CN109400697A - A kind of TCR of PRAME antigen small peptide and its compositions related of identifying - Google Patents

A kind of TCR of PRAME antigen small peptide and its compositions related of identifying Download PDF

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CN109400697A
CN109400697A CN201710707316.8A CN201710707316A CN109400697A CN 109400697 A CN109400697 A CN 109400697A CN 201710707316 A CN201710707316 A CN 201710707316A CN 109400697 A CN109400697 A CN 109400697A
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CN109400697B (en
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李懿
区裕升
林燕梅
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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 LYVDSLFFL derived from PRAME antigen, the antigen small peptide LYVDSLFFL can form compound with HLA A2402 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 of PRAME antigen small peptide and its compositions related of identifying
Technical field
The present invention relates to that can identify the TCR from PRAME antigen small peptide, the invention further relates to transduce above-mentioned TCR to obtain PRAME specificity T cell and they prevention and treatment PRAME related disease in purposes.
Background technique
PRAME is melanoma specific antigen (preferentially expressed antigen of Melanoma, PRAME), 88% it is initial and 95% transfer melanoma in have expression (Ikeda H, et Al.Immunity, 1997,6 (2): 199-208), normal skin tissue and benign melanocyte are not expressed then.PRAME is in cell It is degraded to micromolecule polypeptide after interior generation, and forms compound in conjunction with MHC (main histocompatibility complex) molecule, is in It is delivered to cell surface.LYVDSLFFL is the small peptide derived from PRAME antigen, is a kind of target of PRAME treating correlative diseases. In addition to melanoma, PRAME is also expressed in kinds of tumors, including squamous cell lung carcinoma, breast cancer, clear-cell carcinoma, incidence are swollen Tumor, Huo Jiejin lymphomas, sarcoma and medulloblastoma etc. (van't Veer LJ, et al.Nature, 2002,415 (6871):530-536；Boon K, et al.Oncogene, 2003,22 (48): 7687-7694) in addition, its in leukaemia PRAME also has significant expression, acute lymphoblastic leukemia 17%~42%, acute myeloblastic leukemia 30%~64% (SteinbachD,et al.Cancer Genet Cytogene,2002,138(1):89-91).For controlling for above-mentioned disease It treats, the methods of chemotherapy and radiation treatment can be used, but can all damage to the normal cell of itself.
T cell adoptive immunotherapy is that will there is the reaction-ive T cell of specificity to be transferred in patient body target cell antigen, It is set to play a role for target cell.T cell receptor (TCR) is a kind of memebrane protein on T cell surface, can be identified corresponding The antigen small peptide of target cell surface.In immune system, pass through the TCR and the main histocompatbility of small peptide-of antigen small peptide specificity The combination of complex (pMHC compound) causes T cell and antigen presenting cell (APC) is directly physically contacted, then T cell And other cell membrane surface molecules of both APC just interact, and cause a series of subsequent cell signal transmitting and its His physiological reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, this field skill Art personnel are dedicated to isolating the TCR for having specificity to PRAME antigen small peptide, and the TCR transduceed T cell to obtain pair PRAME 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 PRAME antigen small peptide.
The first aspect of the present invention, provides a kind of T cell receptor (TCR), and the TCR can be with LYVDSLFFL-HLA A2402 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 AVNRVTGGGNKLT (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domain Amino acid sequence is ASSLAQGNNQPQH (SEQ ID NO:15).
In another preferred example, 3 complementary determining regions (CDR) of the TCR α chain variable domain are as follows:
αCDR1-DRVSQS(SEQ ID NO:10)
αCDR2-IYSNGD(SEQ ID NO:11)
αCDR3-AVNRVTGGGNKLT(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-SGHDT(SEQ ID NO:13)
βCDR2-YYEEEE(SEQ ID NO:14)
βCDR3-ASSLAQGNNQPQH(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 includes melanoma and other tumours such as lung squama It is shape cell cancer, breast cancer, clear-cell carcinoma, head and neck neoplasm, Huo Jiejin lymphomas, sarcoma and medulloblastoma, acute Lymphocytic leukemia, acute myeloblastic leukemia etc..
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively that TCR α chain variable domain amino acid sequence, TCR α chain are variable Domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, the TCR α chain amino acid sequence with leader sequence And the TCR α chain nucleotide sequence with leader sequence.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively that TCR β chain variable domain amino acid sequence, TCR β chain are variable Domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, the TCR β chain amino acid sequence with leader sequence And the TCR β chain nucleotide sequence with leader sequence.
Fig. 3 a and Fig. 3 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chain.
Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chain.
Fig. 5 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. 6 a and Fig. 6 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR.
Fig. 7 a and Fig. 7 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR α chain.
Fig. 8 a and Fig. 8 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR β chain.
Fig. 9 a and Fig. 9 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR catenation sequence (linker).
Figure 10 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 11 is BIAcore dynamics figure of the sTCR of the present invention in conjunction with LYVDSLFFL-HLA A2402 compound Spectrum.
Figure 12 is dynamic for BIAcore of the soluble single-chain T CR of the present invention in conjunction with LYVDSLFFL--HLA A2402 compound Mechanics map.
Figure 13 be transduce TCR of the present invention effector cell to the activation contractile studies figure of target cell LCL.
Figure 14 be transduce TCR of the present invention effector cell to the activation contractile studies figure of tumor cell line.
Specific embodiment
The present inventor after extensive and in-depth study, has found and PRAME antigen small peptide LYVDSLFFL (SEQ ID NO:9) the TCR that can be specifically bound, the antigen small peptide LYVDSLFFL can with HLA A2402 formed compound and together by It is presented to cell surface.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.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, for The presentation of antigen has specificity, and different individuals has different MHC, can present small peptide different in a kind of proteantigen to respectively From APC cell surface.The MHC of the mankind is commonly referred to as HLA gene or HLA complex.
T cell receptor (TCR) is the unique of specific antigen peptide of the presentation on main histocompatibility complex (MHC) Receptor.In immune system, T cell is caused by the combination of the TCR and pMHC compound of antigentic specificity and antigen presentation is thin Born of the same parents (APC) are directly physically contacted, and then other cell membrane surface molecules of both T cell and APC just interact, this A series of subsequent cell signal transmitting and other physiological reactions are just caused, so that the T cell of different antigentic specificities Immunological effect is played to its target cell.
TCR be as α chain/β chain or γ chain/δ chain in the form of heterodimer existing for cell membrane surface glycoprotein.? TCR heterodimer is made of α and β chain in 95% T cell, and 5% T cell has the TCR being made of γ and δ chain.It The right heterogeneous dimerization TCR of α β has α chain and β chain, and α chain and β chain constitute the subunit of α β heterodimeric TCR.In a broad sense, α and β are each Chain includes variable region, bonding pad and constant region, and β chain usually contains short variable region also between variable region and bonding pad, but should Variable region is often regarded as a part of bonding pad.Each variable region includes 3 be entrenched in frame structure (framework regions) A CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compound, wherein CDR3 by Variable region and bonding pad recombinate, referred to as hypervariable region.α the and β chain of TCR generally regards that each there are two " structural domains " can be changed as Domain and constant domain, variable domain are made of the variable region connected and bonding pad.The sequence of TCR constant domain can be in international immune genetic It learns and is found in the public database of information system (IMGT), if the constant domain sequence of TCR molecule alpha chain is " TRAC*01 ", TCR divides The constant domain sequence of sub- β chain is " TRBC1*01 " or " TRBC2*01 ".In addition, α the and β chain of TCR also includes transmembrane region and cytoplasm Area, cytoplasmic region are very short.
In the present invention, term " polypeptide of the present invention ", " TCR of the invention ", " T cell receptor of the invention " is interchangeable makes With.
Native interchain disulfide bond and artificial interchain disulfide bond
Natural TCR membrane-proximal region C α and C β interchain exist one group of disulfide bond, the present invention in referred to as " two sulphur of native interchain Key ".In the present invention, by what is be artificially introduced, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond claim For " artificial interchain disulfide bond ".
For convenience of the position of description disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequence in the present invention Position Number by from N-terminal to C-terminal sequence successively carry out Position Number, in TRBC1*01 or TRBC2*01, by from N-terminal to The 60th amino acid of the sequence of C-terminal successively is P (proline), then can describe it as TRBC1*01 or TRBC2*01 in the present invention The Pro60 of exons 1 can also be stated that the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1, for another example It is Q (glutamine) by the 61st amino acid of the sequence from N-terminal to C-terminal successively in TRBC1*01 or TRBC2*01, then it is of the invention In can describe it as the Gln61 of TRBC1*01 or TRBC2*01 exons 1, can also be stated that TRBC1*01 or TRBC2* 61st amino acids of 01 exons 1, other and so on.In the present invention, the amino acid sequence of variable region TRAV and TRBV Position Number, according to the Position Number listed in IMGT.Such as some amino acid in TRAV, the Position Number listed in IMGT is 46, then the 46th amino acids of TRAV, other and so on are described it as in the present invention.In the present invention, the sequence of other amino acid Column position number has specified otherwise, then presses specified otherwise.
Detailed description of the invention
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 LYVDSLFFL-HLA A2402 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-DRVSQS(SEQ ID NO:10)
αCDR2-IYSNGD(SEQ ID NO:11)
αCDR3-AVNRVTGGGNKLT(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-SGHDT(SEQ ID NO:13)
βCDR2-YYEEEE(SEQ ID NO:14)
βCDR3-ASSLAQGNNQPQH(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 PRAME 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 PCT/CN2016/077680 is also disclosed in the α chain variable region of TCR for stability Introducing artificial interchain disulfide bond between β chain constant region can be such that the stability of TCR significantly improves.Therefore, height parent of the invention Artificial interchain disulfide bond can also be contained between the α chain variable region and β chain constant region of power TCR.Specifically, in the α of the TCR The cysteine residues of artificial interchain disulfide bond are formed between chain variable region and β chain constant region instead of the 46th ammonia of TRAV 60th amino acids of base acid and TRBC1*01 or TRBC2*01 exons 1；The 47th amino acids and TRBC1*01 of TRAV or 61 amino acids of TRBC2*01 exons 1；The of the 46th amino acids of TRAV and TRBC1*01 or TRBC2*01 exons 1 61 amino acids；Or TRAV the 47th amino acids and TRBC1*01 or TRBC2*01 exons 1 the 60th amino acids.It is preferred that Ground, such TCR may include all or part of TCR α chain of (I) in addition to its transmembrane domain, and (II) removes its cross-film knot All or part of TCR β chain other than structure domain, wherein (I) and (II) variable domain comprising TCR chain and at least part is constant Domain, α chain and β chain form heterodimer.It is highly preferred that such TCR may include α chain variable domain and β chain variable domain and All or part of β chain constant domain in addition to transmembrane domain, but it does not contain α chain constant domain, the α chain variable domain of the TCR Heterodimer is formed with β chain.
TCR of the invention can also be provided in the form of multivalence complex.Multivalent TCR complex of the invention include two, Three, four or more TCR of the present invention are combined and the polymer that is formed, can such as be generated with four dimerization domains of p53 The compound that the tetramer or multiple TCR of the present invention are formed in conjunction with another molecule.TCR compound of the invention can be used for body Outer or tracking in vivo or targeting present the cell of specific antigen, it can also be used to which generating has other multivalence TCR of such application multiple Close the intermediate of object.
TCR of the invention can be used alone, can also with conjugate with covalent or other modes in conjunction with, preferably with covalently side Formula combines.The conjugate includes that detectable marker (for diagnostic purpose, presents wherein the TCR is used to detect The presence of the cell of LYVDSLFFL-HLA A2402 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-gaccgagtttcccagtcc(SEQ ID NO:16)
αCDR2-atatactccaatggtgac(SEQ ID NO:17)
αCDR3-gccgtgaaccgggtcacgggaggaggaaacaaactcacc(SEQ ID NO:18)
The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR region is as follows:
βCDR1-tctgggcatgacact(SEQ ID NO:19)
βCDR2-tattatgaggaggaagag(SEQ ID NO:20)
βCDR3-gccagcagcttggcacagggtaacaatcagccccagcat(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 PRAME antigen specificity.Ideally, which can should express to continual high levels in T cell.
Cell
The invention further relates to the genetically engineered host cell of carrier or coded sequence of the invention.The host Contain in carrier or chromosome of the invention in cell and is integrated with nucleic acid molecules of the invention.Host cell is selected from: prokaryotic cell And eukaryocyte, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..
In addition, the invention also includes the isolated cell for expressing TCR of the invention, especially T cell.The T cell can spread out It is born from the T cell separated from subject, or can be the mixed cellularity group separated from subject, such as periphery hemolymph is thin A part of born of the same parents (PBL) group.Such as, which can be isolated from peripheral blood mononuclear cells (PBMC), can be CD4+T helper cell Or CD8+Cytotoxic T cell.The cell can be in CD4+T helper cell/CD8+In the mixing group of cytotoxic T cell.Generally Ground, the cell can be activated with antibody (e.g., the antibody of anti-CD3 or anti-CD28), to allow them to more easily receive to turn Dye, such as transfected with the carrier of the nucleotide sequence comprising encoding TCR molecule of the present invention.
Alternatively, cell of the invention can also be or derived from stem cell, such as candidate stem cell (HSC).Gene is turned Moving to HSC not will lead in cell surface expression TCR, because stem cell surface does not express CD3 molecule.However, when stem cell point It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecule will start in thymocyte The surface expression introducing TCR molecule.
There are many method be suitable for being carried out with the DNA or RNA of coding TCR of the present invention T cell transfection (e.g., the such as Robbins, (2008)J.Immunol.180:6116-6131).The T cell for expressing TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat Rev for carrying out adoptive treatment Cancer8 (4): 299-308).
PRAME antigen related disease
The invention further relates to the method with PRAME related disease is treated and/or prevented in subject comprising adoptive Shift the step of PRAME specific T-cells are to the subject.The PRAME specific T-cells can recognize LYVDSLFFL-HLA A2402 compound.
The T cell of PRAME specificity of the invention can be used for treating any presentation PRAME antigen small peptide LYVDSLFFL- The PRAME related disease of HLA A2402 compound.Including but not limited to tumour, such as melanoma, squamous cell lung carcinoma, mammary gland Cancer, clear-cell carcinoma, head and neck neoplasm, Huo Jiejin lymphomas, sarcoma and the white blood of medulloblastoma, acute lymphoblastic Disease, acute myeloblastic leukemia etc..
Treatment method
Can by separation with the patient of PRAME antigen related disease or the T cell of volunteer, and will be of the invention TCR is imported in above-mentioned T cell, is then fed back to the cell that these genetic engineerings are modified in patient body to treat.Therefore, The present invention provides a kind of method for treating PRAME related disease, the T cell including the expression TCR of the present invention that will be separated, preferably Ground, the T cell derive from patient itself, are input in patient body.Generally, the T cell including (1) separation patient, (2) are with originally Invention nucleic acid molecules or the nucleic acid molecules ex vivo transduction T cell that can encode TCR molecule of the present invention, (3) modify genetic engineering T cell be input in patient body.The quantity of separation, transfection and the cell fed back can be determined by doctor.
Main advantages of the present invention are:
(1) TCR of the invention can turn simultaneously in conjunction with PRAME antigen small peptide compound LYVDSLFFL-HLA A2402 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 Russell et al., molecular cloning: laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing house) described in condition, or according to the normal condition proposed by manufacturer.Unless In addition illustrate, otherwise percentage and number are calculated by weight.Unless otherwise stated, otherwise percentage and number are calculated by weight. Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
Embodiment 1 clones PRAME antigen small peptide specific T-cells
Utilize synthesis small peptide LYVDSLFFL (SEQ ID NO.9；Beijing SBS Genetech gene technology Co., Ltd) stimulation come from In the peripheral blood lymphocytes (PBL) for the healthy volunteer that genotype is HLA-A2402.By LYVDSLFFL small peptide and with life The HLA-A2402 renaturation of object element label, prepares pHLA monoploid.(BD is public with the Streptavidin marked with PE for these monoploid Department) 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 carries out monoclonal with limiting dilution assay.Monoclonal cell tetramer staining, screening Double positive colonies.
Since entire experiment flow takes a long time, influence factor is extremely more, and experiment is more complicated, and the performance of cell at all can not Prediction, even across screening layer by layer with stringent detection, the success rate for obtaining corresponding T cell monoclonal is also very low.One As in the case of merely through several batches experiment be difficult obtain have desired activities TCR.
In the present invention, by inventor's in-depth study and a large amount of experiment, double sun of the condition of satisfaction have been finally obtained Property monoclonal cell.Even if successfully screening obtains T cell monoclonal, TCR obtained from this also not necessarily be can satisfy It is required that because in many cases, obtained TCR can not by after success renaturation or renaturation with the affinity of corresponding epitope very Difference, or even can not combine.It needs further to verify in conjunction with activity.
Embodiment 2 obtains the building of the tcr gene and carrier of PRAME antigen small peptide specific T-cell clones
With the antigen small peptide screened in Quick-RNATMMiniPrep (ZYMO research) extracting embodiment 1 The total serum IgE of the restrictive T cell clone of LYVDSLFFL specificity, HLA-A2402.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-DRVSQS(SEQ ID NO:10)
αCDR2-IYSNGD(SEQ ID NO:11)
αCDR3-AVNRVTGGGNKLT(SEQ ID NO:12)
β chain includes the CDR with following amino acid sequence:
βCDR1-SGHDT(SEQ ID NO:13)
βCDR2-YYEEEE(SEQ ID NO:14)
βCDR3-ASSLAQGNNQPQH(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 3PRAME 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 of chain distinguishes as shown in Figure 3a and Figure 3b shows, the amino acid sequence and nucleotide sequence of β chain with nucleotide sequence Respectively as shown in figures 4 a and 4b, the cysteine residues of introducing with overstriking and underline alphabetical indicate.Pass through " molecular cloning Laboratory manual " (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) Described in standard method the objective gene sequence of above-mentioned TCR α and β chain is inserted respectively into expression vector pET28a after synthesizing + (Novagene), the cloning site of upstream and downstream are NcoI and NotI respectively.Insert Fragment is errorless by sequencing confirmation.
The expression vector of TCR α and β chain is converted by chemical transformation respectively and enters expression bacterium BL21 (DE3), bacterium It is grown with LB culture solution, 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 5.
The generation of the soluble single-chain T CR of 4 PRAME antigen small peptide specificity of embodiment
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 figures 6 a and 6b.The amino acid sequence of its α chain variable domain and Nucleotide sequence difference is as shown in figs. 7 a and 7b；The amino acid sequence and nucleotide sequence of its β chain variable domain are respectively such as Fig. 8 a With shown in Fig. 8 b；Amino acid sequence and the nucleotide sequence difference of its linker sequence are as shown in figures 9 a and 9b.
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 5PRAME 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 10.
Embodiment 6 combines characterization
BIAcore analysis
This example demonstrated soluble TCR molecules of the present invention can be special with LYVDSLFFL-HLA A2402 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 LYVDSLFFL-HLA A2402 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 LYVDSLFFL-HLA A2402 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 LYVDSLFFL-HLA A2402 compound is as follows:
A. it purifies
The E.coli bacterium solution for collecting 100ml inducing expression heavy chain or light chain uses 10ml after 4 DEG C of 8000g are centrifuged 10min PBS washing thalline is primary, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) later Thallus is resuspended in concussion, and rotates in room temperature and be incubated for 20min, and later in 4 DEG C, 6000g is centrifuged 15min, discards supernatant, collection is forgiven Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated for 5min；Add 30ml The BugBuster of 10 times of dilution is mixed, and 4 DEG C of 6000g are centrifuged 15min；It discards supernatant, 30ml is added to dilute 10 times of BugBuster Inclusion body is resuspended, mixes, 4 DEG C of 6000g are centrifuged 15min, are repeated twice, and 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 LYVDSLFFL (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 LYVDSLFFL 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 16/60S200HR column (GE General Electric Co. Limited), load 1ml concentrated biotinylation pMHC molecule, then with PBS with 1ml/ The elution of min flow velocity.Biotinylated pMHC molecule occurs in about 55ml as unimodal elution.Merge the group containing protein Point, it is concentrated with Mill ipore super filter tube, 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 LYVDSLFFL-HLA A2402 compound point Not as is illustrated by figs. 11 and 12.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 LYVDSLFFL-HLA A2402 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 verifies the function of the effector cell for the TCR of the present invention that transduces using LCL cell line
ELISPOT scheme
Following tests is carried out to prove activating reaction of the T cell to target cell specificity of TCR transduction.Utilize ELISPOT Readout of the IFN-γ yield of 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 LCL cell.Target cell is prepared in assay medium: 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+T cell of TCR of the present invention of having transduceed, and not with same volunteer Transfect the CD8+T of TCR of the present invention as a control group.Pearl (T cell amplified matter, life are coated with AntiCD3 McAb/CD28 Technologies) stimulate T cell, with the lentiviruses transduction for carrying tcr gene of the present invention, containing 50IU/ml IL-2 with Then these cells are placed in examination until 9-12 days after transduction by the 1640 culture mediums amplification containing 10%FBS of 10ng/ml IL-7 It tests in culture medium, 300g room temperature is centrifuged 10 minutes and is washed.Then cell is resuspended in test culture with final concentration needed for 2 times In base.Same processing negative control effector cell.
The preparation of small peptide solution
Corresponding small peptide is added in respective target cell experiment group, making final concentration of the small peptide in ELISPOT orifice plate is respectively 1 μ G/ml, 0.1 μ g/ml, 0.01 μ g/ml, 0.001 μ g/ml and 0 μ g/ml, in addition, experimental group also adds non-specific small peptide, as right It is the final concentration of 1 μ g/ml of (NC) and non-specific small peptide in ELISPOT orifice plate according to group echo.
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 PRAME TCR 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 PRAME antigen small peptide by ELISPOT The IFN-γ release that the target cell of LYVDSLFFL 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 13, and the T cell for the TCR of the present invention that transduces has very the target cell for loading its special small peptide The target cell of good activating reaction, target cell and the non-specific small peptide of load to unsupported corresponding small peptide does not have activation anti-substantially It answers.
Embodiment 8 verifies the function of the effector cell for the TCR of the present invention that transduces using cell line
ELISPOT experiment is carried out according to ELISPOT scheme described in embodiment 7, verifies this hair of transduceing using cell line The function of the effector cell of bright TCR.
Method
Target cell preparation
Target cell used in this experiment be SW620, SW620-PRAME (PRAME overexpression), WiDr, WiDr-PRAME, K562-A24 and K562-A2.The expression quantity of PRAME antigen in above-mentioned cell line is measured according to nanostring technology, wherein SW620 does not express PRAME antigen substantially, and PRAME is overexpressed in SW620-PRAME, and WiDr does not express PRAME, WiDr-PRAME Middle PRAME is overexpressed, and K562-A24 high expresses PRAME antigen, and also more but its genotype of PRAME expression is in K562-A2 A0201 is not A2402.Therefore, SW620, WiDr and K562-A2 are as control.Target cell will be prepared in assay medium: 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+T cell of TCR of the present invention of having transduceed, and not with same volunteer The CD8+T cell of transduction TCR of the present invention is as a control group (NC).Effector cell is prepared in assay medium: effector cell is dense Degree is adjusted to 2.0 × 104A positive cell/milliliter, every hole take 100 microlitres to obtain 2.0 × 103A positive cell/hole.
ELISPOT
According to the specification that manufacturer provides, prepare orifice plate as described below: dilute by 1:200 with 5 milliliters of sterile PBS of every block of plate It releases anti-human IFN-γ and captures antibody, 50 microlitres of dilution is then captured into antibody etc. point, each hole is added.Orifice plate mistake is incubated at 4 DEG C Night.After incubation, orifice plate is washed to remove extra capture antibody.200 PBS containing 5%FBS are added, the lower incubation orifice plate 2 of temperature is small When to close orifice plate.It outwells confining liquid, bullet and pats ELISPOT orifice plate to remove the confining liquid 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 cells (obtain in total about 2.0 × 103A positive cell/hole and control effector 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 5%FBS 1:200 dilution detection antibody, is added each hole by 50 microlitres/hole.It incubates orifice plate 2 hours at room temperature, then washs 3 with washing buffer It is secondary, orifice plate is patted on paper handkerchief to remove excessive washing buffer.
Streptavidin-alkaline phosphatase is diluted by 1:100 with the PBS containing 5%FBS, by 50 microlitres of diluted strepto-s Avidin-alkaline phosphatase is added each hole and incubates orifice plate 1 hour at room temperature.Then 3 PBS are washed with washing buffer to wash It washs 3 times, pats orifice plate on paper handkerchief to remove excessive washing buffer and PBS.It is added what kit provided after washing Develop in 50 microlitres/hole of BCIP/NBT solution.It is protected from light during development with masking foil covering orifice plate, stands 5-15 minutes.? Spot of conventional detection development orifice plate, determines the Best Times for terminating reaction during this.It removes BCIP/NBT solution and is steamed with double Water rinses orifice plate to stop developing reaction, then drying removes orifice plate bottom, is dried at room temperature for orifice plate until each hole is complete White drying 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 of verifying (as described above) TCR transduction of the present invention is tested to positive cell line SW620- by ELISPOT PRAME, WiDr-PRAME, K562-A24, which react, discharges IFN-γ, does not have on negative cells SW620, WiDr and K526-A2 Function.The ELSPOT amount of speckle observed in each hole is drawn using graphpad prism6.
Experimental result is as shown in figure 14, and the T cell for the TCR of the present invention that transduces has the target cell of expression PRAME very strong sharp Reaction living, the target cell different to the target cell or genotype of not expressing PRAME do not have activating reaction.Meanwhile this hair of not transduceing The NC group of the T cell of bright TCR is to 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 of PRAME antigen small peptide and its compositions related of identifying
<130> P2017-1198
<160> 37
<170> PatentIn version 3.5
<210> 1
<211> 114
<212> PRT
<213> artificial sequence
<220>
<223>TCR α chain variable domain
<400> 1
Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Arg Val Thr Gly
85 90 95
Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Lys Val Glu
100 105 110
Leu Asn
<210> 2
<211> 342
<212> DNA
<213> artificial sequence
<220>
<223>TCR α chain variable domain
<400> 2
cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
ctcaactgca cttacagtga ccgagtttcc cagtccttct tctggtacag acaatattct 120
gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gtgaaccggg tcacgggagg aggaaacaaa 300
ctcacctttg ggacaggcac tcagctaaaa gtggaactca at 342
<210> 3
<211> 254
<212> PRT
<213> artificial sequence
<220>
<223>TCR α chain
<400> 3
Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Arg Val Thr Gly
85 90 95
Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Lys Val Glu
100 105 110
Leu Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser
115 120 125
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln
130 135 140
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys
145 150 155 160
Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val
165 170 175
Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn
180 185 190
Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys
195 200 205
Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
210 215 220
Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val
225 230 235 240
Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 4
<211> 762
<212> DNA
<213> artificial sequence
<220>
<223>TCR α chain
<400> 4
cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
ctcaactgca cttacagtga ccgagtttcc cagtccttct tctggtacag acaatattct 120
gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gtgaaccggg tcacgggagg aggaaacaaa 300
ctcacctttg ggacaggcac tcagctaaaa gtggaactca atatccagaa ccctgaccct 360
gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 420
tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480
actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540
aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600
ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 660
acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 720
gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 762
<210> 5
<211> 114
<212> PRT
<213> artificial sequence
<220>
<223>TCR β chain variable domain
<400> 5
Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys Thr Arg Gly
1 5 10 15
Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His Asp Thr Val
20 25 30
Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe Ile Phe Gln
35 40 45
Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro Asp Arg Phe
50 55 60
Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn Val Asn Ala
65 70 75 80
Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Leu Ala
85 90 95
Gln Gly Asn Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser
100 105 110
Ile Leu
<210> 6
<211> 342
<212> DNA
<213> artificial sequence
<220>
<223>TCR β chain variable domain
<400> 6
gacgctggag tcacccaaag tcccacacac ctgatcaaaa cgagaggaca gcaagtgact 60
ctgagatgct ctcctaagtc tgggcatgac actgtgtcct ggtaccaaca ggccctgggt 120
caggggcccc agtttatctt tcagtattat gaggaggaag agagacagag aggcaacttc 180
cctgatcgat tctcaggtca ccagttccct aactatagct ctgagctgaa tgtgaacgcc 240
ttgttgctgg gggactcggc cctctatctc tgtgccagca gcttggcaca gggtaacaat 300
cagccccagc attttggtga tgggactcga ctctccatcc ta 342
<210> 7
<211> 293
<212> PRT
<213> artificial sequence
<220>
<223>TCR β chain
<400> 7
Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys Thr Arg Gly
1 5 10 15
Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His Asp Thr Val
20 25 30
Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe Ile Phe Gln
35 40 45
Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro Asp Arg Phe
50 55 60
Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn Val Asn Ala
65 70 75 80
Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Leu Ala
85 90 95
Gln Gly Asn Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser
100 105 110
Ile Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe
115 120 125
Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val
130 135 140
Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp
145 150 155 160
Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro
165 170 175
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser
180 185 190
Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe
195 200 205
Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
210 215 220
Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
225 230 235 240
Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val
245 250 255
Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu
260 265 270
Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg
275 280 285
Lys Asp Ser Arg Gly
290
<210> 8
<211> 879
<212> DNA
<213> artificial sequence
<220>
<223>TCR β chain
<400> 8
gacgctggag tcacccaaag tcccacacac ctgatcaaaa cgagaggaca gcaagtgact 60
ctgagatgct ctcctaagtc tgggcatgac actgtgtcct ggtaccaaca ggccctgggt 120
caggggcccc agtttatctt tcagtattat gaggaggaag agagacagag aggcaacttc 180
cctgatcgat tctcaggtca ccagttccct aactatagct ctgagctgaa tgtgaacgcc 240
ttgttgctgg gggactcggc cctctatctc tgtgccagca gcttggcaca gggtaacaat 300
cagccccagc attttggtga tgggactcga ctctccatcc tagaggacct gaacaaggtg 360
ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420
gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480
gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 540
cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 600
tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660
gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720
ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 780
atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 840
gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 879
<210> 9
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223>antigen small peptide
<400> 9
Leu Tyr Val Asp Ser Leu Phe Phe Leu
1 5
<210> 10
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> α CDR1
<400> 10
Asp Arg Val Ser Gln Ser
1 5
<210> 11
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> α CDR2
<400> 11
Ile Tyr Ser Asn Gly Asp
1 5
<210> 12
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> α CDR3
<400> 12
Ala Val Asn Arg Val Thr Gly Gly Gly Asn Lys Leu Thr
1 5 10
<210> 13
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> β CDR1
<400> 13
Ser Gly His Asp Thr
1 5
<210> 14
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> β CDR2
<400> 14
Tyr Tyr Glu Glu Glu Glu
1 5
<210> 15
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> β CDR3
<400> 15
Ala Ser Ser Leu Ala Gln Gly Asn Asn Gln Pro Gln His
1 5 10
<210> 16
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> α CDR1
<400> 16
gaccgagttt cccagtcc 18
<210> 17
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> α CDR2
<400> 17
atatactcca atggtgac 18
<210> 18
<211> 39
<212> DNA
<213> artificial sequence
<220>
<223> α CDR3
<400> 18
gccgtgaacc gggtcacggg aggaggaaac aaactcacc 39
<210> 19
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> β CDR1
<400> 19
tctgggcatg acact 15
<210> 20
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> β CDR2
<400> 20
tattatgagg aggaagag 18
<210> 21
<211> 39
<212> DNA
<213> artificial sequence
<220>
<223> β CDR3
<400> 21
gccagcagct tggcacaggg taacaatcag ccccagcat 39
<210> 22
<211> 275
<212> PRT
<213> artificial sequence
<220>
<223>with the TCR α chain of leader sequence
<400> 22
Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser
1 5 10 15
Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu
20 25 30
Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp
35 40 45
Arg Val Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser
50 55 60
Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly
65 70 75 80
Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu
85 90 95
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val
100 105 110
Asn Arg Val Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr
115 120 125
Gln Leu Lys Val Glu Leu Asn 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
<220>
<223>with the TCR α chain of leader sequence
<400> 23
atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60
caacagaagg aggtggagca gaattctgga cccctcagtg ttccagaggg agccattgcc 120
tctctcaact gcacttacag tgaccgagtt tcccagtcct tcttctggta cagacaatat 180
tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 240
aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 300
cagcccagtg attcagccac ctacctctgt gccgtgaacc gggtcacggg aggaggaaac 360
aaactcacct ttgggacagg cactcagcta aaagtggaac tcaatatcca 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> 312
<212> PRT
<213> artificial sequence
<220>
<223>with the TCR β chain of leader sequence
<400> 24
Met Gly Pro Gly Leu Leu Cys Trp Ala Leu Leu Cys Leu Leu Gly Ala
1 5 10 15
Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys
20 25 30
Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His
35 40 45
Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe
50 55 60
Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro
65 70 75 80
Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn
85 90 95
Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110
Ser Leu Ala Gln Gly Asn Asn Gln Pro Gln His Phe Gly Asp Gly Thr
115 120 125
Arg Leu Ser Ile Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val
130 135 140
Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala
145 150 155 160
Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu
165 170 175
Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp
180 185 190
Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys
195 200 205
Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg
210 215 220
Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp
225 230 235 240
Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala
245 250 255
Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln
260 265 270
Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys
275 280 285
Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met
290 295 300
Val Lys Arg Lys Asp Ser Arg Gly
305 310
<210> 25
<211> 936
<212> DNA
<213> artificial sequence
<220>
<223>with the TCR β chain of leader sequence
<400> 25
atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60
gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120
agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180
gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240
gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300
ttgctggggg actcggccct ctatctctgt gccagcagct tggcacaggg taacaatcag 360
ccccagcatt ttggtgatgg gactcgactc tccatcctag aggacctgaa caaggtgttc 420
ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480
acactggtat gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540
aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600
gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660
cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720
gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780
agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840
ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900
ctgatggcca tggtcaagag aaaggattcc agaggc 936
<210> 26
<211> 207
<212> PRT
<213> artificial sequence
<220>
<223>sTCR α chain
<400> 26
Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Arg Val Thr Gly
85 90 95
Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Lys Val Glu
100 105 110
Leu Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser
115 120 125
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln
130 135 140
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys
145 150 155 160
Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val
165 170 175
Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn
180 185 190
Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
<210> 27
<211> 621
<212> DNA
<213> artificial sequence
<220>
<223>sTCR α chain
<400> 27
cagaaagaag tggaacagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
ctcaactgca cttacagtga ccgagtttcc cagtccttct tctggtacag acaatattct 120
gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gtgaaccggg tcacgggagg aggaaacaaa 300
ctcacctttg ggacaggcac tcagctaaaa gtggaactca atatccagaa ccctgaccct 360
gccgtgtacc agctgagaga ctctaagtcg agtgacaagt ctgtctgcct attcaccgat 420
tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480
tgtgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540
aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600
ttccccagcc cagaaagttc c 621
<210> 28
<211> 244
<212> PRT
<213> artificial sequence
<220>
<223>sTCR β chain
<400> 28
Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys Thr Arg Gly
1 5 10 15
Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His Asp Thr Val
20 25 30
Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe Ile Phe Gln
35 40 45
Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro Asp Arg Phe
50 55 60
Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn Val Asn Ala
65 70 75 80
Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Leu Ala
85 90 95
Gln Gly Asn Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser
100 105 110
Ile Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe
115 120 125
Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val
130 135 140
Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp
145 150 155 160
Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro
165 170 175
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser
180 185 190
Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe
195 200 205
Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
210 215 220
Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
225 230 235 240
Gly Arg Ala Asp
<210> 29
<211> 732
<212> DNA
<213> artificial sequence
<220>
<223>sTCR β chain
<400> 29
gatgcgggcg tgacccaaag tcccacacac ctgatcaaaa cgagaggaca gcaagtgact 60
ctgagatgct ctcctaagtc tgggcatgac actgtgtcct ggtaccaaca ggccctgggt 120
caggggcccc agtttatctt tcagtattat gaggaggaag agagacagag aggcaacttc 180
cctgatcgat tctcaggtca ccagttccct aactatagct ctgagctgaa tgtgaacgcc 240
ttgttgctgg gggactcggc cctctatctc tgtgccagca gcttggcaca gggtaacaat 300
cagccccagc attttggtga tgggactcga ctctccatcc tagaggacct gaaaaacgtg 360
ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420
gccacactgg tgtgcctggc caccggtttc taccccgacc acgtggagct gagctggtgg 480
gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540
cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600
tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660
gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720
ggtagagcag ac 732
<210> 30
<211> 251
<212> PRT
<213> artificial sequence
<220>
<223>single-stranded TCR
<400> 30
Gly Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu
1 5 10 15
Gly Ala Thr Val Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln
20 25 30
Ser Phe Phe Trp Tyr Arg Gln Tyr Pro Gly Lys Ser Pro Glu Leu Ile
35 40 45
Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala
50 55 60
Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Val
65 70 75 80
Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Arg Val Thr
85 90 95
Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Thr Val
100 105 110
Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu
115 120 125
Gly Gly Gly Ser Glu Gly Gly Thr Gly Asp Ala Gly Val Thr Gln Ser
130 135 140
Pro Thr His Leu Ser Lys Pro Glu Gly Ala Gln Val Thr Leu Arg Cys
145 150 155 160
Ser Pro Lys Ser Gly His Asp Thr Val Ser Trp Tyr Gln Gln Ala Pro
165 170 175
Gly Gln Gly Pro Gln Phe Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg
180 185 190
Gln Arg Gly Asn Phe Pro Asp Arg Phe Ser Gly His Gln Phe Pro Asn
195 200 205
Tyr Ser Ser Glu Leu Asn Ile Asn Ala Leu Ser Pro Gly Asp Ser Ala
210 215 220
Leu Tyr Leu Cys Ala Ser Ser Leu Ala Gln Gly Asn Asn Gln Pro Gln
225 230 235 240
His Phe Gly Asp Gly Thr Arg Leu Ser Ile Thr
245 250
<210> 31
<211> 753
<212> DNA
<213> artificial sequence
<220>
<223>single-stranded TCR
<400> 31
ggccagaaag aagtggaaca gaacagcggc ccgctgagcg tgccggaagg cgcgaccgtg 60
agcctgaact gcacctatag cgatcgcgtg agccagagct ttttttggta tcgccagtat 120
ccgggcaaaa gcccggaact gattatgagc atttatagca acggcgataa agaagatggc 180
cgctttaccg cgcagctgaa caaagcgagc cagtatgtga gcctgctgat tcgcgatgtg 240
cagccgagcg atagcgcgac ctatctgtgc gcggtgaacc gcgtgaccgg cggcggcaac 300
aaactgacct ttggcaccgg cacccagctg accgtggaag gcggcggcag cgaaggcggc 360
ggcagcgaag gcggcggcag cgaaggcggc ggcagcgaag gcggcaccgg cgatgcgggc 420
gtgacccaga gcccgaccca tctgagcaaa ccggaaggcg cgcaggtgac cctgcgctgc 480
agcccgaaaa gcggccatga taccgtgagc tggtatcagc aggcgccggg ccagggcccg 540
cagtttattt ttcagtatta tgaagaagaa gaacgccagc gcggcaactt tccggatcgc 600
tttagcggcc atcagtttcc gaactatagc agcgaactga acattaacgc gctgagcccg 660
ggcgatagcg cgctgtatct gtgcgcgagc agcctggcgc agggcaacaa ccagccgcag 720
cattttggcg atggcacccg cctgagcatt acc 753
<210> 32
<211> 113
<212> PRT
<213> artificial sequence
<220>
<223>single-stranded TCR α chain
<400> 32
Gly Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu
1 5 10 15
Gly Ala Thr Val Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln
20 25 30
Ser Phe Phe Trp Tyr Arg Gln Tyr Pro Gly Lys Ser Pro Glu Leu Ile
35 40 45
Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala
50 55 60
Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Val
65 70 75 80
Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Arg Val Thr
85 90 95
Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Thr Val
100 105 110
Glu
<210> 33
<211> 339
<212> DNA
<213> artificial sequence
<220>
<223>single-stranded TCR α chain
<400> 33
ggccagaaag aagtggaaca gaacagcggc ccgctgagcg tgccggaagg cgcgaccgtg 60
agcctgaact gcacctatag cgatcgcgtg agccagagct ttttttggta tcgccagtat 120
ccgggcaaaa gcccggaact gattatgagc atttatagca acggcgataa agaagatggc 180
cgctttaccg cgcagctgaa caaagcgagc cagtatgtga gcctgctgat tcgcgatgtg 240
cagccgagcg atagcgcgac ctatctgtgc gcggtgaacc gcgtgaccgg cggcggcaac 300
aaactgacct ttggcaccgg cacccagctg accgtggaa 339
<210> 34
<211> 114
<212> PRT
<213> artificial sequence
<220>
<223>single-stranded TCR β chain
<400> 34
Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ser Lys Pro Glu Gly
1 5 10 15
Ala Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His Asp Thr Val
20 25 30
Ser Trp Tyr Gln Gln Ala Pro Gly Gln Gly Pro Gln Phe Ile Phe Gln
35 40 45
Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro Asp Arg Phe
50 55 60
Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn Ile Asn Ala
65 70 75 80
Leu Ser Pro Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Leu Ala
85 90 95
Gln Gly Asn Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser
100 105 110
Ile Thr
<210> 35
<211> 342
<212> DNA
<213> artificial sequence
<220>
<223>single-stranded TCR β chain
<400> 35
gatgcgggcg tgacccagag cccgacccat ctgagcaaac cggaaggcgc gcaggtgacc 60
ctgcgctgca gcccgaaaag cggccatgat accgtgagct ggtatcagca ggcgccgggc 120
cagggcccgc agtttatttt tcagtattat gaagaagaag aacgccagcg cggcaacttt 180
ccggatcgct ttagcggcca tcagtttccg aactatagca gcgaactgaa cattaacgcg 240
ctgagcccgg gcgatagcgc gctgtatctg tgcgcgagca gcctggcgca gggcaacaac 300
cagccgcagc attttggcga tggcacccgc ctgagcatta cc 342
<210> 36
<211> 24
<212> PRT
<213> artificial sequence
<220>
<223>single-stranded TCR catenation 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
<220>
<223>single-stranded TCR catenation sequence
<400> 37
ggcggcggca gcgaaggcgg cggcagcgaa ggcggcggca gcgaaggcgg cggcagcgaa 60
ggcggcaccg gc 72

Claims (10)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be with LYVDSLFFL-HLAA2402 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 AVNRVTGGGNKLT (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domain Amino acid sequence is ASSLAQGNNQPQH (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-DRVSQS(SEQ ID NO:10)
αCDR2-IYSNGD(SEQ ID NO:11)
αCDR3-AVNRVTGGGNKLT(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-SGHDT(SEQ ID NO:13)
βCDR2-YYEEEE(SEQ ID NO:14)
βCDR3-ASSLAQGNNQPQH(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.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022063966A1 (en) * 2020-09-24 2022-03-31 Medigene Immunotherapies Gmbh Prame specific t-cell receptors and uses thereof
WO2024101994A1 (en) * 2022-11-11 2024-05-16 ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC) T cell receptors directed against cancer-associated antigens and uses thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1357006A (en) * 1999-06-18 2002-07-03 路德维哥癌症研究院 Mage-A1 peptides presented by HLA class II molecules
CN101160524A (en) * 2005-02-24 2008-04-09 西米尼斯公司 Compositions and methods for classifying biological samples
CN101668770A (en) * 2007-01-15 2010-03-10 葛兰素史密丝克莱恩生物有限公司 Vaccine
CN102575284A (en) * 2009-03-17 2012-07-11 MDx健康公司 Improved detection of gene expression
CN106188274A (en) * 2015-05-06 2016-12-07 广州市香雪制药股份有限公司 Identify the φt cell receptor of RHAMM antigen small peptide
CN106459178A (en) * 2015-09-09 2017-02-22 广州市香雪制药股份有限公司 T cell receptor having high affinity targeting rhamm antigen short chain polypeptide
CN106478809A (en) * 2015-11-06 2017-03-08 广州市香雪制药股份有限公司 The TCR of identification DAGE small peptide

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1357006A (en) * 1999-06-18 2002-07-03 路德维哥癌症研究院 Mage-A1 peptides presented by HLA class II molecules
CN101160524A (en) * 2005-02-24 2008-04-09 西米尼斯公司 Compositions and methods for classifying biological samples
CN101668770A (en) * 2007-01-15 2010-03-10 葛兰素史密丝克莱恩生物有限公司 Vaccine
CN102575284A (en) * 2009-03-17 2012-07-11 MDx健康公司 Improved detection of gene expression
CN106188274A (en) * 2015-05-06 2016-12-07 广州市香雪制药股份有限公司 Identify the φt cell receptor of RHAMM antigen small peptide
CN106459178A (en) * 2015-09-09 2017-02-22 广州市香雪制药股份有限公司 T cell receptor having high affinity targeting rhamm antigen short chain polypeptide
CN106478809A (en) * 2015-11-06 2017-03-08 广州市香雪制药股份有限公司 The TCR of identification DAGE small peptide

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AARON Y CHANG等: "A therapeutic T cell receptor mimic antibody targets tumor-associated PRAME peptide/HLA-I antigens", 《J CLIN INVEST.》 *
J H KESSLER等: "Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis", 《J EXP MED.》 *
XIAOMING SUN等: "Effects of a Single Escape Mutation on T Cell and HIV-1 Co-adaptation", 《CELL REPORTS》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022063966A1 (en) * 2020-09-24 2022-03-31 Medigene Immunotherapies Gmbh Prame specific t-cell receptors and uses thereof
WO2024101994A1 (en) * 2022-11-11 2024-05-16 ACADEMISCH ZIEKENHUIS LEIDEN (h.o.d.n. LUMC) T cell receptors directed against cancer-associated antigens and uses thereof

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