GE General Electric Co. Limited) using Akta, pre-equilibrates HiPrepTM with filtered PBS 16/60S200HR 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.
5 PRAME specific t-cell receptor slow virus of embodiment packaging transfects PRAME TCR with primary T cells
(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 plasmid (one kind containing pLenti-PRAME TRA-2A-TRB-IRES-NGFR described in embodiment 2 Slow virus carrier, and 3 kinds of plasmids containing other components necessary to building infectiousness but non-replicating lentiviral particle) turn Contaminate 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-PRAME TRA-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: 22.5 micrograms: 15 micrograms: 15 micrograms: 7.5 micrograms.Transfection The ratio of reagent PEI-MAX and plasmid is 2:1, and the usage amount of each plate is 114.75 micrograms.Concrete operations are as follows: expression matter Grain and packaging plasmid are added in 1800 microlitres of OPTI-MEM (Ji Bu can company (Gibco), catalog number (Cat.No.) 31985-070) culture medium and mix It closes uniformly, being stored at room temperature 5 minutes becomes DNA mixed liquor；Corresponding amount PEI is taken to mix with 1800 microlitres of OPTI-MEM culture mediums Even, being stored at room temperature 5 minutes becomes PEI mixed liquor.DNA mixed liquor and PEI mixed liquor are mixed and are being stored at room temperature 30 Minute, then 3150 microlitres of OPTI-MEM culture mediums are added, it is added to has been converted into 11.25 milliliters of OPTI-MEM after mixing 293T/17 cell in, shake gently culture dish, make culture medium be uniformly mixed, 37 DEG C/5%CO2Lower culture.It is small to transfect 5-7 When, remove transfection media, change into containing 10% fetal calf serum DMEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt)) complete medium, 37 DEG C/5%CO2Lower culture.Training of the collection containing wrapped slow virus in 3rd and the 4th day Support base supernatant.For the slow virus of harvest packaging, collected culture supernatant 3000g is centrifuged 15 minutes removal cell fragments, It filters through 0.45 micron filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB), finally uses again The concentration tube (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) UFC905096) of 50KD interception is concentrated, and is removed Most of supernatant is finally concentrated to 1 milliliter, freezes for -80 DEG C after equal portions packing.Pseudovirus sample is taken to carry out virus titer survey Fixed, 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 PRAME 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=3:1.
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 PRAME 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 10%FBS that 50IU/ml IL-2 and 10ng/ml IL-7 is added 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
PRAME 176-184EVLVDLFLK small peptide and the HLA-A*1101 renaturation for having biotin labeling, preparation pHLA are mono- Aggressiveness.The Streptavidin (BD) that these single aggressiveness are marked with PE is combined into the tetramer of PE label, referred to as PRAME- tetramer-PE.The T cell for expressing PRAME specific t-cell receptor gene can be labeled as positive cell by this tetramer. T cell sample transduced in (b) is mixed with PRAME-tetramer-PE and is incubated on ice 30 minutes, then addition resists small Mouse β chain-APC antibody continues to be incubated for 15 minutes on ice.Sample uses Millipore after cleaning 2 times with the PBS containing 2%FBS Guava or BD Arial detection or sorting express PRAME specific t-cell receptor gene PRAME-tetramer-PE and The double positive T cells of anti-mouse β chain-APC, data analysis use 3.1 software of guavaSoft ((Merck Mi Libo (Merck Millipore)) or FlowJo software (Tree Star Inc, Ashland, OR) is analyzed.
Through testing and analyzing, as a result as shown in figure 8, being dyed with PRAME-tetramer-PE and anti-mouse β chain-APC antibody Afterwards, the blank control group T cell without TCR slow-virus infection is double positive without PRAME-tetramer-PE and anti-mouse β chain-APC Cell, and the PRAME-tetramer-PE and anti-mouse β chain-APC that expression TCR occur through the T cell of TCR slow-virus infection are double Positive cell, only a small amount of nonspecific double sun when being dyed with other tetramer-PE of non-PRAME-tetramer-PE Property cell.
Embodiment 6 verifies PRAME specificity TCR function
6.1 ELISPOT schemes
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 of Epstein-Barr viral (EBV) conversion (LCLs).B95-8 cell is induced through myristoyl acetic acid phorbol ester (TPA) produces the culture medium supernatant containing EBV, and 4 DEG C/600g It is centrifuged 10 minutes removal impurity, then crosses 0.22 micron filter, equal part dispenses -70 DEG C of preservations.It is HLA-A11/ from genotype The peripheral blood lymphocytes (PBL) of the healthy volunteer of A02/A24 (including homozygote and heterozygote), taking 10 milliliters of concentration is 2 ×107The PBL suspension of/milliliter is added after cyclosporin in 25 square centimeters of culture bottle in 37 DEG C/CO2It is incubated in incubator It educates 1 hour, quick-thawing portion EBV, is added in above-mentioned cell by 1/10 dilution, gently shakes up and culture bottle is uprightly placed in 37℃/CO2It is cultivated in incubator.10 milliliters of culture mediums of addition continue to cultivate after culture 12 days, and further expansion is trained after about 30 days It supports and carries out flow cytometer detection, wherein CD19+CD23hiCD58+For B-lymphoblastoid cell lines (LCLs).This ELISPOT test Using HLA-A11 as specificity target cell.
Effector cell's preparation
Effector cell's (T cell) of this test is to express PRAME 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 PRAME 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.
PRAME T Cell (T cell of PRAME TCR transduction, effector cell), T cell (it is non-to express other The T cell of EVLVDLFLK small peptide specificity TCR) and LCLs A24/A11 (target cell) prepared according to described in embodiment 3, and Corresponding small peptide is added in corresponding experimental group, wherein PPRAME176-184EVLVDLFLK small peptide, PN1、PN2、PN3It is special for non-PRAME TCR Different bound short peptide.
Then all components of test are added by ELISPOT orifice plate using following sequence:
130 microlitres of target cells, 77000 cells/mls (obtain about 10000 target cell/holes in total).
50 microlitres of effector cells's (1000 bis- positive T cells of PRAME TCR).
20 microlitre 10-5The PRAME 176-184EVLVDLFLK/ non-specificity small peptide solution (final concentration of 10 of mol/L-6Mol/L).
All holes prepare addition in triplicate.
Then (37 DEG C/5%CO overnight of orifice plate are incubated2) second day, 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 1:100 with the PBS containing 10%FBS, by 100 microlitres of diluted chains Mould Avidin-alkaline phosphatase is added each hole and incubates orifice plate 1 hour at room temperature.Then 3 PBS are washed with washing buffer Washing 2 times, pats orifice plate on paper handkerchief to remove excessive washing buffer and PBS.Kit is added after washing to provide 100 microlitres/hole of BCIP/NBT solution develop.It is protected from light during development with masking foil covering orifice plate, stands 5-15 minutes. The spot of conventional detection development orifice plate during this period, determines the Best Times for terminating reaction.Remove BCIP/NBT solution and with pair It steams water and rinses orifice plate to stop developing reaction, then drying removes orifice plate bottom, be dried at room temperature for orifice plate until each hole It is completely dried, 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 PRAME TCR transduction is tested to load PRAME176- by ELISPOT The IFN-γ release that the target cell of 184EVLVDLFLK small peptide and non-specific small peptide reacts.Utilize graphpad prism6 Draw the ELSPOT amount of speckle observed in each hole.
Experimental result as shown in figure 9, PRAME T Cell cell (effector cell) individually with LCL cell (target cell) when release It is seldom to put IFN-γ.
PRAME T Cell (effector cell) can be with addition PPRAMELCL cell react and release more IFN-γ.It says Bright TCR of the present invention can identify PPRAMESmall peptide, therefore can mediate T cell activation.
IFN-γ release is seldom when PRAME T Cell (effector cell) adds the LCL cell of non-specific small peptide.
T Cell (negative control effector cell) adds PPRAMELCL cell when IFN-γ release it is seldom.Illustrate that other are non- Specificity TCR cannot identify PPRAMESmall peptide, therefore it is unable to mediate T cell activation.
6.2 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
CytoTox Non-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), without phenol red 95%RPMI 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) 11835-030).
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
This test is target cell using tetra- plants of tumor cell lines of K562-A24, K562-A11, SK-MEL-5 and A375.It is trying Test and prepare target cell in culture medium: target cell concentration is adjusted to 200/milliliter, and every hole takes 100 microlitres to obtain 2 × 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 PRAME specificity TCR through flow cytometry in embodiment 3 CD8+T cell.Effector cell and target cell ratio use 2.5:1. and 1.25:1.If it is special to express other non-PRAME small peptides The CD8 of property TCR+T cell adds target cell to be control group (2.5:1).
The CD8 of PRAME specificity TCR transduction+T cell specific killing test neoplastic cell
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 -100ul target cell (preparation as described above)
Each hole is added in -100ul effector cell (preparation as described above)
Prepare control group as described below:
The spontaneous release of effector cell: 100ul effector cell.
The spontaneous release of target cell: 100ul target cell.
Target cell maximum release: 100ul target cell.
Culture medium control: 200ul culture medium.
Volume correction control: 200ul culture medium+20ul lysate (10X) (is added) before detection
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, 10 × lysate is added in target cell maximum release control wells And culture about 45 minutes is placed, 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 the corresponding of flat 96 orifice plate Hole.Using test buffer (12ml) reconstituted substrate mixture, then plus 50ul is to each hole of plate.Plate close the lid after in yin Dark at room temperature incubates 30 minutes.Each hole of plate is added to terminate reaction in 50ul stop bath.It is added after stop bath in 1 hour Count the absorbance of record 490nm.
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, caused by the release of target cell maximum deducts volume correction control to correct since lysate (10X) is added Volume change.
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
It is special to loading by the non-radioactive cell toxicity detection T cell (as described above) for examining PRAME TCR transduction Property target cell react LDH release.490nm visible light light absorption value in each hole, which is drawn, using graphpad prism6 substitutes into public affairs Percentage of cytotoxicity value after formula calculating.
Experimental data statistical result is as shown in Figure 10, as effect target ratio increases, the CD8 of PRAME TCR transduction+T cell (PRAME T Cell) increases K562-A11 the and SK-MEL-5 killing functions of immunocytes of HLA A11 and expression PRAME antigen；It is right Non-specific cell K562-A24 and A375 makees without killing.Express the CD8 of other non-PRAME small peptide specificity TCRs+T cell pair HLA A11 and the K562-A11 killing rate for expressing PRAME antigen are significantly lower than the T cell group of PRAME TCR transduction.This test knot Fruit illustrate TCR of the present invention can mediate T cell specific killing target cell, but other non-PRAME small peptide specificity TCRs are then not It can mediate T cell killing target cell.
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 PRAME antigen small peptide is identified
<130> P2017-2277
<160> 29
<170> PatentIn version 3.5
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Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly
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Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala
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Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser
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Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Ser Gly Gly
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Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro Asn Gly Tyr
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Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Glu Leu
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Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Leu Ser
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Gly Arg Leu Gly Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val
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Leu
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Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly
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Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala
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Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser
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Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Ser Gly Gly
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Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His Pro
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Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys
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Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr
130 135 140
Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr
145 150 155 160
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala
165 170 175
Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser
180 185 190
Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp
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Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe
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Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala
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Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
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Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro Asn Gly Tyr
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Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Glu Leu
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Gly Arg Leu Gly Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val
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Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu
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Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
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Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val
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Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
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Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 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 Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu
245 250 255
Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr
260 265 270
Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys
275 280 285
Asp Ser Arg Gly
290
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Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp
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Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val
20 25 30
Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala
35 40 45
Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr
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Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg
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Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile
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Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr
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Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile
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Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg
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Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp
145 150 155 160
Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr
165 170 175
Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser
180 185 190
Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe
195 200 205
Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser
210 215 220
Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn
225 230 235 240
Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu
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Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265 270
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Met Ser Ile Ser Leu Leu Cys Cys Ala Ala Phe Pro Leu Leu Trp Ala
1 5 10 15
Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Arg Ile Leu
20 25 30
Lys Ile Gly Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His
35 40 45
Asn Tyr Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu
50 55 60
Ile Tyr Tyr Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro
65 70 75 80
Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg
85 90 95
Leu Glu Leu Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser
100 105 110
Ser Leu Ser Gly Arg Leu Gly Glu Gln Phe Phe Gly Pro Gly Thr Arg
115 120 125
Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala
130 135 140
Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
145 150 155 160
Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser
165 170 175
Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro
180 185 190
Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu
195 200 205
Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn
210 215 220
His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
225 230 235 240
Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu
245 250 255
Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln
260 265 270
Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala
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Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val
290 295 300
Lys Arg Lys Asp Ser Arg Gly
305 310
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Ala Ala Ala Cys Ala Gly Gly Ala Gly Gly Thr Gly Ala Cys Gly Cys
1 5 10 15
Ala Gly Ala Thr Thr Cys Cys Thr Gly Cys Ala Gly Cys Thr Cys Thr
20 25 30
Gly Ala Gly Thr Gly Thr Cys Cys Cys Ala Gly Ala Ala Gly Gly Ala
35 40 45
Gly Ala Ala Ala Ala Cys Thr Thr Gly Gly Thr Thr Cys Thr Cys Ala
50 55 60
Ala Cys Thr Gly Cys Ala Gly Thr Thr Thr Cys Ala Cys Thr Gly Ala
65 70 75 80
Thr Ala Gly Cys Gly Cys Thr Ala Thr Thr Thr Ala Cys Ala Ala Cys
85 90 95
Cys Thr Cys Cys Ala Gly Thr Gly Gly Thr Thr Thr Ala Gly Gly Cys
100 105 110
Ala Gly Gly Ala Cys Cys Cys Thr Gly Gly Gly Ala Ala Ala Gly Gly
115 120 125
Thr Cys Thr Cys Ala Cys Ala Thr Cys Thr Cys Thr Gly Thr Thr Gly
130 135 140
Cys Thr Thr Ala Thr Thr Cys Ala Gly Thr Cys Ala Ala Gly Thr Cys
145 150 155 160
Ala Gly Ala Gly Ala Gly Ala Gly Cys Ala Ala Ala Cys Ala Ala Gly
165 170 175
Thr Gly Gly Ala Ala Gly Ala Cys Thr Thr Ala Ala Thr Gly Cys Cys
180 185 190
Thr Cys Gly Cys Thr Gly Gly Ala Thr Ala Ala Ala Thr Cys Ala Thr
195 200 205
Cys Ala Gly Gly Ala Cys Gly Thr Ala Gly Thr Ala Cys Thr Thr Thr
210 215 220
Ala Thr Ala Cys Ala Thr Thr Gly Cys Ala Gly Cys Thr Thr Cys Thr
225 230 235 240
Cys Ala Gly Cys Cys Thr Gly Gly Thr Gly Ala Cys Thr Cys Ala Gly
245 250 255
Cys Cys Ala Cys Cys Thr Ala Cys Cys Thr Cys Thr Gly Thr Gly Cys
260 265 270
Thr Gly Cys Thr Ala Cys Ala Thr Cys Ala Gly Gly Ala Gly Gly Ala
275 280 285
Ala Gly Cys Thr Ala Cys Ala Thr Ala Cys Cys Thr Ala Cys Ala Thr
290 295 300
Thr Thr Gly Gly Ala Ala Gly Ala Gly Gly Ala Ala Cys Cys Ala Gly
305 310 315 320
Cys Cys Thr Thr Ala Thr Thr Gly Thr Thr Cys Ala Thr Cys Cys Gly
325 330 335
<210> 8
<211> 339
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 8
Ala Ala Thr Gly Cys Thr Gly Gly Thr Gly Thr Cys Ala Cys Thr Cys
1 5 10 15
Ala Gly Ala Cys Cys Cys Cys Ala Ala Ala Ala Thr Thr Cys Cys Gly
20 25 30
Cys Ala Thr Cys Cys Thr Gly Ala Ala Gly Ala Thr Ala Gly Gly Ala
35 40 45
Cys Ala Gly Ala Gly Cys Ala Thr Gly Ala Cys Ala Cys Thr Gly Cys
50 55 60
Ala Gly Thr Gly Thr Ala Cys Cys Cys Ala Gly Gly Ala Thr Ala Thr
65 70 75 80
Gly Ala Ala Cys Cys Ala Thr Ala Ala Cys Thr Ala Cys Ala Thr Gly
85 90 95
Thr Ala Cys Thr Gly Gly Thr Ala Thr Cys Gly Ala Cys Ala Ala Gly
100 105 110
Ala Cys Cys Cys Ala Gly Gly Cys Ala Thr Gly Gly Gly Gly Cys Thr
115 120 125
Gly Ala Ala Gly Cys Thr Gly Ala Thr Thr Thr Ala Thr Thr Ala Thr
130 135 140
Thr Cys Ala Gly Thr Thr Gly Gly Thr Gly Cys Thr Gly Gly Thr Ala
145 150 155 160
Thr Cys Ala Cys Thr Gly Ala Thr Ala Ala Ala Gly Gly Ala Gly Ala
165 170 175
Ala Gly Thr Cys Cys Cys Gly Ala Ala Thr Gly Gly Cys Thr Ala Cys
180 185 190
Ala Ala Cys Gly Thr Cys Thr Cys Cys Ala Gly Ala Thr Cys Ala Ala
195 200 205
Cys Cys Ala Cys Ala Gly Ala Gly Gly Ala Thr Thr Thr Cys Cys Cys
210 215 220
Gly Cys Thr Cys Ala Gly Gly Cys Thr Gly Gly Ala Gly Thr Thr Gly
225 230 235 240
Gly Cys Thr Gly Cys Thr Cys Cys Cys Thr Cys Cys Cys Ala Gly Ala
245 250 255
Cys Ala Thr Cys Thr Gly Thr Gly Thr Ala Cys Thr Thr Cys Thr Gly
260 265 270
Thr Gly Cys Cys Ala Gly Cys Ala Gly Cys Cys Thr Thr Ala Gly Cys
275 280 285
Gly Gly Gly Ala Gly Gly Thr Thr Gly Gly Gly Thr Gly Ala Gly Cys
290 295 300
Ala Gly Thr Thr Cys Thr Thr Cys Gly Gly Gly Cys Cys Ala Gly Gly
305 310 315 320
Gly Ala Cys Ala Cys Gly Gly Cys Thr Cys Ala Cys Cys Gly Thr Gly
325 330 335
Cys Thr Ala
<210> 9
<211> 759
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 9
aaacaggagg tgacgcagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60
ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120
gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180
agacttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240
cagcctggtg actcagccac ctacctctgt gctgctacat caggaggaag ctacatacct 300
acatttggaa gaggaaccag ccttattgtt catccgtata tccagaaccc tgaccctgcc 360
gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420
gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480
gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540
tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600
cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 660
aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 720
gggtttaatc tgctcatgac gctgcggctg tggtccagc 759
<210> 10
<211> 6
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 10
Asp Ser Ala Ile Tyr Asn
1 5
<210> 11
<211> 7
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 11
Ile Gln Ser Ser Gln Arg Glu
1 5
<210> 12
<211> 11
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 12
Ala Ala Thr Ser Gly Gly Ser Tyr Ile Pro Thr
1 5 10
<210> 13
<211> 5
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 13
Met Asn His Asn Tyr
1 5
<210> 14
<211> 6
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 14
Ser Val Gly Ala Gly Ile
1 5
<210> 15
<211> 12
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 15
Ala Ser Ser Leu Ser Gly Arg Leu Gly Glu Gln Phe
1 5 10
<210> 16
<211> 816
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 16
atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60
caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctc 120
aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180
aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240
cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300
cctggtgact cagccaccta cctctgtgct gctacatcag gaggaagcta catacctaca 360
tttggaagag gaaccagcct tattgttcat ccgtatatcc agaaccctga ccctgccgtg 420
taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480
tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 540
ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 600
gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 660
agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 720
ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780
tttaatctgc tcatgacgct gcggctgtgg tccagc 816
<210> 17
<211> 876
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 17
aatgctggtg tcactcagac cccaaaattc cgcatcctga agataggaca gagcatgaca 60
ctgcagtgta cccaggatat gaaccataac tacatgtact ggtatcgaca agacccaggc 120
atggggctga agctgattta ttattcagtt ggtgctggta tcactgataa aggagaagtc 180
ccgaatggct acaacgtctc cagatcaacc acagaggatt tcccgctcag gctggagttg 240
gctgctccct cccagacatc tgtgtacttc tgtgccagca gccttagcgg gaggttgggt 300
gagcagttct tcgggccagg gacacggctc accgtgctag aggacctgaa aaacgtgttc 360
ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 420
acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 480
aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 540
gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 600
cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 660
gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 720
agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 780
ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 840
ctgatggcca tggtcaagag aaaggattcc agaggc 876
<210> 18
<211> 933
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 18
atgagcatca gcctcctgtg ctgtgcagcc tttcctctcc tgtgggcagg tccagtgaat 60
gctggtgtca ctcagacccc aaaattccgc atcctgaaga taggacagag catgacactg 120
cagtgtaccc aggatatgaa ccataactac atgtactggt atcgacaaga cccaggcatg 180
gggctgaagc tgatttatta ttcagttggt gctggtatca ctgataaagg agaagtcccg 240
aatggctaca acgtctccag atcaaccaca gaggatttcc cgctcaggct ggagttggct 300
gctccctccc agacatctgt gtacttctgt gccagcagcc ttagcgggag gttgggtgag 360
cagttcttcg ggccagggac acggctcacc gtgctagagg acctgaaaaa cgtgttccca 420
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480
ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 540
gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600
ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660
aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720
tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 780
gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 840
tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 900
atggccatgg tcaagagaaa ggattccaga ggc 933
<210> 19
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 19
gatagcgcta tttacaac 18
<210> 20
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 20
attcagtcaa gtcagagaga g 21
<210> 21
<211> 33
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 21
gctgctacat caggaggaag ctacatacct aca 33
<210> 22
<211> 15
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 22
atgaaccata actac 15
<210> 23
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 23
tcagttggtg ctggtatc 18
<210> 24
<211> 36
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 24
gccagcagcc ttagcgggag gttgggtgag cagttc 36
<210> 25
<211> 207
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 25
Met Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu
1 5 10 15
Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr
20 25 30
Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu
35 40 45
Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn
50 55 60
Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala
65 70 75 80
Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Ser Gly
85 90 95
Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val His
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
Cys 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
195 200 205
<210> 26
<211> 621
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 26
atgaaacagg aagttaccca gattcctgca gctctgagtg tcccagaagg agaaaacttg 60
gttctcaact gcagtttcac tgatagcgct atttacaacc tccagtggtt taggcaggac 120
cctgggaaag gtctcacatc tctgttgctt attcagtcaa gtcagagaga gcaaacaagt 180
ggaagactta atgcctcgct ggataaatca tcaggacgta gtactttata cattgcagct 240
tctcagcctg gtgactcagc cacctacctc tgtgctgcta catcaggagg aagctacata 300
cctacatttg gaagaggaac cagccttatt gttcatccgt atatccagaa ccctgaccct 360
gccgtgtacc agctgagaga ctctaagtcg agtgacaagt ctgtctgcct attcaccgat 420
tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480
tgtgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540
aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600
ttccccagcc cagaaagttc c 621
<210> 27
<211> 244
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 27
Met Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Arg Ile Leu Lys Ile
1 5 10 15
Gly Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His Asn Tyr
20 25 30
Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu Ile Tyr
35 40 45
Tyr Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro Asn Gly
50 55 60
Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Glu
65 70 75 80
Leu Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Ser Gly Arg Leu Gly Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr
100 105 110
Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe
115 120 125
Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val
130 135 140
Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp
145 150 155 160
Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro
165 170 175
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser
180 185 190
Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe
195 200 205
Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
210 215 220
Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
225 230 235 240
Gly Arg Ala Asp
<210> 28
<211> 732
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 28
atgaacgcgg gcgtgaccca gaccccaaaa ttccgcatcc tgaagatagg acagagcatg 60
acactgcagt gtacccagga tatgaaccat aactacatgt actggtatcg acaagaccca 120
ggcatggggc tgaagctgat ttattattca gttggtgctg gtatcactga taaaggagaa 180
gtcccgaatg gctacaacgt ctccagatca accacagagg atttcccgct caggctggag 240
ttggctgctc cctcccagac atctgtgtac ttctgtgcca gcagccttag cgggaggttg 300
ggtgagcagt tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg 360
ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420
gccacactgg tgtgcctggc caccggtttc taccccgacc acgtggagct gagctggtgg 480
gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540
cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600
tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660
gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720
ggtagagcag ac 732
<210> 29
<211> 9
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 29
Glu Val Leu Val Asp Leu Phe Leu Lys
1 5

Claims (10)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be with EVLVDLFLK-HLA-A*1101 compound knot It closes.
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 AATSGGSYIPT (SEQ ID NO:12)；And/or it is described The amino acid sequence of the CDR3 of TCR β chain variable domain is ASSLSGRLGEQF (SEQ ID NO:15).
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-DSAIYN (SEQ ID NO:10)
αCDR2-IQSSQRE (SEQ ID NO:11)
αCDR3-AATSGGSYIPT (SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-MNHNY (SEQ ID NO:13)
βCDR2-SVGAGI (SEQ ID NO:14)
βCDR3-ASSLSGRLGEQF (SEQ ID NO:15)。
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.
CN201810210194.6A 2018-03-14 2018-03-14 T cell receptor recognizing PRAME antigen short peptide Active CN110272482B (en)

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Publication number Priority date Publication date Assignee Title
CN111647069A (en) * 2020-06-17 2020-09-11 深圳豪石生物科技有限公司 Improved TCR and application thereof
CN111718409A (en) * 2020-06-17 2020-09-29 深圳豪石生物科技有限公司 T cell receptor and application thereof
WO2021068938A1 (en) * 2019-10-10 2021-04-15 香雪生命科学技术（广东）有限公司 T cell receptor recognising kras mutation and encoding sequence thereof
CN112759641A (en) * 2019-11-01 2021-05-07 香雪生命科学技术（广东）有限公司 High-affinity TCR for recognizing KrasG12V
CN115073584A (en) * 2022-05-07 2022-09-20 深圳普瑞金生物药业股份有限公司 TCR for specifically recognizing PRAME antigen peptide and application thereof

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WO2021068938A1 (en) * 2019-10-10 2021-04-15 香雪生命科学技术（广东）有限公司 T cell receptor recognising kras mutation and encoding sequence thereof
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CN112759641B (en) * 2019-11-01 2023-01-20 香雪生命科学技术（广东）有限公司 High-affinity TCR for recognizing Kras G12V
CN111647069A (en) * 2020-06-17 2020-09-11 深圳豪石生物科技有限公司 Improved TCR and application thereof
CN111718409A (en) * 2020-06-17 2020-09-29 深圳豪石生物科技有限公司 T cell receptor and application thereof
CN111718409B (en) * 2020-06-17 2021-03-02 深圳豪石生物科技有限公司 T cell receptor and application thereof
CN111647069B (en) * 2020-06-17 2022-06-21 深圳豪石生物科技有限公司 Improved TCR and application thereof
CN115073584A (en) * 2022-05-07 2022-09-20 深圳普瑞金生物药业股份有限公司 TCR for specifically recognizing PRAME antigen peptide and application thereof
CN115073584B (en) * 2022-05-07 2023-10-20 溧阳瑅赛生物医药有限公司 TCR capable of specifically recognizing PRAME antigen peptide and application thereof
WO2023216440A1 (en) * 2022-05-07 2023-11-16 溧阳瑅赛生物医药有限公司 Tcr specifically recognizing prame antigen peptide and use thereof

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