CN110407926A - A kind of TCR and its coded sequence identifying LMP1 antigen small peptide - Google Patents

A kind of TCR and its coded sequence identifying LMP1 antigen small peptide Download PDF

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CN110407926A
CN110407926A CN201810386338.3A CN201810386338A CN110407926A CN 110407926 A CN110407926 A CN 110407926A CN 201810386338 A CN201810386338 A CN 201810386338A CN 110407926 A CN110407926 A CN 110407926A
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CN110407926B (en
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李懿
胡静
相瑞瑞
孙含丽
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Xiangxue Life Science Technology Guangdong Co ltd
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Guangdong Xiangxue Precision Medical Technology Co Ltd
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Abstract

The present invention provides the T cell receptor (TCR) that one kind can specifically bind the small peptide MLWRLGATI derived from LMP1 antigen, the antigen small peptide MLWRLGATI can form compound with HLA A0201 and be presented to cell surface together.Carrier the present invention also provides the nucleic acid molecules for encoding the TCR and comprising the nucleic acid molecules.In addition, the present invention also provides the cells for the TCR of the present invention that transduces.

Description

A kind of TCR and its coded sequence identifying LMP1 antigen small peptide
Technical field
The present invention relates to can identify that the invention further relates to transductions from the TCR and its coded sequence of LMP1 antigen small peptide Above-mentioned TCR come the T cell of LMP1 specificity obtained and they preventing and treating the purposes in LMP1 related disease.
Background technique
EBV is a kind of generally existing nerpes vinrus hominis of global range.Studies have shown that is more than in 95% adult human body Containing the antibody for being directed to this virus, this also means that them in a certain stage by this virus infection mistake.It is most of to be felt The internal all one's life of the people contaminated can all have EBV, generally seldom go wrong.However, in some cases, EBV and some cancers The generation of disease is related, including Burkitt lymphoma (Burkitt ' s lymphoma), Hodgkin lymphoma (Hodgkin Lymphoma), lymphocytic hyperplasia disease (PTLD) or nasopharyngeal carcinoma etc. after EBV positive graft.For example, LMP1 belongs to the incubation period film of EBV Albumen can express (Raab-Traub N.Epstein-Barr virus in the by most nasopharyngeal carcinoma cells pathogenesis of NPC[J].Semin Cancer Biol,2002,12(6):431-441.).LMP1 gives birth in the cell It is degraded to micromolecule polypeptide after, and forms compound in conjunction with MHC (main histocompatibility complex) molecule, is presented to Cell surface.MLWRLGATI (SEQ ID NO:9) is the small peptide derived from LMP1 antigen, is the one of LMP1 treating correlative diseases Kind target.Treatment for above-mentioned disease can use the methods of chemotherapy and radiation treatment, but all can be to the normal thin of itself Born of the same parents damage.
T cell adoptive immunotherapy is that will there is the reaction-ive T cell of specificity to be transferred in patient body target cell antigen, It is set to play a role for target cell.T cell receptor (TCR) is a kind of memebrane protein on T cell surface, can be identified corresponding The antigen small peptide of target cell surface.In immune system, pass through the TCR and the main histocompatbility of small peptide-of antigen small peptide specificity The combination of complex (pMHC compound) causes T cell and antigen presenting cell (APC) is directly physically contacted, then T cell And other cell membrane surface molecules of both APC just interact, and cause a series of subsequent cell signal transmitting and its His physiological reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, this field skill Art personnel are dedicated to isolating the TCR for having specificity to LMP1 antigen small peptide, and the TCR transduceed T cell to obtain pair LMP1 antigen small peptide has the T cell of specificity, so that them be made to play a role in cellular immunotherapy.
Summary of the invention
The purpose of the present invention is to provide a kind of T cell receptors for identifying LMP1 antigen small peptide.
The first aspect of the present invention, provides a kind of T cell receptor (TCR), and the TCR can be with MLWRLGATI-HLA A0201 compound combines.
In another preferred example, the TCR includes TCR α chain variable domain and TCR β chain variable domain, the TCR α chain variable domain CDR3 amino acid sequence be ALNAGAGNMLT (SEQ ID NO:12)；And/or the ammonia of the CDR3 of the TCR β chain variable domain Base acid sequence is SASETSGSYEQF (SEQ ID NO:15).
In another preferred example, 3 complementary determining regions (CDR) of the TCR α chain variable domain are as follows:
αCDR1-TRDTTYY(SEQ ID NO:10)
αCDR2-RNSFDEQN(SEQ ID NO:11)
αCDR3-ALNAGAGNMLT(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-SASETSGSYEQF(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:5 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:5.
In another preferred example, the TCR is α β heterodimer, and it includes TCR α chain constant region TRAC*01 and TCR β Chain constant region TRBC1*01 or TRBC2*01.
In another preferred example, the α chain amino acid sequence of the TCR is the β chain ammonia of the SEQ ID NO:3 and/or TCR Base acid sequence is SEQ ID NO:7.
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 is in α chain variable region amino acid the 11st, 13,19,21,53,76,89,91 or There is one or more dash forward in 94 and/or α chain J gene small peptide amino acid inverse the 3rd, 5th reciprocal or 7th reciprocal Become；And/or the TCR is in β chain variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th and/or β chain J In gene small peptide amino acid inverse the 2nd, 4th reciprocal or 6th reciprocal there is one or more to be mutated, wherein amino acid position Number is set by the Position Number listed in IMGT (international immunogenetics information system).
In another preferred example, the α chain variable domain amino acid sequence of the TCR includes SEQ ID NO:32 and/or described The β chain variable domain amino acid sequence of TCR includes SEQ ID NO:34.
In another preferred example, the amino acid sequence of the TCR is SEQ ID NO:30.
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 the β chain of the SEQ ID NO:26 and/or TCR Amino acid sequence is SEQ ID NO:28.
In another preferred example, artificial interchain disulfide bond is contained between the α chain variable region of the TCR and β chain constant region.
In another preferred example, 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.
The second aspect of the present invention provides a kind of multivalent TCR complex, and it includes at least two TCR molecules, and its In at least one TCR molecule be first aspect present invention described in TCR.
The third aspect of the present invention, provides a kind of nucleic acid molecules, and the nucleic acid molecules include to encode first party of the present invention The nucleic acid sequence of TCR molecule or its complementary series described in face.
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chain variable domain: 2 or SEQ ID NO:33.
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID of coding TCR β chain variable domain NO:6 or SEQ ID NO:35.
In another preferred example, the nucleic acid molecules include coding TCR α chain nucleotide sequence SEQ ID NO:4 and/or Nucleotide sequence SEQ ID NO:8 comprising encoding TCR β chain.
The fourth aspect of the present invention, provides a kind of carrier, and the carrier contains core described in third aspect present invention Acid molecule；Preferably, the carrier is viral vectors；It is highly preferred that the carrier is slow virus carrier.
The fifth aspect of the present invention provides a kind of isolated host cell, contains the present invention in the host cell Nucleic acid molecules described in the third aspect present invention of external source are integrated in carrier described in fourth aspect or genome.
The sixth aspect of the present invention provides a kind of cell, nucleic acid described in the cell transduction third aspect present invention Carrier described in molecule or fourth aspect present invention；Preferably, the cell is T cell or stem cell.
The seventh aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains pharmaceutically acceptable load TCR described in body and first aspect present invention, TCR compound described in second aspect of the present invention, third aspect present invention institute Cell described in carrier described in the nucleic acid molecules stated, fourth aspect present invention or sixth aspect present invention.
The eighth aspect of the present invention provides T cell receptor described in first aspect present invention or second aspect of the present invention Nucleic acid molecules described in the TCR compound, third aspect present invention, carrier or this hair described in fourth aspect present invention The purposes of cell described in bright 6th aspect, is used to prepare the drug for the treatment of tumour or autoimmune disease.
The ninth aspect of the present invention provides a kind of method for treating disease, including suitable to object in need for the treatment of application TCR compound, third of the present invention described in T cell receptor described in the first aspect present invention of amount or second aspect of the present invention Nucleic acid molecules described in aspect, cell or this hair described in carrier or sixth aspect present invention described in fourth aspect present invention Pharmaceutical composition described in bright 7th aspect；
Preferably, the disease is tumour, and the preferably described tumour is hepatocellular carcinoma.
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, In This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively that TCR α chain variable domain amino acid sequence, TCR α chain are variable Domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, the TCR α chain amino acid sequence with leader sequence And the TCR α chain nucleotide sequence with leader sequence.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively that TCR β chain variable domain amino acid sequence, TCR β chain are variable Domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, the TCR β chain amino acid sequence with leader sequence And the TCR β chain nucleotide sequence with leader sequence.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chain.
Fig. 5 a and Fig. 5 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chain.
Fig. 6 is the glue figure of the sTCR obtained after purification.Leftmost side swimming lane is to go back virgin rubber, and intermediate swimming lane is molecular weight It marks (marker), rightmost side swimming lane is non-reduced glue.
Fig. 7 a and Fig. 7 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR.
Fig. 8 a and Fig. 8 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR α chain variable domain.
Fig. 9 a and Fig. 9 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR β chain variable domain.
Figure 10 a and Figure 10 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR catenation sequence (linker).
Figure 11 is the glue figure of the soluble single-chain T CR obtained after purification.Left side swimming lane is molecular weight marker (marker), right Breathing arm road is non-reduced glue.
Figure 12 is BIAcore dynamics figure of the sTCR of the present invention in conjunction with MLWRLGATI-HLA A0201 compound Spectrum.
Figure 13 is BIAcore power of the soluble single-chain T CR of the present invention in conjunction with MLWRLGATI-HLA A0201 compound Learn map.
The ELISPOT for the T cell clone that Figure 14 is activates functional verification result.
Figure 15 is the ELISPOT activation functional verification result of the effector cell of transduction TCR of the invention.
Specific embodiment
The present inventor after extensive and in-depth study, have found with LMP1 antigen small peptide MLWRLGATI (SEQ ID NO: 9) TCR that can be specifically bound, the antigen small peptide MLWRLGATI can form compound with HLA A0201 and be in together It is delivered to cell surface.Carrier the present invention also provides the nucleic acid molecules for encoding the TCR and comprising the nucleic acid molecules.Separately Outside, 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.In 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
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 MLWRLGATI-HLA A0201 compound.Preferably, the TCR molecule is separation or purifying 's.α the and β chain of the TCR respectively has 3 complementary determining regions (CDR).
It is preferably carried out in mode at of the invention one, the α chain of the TCR includes with following amino acid sequence CDR:
αCDR1-TRDTTYY(SEQ ID NO:10)
αCDR2-RNSFDEQN(SEQ ID NO:11)
αCDR3-ALNAGAGNMLT(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-SASETSGSYEQF(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:5, 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:5.It is more excellent Selection of land, the β chain variable domain amino acid sequence of the TCR molecule are SEQ ID NO:5.
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:5.It is highly preferred that the β chain variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:5.
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 and/or β chain is SEQ ID NO:7.
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 LMP1 antigen small peptide TCR。
To obtain sTCR, on the one hand, TCR of the present invention can be to be introduced between the residue of itself α and β chain constant domain The TCR of artificial disulfide bond.Cysteine residues form artificial interchain disulfide bond between α the and β chain constant domain of the TCR.Half Guang Histidine residue can be substituted in other amino acid residues of appropriate site in natural TCR to form artificial interchain disulfide bond.For example, Replace the Thr48 of TRAC*01 exons 1 and replaces the cysteine residues of the Ser57 of TRBC1*01 or TRBC2*01 exons 1 To form disulfide bond.It introduces cysteine residues and may also is that TRAC*01 exons 1 with other sites for forming disulfide bond The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1；The Tyr10 and TRBC1*01 of TRAC*01 exons 1 or The Ser17 of TRBC2*01 exons 1；Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1 Asp59；The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；TRAC*01 exons 1 Arg53 and TRBC1*01 or TRBC2*01 exons 1 Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1 or The Ala19 of TRBC2*01 exons 1；Or Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1 Glu20.I.e. cysteine residues are instead of any group of site in above-mentioned α and β chain constant domain.It can be in TCR constant domain of the present invention One or more C-terminals truncate most 50 or most 30 or most 15 or most 10 or most 8 or less Amino acid can also be by the way that day will be formed so that it does not include cysteine residues to achieve the purpose that lack natural disulphide bonds The cysteine residues of right disulfide bond sport another amino acid to reach above-mentioned purpose.
As described above, TCR of the invention may be embodied in the artificial disulfide bond introduced between the residue of itself α and β chain constant domain. It should be noted that the artificial disulfide bond with or without introducing described above between constant domain, TCR of the invention can be constant containing TRAC Domain sequence and TRBC1 or TRBC2 constant domain sequence.The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequence of TCR can It is connected by the natural disulphide bonds being present in TCR.
To obtain sTCR, on the other hand, TCR of the present invention further includes the TCR to mutate in its hydrophobic core region, The mutation of these hydrophobic core regions is preferably capable making the stability-enhanced mutation of sTCR of the present invention, such as in publication number Described in patent document for WO2014/206304.Such TCR can mutate in its following hydrophobic core position of variable domain: (α and/or β chain) variable region amino acid the 11st, 13,19,21,53,76,89,91,94 and/or α chain J gene (TRAJ) small peptide Amino acid position the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptide amino acid position reciprocal is 2nd, 4,6 reciprocal, wherein ammonia The Position Number of base acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).Those skilled in the art Member knows above-mentioned international immunogenetics information system, and the amino acid residue that different TCR can be obtained according to the database exists Position Number in IMGT.
The TCR that hydrophobic core region mutates in the present invention can be by α and the β chain of a flexible peptide chain link TCR can Variable domain and the solvable single-stranded TCR of stability constituted.It should be noted that in the present invention flexible peptide chain can be any suitable connection TCR α and The peptide chain of β chain variable domain.Single chain soluble TCR, α the chain variable domain amino acid sequence such as constructed in the embodiment of the present invention 4 For SEQ ID NO:32, the nucleotides sequence of coding is classified as SEQ ID NO:33；β chain variable domain amino acid sequence is SEQ ID NO: 34, the nucleotides sequence of coding is classified as SEQ ID NO:35.
In addition, patent document 201510260322.4 is also disclosed in the α chain variable region of TCR and β for stability Artificial interchain disulfide bond is introduced between chain constant region can be such that the stability of TCR significantly improves.Therefore, high-affinity of the invention Artificial interchain disulfide bond can also be contained between the α chain variable region and β chain constant region of TCR.Specifically, the α chain of the TCR can Become between area and β chain constant region and forms the cysteine residues of artificial interchain disulfide bond instead of the 46th amino acids of TRAV With the 60th amino acids of 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 MLWRLGATI-HLA A0201 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:
αCDR1-acccgtgatactacttattac(SEQ ID NO:16)
αCDR2-cggaactcttttgatgagcaaaat(SEQ ID NO:17)
αCDR3-gctctgaacgccggggcaggcaacatgctcacc(SEQ ID NO:18)
The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR region is as follows:
βCDR1-gactttcaggccacaact(SEQ ID NO:19)
βCDR2-tccaatgagggctccaaggcc(SEQ ID NO:20)
βCDR3-agtgctagtgagactagcgggagctatgagcagttc(SEQ ID NO:21)
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:16, SEQ ID NO:17 and SEQ ID NO:18, 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:19, SEQ ID NO:20 and SEQ ID NO:21.
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:2 and/or coding TCR β chain variable domain of the present invention includes SEQ ID NO:6.Alternatively, the nucleotide sequence of the nucleic acid molecules of the present invention of coding TCR α chain variable domain of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequence of nucleic acid molecules of the present invention of coding TCR β chain variable domain of the present invention include SEQ ID NO:35.More Preferably, the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:4 and/or SEQ ID NO:8.Alternatively, of the invention The nucleotides sequence of nucleic acid molecules is classified as SEQ ID NO:31.
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:2.
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 LMP1 antigentic specificity.Ideally, which can should express to continual high levels in T cell.
Cell
The invention further relates to the genetically engineered host cell of carrier or coded sequence of the invention.The host Contain in carrier or chromosome of the invention in cell and is integrated with nucleic acid molecules of the invention.Host cell is selected from: prokaryotic cell And eukaryocyte, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..
In addition, the invention also includes the isolated cell for expressing TCR of the invention, especially T cell.The T cell can spread out It is born from the T cell separated from subject, or can be the mixed cellularity group separated from subject, such as periphery hemolymph is thin A part of born of the same parents (PBL) group.Such as, which can be isolated from peripheral blood mononuclear cells (PBMC), can be CD4+T helper cell Or CD8+Cytotoxic T cell.The cell can be in CD4+T helper cell/CD8+In the mixing group of cytotoxic T cell.Generally Ground, the cell can be activated with antibody (e.g., the antibody of anti-CD3 or anti-CD28), to allow them to more easily receive to turn Dye, such as transfected with the carrier of the nucleotide sequence comprising encoding TCR molecule of the present invention.
Alternatively, cell of the invention can also be or derived from stem cell, such as candidate stem cell (HSC).Gene is turned Moving to HSC not will lead in cell surface expression TCR, because stem cell surface does not express CD3 molecule.However, when stem cell point It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecule will start in thymocyte The surface expression introducing TCR molecule.
There are many method be suitable for being carried out with the DNA or RNA of coding TCR of the present invention T cell transfection (e.g., the such as Robbins, (2008)J.Immunol.180:6116-6131).The T cell for expressing TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat Rev for carrying out adoptive treatment Cancer8 (4): 299-308).
LMP1 antigen-related disease
The invention further relates to the method with LMP1 related disease is treated and/or prevented in subject comprising adoptive Shift the step of LMP1 specific T-cells are to the subject.The LMP1 specific T-cells can recognize MLWRLGATI-HLA A0201 compound.
The T cell of LMP1 specificity of the invention can be used for treating any presentation LMP1 antigen small peptide MLWRLGATI-HLA The LMP1 related disease of A0201 compound.Including but not limited to tumour, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin lymphoma Deng.
Treatment method
Can by separation with the patient of LMP1 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 LMP1 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 are:
(1) TCR of the invention can turn simultaneously in conjunction with LMP1 antigen small peptide compound MLWRLGATI-HLA A0201 The cell for having led TCR of the present invention can have very strong lethal effect by specific activation and to target cell.
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 Russel l et al., molecular cloning: laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing house) described in condition, or according to proposed by manufacturer Condition.Unless otherwise stated, otherwise percentage and number are calculated by weight.Unless otherwise stated, otherwise percentage and number It calculates by weight.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
Embodiment 1 clones LMP1 antigen small peptide specific T-cells
Utilize synthesis small peptide MLWRLGATI (SEQ ID NO.:9；Beijing SBS Genetech gene technology Co., Ltd) it stimulates and From in the peripheral blood lymphocytes (PBL) for the healthy volunteer that genotype is HLA-A0201.By MLWRLGATI small peptide with have The HLA-A0201 renaturation of biotin labeling prepares pHLA monoploid.These monoploid and the Streptavidin (BD marked with PE Company) it is combined into the tetramer of PE label, sort the tetramer and anti-CD8-APC double positive cells.The cell of sorting is expanded, And secondary sorting is carried out according to the above method, then monoclonal is carried out with limiting dilution assay.Monoclonal cell tetramer staining, sieve The double positive colonies chosen are as shown in Figure 3.By the double positive colonies screened layer by layer, it is also necessary to meet further function Test.
The function and specificity of T cell clone are further detected by ELISPOT experiment.Those skilled in the art are known The method for testing detection cell function using ELISPOT.The present embodiment IFN-γ ELISPOT experiment used in effector cell be The T cell clone obtained in the present invention, target cell are the T2 cell for having loaded small peptide of the present invention, and control group is short to have loaded other The T2 cell of the T2 cell of peptide and unsupported any small peptide.
Prepare ELISPOT plate first, ELISPOT experimental procedure is as follows: in the following order adding each component of test Enter ELISPOT plate: 40 μ l T2 cells 5 × 105A cells/ml (i.e. 20,000 T2 cells/wells), 40 μ l effector cells After (2000 T cell clone/holes), 20 μ l specificity small peptides are added in experimental group, and 20 μ l non-specificity small peptides are added in control group, empty White group of 20 μ l culture medium (test medium) of addition, and 2 multiple holes are set.Then be incubated overnight (37 DEG C, 5%CO2).Then washing Plate simultaneously carries out secondary detection and colour developing, dry plate 1 hour, recycles immunodotting plate reader (ELISPOT READER system；AID company) count the spot formed on film.Experimental result is as shown in figure 14, and obtained specific antigen is special Specific T cell clone has a specific reaction to the T2 cell for loading small peptide of the present invention, and to other unrelated peptides of load and unsupported The T2 cell of small peptide does not react substantially.
Embodiment 2 obtains the building of the tcr gene and carrier of LMP1 antigen small peptide specific T-cell clones
Use Quick-RNATMThe antigen small peptide screened in MiniPrep (ZYMO research) extracting embodiment 1 The total serum IgE of the restrictive T cell clone of MLWRLGATI specificity, HLA-A0201.The synthesis of cDNA is using clontech's SMART RACE cDNA amplification kit, the primer of use are designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned It is sequenced on to carrier T (TAKARA).It should be noted that the sequence is complementary series, introne is not included.Through being sequenced, this pair sun Property clonal expression TCR α chain and β chain-ordering structure distinguish as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e With Fig. 1 f be respectively 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 sequence with leader sequence and the TCR α chain nucleotide with leader sequence Sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively 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 sequence with leader sequence with And the TCR β chain nucleotide sequence with leader sequence.
Identified, α chain includes the CDR with following amino acid sequence:
αCDR1-TRDTTYY(SEQ ID NO:10)
αCDR2-RNSFDEQN(SEQ ID NO:11)
αCDR3-ALNAGAGNMLT(SEQ ID NO:12)
β chain includes the CDR with following amino acid sequence:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-SASETSGSYEQF(SEQ ID NO:15)。
The full-length gene of TCR α chain and β chain is cloned into Lentiviral respectively by overlapping (overlap) PCR pLenti(addgene).Specifically: it is attached the full-length gene of TCR α chain and TCR β chain to obtain TCR with overlap PCR α -2A-TCR β segment.It connects Lentiviral and TCR α -2A-TCR β digestion to obtain pLenti-TRA-2A-TRB- IRES-NGFR plasmid.It is used as control, while the also slow virus carrier pLenti-eGFP of building expression eGFP.It uses again later 293T/17 packs pseudovirus.
Expression, refolding and the purifying of the solvable TCR of embodiment 3LMP1 antigen small peptide specificity
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.Pass through " Molecular Cloning: A Laboratory room handbook " (Molecular Cloning a Laboratory Manual) standard method described in (third edition, Sambrook and Russell) is by the target gene sequence of above-mentioned TCR α and β chain Column be inserted respectively into after synthesizing expression vector pET28a+ (Novagene), the cloning site of upstream and downstream be respectively NcoI and NotI.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, in OD600It is induced when=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), 20mM Tris (pH 8.1) in.
Dissolved TCR α and β chain are quickly mixed in 5M urea, 0.4M arginine, 20mM Tris with the mass ratio of 1:1 (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Solution is placed in dialysis (4 DEG C) in the deionized water of 10 times of volumes afterwards, changes deionized water into buffer (20mM after 12 hours Tris, pH 8.0) continue at 4 DEG C of dialysis 12 hours.Solution after the completion of dialysis after 0.45 μM of membrane filtration, by yin from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains the successful α and β dimer of renaturation TCR is confirmed by SDS-PAGE glue.TCR then pass 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 by BCA method It determines.The SDS-PAGE glue figure for the sTCR that the present invention obtains is as shown in Figure 6.
The generation of the soluble single-chain T CR of embodiment 4LMP1 antigen small peptide specificity
According to patent document WO2014/206304, using the method for rite-directed mutagenesis by TCR α and β in embodiment 2 The variable domain of chain has been built into the stable soluble single-chain T CR molecule connected with flexible small peptide (l inker).This is single-stranded Amino acid sequence and the nucleotide sequence difference of TCR molecule are as shown in figs. 7 a and 7b.The amino acid sequence of its α chain variable domain and Nucleotide sequence difference is as figures 8 a and 8 b show；The amino acid sequence and nucleotide sequence of its β chain variable domain are respectively such as Fig. 9 a With shown in Fig. 9 b；Amino acid sequence and the nucleotide sequence difference of its l inker sequence are as as-shown-in figures 10 a and 10b.
By target gene through I double digestion of Nco I and Not, it is connect with the pET28a carrier by Nco I and I double digestion of Not. Connection product is converted to E.coli DH5 α, is coated with the LB plate containing kanamycins, 37 DEG C of inversion overnight incubations, the picking positive gram Grand progress PCR screening, is sequenced positive recombinant, determines that sequence correctly extracts recombinant plasmid transformed to E.coli afterwards BL21 (DE3), for expressing.
Expression, renaturation and the purifying of the soluble single-chain T CR of embodiment 5LMP1 antigen small peptide specificity
BL21 (DE 3) bacterium colony containing recombinant plasmid pET28a- template strand prepared in embodiment 4 is all inoculated in In LB culture medium containing kanamycin, 37 DEG C culture to OD600 be 0.6-0.8, be added IPTG to final concentration of 0.5mM, 37 DEG C continue to cultivate 4h.5000rpm is centrifuged 15min and harvests cell precipitate, is cracked with Bugbuster Master Mix (Merck) Cell precipitate, 6000rpm is centrifuged 15min and recycles inclusion body, then is washed with Bugbuster (Merck) to remove cell Fragment and membrane component, 6000rpm are centrifuged 15min, collect inclusion body.By solubilization of inclusion bodies in buffer (20mM Tris-HCl PH 8.0,8M urea) in, high speed centrifugation removes insoluble matter, is dispensed after supernatant BCA standard measure, standby in -80 DEG C of preservations With.
In the single-stranded TCR inclusion body protein dissolved to 5mg, 2.5mL buffer (6M Gua-HCl, 50mM Tris- is added HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration of 10mM, 37 DEG C of processing 30min.With injection Device is to 125mL renaturation buffer (100mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) in treated single-stranded TCR, 4 DEG C of stirrings are added dropwise Then renaturation solution is packed into the cellulose membrane bag filter that interception is 4kDa by 10min, bag filter is placed in the water of 1L pre-cooling, and 4 DEG C It is slowly stirred overnight.After 17 hours, by dialyzate change into 1L pre-cooling buffer (20mM Tris-HCl pH 8.0), 4 DEG C after Continuous dialysis 8h, then changes dialyzate into identical fresh buffer and continues dialysed overnight.After 17 hours, sample is filtered through 0.45 μm Film filtering, by anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgas, with 20mM Tris-HCl The 0-1M NaCl linear gradient elution liquid purifying protein that pH 8.0 is prepared, the elution fraction of collection carry out SDS-PAGE analysis, packet It is further carried out with solvent resistant column (Superdex 7510/300, GE Healthcare) after component concentration containing single-stranded TCR Purifying, target components also carry out SDS-PAGE analysis.
Elution fraction for BIAcore analysis further uses gel filtration to test its purity.Condition are as follows: chromatographic column Agilent Bio SEC-3 (7.8 × 300mm of 300A, φ), mobile phase be 150mM phosphate buffer, flow velocity 0.5mL/min, 25 DEG C of column temperature, ultraviolet detection wavelength 214nm.
The SDS-PAGE glue figure for the soluble single-chain T CR that the present invention obtains is as shown in figure 11.
Embodiment 6 combines characterization
BIAcore analysis
This example demonstrated soluble TCR molecules of the present invention can be special with MLWRLGATI-HLA A0201 compound The opposite sex combines.
Using BIAcore T200 real-time analyzer detect TCR molecule obtained in embodiment 3 and embodiment 5 with The combination activity of MLWRLGATI-HLA A0201 compound.It is slow that coupling is added in the antibody (GenScript) of anti-Streptavidin Antibody, is then flowed through the CM5 chip activated in advance with EDC and NHS, made by fliud flushing (10mM sodium-acetate buffer, pH 4.77) Antibody is fixed on chip surface, finally closes unreacted activating surface with the hydrochloric acid solution of ethanol amine, completes coupling process, even Connection horizontal about 15,000RU.
The Streptavidin of low concentration is set to flow through the chip surface of coated antibody, then by MLWRLGATI-HLA A0201 compound flows through sense channel, and another channel is as reference channel, then by the biotin of 0.05mM with the stream of 10 μ L/min Speed flows through chip 2min, closes the remaining binding site of Streptavidin.
The preparation process of above-mentioned MLWRLGATI-HLA A0201 compound is as follows:
A. it purifies
The E.col i 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 add 30ml 20mM Tris-HCl pH 8.0 that packet is resuspended Contain body, mix, 4 DEG C of 6000g are centrifuged 15min, finally dissolve inclusion body, SDS-PAGE detection with 20mM Tris-HCl 8M urea Inclusion body purity, BCA kit survey concentration.
B. renaturation
The small peptide MLWRLGATI (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 8M urea, 20mM Tris pH 8.0,10mM DTT dissolve, and are added before renaturation 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA are further denaturalized.Renaturation is added with 25mg/L (final concentration) in MLWRLGATI peptide Buffer (0.4M L-arginine, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced form Glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, Heavy chain is added in three times, 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 20mM Tris 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 Mill ipore 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 HiPrep with filtered PBSTM 16/60 S200HR column (GE General Electric Co. Limited), the concentrated biotinylation pMHC molecule of load 1ml, then with PBS with The elution of 1ml/min flow velocity.Biotinylated pMHC molecule occurs in about 55ml as unimodal elution.Merging contains protein Component is concentrated with Mill ipore super filter tube, and BCA method (Thermo) measures protein concentration, and protease inhibitors is added The packing of biotinylated pMHC molecule is stored in -80 DEG C by cocktail (Roche).
Using BIAcore Evaluation software computational dynamics parameter, obtain the TCR molecule of solubility of the invention with And the soluble single-chain T CR molecule that constructs of the present invention kinetic profile in conjunction with MLWRLGATI-HLA A0201 compound point Not not as shown in Figure 12 and Figure 13.Map shows that the soluble TCR molecules and soluble single-chain T CR molecule that the present invention obtains are all It can be in conjunction with MLWRLGATI-HLA A0201 compound.Meanwhile solubility of the invention also is had detected using the above method The combination of TCR molecule and other several irrelevant antigen small peptides and HLA compound is active, as the result is shown TCR molecule of the present invention and its His irrelevant antigen is without combination.
Embodiment 7 transduce TCR of the present invention T cell activation experiment
Building includes the slow virus carrier of TCR target gene of the present invention, and T cell of transduceing carries out ELISPOT functional verification examination It tests.
ELISPOT scheme
Following tests is carried out to prove activating reaction of the T cell to target cell specificity of TCR transduction of the present invention.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 (PBST): 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
Target cell used in this experiment is the T2 cell for having loaded special small peptide.It is thin that target is prepared in assay medium Born of the same parents: target cell concentration is adjusted to 2.0 × 105A/milliliter, every hole take 100 microlitres to obtain 2.0 × 104A cells/well.
Effector cell's preparation
Effector cell's (T cell) of this experiment is the CD8 for expressing LMP1 antigen small peptide specificity TCR of the present invention+T cell, And with the CD8 of same volunteer's untransfected TCR of the present invention+T is as a control group.With AntiCD3 McAb/CD28 coating pearl (T cell amplification Object, life technologies) stimulation T cell, with the lentiviruses transduction for carrying LMP1 antigen small peptide specificity TCR gene, In The 1640 culture mediums amplification containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 is 9-12 days after transduction, so These cells are placed in test medium afterwards, 300g room temperature is centrifuged 10 minutes and is washed.Then by cell with 2 × required end Concentration is resuspended in test medium.Same processing negative control effector cell.
The preparation of small peptide solution
Corresponding small peptide is added in corresponding target cell (T2) experimental group, makes final concentration of 1 μ of the small peptide in ELISPOT orifice plate g/ml。
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, and incubate orifice plate 2 hours at room temperature to close orifice plate.Then culture medium is washed away from orifice plate, by light on paper Bullet and ELISPOT orifice plate is patted to remove the washing buffer of any remnants.
Then all components of test are added by ELISPOT orifice plate using following sequence:
100 microlitres of target cell 2*105A cells/ml (obtains about 2*10 in total4A target cell/hole).
100 microlitres of effector cell (1*104A control effector cell/hole and LMP1TCR positive T cell/hole).
All holes prepare addition in duplicate.
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 it is pressed with the PBS containing 10%FBS 1:200 dilution detection antibody, is added each hole by 100 microlitres/hole.It incubates orifice plate 2 hours, then is washed with washing buffer at room temperature 3 times, 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 4 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 TCR transduction of the present invention is tested to load LMP1 antigen small peptide by ELISPOT The IFN-γ release that the target cell of MLWRLGATI reacts.It is drawn using graphpad prism6 and to be observed in each hole ELSPOT amount of speckle.
Experimental result is as shown in figure 15, and the T cell for the TCR of the present invention that transduces has very the target cell for loading its special small peptide Good activating reaction, and the T cell for other TCR that transduce to corresponding target cell substantially without activating reaction.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Guangdong perfume (or spice) avenges accurate medical technology Co., Ltd
<120>a kind of TCR and its coded sequence for identifying LMP1 antigen small peptide
<130> P2018-0692
<160> 37
<170> PatentIn version 3.5
<210> 1
<211> 115
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 1
Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser Val Val Glu Lys
1 5 10 15
Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr
20 25 30
Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu Leu Val Phe Leu
35 40 45
Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr
50 55 60
Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr
65 70 75 80
Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala
85 90 95
Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val
100 105 110
Lys Pro His
115
<210> 2
<211> 345
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 60
ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 120
ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 180
agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 240
gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 300
atgctcacct ttggaggggg aacaaggtta atggtcaaac cccat 345
<210> 3
<211> 255
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 3
Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser Val Val Glu Lys
1 5 10 15
Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr
20 25 30
Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu Leu Val Phe Leu
35 40 45
Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr
50 55 60
Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr
65 70 75 80
Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala
85 90 95
Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val
100 105 110
Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp
115 120 125
Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser
130 135 140
Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp
145 150 155 160
Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
165 170 175
Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn
180 185 190
Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu
210 215 220
Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys
225 230 235 240
Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250 255
<210> 4
<211> 765
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 60
ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 120
ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 180
agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 240
gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 300
atgctcacct ttggaggggg aacaaggtta atggtcaaac cccatatcca gaaccctgac 360
cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 420
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480
aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600
ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 660
gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 720
gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 765
<210> 5
<211> 116
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 5
Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly
1 5 10 15
Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr
20 25 30
Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala
35 40 45
Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys
50 55 60
Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
65 70 75 80
Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala
85 90 95
Ser Glu Thr Ser Gly Ser Tyr Glu Gln Phe Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu
115
<210> 6
<211> 348
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
ggtgctgtcg tctctcaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60
atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120
aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180
ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240
gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctag tgagactagc 300
gggagctatg agcagttctt cgggccaggg acacggctca ccgtgcta 348
<210> 7
<211> 295
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 7
Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly
1 5 10 15
Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr
20 25 30
Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala
35 40 45
Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys
50 55 60
Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
65 70 75 80
Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala
85 90 95
Ser Glu Thr Ser Gly Ser Tyr Glu Gln Phe Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala
115 120 125
Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
130 135 140
Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser
145 150 155 160
Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro
165 170 175
Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu
180 185 190
Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn
195 200 205
His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
210 215 220
Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu
225 230 235 240
Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln
245 250 255
Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala
260 265 270
Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val
275 280 285
Lys Arg Lys Asp Ser Arg Gly
290 295
<210> 8
<211> 885
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 8
ggtgctgtcg tctctcaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60
atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120
aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180
ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240
gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctag tgagactagc 300
gggagctatg agcagttctt cgggccaggg acacggctca ccgtgctaga ggacctgaaa 360
aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420
caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 480
tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 540
gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc 600
accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660
gagaatgacg agtggaccca ggatagggcc aaacctgtca cccagatcgt cagcgccgag 720
gcctggggta gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct 780
gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt 840
gccctcgtgc tgatggccat ggtcaagaga aaggattcca gaggc 885
<210> 9
<211> 9
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 9
Met Leu Trp Arg Leu Gly Ala Thr Ile
1 5
<210> 10
<211> 7
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 10
Thr Arg Asp Thr Thr Tyr Tyr
1 5
<210> 11
<211> 8
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 11
Arg Asn Ser Phe Asp Glu Gln Asn
1 5
<210> 12
<211> 11
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 12
Ala Leu Asn Ala Gly Ala Gly Asn Met Leu Thr
1 5 10
<210> 13
<211> 6
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 13
Asp Phe Gln Ala Thr Thr
1 5
<210> 14
<211> 7
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 14
Ser Asn Glu Gly Ser Lys Ala
1 5
<210> 15
<211> 12
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 15
Ser Ala Ser Glu Thr Ser Gly Ser Tyr Glu Gln Phe
1 5 10
<210> 16
<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 16
acccgtgata ctacttatta c 21
<210> 17
<211> 24
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 17
cggaactctt ttgatgagca aaat 24
<210> 18
<211> 33
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 18
gctctgaacg ccggggcagg caacatgctc acc 33
<210> 19
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 19
gactttcagg ccacaact 18
<210> 20
<211> 21
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 20
tccaatgagg gctccaaggc c 21
<210> 21
<211> 36
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 21
agtgctagtg agactagcgg gagctatgag cagttc 36
<210> 22
<211> 275
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 22
Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val
1 5 10 15
Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser
20 25 30
Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg
35 40 45
Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu
50 55 60
Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile
65 70 75 80
Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn
85 90 95
Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys
100 105 110
Ala Leu Asn Ala Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr
115 120 125
Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr
130 135 140
Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr
145 150 155 160
Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val
165 170 175
Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys
180 185 190
Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala
195 200 205
Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser
210 215 220
Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr
225 230 235 240
Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile
245 250 255
Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu
260 265 270
Trp Ser Ser
275
<210> 23
<211> 825
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 23
atgctgactg ccagcctgtt gagggcagtc atagcctcca tctgtgttgt atccagcatg 60
gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 120
ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 180
ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 240
agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 300
gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 360
atgctcacct ttggaggggg aacaaggtta atggtcaaac cccatatcca gaaccctgac 420
cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 480
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 540
aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 600
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 660
ttcttcccca gcccagaaag ttcctgtgat gtcaagctgg tcgagaaaag ctttgaaaca 720
gatacgaacc taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa 780
gtggccgggt ttaatctgct catgacgctg cggctgtggt ccagc 825
<210> 24
<211> 309
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 24
Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ser Gly Leu Gly Ala
1 5 10 15
Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser
20 25 30
Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe
35 40 45
Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser
50 55 60
Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys
65 70 75 80
Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr
85 90 95
Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ser Glu
100 105 110
Thr Ser Gly Ser Tyr Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr
115 120 125
Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe
130 135 140
Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val
145 150 155 160
Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp
165 170 175
Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro
180 185 190
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser
195 200 205
Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe
210 215 220
Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
225 230 235 240
Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
245 250 255
Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val
260 265 270
Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu
275 280 285
Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg
290 295 300
Lys Asp Ser Arg Gly
305
<210> 25
<211> 927
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 25
atgctgctgc ttctgctgct tctggggcca ggctccgggc ttggtgctgt cgtctctcaa 60
catccgagct gggttatctg taagagtgga acctctgtga agatcgagtg ccgttccctg 120
gactttcagg ccacaactat gttttggtat cgtcagttcc cgaaacagag tctcatgctg 180
atggcaactt ccaatgaggg ctccaaggcc acatacgagc aaggcgtcga gaaggacaag 240
tttctcatca accatgcaag cctgaccttg tccactctga cagtgaccag tgcccatcct 300
gaagacagca gcttctacat ctgcagtgct agtgagacta gcgggagcta tgagcagttc 360
ttcgggccag ggacacggct caccgtgcta gaggacctga aaaacgtgtt cccacccgag 420
gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggtg 480
tgcctggcca caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag 540
gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc cgccctcaat 600
gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660
cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720
caggataggg ccaaacctgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780
tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag 840
atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt gctgatggcc 900
atggtcaaga gaaaggattc cagaggc 927
<210> 26
<211> 208
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 26
Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser Val Val Glu Lys
1 5 10 15
Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr
20 25 30
Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu Leu Val Phe Leu
35 40 45
Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr
50 55 60
Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr
65 70 75 80
Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala
85 90 95
Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val
100 105 110
Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp
115 120 125
Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser
130 135 140
Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp
145 150 155 160
Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
165 170 175
Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn
180 185 190
Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
<210> 27
<211> 624
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 27
gcgcagaaag tgacccaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 60
ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 120
ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 180
agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 240
gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgaacgccgg ggcaggcaac 300
atgctcacct ttggaggggg aacaaggtta atggtcaaac cccatatcca gaaccctgac 360
cctgccgtgt accagctgag agactctaag tcgagtgaca agtctgtctg cctattcacc 420
gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 480
aaatgtgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 540
aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 600
ttcttcccca gcccagaaag ttcc 624
<210> 28
<211> 246
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 28
Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly
1 5 10 15
Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr
20 25 30
Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala
35 40 45
Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys
50 55 60
Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
65 70 75 80
Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala
85 90 95
Ser Glu Thr Ser Gly Ser Tyr Glu Gln Phe Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala
115 120 125
Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
130 135 140
Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser
145 150 155 160
Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro
165 170 175
Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu
180 185 190
Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn
195 200 205
His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
210 215 220
Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu
225 230 235 240
Ala Trp Gly Arg Ala Asp
245
<210> 29
<211> 738
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 29
ggtgcagttg ttagccaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60
atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120
aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180
ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240
gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctag tgagactagc 300
gggagctatg agcagttctt cgggccaggg acacggctca ccgtgctaga ggacctgaaa 360
aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 420
caaaaggcca cactggtgtg cctggccacc ggtttctacc ccgaccacgt ggagctgagc 480
tggtgggtga atgggaagga ggtgcacagt ggggtctgca cagacccgca gcccctcaag 540
gagcagcccg ccctcaatga ctccagatac gctctgagca gccgcctgag ggtctcggcc 600
accttctggc aggacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 660
gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag 720
gcctggggta gagcagac 738
<210> 30
<211> 254
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 30
Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Leu Ser Val Pro Glu Gly
1 5 10 15
Glu Asp Val Thr Ile Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr
20 25 30
Tyr Leu Phe Trp Tyr Arg Gln Asp Pro Gly Gly Glu Leu Val Phe Leu
35 40 45
Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr
50 55 60
Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr
65 70 75 80
Ala Val Gln Pro Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala
85 90 95
Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Ser Val
100 105 110
Lys Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
115 120 125
Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Gly Ala Val Val Ser Gln
130 135 140
His Pro Ser His Leu Ser Val Lys Ser Gly Thr Ser Val Lys Leu Glu
145 150 155 160
Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln
165 170 175
Asp Pro Gly Gln Ser Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser
180 185 190
Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Arg Phe Leu Ile Asn
195 200 205
His Ala Ser Leu Thr Leu Ser Thr Leu Thr Ile Thr Ser Val His Pro
210 215 220
Glu Asp Ser Ser Phe Tyr Phe Cys Ser Ala Ser Glu Thr Ser Gly Ser
225 230 235 240
Tyr Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Asp
245 250
<210> 31
<211> 762
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 31
gctcaaaaag ttactcaggc ccagaccgaa ctgagcgttc cggaaggcga agatgttacc 60
attgattgtg tgtatgaaac ccgcgatacc acctattatc tgttttggta tcgtcaggat 120
ccgggcggcg aactggtttt tctgattcgc cgtaatagtt ttgatgaaca gaatgaaatc 180
agcggtcgtt atagctggaa ttttcagaaa agtaccagca gctttaattt taccattacc 240
gcagttcagc cggtggatag tgccgtttat ttttgtgccc tgaatgcagg cgccggtaat 300
atgctgacct ttggtggcgg cacccgcctg agtgtgaaac cgggtggtgg cagcgaaggt 360
ggcggtagtg aaggcggcgg cagtgaaggt ggtggctcag aaggcggcac cggtggtgca 420
gtggtgagcc agcatccgag tcatctgagc gttaaaagcg gcaccagtgt taaactggaa 480
tgtcgtagcc tggattttca ggcaaccacc atgttttggt atcgccagga tccgggtcag 540
agtctgatgc tgatggcaac cagtaatgaa ggtagtaaag caacctatga acagggtgtt 600
gaaaaagatc gctttctgat taatcatgcc agtctgaccc tgagcaccct gaccattacc 660
agtgttcatc cggaagatag cagtttttat ttttgcagtg caagcgaaac cagcggtagt 720
tatgaacagt ttttcggtcc gggcacccgc ttaaccgtgg at 762
<210> 32
<211> 114
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 32
Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Leu Ser Val Pro Glu Gly
1 5 10 15
Glu Asp Val Thr Ile Asp Cys Val Tyr Glu Thr Arg Asp Thr Thr Tyr
20 25 30
Tyr Leu Phe Trp Tyr Arg Gln Asp Pro Gly Gly Glu Leu Val Phe Leu
35 40 45
Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr
50 55 60
Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr
65 70 75 80
Ala Val Gln Pro Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Asn Ala
85 90 95
Gly Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Ser Val
100 105 110
Lys Pro
<210> 33
<211> 342
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 33
gctcaaaaag ttactcaggc ccagaccgaa ctgagcgttc cggaaggcga agatgttacc 60
attgattgtg tgtatgaaac ccgcgatacc acctattatc tgttttggta tcgtcaggat 120
ccgggcggcg aactggtttt tctgattcgc cgtaatagtt ttgatgaaca gaatgaaatc 180
agcggtcgtt atagctggaa ttttcagaaa agtaccagca gctttaattt taccattacc 240
gcagttcagc cggtggatag tgccgtttat ttttgtgccc tgaatgcagg cgccggtaat 300
atgctgacct ttggtggcgg cacccgcctg agtgtgaaac cg 342
<210> 34
<211> 116
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 34
Gly Ala Val Val Ser Gln His Pro Ser His Leu Ser Val Lys Ser Gly
1 5 10 15
Thr Ser Val Lys Leu Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr
20 25 30
Met Phe Trp Tyr Arg Gln Asp Pro Gly Gln Ser Leu Met Leu Met Ala
35 40 45
Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys
50 55 60
Asp Arg Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
65 70 75 80
Ile Thr Ser Val His Pro Glu Asp Ser Ser Phe Tyr Phe Cys Ser Ala
85 90 95
Ser Glu Thr Ser Gly Ser Tyr Glu Gln Phe Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Asp
115
<210> 35
<211> 348
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 35
ggtgcagtgg tgagccagca tccgagtcat ctgagcgtta aaagcggcac cagtgttaaa 60
ctggaatgtc gtagcctgga ttttcaggca accaccatgt tttggtatcg ccaggatccg 120
ggtcagagtc tgatgctgat ggcaaccagt aatgaaggta gtaaagcaac ctatgaacag 180
ggtgttgaaa aagatcgctt tctgattaat catgccagtc tgaccctgag caccctgacc 240
attaccagtg ttcatccgga agatagcagt ttttattttt gcagtgcaag cgaaaccagc 300
ggtagttatg aacagttttt cggtccgggc acccgcttaa ccgtggat 348
<210> 36
<211> 24
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 36
Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly
1 5 10 15
Gly Gly Ser Glu Gly Gly Thr Gly
20
<210> 37
<211> 72
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 37
ggtggtggca gcgaaggtgg cggtagtgaa ggcggcggca gtgaaggtgg tggctcagaa 60
ggcggcaccg gt 72

Claims (10)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be with MLWRLGATI-HLA A0201 compound knot It closes；Preferably, the TCR includes TCR α chain variable domain and TCR β chain variable domain, which is characterized in that the TCR α chain variable domain CDR3 amino acid sequence be ALNAGAGNMLT (SEQ ID NO:12)；And/or the ammonia of the CDR3 of the TCR β chain variable domain Base acid sequence is SASETSGSYEQF (SEQ ID NO:15)；
It is highly preferred that 3 complementary determining regions (CDR) of the TCR α chain variable domain are as follows:
αCDR1-TRDTTYY(SEQ ID NO:10)
αCDR2-RNSFDEQN(SEQ ID NO:11)
αCDR3-ALNAGAGNMLT(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-SASETSGSYEQF(SEQ ID NO:15)。
2. TCR as described in claim 1, which is characterized in that described it includes TCR α chain variable domain and TCR β chain variable domain TCR α chain variable domain is the amino acid sequence for having at least 90% sequence identity with SEQ ID NO:1；And/or the TCR β chain Variable domain is the amino acid sequence for having at least 90% sequence identity with SEQ ID NO:5.
3. TCR as described in claim 1, which is characterized in that the α chain of the TCR and/or the end C- or N- of β chain are combined with Conjugate；Preferably, the conjugate in conjunction with the T cell receptor is detectable marker, therapeutic agent, PK modified part or appoints The combination of what these substance；Preferably, the therapeutic agent is anti-CD 3 antibodies.
4. a kind of multivalent TCR complex, which is characterized in that contain at least two TCR molecule, and at least one TCR therein Molecule is TCR described in any one of the claims.
5. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules include to encode TCR described in any of the above-described claim points The nucleic acid sequence or its complementary series of son；
Preferably, the nucleic acid molecules include nucleotide sequence SEQ ID NO:2 or the SEQ ID of coding TCR α chain variable domain NO:33；And/or
The nucleic acid molecules include the nucleotide sequence SEQ ID NO:6 or SEQ ID NO:35 of coding TCR β chain variable domain.
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 contain the carrier described in claim 6 in the host cell Or nucleic acid molecules described in the claim 5 of external source are integrated in chromosome.
8. a kind of cell, which is characterized in that institute in nucleic acid molecules described in the cell transduction claim 5 or claim 6 State 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 TCR described in any one of 1-3, TCR compound described in claim 4, nucleic acid molecules described in claim 5 or power Benefit requires cell described in 8.
10. TCR compound or right described in T cell receptor of any of claims 1-3 or claim 4 It is required that the purposes of cell described in 8, which is characterized in that be used to prepare the drug for the treatment of tumour or autoimmune disease.
CN201810386338.3A 2018-04-26 2018-04-26 TCR for identifying LMP1 antigen short peptide and coding sequence thereof Active CN110407926B (en)

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Citations (4)

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US20170056473A1 (en) * 2015-09-01 2017-03-02 The Catholic University Of Korea Industry-Academic Cooperation Foundation Ebv antigen specific t-cell receptor and use thereof
CN107001444A (en) * 2014-12-17 2017-08-01 中国科学院广州生物医药与健康研究院 Recognize the φt cell receptor of Epstein-Barr virus small peptide

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AU8200098A (en) * 1997-07-10 1999-02-08 Council Of The Queensland Institute Of Medical Research, The Ctl epitopes from ebv
WO2011062560A1 (en) * 2009-11-19 2011-05-26 National University Of Singapore Method for producing t cell receptor-like monoclonal antibodies and uses thereof
US20120294874A1 (en) * 2009-11-19 2012-11-22 Paul Anthony Macary Method for Producing T Cell Receptor-Like Monoclonal Antibodies and Uses Thereof
CN107001444A (en) * 2014-12-17 2017-08-01 中国科学院广州生物医药与健康研究院 Recognize the φt cell receptor of Epstein-Barr virus small peptide
US20170056473A1 (en) * 2015-09-01 2017-03-02 The Catholic University Of Korea Industry-Academic Cooperation Foundation Ebv antigen specific t-cell receptor and use thereof

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WANG, LW等: "Epstein-Barr Virus LMP1-Mediated Oncogenicity", 《JOURNAL OF VIROLOGY》 *
邵红伟,郭亨彭,黄晓露等: "单链TCR分子的构建与酵母表面展示", 《广东药学院学报》 *

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