CN108659114A - Identify the TCR of PASD1 antigen small peptides - Google Patents

Identify the TCR of PASD1 antigen small peptides Download PDF

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CN108659114A
CN108659114A CN201710214669.4A CN201710214669A CN108659114A CN 108659114 A CN108659114 A CN 108659114A CN 201710214669 A CN201710214669 A CN 201710214669A CN 108659114 A CN108659114 A CN 108659114A
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CN108659114B (en
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李懿
陈安安
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Guangzhou Institute of Biomedicine and Health of CAS
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Abstract

The present invention relates to a kind of TCR of identification PASD1 antigen small peptides.The present invention provides a kind of T cell receptor (TCR) that can specifically bind the small peptide QLEERTWLL derived from PASD1 antigens, the antigen small peptide QLEERTWLL can form compound with HLA A0201 and be presented to cell surface together.The present invention also provides encode the nucleic acid molecules of the TCR and include the carrier of the nucleic acid molecules.In addition, the present invention also provides the cells for the TCR of the present invention that transduces.

Description

Identify the TCR of PASD1 antigen small peptides
Technical field
The present invention relates to that can identify the TCR from PASD1 antigen small peptides, the invention further relates to transduce above-mentioned TCR to obtain PASD1 specificity T cell and their purposes in preventing and treating PASD1 relevant diseases.
Background technology
PASD1 is degraded to micromolecule polypeptide, and and MHC as a kind of autochthonous tumor antigen after generating in the cell (main histocompatibility complex) molecule combines and forms compound, is presented to cell surface.Studies have shown that QLEERTWLL is Small peptide derived from PASD1.PASD1 antigens are common in diffusivity large B cell lymphoid tumor and Huppert's disease, and except testis (Joseph-Pietras D1, Gao Y, Zojer N, Ait-Tahar K, Banham are not expressed in outer most normal structures AH,Pulford K,Rice J,Savelyeva N,Sahota SS.Leukemia.2010 Nov；24(11):1951-9； Cooper CD,Liggins AP,Ait-Tahar K,Roncador G,Banham AH,Pulford K.PASD1, Leukemia 2006；20:2172–2174.).The side such as chemotherapy and radiation treatment may be used in treatment for above-mentioned disease Method, but the normal cell of itself can all be damaged.
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 TCR and the main histocompatbility of small peptide-of antigen small peptide specificity The combination of complex (pMHC compounds) causes T cell to be directly physically contacted with antigen presenting cell (APC), then T cell And other cell membrane surface molecules of both APC just interact, and cause a series of subsequent cell signals transmission 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 PASD1 antigen small peptides, so that it is played a role, or the TCR is transduceed T cell has PASD1 antigen small peptides specific T cell to obtain, to make them play work in cellular immunotherapy With.
Invention content
The purpose of the present invention is to provide a kind of T cell receptors of identification PASD1 antigen small peptides.
The first aspect of the present invention, provides a kind of T cell receptor (TCR), and the TCR can be with QLEERTWLL-HLA A0201 compounds combine.
In another preferred example, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chain variable domains CDR3 amino acid sequence be CAMRAIQGAQKLVF (SEQ ID NO.12)；And/or the CDR3 of the TCR β chain variable domains Amino acid sequence be CASSRGQGEAFF (SEQ ID NO.15).
In another preferred example, 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
αCDR1-NSAFQY(SEQ ID NO.10)
αCDR2-TYSSGN(SEQ ID NO.11)
αCDR3-CAMRAIQGAQKLVF(SEQ ID NO.12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
βCDR1-PRHDT(SEQ ID NO.13)
βCDR2-FYEKMQ(SEQ ID NO.14)
βCDR3-CASSRGQGEAFF(SEQ ID NO.15)。
In another preferred example, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chain variable domains To have the amino acid sequence of at least 90% sequence identity with SEQ ID NO.1；And/or the TCR β chain variable domains be with SEQ ID NO：5 amino acid sequences 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 heterodimers, 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 sequences of the TCR are SEQ ID NO：The β chain ammonia of the 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 by peptide catenation sequence with β chains variable domain by α chains variable domain.
In another preferred example, the TCR is in α chains 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 peptides amino acid inverses the 3rd, 5th reciprocal or 7th reciprocal Become；And/or the TCR is in β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th and/or β chains 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 TCR includes (a) all or part of TCR α chains in addition to transmembrane domain；And (b) all or part of TCR β chains 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 domains 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 1s；
The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；With
The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.
In another preferred example, the α chain amino acid sequences of the TCR are the β chains 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 chains variable region of the TCR and β chain constant regions.
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 1s；
The 47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1s；
The 46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s；Or
The 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s.
In another preferred example, the TCR is comprising α chains variable domain and β chains variable domain and in addition to transmembrane domain All or part of β chains constant domain, but it does not contain α chain constant domains, α chains variable domain and the β chains of the TCR form heterogeneous dimerization Body.
In another preferred example, the α chains of the TCR and/or the ends C- or N- of β chains are combined with conjugate.
In another preferred example, the conjugate combined with the T cell receptor is detectable marker, therapeutic agent, PK are repaiied 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 At least one of TCR molecules 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 or its complementary series of TCR molecules described in face.
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chain variable domains： 2。
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID of coding TCR β chain variable domains NO：6.
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chains：4 and/or Include the nucleotide sequence SEQ ID NO of coding TCR β chains：8.
The fourth aspect of the present invention, provides a kind of carrier, and the carrier contains the 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 host cell of separation, contains the present invention in the host cell The nucleic acid molecules described in the third aspect present invention of external source are integrated in carrier or genome described in fourth aspect.
The sixth aspect of the present invention provides a kind of cell, the 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 The TCR compounds described in TCR, second aspect of the present invention, third aspect present invention institute described in body and first aspect present invention Carrier described in the nucleic acid molecules stated, fourth aspect present invention or the cell described in sixth aspect present invention.
The eighth aspect of the present invention provides T cell receptor or second aspect of the present invention described in first aspect present invention Nucleic acid molecules described in the TCR compounds, 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 gives object application in need for the treatment of suitable The TCR compounds described in T cell receptor or second aspect of the present invention described in the first aspect present invention of amount, third of the present invention The carrier described in nucleic acid molecules, fourth aspect present invention described in aspect or the cell described in sixth aspect present invention or this hair Pharmaceutical composition described in bright 7th aspect；
Preferably, the disease is tumour, and the preferably described tumour includes diffusivity large B cell lymphoid tumor, multiple Myeloma, melanoma and other entity tumors such as gastric cancer, lung cancer, cancer of the esophagus, carcinoma of urinary bladder, head and neck squamous cell carcinoma, preceding Row gland cancer, breast cancer, colon cancer, oophoroma etc..
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively that TCR α chains variable domain amino acid sequence, TCR α chains are variable Domain nucleotide sequence, TCR α chain amino acid sequences, TCR α chains nucleotide sequence, the TCR α chain amino acid sequences with targeting sequencing And the TCR α chain nucleotide sequences with targeting sequencing.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively that TCR β chains variable domain amino acid sequence, TCR β chains are variable Domain nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, the TCR β chain amino acid sequences with targeting sequencing And the TCR β chain nucleotide sequences with targeting sequencing.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chains.
Fig. 5 a and Fig. 5 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chains.
Fig. 6 is the glue figure of the sTCR obtained after purification.Rightmost side swimming lane is to go back virgin rubber, and intermediate swimming lane is molecular weight It marks (marker), leftmost side swimming lane is non-reduced glue.
Fig. 7 is the ProteOn dynamics figures that sTCR of the present invention is combined with QLEERTWLL-HLA A0201 compounds Spectrum.
Fig. 8 is that T cell receptor slow virus packaging transfects testing result with primary T cells.
Fig. 9 is ELISPOT testing inspection results.
Figure 10 be transduce TCR of the present invention effector cell non-radioactive cell toxicity test testing result.
Specific implementation mode
The present inventor after extensive and in-depth study, has found and PASD1 antigen small peptide QLEERTWLL (SEQ ID NO.9) the TCR that can be specifically bound, the antigen small peptide QLEERTWLL can be formed with HLA A0201 compound and together by It is presented to cell surface.The present invention also provides encode the nucleic acid molecules of the TCR and include the carrier of 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 surfaces.The MHC of the mankind is commonly referred to as HLA genes or HLA complexs.
T cell receptor (TCR) is the unique of specific antigen peptide of the presentation in main histocompatibility complex (MHC) Receptor.In immune system, T cell is caused by the combination of the TCR and pMHC compounds 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 signals transmission 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 by α chains/β chains or γ chains/δ chains in the form of heterodimer existing for cell membrane surface glycoprotein. TCR heterodimers are made of α and β chains in 95% T cell, and 5% T cell has the TCR being made of γ and δ chains.It There is the heterogeneous dimerization TCR of right α β α chains and β chains, α chains and β chains to constitute the subunit of α β heterodimerics TCR.In a broad sense, α and β are each Chain includes variable region, bonding pad and constant region, and β chains usually contain short variable region also between variable region and bonding pad, but should Variable region is often regarded as a part for 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 compounds, wherein CDR3 by Variable region and bonding pad recombinate, and are referred to as hypervariable region.α the and β chains of TCR generally regard 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 domains 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 chains is " TRAC*01 ", TCR divides The constant domain sequence of sub- β chains is " TRBC1*01 " or " TRBC2*01 ".In addition, α the and β chains of TCR also include 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
In the membrane-proximal region C α of natural TCR, there are one group of disulfide bond with C β interchains, are known as " two sulphur of native interchain in of the invention 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 sequences 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 1s, for another example Be Q (glutamine) by the 61st amino acid of 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 1s, 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 are described it as in the present invention, other and so on.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 molecules
In antigen processing pathways, antigen is degraded in the cell, is then carried to cell surface by MHC molecule.T is thin Born of the same parents' receptor can identify the peptide-MHC compounds of Antigen Presenting Cell surface.Therefore, the first aspect of the present invention provides one kind It can be in conjunction with the TCR molecules of QLEERTWLL-HLA A0201 compounds.Preferably, the TCR molecules are separation or purifying 's.α the and β chains of the TCR respectively have 3 complementary determining regions (CDR).
One in the present invention is preferably carried out in mode, and the α chains of the TCR include with following amino acid sequence CDR：
αCDR1-NSAFQY(SEQ ID NO.10)
αCDR2-TYSSGN(SEQ ID NO.11)
αCDR3-CAMRAIQGAQKLVF(SEQ ID NO.12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
βCDR1-PRHDT(SEQ ID NO.13)
βCDR2-FYEKMQ(SEQ ID NO.14)
βCDR3-CASSRGQGEAFF(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 present invention, those skilled in the art are according to CDR region disclosed by the invention It can design or synthesize the TCR molecules with corresponding function.Therefore, TCR molecules of the present invention refer to comprising above-mentioned α and/or β The TCR molecules of chain CDR region sequence and any suitable frame structure.TCR α chain variable domains of the present invention are 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 chains of the present invention can Variable domain be and SEQ ID NO：5 have at least 90%, preferably 95%, the amino acid sequence of more preferably 98% sequence identity Row.
In the preference of the present invention, TCR molecules of the invention are the heterodimers being made of α and β chains.Specifically Ground, on the one hand the α chains of the heterogeneous dimerization TCR molecules include variable domain and constant domain, the α chains variable domain amino acid sequence packet CDR1 (SEQ ID NO containing above-mentioned α chains：10)、CDR2(SEQ ID NO：And CDR3 (SEQ ID NO.12) 11).Preferably, The TCR molecules include α chain variable domain amino acid sequence SEQ ID NO.1.It is highly preferred that the α chain variable domains of the TCR molecules Amino acid sequence is SEQ ID NO.1.On the other hand, the β chains of the heterogeneous dimerization TCR molecules include variable domain and constant domain, The β chains variable domain amino acid sequence includes CDR1 (SEQ ID NO.13), CDR2 (the SEQ ID NO of above-mentioned β chains：14) and CDR3(SEQ ID NO.15).Preferably, the TCR molecules include β chain variable domain amino acid sequence SEQ ID NO.5.It is more excellent The β chain variable domain amino acid sequences of selection of land, the TCR molecules are SEQ ID NO.5.
In the preference of the present invention, TCR molecules of the invention are the portions by some or all of α chains and/or β chains 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 Structure includes the single chain TCR molecules in the areas CDRs of the present invention.Specifically, the single chain TCR molecules include V α, V β and C β, preferably According to being linked in sequence from N-terminal to C-terminal.
The α chain variable domain amino acid sequences of the single chain TCR molecules include CDR1 (the SEQ ID NO of above-mentioned α chains：10)、 CDR2(SEQ ID NO：And CDR3 (SEQ ID NO 11):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 sequences of the single chain TCR molecules are SEQ ID NO.1.The β chain variable domain amino acid sequences of the single chain TCR molecules include CDR1 (the SEQ ID NO of above-mentioned β chains：13)、CDR2 (SEQ ID NO：And CDR3 (SEQ ID NO 14)：15).Preferably, the single chain TCR molecules include β chain variable domain amino acids Sequence SEQ ID NO.5.It is highly preferred that the β chain variable domain amino acid sequences of the single chain TCR molecules are SEQ ID NO.5.
In the preference of the present invention, the constant domain of TCR molecules 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 alphas chain of the present invention can be " TRAC*01 ", TCR points The constant domain sequence of sub- β chains can be " TRBC1*01 " or " TRBC2*01 ".The amino acid sequence provided in the TRAC*01 of IMGT The 53rd be Arg, be expressed as herein：The Arg53 of TRAC*01 exons 1s, 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 chains 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 molecules do 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 therapeutic agent (such as cytotoxin compounds or immunostimulating compound) being 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 PASD1 antigen small peptides TCR。
To obtain sTCR, on the one hand, TCR of the present invention can 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 domains 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 cysteine residues of the Thr48 of TRAC*01 exons 1s and the Ser57 of substitution TRBC1*01 or TRBC2*01 exons 1s To form disulfide bond.Cysteine residues are introduced to can also be with other sites for forming disulfide bond：TRAC*01 exons 1s The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1s；The Tyr10 and TRBC1*01 of TRAC*01 exons 1s or The Ser17 of TRBC2*01 exons 1s；Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s Asp59；The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 exons 1s Arg53 and TRBC1*01 or TRBC2*01 exons 1s Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1s or The Ala19 of TRBC2*01 exons 1s；Or Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s Glu20.I.e. cysteine residues are instead of any group of site in above-mentioned α and β chain constant domains.It can be in TCR constant domains 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 so that its not including cysteine residues come achieve the purpose that lack natural disulphide bonds, also can be by the way that day will be formed The cysteine residues of right disulfide bond sport another amino acid to reach above-mentioned purpose.
As described above, the TCR of the present 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, it is constant that TCR of the invention can contain TRAC Domain sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences 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 chains) variable region amino acid the 11st, 13,19,21,53,76,89,91,94 and/or α chain J genes (TRAJ) small peptide Amino acid position the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptides 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).People 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 domains.
In addition, for stability, patent document PCT/CN2016/077680 is also disclosed in the α chains variable region of TCR Introducing artificial interchain disulfide bond between β chain constant regions 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 chains variable region and β chain constant regions of power TCR.Specifically, in the α of the TCR Formed between chain variable region and β chain constant regions the cysteine residues of artificial interchain disulfide bond instead of：The 46th ammonia of TRAV 60th amino acids of base acid and TRBC1*01 or TRBC2*01 exons 1s；The 47th amino acids and TRBC1*01 of TRAV or 61 amino acids of TRBC2*01 exons 1s；The 46th amino acids of TRAV and the of TRBC1*01 or TRBC2*01 exons 1s 61 amino acids；Or the 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s.It is preferred that Ground, such TCR can include all or part of TCR α chains of (I) in addition to its transmembrane domain, and (II) removes its cross-film knot All or part of TCR β chains other than structure domain, wherein (I) and (II) variable domain comprising TCR chains and at least part is constant Domain, α chains form heterodimer with β chains.It is highly preferred that such TCR can include α chains variable domain and β chains variable domain and All or part of β chains constant domain in addition to transmembrane domain, but it does not contain α chain constant domains, the α chain variable domains of the TCR Heterodimer is formed with β chains.
The present invention TCR can also multivalence complex form provide.The present invention multivalent TCR complex 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 tetramer or multiple TCR of the present invention and another molecule in conjunction with and the compound that is formed.The TCR compounds of the present 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 it is multiple to generate other multivalence TCR with such application Close the intermediate of object.
The TCR of the present invention can be used alone, and can also be combined with covalent or other modes with conjugate, 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 QLEERTWLL-HLA A0201 compounds), therapeutic agent, PK (protein kinase) modified parts or it is any more than The combination of these substances combines or coupling.
Detectable marker for diagnostic purposes includes but 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.
The therapeutic agent that can be combined or be coupled with TCR of the present invention includes but not limited to：1. radionuclide (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 segments (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, it is beautiful Chemical Society of 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- diaphorases (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
In addition, the TCR of the present invention can also be comprising derived from the heterozygosis TCR more than a kind of species sequence.For example, grinding Study carefully display Muridae TCR can more effectively to express than people TCR in human T-cell.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 adoptive T cell treatment 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 correspondence 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 molecules or part thereof, institute It can be one or more CDR, the variable domain and α chains and/or β chains of α and/or β chains to state part.
The nucleotide sequence for encoding first aspect present invention TCR molecule alpha chain CDR regions is as follows：
αCDR1-aacagtgcttttcaatac(SEQ ID NO.16)
αCDR2-acatactccagtggtaac(SEQ ID NO.17)
αCDR3-tgtgcaatgagggcaattcagggagcccagaagctggtattt(SEQ ID NO.18)
The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR regions is as follows：
βCDR1-cctagacacgacact(SEQ ID NO.19)
βCDR2-ttttatgaaaagatgcag(SEQ ID NO.20)
βCDR3-tgtgccagcagccgcggacagggtgaagctttcttt(SEQ ID NO.21)
Therefore, the nucleotide sequence for encoding the nucleic acid molecules of the present invention of TCR α chains of the present invention includes SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, and/or the nucleotide sequence of the nucleic acid molecules of the present invention of coding TCR β chains 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 can include or do 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 domains of the present invention The nucleotide sequence for including SEQ ID NO.2 and/or the nucleic acid molecules of the present invention of coding TCR β chain variable domains of the present invention includes SEQ ID NO.6..It is highly preferred that the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO.4 and/or SEQ ID NO.8..
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 nucleic acid sequence shown in attached drawing of the present invention or degeneracy.With Example illustrates one of in the present invention, and " variant of degeneracy " refers to protein sequence of the coding with SEQ ID NO.1, But with the differentiated nucleic acid sequence of sequence of 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 according to the type of cell to increase expression quantity.Mammalian cell and various other The codon usage table of biology is well known to those skilled in the art.
The present invention nucleic acid molecules full length sequence or its segment 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 completely by chemical synthesis come obtain encoding 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
The invention further relates to the carrier for the nucleic acid molecules for including the present invention, including expression vector can in vivo or body The construct of outer expression.Common carrier includes bacterial plasmid, bacteriophage and animals and plants virus.
Viral delivery systems include but not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, Retroviral vector, slow virus carrier, baculovirus vector.
Preferably, the nucleotide of the present invention can be transferred in cell by carrier, such as in T cell so that the cell table Up to the TCR of PASD1 antigentic specificities.Ideally, which can should express to continual high levels in T cell.
Cell
The invention further relates to the carriers or the genetically engineered host cell of coded sequence with the present invention.The host The nucleic acid molecules of the present invention are integrated in carrier or chromosome containing the present invention in cell.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 cell of the separation of the TCR of the expression present invention, especially T cell.The T cell can spread out It is born from the T cell detached from subject, or can be the mixed cellularity group detached from subject, such as periphery hemolymph is thin The 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 use antibody (e.g., the antibody of anti-CD3 or anti-CD28) to activate, to allow them to more easily receive to turn Dye, such as transfected with the carrier comprising the nucleotide sequence for encoding TCR molecules of the present invention.
Alternatively, cell of the invention can also be or be derived from stem cell, such as candidate stem cell (HSC).Gene is turned Moving to HSC will not cause to express TCR in cell surface, because stem cell surface does not express CD3 molecules.However, when stem cell point It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecules will start in thymocyte The surface expression introducing TCR molecules.
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 of expression 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).
PASD1 antigen-related diseases
The invention further relates to the method with PASD1 relevant diseases is treated and/or prevented in subject comprising adoptive Shift the step of PASD1 specific T-cells are to the subject.The PASD1 specific T-cells can recognize that QLEERTWLL-HLA A0201 compounds.
The T cell of the PASD1 specificity of the present invention can be used for treating any presentation PASD1 antigen small peptides QLEERTWLL- The PASD1 relevant diseases of HLA A0201 compounds.Including but not limited to tumour, such as melanoma and other entity tumors are such as Gastric cancer, lung cancer, cancer of the esophagus, carcinoma of urinary bladder, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, oophoroma etc..
Therapy
Can by separation with the patient of PASD1 antigen-related diseases or the T cell of volunteer, and by the present 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 methods for treating PASD1 relevant diseases, include the T cell for the expression TCR of the present invention that will be detached, preferably Ground, the T cell derive from patient itself, are input in patient body.Usually, include the T cell of (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 molecules 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 be combined with PASD1 antigen small peptide compound QLEERTWLL-HLA A0201, be turned simultaneously 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 houses) 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. Experiment material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
1 clone-specific T cell of embodiment
Coming from genotype using synthesis small peptide QLEERTWLL (the Nanjing bio tech ltd Jin Sikang) stimulations is The peripheral blood lymphocytes (PBL) of the healthy volunteer of HLA-A02.By QLEERTWLL small peptides and carry biotin labeling HLA-A*0201 renaturation prepares the mono- aggressiveness of pHLA.These single aggressiveness are combined into the Streptavidin (BD companies) marked with PE The tetramer of PE labels, sorts the tetramer and anti-CD8-APC double positive cells.The cell of sorting is expanded, and as stated above Secondary sorting is carried out, then carries out Colony Culture with limiting dilution assay.Monoclonal cell tetramer staining, what is screened is double Positive colony is as shown in Figure 3.
Embodiment 2 obtains the structure of the tcr gene and carrier of PASD1 specific T-cell clones
Use Quick-RNATMThe QLEERTWLL screened in MiniPrep (ZYMO research) extracting embodiments 1 is special Property, HLA-A02 restrictive T cell clones total serum IgE.The synthesis of cDNA is expanded using the SMART RACE cDNA of clontech Increase kit, the primer of use is designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned into carrier T (TAKARA) On be sequenced.Through sequencing, the α chains and β chain-ordering structures difference of the TCR of double positive colony expression is as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c and Fig. 1 d are respectively TCR α chains variable domain amino acid sequence, TCR α chain variable domains nucleotide sequence, TCR α Chain amino acid sequence and TCR α chain nucleotide sequences；Fig. 2 a, Fig. 2 b, Fig. 2 c and Fig. 2 d are respectively TCR β chain variable domain amino acid sequences Row, TCR β chain variable domains nucleotide sequence, TCR β chain amino acid sequences and TCR β chain nucleotide sequences.
Identified, α chains include the CDR with following amino acid sequence：
αCDR1-NSAFQY(SEQ ID NO.10)
αCDR2-TYSSGN(SEQ ID NO.11)
αCDR3-CAMRAIQGAQKLVF(SEQ ID NO.12)
β chains include the CDR with following amino acid sequence：
βCDR1-PRHDT(SEQ ID NO.13)
βCDR2-FYEKMQ(SEQ ID NO.14)
βCDR3-CASSRGQGEAFF(SEQ ID NO.15)。
By overlapping (overlap) PCR respectively by the variable domain of TCR α chains and β chains respectively with mouse TCR α chains and β chains Conservative domain is spliced into full-length gene and is connected to Lentiviral pLenti (addgene).Specially：Use overlap The full-length gene of TCR α chains and TCR β chains is attached to obtain TCR α -2A-TCR β segments by PCR.By Lentiviral and TCR α -2A-TCR β digestions connect to obtain pLenti-PASD1TRA-2A-TRB-IRES-NGFR plasmids.It uses as a contrast, simultaneously Also the slow virus carrier pLenti-eGFP of structure expression eGFP.Later pseudovirus is packed with 293T/17 again.
Expression, refolding and the purifying of the solvable TCR of embodiment 3PASD1 antigens small peptide specificity
To obtain soluble TCR molecules, α the and β chains of TCR molecules of the invention can include only its variable domain and portion respectively Point constant domain, and a cysteine residues are introduced in the constant domain of α and β chains respectively to form artificial interchain disulfide bond, The position for introducing cysteine residues is respectively the Ser57 of the Thr48 and TRBC2*01 exons 1s of TRAC*01 exons 1s；Its α The amino acid sequence of chain is distinguished as shown in figures 4 a and 4b with nucleotide sequence, the amino acid sequence and nucleotide sequence of β chains Respectively as shown in figure 5 a and 5b, the cysteine residues of introducing with overstriking and underline alphabetical indicate.Pass through《Molecular cloning Laboratory manual》(Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) Described in standard method the objective gene sequence of above-mentioned TCR α and β chains are inserted respectively into expression vector pET28a after synthesis The cloning site of+(Novagene), upstream and downstream are NcoI and NotI respectively.Insert Fragment confirms errorless by sequencing.
The expression vector of TCR α and β chains is converted by chemical transformation respectively and enters expression bacterium BL21 (DE3), bacterium It is grown with LB culture solutions, in OD600It is induced with final concentration 0.5mM IPTG when=0.6, the packet formed after α the and β chains 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 hydrochlorides, 10mM dithiothreitol (DTT)s (DTT), 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris (pH 8.1) in.
Dissolved TCR α and β chains are with 1：1 mass ratio is quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (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, deionized water is changed into buffer solution (20mM after 12 hours Tris, pH 8.0) continue at 4 DEG C dialyse 12 hours.Solution after the completion of dialysis after 0.45 μM of membrane filtration, by the moon from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains the successful α and β dimers of renaturation TCR is confirmed by SDS-PAGE glue.TCR then by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) it is further purified.TCR purity after purification is measured by SDS-PAGE is more than 90%, and concentration is by BCA methods It determines.The SDS-PAGE glue figures for the sTCR that the present invention obtains are as shown in Figure 6.
Embodiment 4 combines characterization
ProteOn is analyzed
This example demonstrated soluble TCR molecules of the present invention can be special with QLEERTWLL-HLA A0201 compounds The opposite sex combines.
The TCR molecules and QLEERTWLL- obtained in embodiment 3 is detected using ProteOn XPR36 real-time analyzers The combination activity of HLA A0201 compounds.The respective channel of CM5 chips is activated using EDC/NHS, by 50 μ g/ml strepto-s Avidin (being dissolved in 10mM acetate buffer solutions (pH 4.5)) is fixed in the respective channel of CM5 chips.It is molten to inject 1M ethanol amines Liquid (pH 8.0) closes unreacted activating surface, completes coupling process.The coupling amount of Streptavidin is generally higher than 1000RU. TCR flows through the chip for being coated with pMHC, binding time 30-60s, Dissociation time 30-60s with several various concentrations.
The preparation process of above-mentioned QLEERTWLL-HLA A0201 compounds is as follows：
A. it purifies
The E.coli bacterium solutions for collecting 100ml induced expressions heavy chain or light chain use 10ml after 4 DEG C of 8000g centrifuge 10min PBS washing thallines are primary, use 5ml BugBuster Master Mix Extraction Reagents (Merck) violent later Thalline is resuspended in concussion, and is rotated in room temperature and be incubated 20min, and later in 4 DEG C, 6000g centrifuges 15min, discards supernatant, collection is forgiven Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Add 30ml The BugBuster of 10 times of dilution, mixing, 4 DEG C of 6000g centrifuge 15min；It discards supernatant, 30ml is added to dilute 10 times of BugBuster Inclusion body, mixing is resuspended, 4 DEG C of 6000g centrifuge 15min, are repeated twice, and add 30ml 20mM Tris-HCl pH 8.0 that packet is resuspended Contain body, mixing, 4 DEG C of 6000g centrifuge 15min, and 20mM Tris-HCl 8M urea is finally used to dissolve inclusion body, SDS-PAGE detections Inclusion body purity, BCA kits survey concentration.
B. renaturation
The small peptide QLEERTWLL (the Nanjing bio tech ltd Jin Sikang) 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 hydrochlorides, 10mM sodium acetates, 10mM EDTA are further denaturalized.Renaturation is added with 25mg/L (final concentration) in QLEERTWLL peptides Buffer solution (0.4M L-arginines, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced forms 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, 8h/ times), renaturation carries out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detections 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 replaces buffer solution and come twice Fully reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Instrument (the general electricity of GE is purified using Akta Gas company), the 0-400mM NaCl linear gradient liquid that 20mM Tris pH 8.0 are prepared elutes albumen, and pMHC is about in 250mM It is eluted at NaCl, collects all peak components, SDS-PAGE detects purity.
D. biotinylation
With Millipore super filter tubes by the pMHC molecular concentrations 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- are 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 concentrations after being marked biotinylation with Millipore super filter tubes are to 1ml, using gel permeation chromatography The pMHC of purifying biological element pre-equilibrates HiPrep using Akta purifying instrument (GE General Electric Co. Limited) with filtered PBSTM Then 16/60S200HR columns (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated load 1ml use PBS with 1ml/ Min flow velocitys elute.Biotinylated pMHC molecules occur in about 55ml as unimodal elution.Merge the group containing protein Point, it is concentrated with Millipore super filter tubes, BCA methods (Thermo) measure protein concentration, and protease inhibitors cocktail is added (Roche) packing of biotinylated pMHC molecules is stored in -80 DEG C.
Computational dynamics parameter obtains the TCR molecules and QLEERTWLL-HLA A0201 compound knots of solubility of the invention The kinetic profile difference of conjunction is as shown in Figure 7.Collection of illustrative plates shows, the soluble TCR molecules that the present invention obtains can be with QLEERTWLL-HLA A0201 compounds combine.Meanwhile the TCR molecules of solubility of the invention are also had detected using the above method With the combination activity of other several irrelevant antigen small peptides and HLA compounds, as a result show that TCR molecules of the present invention are unrelated with other anti- Original is without combination.
Embodiment 5T cell receptor slow virus is packed to be transfected with primary T cells
(a) (Express-In-mediated transient are transiently transfected by the quick mediation of 293T/17 cells Transfection slow virus) is prepared
Utilize slow virus of the third generation slow virus packaging system packaging containing the gene of TCR needed for coding.It is situated between using quick Lead transient transfection (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)) with 4 kinds of plasmids, (one kind containing pLenti-PASD1TRA-2A-TRB-IRES-NGFR described in embodiment 2 is slow Viral vectors, and 3 kinds of plasmids containing other components necessary to structure infectiousness but non-replicating lentiviral particle) transfection 293T/17 cells.
To be transfected, the 0th day kind cell, in 15 cm dishes, kind upper 1.7 × 107A 293T/17 cells make thin Born of the same parents are evenly distributed on culture dish, and degree of converging is slightly above 50%.1st day transfected plasmids pack pLenti-PASD1TRA-2A- TRB-IRES-NGFR and pLenti-eGFP pseudovirus, by the above expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV It is as follows with the dosage of pMD.2G mixings, a 15 cm diameter plates：22.5 micrograms：15 micrograms:15 micrograms：7.5 microgram.Transfection The ratio of reagent PEI-MAX and plasmid is 2:1, the usage amount of each plate is 114.75 micrograms.Concrete operations are：Expression matter 1800 microlitres of OPTI-MEM are added (in (Ji Bu can company (Gibco), catalog number (Cat.No.) 31985-070) culture medium in grain and packaging plasmid It is uniformly mixed, being stored at room temperature 5 minutes becomes DNA mixed liquors；Corresponding amount PEI is taken to be mixed with 1800 microlitres of OPTI-MEM culture mediums Even, being stored at room temperature 5 minutes becomes PEI mixed liquors.DNA mixed liquors and PEI mixed liquors are mixed and are being stored at room temperature 30 Minute, then 3150 microlitres of OPTI-MEM culture mediums are added, it is added to has been converted to 11.25 milliliters of OPTI-MEM after mixing 293T/17 cells in, gently shake culture dish, make culture medium be uniformly mixed, 37 DEG C/5%CO2Lower culture.It is small to transfect 5-7 When, remove transfection media, change into containing 10% fetal calf serum DMEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt)) complete medium, 37 DEG C/5%CO2Lower culture.Collect the training containing wrapped slow virus within 3rd and the 4th day Support base supernatant.For the slow virus of harvest packaging, collected culture supernatant 3000g is centrifuged 15 minutes removal cell fragments, It filters through 0.22 micron filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB), finally uses again The concentration tube (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) UFC905096) of 50KD interceptions is concentrated, and is removed Most of supernatant is finally concentrated to 1 milliliter, is frozen for -80 DEG C after equal portions packing.Pseudovirus sample is taken to carry out virus titer survey Fixed, step is with reference to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit specification.It uses as a contrast, while also packet turns The pseudovirus of pLenti-eGFP.
(b) with the lentiviruses transduction primary T cells containing T cell receptor gene of the present invention
It is separated to CD8 from the blood of healthy volunteer+T cell, then with the lentiviruses transduction packed.It is thin to count these Born of the same parents, in 48 orifice plates, containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10%FBS (Ji Bu can company (Gibco), Catalog number (Cat.No.) C10010500BT) 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture mediums in 1 × 106 A cells/ml (0.5 milliliter/hole) and pre-wash AntiCD3 McAb/CD28 antibody-coating globule (T cell amplified matter, Lifetechnologies, catalog number (Cat.No.)11452D) be incubated overnight stimulation altogether, cell：Pearl=3：1.
After stimulation overnight, according to the virus titer that p24ELISA kits are measured, it is added in the ratio of MOI=10 dense The slow virus of the PASD1 specific t-cell receptor genes of contracting, 32 DEG C, 900g centrifugations infection 1 hour.It is removed after infection slow Cell is resuspended with 1640 culture mediums containing 10%FBS that 50IU/ml IL-2 and 10ng/ml IL-7 is added in virus infection liquid, and 37 DEG C/5%CO2Lower culture 3 days.Transduction counts cell, diluting cells to 0.5 × 10 after 3 days6A cells/ml.It counts within every two days The fresh culture containing 50IU/ml IL-2 and 10ng/ml IL-7 is replaced or be added to cell, maintain cell 0.5 × 106-1×106A cells/ml.Flow cytometry cell was begun through from the 3rd day, is tried for function since the 5th day It tests (for example, ELISPOT and non-radioactive cell toxicity detection of IFN-γ release).Slow down point since the 10th day or in cell It splits when becoming smaller with size, stored frozen etc. divides cell, at least 4 × 106A cell/pipe (1 × 107A cells/ml, 90% FBS/10%DMSO).
(c) primary T cells of tetramer staining TCR transductions
PASD1PX248 383-391QLEERTWLL small peptides are prepared with the HLA-A*0201 renaturation of biotin labeling The mono- aggressiveness of pHLA.The Streptavidin (BD) that these single aggressiveness are marked with PE is combined into the tetramer of PE labels, referred to as PX248- tetramer-PE.The T cell for expressing T cell receptor gene of the present invention can be labeled as positive cell by this tetramer.(b) In transduced T cell sample mix and be incubated on ice 30 minutes with PX248-tetramer-PE, anti-mouse β chains-are then added APC antibody continues to be incubated 15 minutes on ice.Sample clean 2 times with the PBS containing 2%FBS after with Millipore guava or BD Arial detections or sorting express the PX248-tetramer-PE and anti-mouse β of PASD1 specific t-cell receptor genes The double positive T cells of chain-APC, data analysis use 3.1 softwares of guavaSoft ((Merck Mi Libo (Merck Millipore)) or FlowJo softwares (Tree Star Inc, Ashland, OR) are analyzed.
It analyzes after testing, the results are shown in Figure 8, is dyed with PX248-tetramer-PE and anti-mouse β chain-APC antibody Afterwards, the blank control group T cell without TCR slow-virus infections is double positive without PX248-tetramer-PE and anti-mouse β chains-APC Cell, and the PX248-tetramer-PE and anti-mouse β chains-APC that occur expression TCR through the T cell of TCR slow-virus infections are double Positive cell, only a small amount of nonspecific double sun when being dyed with other tetramer-PE of non-PX248-tetramer-PE Property cell.
The ELISPOT functional verifications of 6 TCR of the present invention of embodiment
ELISPOT schemes
Following tests is carried out to prove the activation that target cell specificity reacts of T cell of TCR- transductions.It utilizes Readout of the IFN-γ yield of ELISPOT testing inspections as t cell activation.
Reagent
Test medium：10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), 1640 (Ji Bu of RPMI Can company (Gibco), catalog number (Cat.No.) C11875500bt)
Washing buffer：0.01M PBS/0.05% polysorbas20s
PBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT)
96 orifice plates of PVDF ELISPOT (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) MSIPS4510)
People's IFN-γ ELISPOT PVDF- enzyme reagent kits (BD) (capture equipped with required every other reagent and detection are anti- Body, Streptavidin-alkaline phosphatase and BCIP/NBT solution)
Method
It is prepared by target cell
The target cell of the present embodiment is the B-lymphoblastoid cell lines of Epstein-Barr viral (EBV) conversion (LCLs).B95-8 cells are induced through myristoyl acetic acid phorbol ester (TPA) produces the culture medium supernatant containing EBV, 4 DEG C/600g 10 minutes removal impurity of centrifugation, then crosses 0.22 micron filter, and decile dispenses -70 DEG C of preservations.It is HLA-A11/ from genotype The peripheral blood lymphocytes (PBL) of the healthy volunteer of A02/A24 (including homozygote and heterozygote), takes 10 milliliters a concentration of 2 ×107The PBL suspension of/milliliter is added after cyclosporin in 25 square centimeters of culture bottle in 37 DEG C/CO2It is incubated in incubator It educates 1 hour, quick-thawing portion EBV, is added in above-mentioned cell by 1/10 dilution, gently shakes up and culture bottle is uprightly placed in 37℃/CO2It is cultivated in incubator.10 milliliters of culture mediums of addition continue to cultivate after culture 12 days, further expand training after about 30 days It supports and carries out flow cytometer detection, wherein CD19+CD23hiCD58+For B-lymphoblastoid cell lines (LCLs).This ELISPOT is tested Using HLA-A02 as specificity target cell.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is to express PASD1 specificity TCRs through flow cytometry in embodiment 3 CD8+T cell, and with the CD8 of same volunteer+T is as negative control effector cell.It is coated with pearl (T cell with AntiCD3 McAb/CD28 Amplified matter, LifeTechnologies) stimulation T cell, with lentiviruses transduction (the foundation embodiment for carrying tcr gene of the present invention 5), the 9-12 after transduction is expanded in 1640 culture mediums containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 These cells, are then placed in test medium by it, and 300g room temperature is centrifuged 10 minutes and washed.Then by cell with 2 × Required final concentration is resuspended in test medium.Same processing negative control effector cell.
ELISPOT
According to the specification that manufacturer provides, prepare orifice plate as described below：1 is pressed with 10 milliliters of sterile PBS of every block of plate：200 It dilutes anti-human IFN-γ and captures antibody, 100 microlitres of dilution, which is then captured antibody etc. point, is added each hole.It is incubated orifice plate at 4 DEG C Overnight.After incubation, washing orifice plate is to remove extra capture antibody.The RPMI 1640 that 10%FBS is contained in 100 microlitres/hole is added Culture medium simultaneously incubates orifice plate 2 hours to close orifice plate at room temperature.Then culture medium is washed away from orifice plate, by being flicked on paper With ELISPOT orifice plates are patted to remove the washing buffer of any remnants.
QL6CD8+T cell (T cell) (T cell of TCR transductions of the present invention, effector cell), CD8+T cells are (negative right According to effector cell) and LCL A02/LCL A24 (target cell) be added pair according to preparing described in embodiment 3, and in corresponding experimental group It is PASD1PX248 383-391QLEERTWLL small peptides to answer small peptide, wherein PX248, and PA02-1, PA24-1 are non-present invention TCR Specific bond small peptide.
Then all components of experiment are added by ELISPOT orifice plates using following sequence：
130 microlitres of target cells, 77000 cells/mls (obtain about 10000 target cell/holes in total).
50 microlitres of effector cells's (1000 bis- positive T cells of PASD1TCR).
20 microlitre 10-4The PASD1PX248 383-391QLEERTWLL/ non-specificity small peptide solution (final concentrations of mol/L It is 10-5Mol/L).
All holes prepare addition in triplicate.
Then (37 DEG C/5%CO overnight of orifice plate is incubated2) second day, culture medium is abandoned, washs orifice plate 2 times with distilled water, then use Washing buffer is washed 3 times, is patted on paper handkerchief to remove remaining washing buffer.Then dilute with the PBS containing 10%FBS Detection primary antibody is released, each hole is added by 100 microlitres/hole.Orifice plate is incubated at room temperature 2 hours, then washed 3 times with washing buffer, Orifice plate is patted on paper handkerchief to remove excessive washing buffer.
1 is pressed with the PBS containing 10%FBS：100 dilution Streptavidin-alkaline phosphatases, by 100 microlitres of diluted chains Mould Avidin-alkaline phosphatase is added each hole and incubates orifice plate at room temperature 1 hour.Then 3 PBS are washed with washing buffer Washing 2 times, pats orifice plate to remove excessive washing buffer and PBS on paper handkerchief.Kit is added after washing to provide 100 microlitres/hole of BCIP/NBT solution develop.It is protected from light with masking foil covering orifice plate during development, 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 PASD1 TCR to transduce is tested to loading PASD1PX248 by ELISPOT The IFN-γ release that the target cell of 383-391 QLEERTWLL small peptides and the target cell of non-specific small peptide react.It utilizes Graphpad prism6 draw the ELSPOT amount of speckle observed in each hole.
Experimental result is as shown in figure 9, PASD1 CD8+T cell (QL6 CD8+T Cell, effector cell) it is individually thin with LCL It is seldom that IFN-γ is discharged when born of the same parents (target cell).
PASD1 CD8+T cell (QL6 CD8+T Cell, effector cell) it can be risen instead with the LCLA02 cells of addition PX248 More IFN-γ should be released.
PASD1 CD8+T cell (QL6 CD8+T Cell, effector cell) the non-specific small peptide of addition LCL cells when IFN-γ release is seldom.
CD8+T cell (CD8+T Cell, negative control effector cell) addition PX248 LCLA02 cells when IFN-γ release It puts seldom.
The non-radioactive cell toxicity test of 7 TCR of the present invention of embodiment
The experiment is the colorimetric alternate test of 51Cr release cell toxicity tests, quantitative determines the breast discharged after cell cracking Acidohydrogenase (LDH).The LDH of release in the medium is detected using the enzyme reaction of coupling in 30 minutes, LDH can in enzyme reaction A kind of tetrazolium salts (INT) are made to be converted into red formazans (formazan).The amount of the red product of generation and the cell number of cracking It is directly proportional.Collection 490nm visible light extinction Value Datas can be collected with 96 hole read plates of standard.
Material
CytoToxNon-radioactive cell toxicity detection (Pu Luomaige companies, G1780) contains substrate mixture, experiment Buffer solution, cracked solution and stop buffer.
Test medium：10%FBS (it is heat-inactivated, Ji Bu can company, catalog number (Cat.No.) 16000-044), without phenol red 95%RPMI 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) 11835-030).
Micropore round bottom tissue culturing plates with 96 hole (Nucor Corporation (Nunc), catalog number (Cat.No.) 163320)
96 hole immuno plate Maxisorb (Nucor Corporation (Nunc), catalog number (Cat.No.) 442404)
Method
It is prepared by target cell
This experiment is target cell using tri- plants of tumor cell lines of U266B1, NCI-H1299 and A375.In test medium Prepare target cell：Target cell concentration is adjusted to 334/milliliter, and 45 microlitres are taken per hole to obtain 1.5 × 104A cells/well.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is to express PASD1 specificity TCRs through flow cytometry in embodiment 3 CD8+T cell.Effector cell uses 10 with target cell ratio：1.、5:1、2.5:1 and 1.25:1.If homologous CD8+T cell adds Target cell control group (10:1).
The CD8 of PASD1 specificity TCRs transduction+T cell specific killing test neoplastic cell
Experiment prepares
All components of experiment are added by micropore round bottom tissue culturing plates with 96 hole using following sequence：
Each hole is added in -45ul target cells (preparing as described above)
Each hole is added in -45ul effector cells (preparing as described above)
Prepare control group as described below：
The spontaneous release of effector cell：Only 45ul effector cell.
The spontaneous release of target cell：Only 45ul target cells.
Target cell maximum discharges：Only 45ul target cells.
Culture medium compares：Only 90ul culture mediums.
All holes prepare in triplicate, and final volume is 90ul (inadequate is supplied with culture medium).
37 DEG C incubate 24 hours.Before collecting all hole supernatants, target cell maximum release control wells are placed into cell at -70 DEG C About 30 minutes, then melt 15 minutes at 37 DEG C, so that target cell all cracks.
Tablet is centrifuged in 250g 4 minutes.The 50ul supernatants in each hole of test panel are transferred to 96 hole immuno plates The corresponding aperture of Maxisorb plates.Using experiment buffer solution (12ml) reconstituted substrate mixture, then add 50ul to each hole of tablet.It is flat Plate close the lid after in shady place incubation at room temperature 30 minutes.Each hole of tablet is added to terminate reaction in 50ul stop baths.It is added The absorbance of the record of inside counting in 1 hour 490nm after stop bath.
Result of calculation
From experimental group, the spontaneous release group of target cell and effector cell culture medium is deducted from all absorbance values of release group The absorbance value of background.
It brings the corrected value obtained among the above into following formula, calculates each effect target than generated cytotoxicity Percentage.
Cytotoxicity=100 % × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximum-target cell from Hair)
As a result
It is thin to specific target by the non-radioactive cell toxicity detection T cell (as described above) for examining PASD1TCR transductions The LDH releases that born of the same parents react.490nm visible light light absorption values in each hole are drawn using graphpad prism6.
Experimental data statistical result is as shown in Figure 10, as effect target ratio increases, the T cell of PASD1TCR transductions (QL6CD8+T Cell) lethal effect of specificity target cell U266B1 is enhanced；To non-specific target cell NCI-H1299 and A375 lethal effects are very weak.Homologous CD8+T(CD8+T Cell) cell to target cell U266B1 killing rates be significantly lower than PASD1TCR The T cell group of transduction.
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 be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>Identify the TCR of PASD1 antigen small peptides
<130> P2017-0445
<160> 29
<170> PatentIn version 3.5
<210> 1
<211> 112
<212> PRT
<213>Artificial sequence
<220>
<223>TCR α chain variable domains
<400> 1
Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr
20 25 30
Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met
35 40 45
Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Met Arg Ala Ile Gln Gly
85 90 95
Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg Leu Thr Ile Asn Pro
100 105 110
<210> 2
<211> 336
<212> DNA
<213>Artificial sequence
<220>
<223>TCR α chain variable domains
<400> 2
cagaaggagg tggagcagga tcctggacca ctcagtgttc cagagggagc cattgtttct 60
ctcaactgca cttacagcaa cagtgctttt caatacttca tgtggtacag acagtattcc 120
agaaaaggcc ctgagttgct gatgtacaca tactccagtg gtaacaaaga agatggaagg 180
tttacagcac aggtcgataa atccagcaag tatatctcct tgttcatcag agactcacag 240
cccagtgatt cagccaccta cctctgtgca atgagggcaa ttcagggagc ccagaagctg 300
gtatttggcc aaggaaccag gctgactatc aaccca 336
<210> 3
<211> 253
<212> PRT
<213>Artificial sequence
<220>
<223>TCR α chains
<400> 3
Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr
20 25 30
Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met
35 40 45
Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Met Arg Ala Ile Gln Gly
85 90 95
Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg Leu Thr Ile Asn Pro
100 105 110
Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys
115 120 125
Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr
130 135 140
Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr
145 150 155 160
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala
165 170 175
Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser
180 185 190
Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp
195 200 205
Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe
210 215 220
Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala
225 230 235 240
Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 4
<211> 759
<212> DNA
<213>Artificial sequence
<220>
<223>TCR α chains
<400> 4
cagaaggagg tggagcagga tcctggacca ctcagtgttc cagagggagc cattgtttct 60
ctcaactgca cttacagcaa cagtgctttt caatacttca tgtggtacag acagtattcc 120
agaaaaggcc ctgagttgct gatgtacaca tactccagtg gtaacaaaga agatggaagg 180
tttacagcac aggtcgataa atccagcaag tatatctcct tgttcatcag agactcacag 240
cccagtgatt cagccaccta cctctgtgca atgagggcaa ttcagggagc ccagaagctg 300
gtatttggcc aaggaaccag gctgactatc aacccaaata tccagaaccc tgaccctgcc 360
gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420
gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480
gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540
tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600
cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 660
aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 720
gggtttaatc tgctcatgac gctgcggctg tggtccagc 759
<210> 5
<211> 111
<212> PRT
<213>Artificial sequence
<220>
<223>TCR β chain variable domains
<400> 5
Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg
1 5 10 15
Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val
20 25 30
Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser
35 40 45
Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe
50 55 60
Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser
65 70 75 80
Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly
85 90 95
Gln Gly Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val
100 105 110
<210> 6
<211> 333
<212> DNA
<213>Artificial sequence
<220>
<223>TCR β chain variable domains
<400> 6
gctgctggag tcatccagtc cccaagacat ctgatcaaag aaaagaggga aacagccact 60
ctgaaatgct atcctatccc tagacacgac actgtctact ggtaccagca gggtccaggt 120
caggaccccc agttcctcat ttcgttttat gaaaagatgc agagcgataa aggaagcatc 180
cctgatcgat tctcagctca acagttcagt gactatcatt ctgaactgaa catgagctcc 240
ttggagctgg gggactcagc cctgtacttc tgtgccagca gccgcggaca gggtgaagct 300
ttctttggac aaggcaccag actcacagtt gta 333
<210> 7
<211> 288
<212> PRT
<213>Artificial sequence
<220>
<223>TCR β chains
<400> 7
Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg
1 5 10 15
Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val
20 25 30
Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser
35 40 45
Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe
50 55 60
Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser
65 70 75 80
Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly
85 90 95
Gln Gly Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val Glu
100 105 110
Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser
115 120 125
Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala
130 135 140
Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly
145 150 155 160
Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu
165 170 175
Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg
180 185 190
Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln
195 200 205
Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg
210 215 220
Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala
225 230 235 240
Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala
245 250 255
Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val
260 265 270
Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe
275 280 285
<210> 8
<211> 864
<212> DNA
<213>Artificial sequence
<220>
<223>TCR β chains
<400> 8
gctgctggag tcatccagtc cccaagacat ctgatcaaag aaaagaggga aacagccact 60
ctgaaatgct atcctatccc tagacacgac actgtctact ggtaccagca gggtccaggt 120
caggaccccc agttcctcat ttcgttttat gaaaagatgc agagcgataa aggaagcatc 180
cctgatcgat tctcagctca acagttcagt gactatcatt ctgaactgaa catgagctcc 240
ttggagctgg gggactcagc cctgtacttc tgtgccagca gccgcggaca gggtgaagct 300
ttctttggac aaggcaccag actcacagtt gtagaggacc tgaacaaggt gttcccaccc 360
gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 420
gtgtgcctgg ccacaggctt cttccccgac cacgtggagc tgagctggtg ggtgaatggg 480
aaggaggtgc acagtggggt cagcacggac ccgcagcccc tcaaggagca gcccgccctc 540
aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 600
ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 660
acccaggata gggccaaacc cgtcacccag atcgtcagcg ccgaggcctg gggtagagca 720
gactgtggct ttacctcggt gtcctaccag caaggggtcc tgtctgccac catcctctat 780
gagatcctgc tagggaaggc caccctgtat gctgtgctgg tcagcgccct tgtgttgatg 840
gccatggtca agagaaagga tttc 864
<210> 9
<211> 9
<212> PRT
<213>Artificial sequence
<220>
<223>Antigen small peptide
<400> 9
Gln Leu Glu Glu Arg Thr Trp Leu Leu
1 5
<210> 10
<211> 6
<212> PRT
<213>Artificial sequence
<220>
<223> α CDR1
<400> 10
Asn Ser Ala Phe Gln Tyr
1 5
<210> 11
<211> 6
<212> PRT
<213>Artificial sequence
<220>
<223> α CDR2
<400> 11
Thr Tyr Ser Ser Gly Asn
1 5
<210> 12
<211> 14
<212> PRT
<213>Artificial sequence
<220>
<223> α CDR3
<400> 12
Cys Ala Met Arg Ala Ile Gln Gly Ala Gln Lys Leu Val Phe
1 5 10
<210> 13
<211> 5
<212> PRT
<213>Artificial sequence
<220>
<223> β CDR1
<400> 13
Pro Arg His Asp Thr
1 5
<210> 14
<211> 6
<212> PRT
<213>Artificial sequence
<220>
<223> β CDR2
<400> 14
Phe Tyr Glu Lys Met Gln
1 5
<210> 15
<211> 12
<212> PRT
<213>Artificial sequence
<220>
<223> β CDR3
<400> 15
Cys Ala Ser Ser Arg Gly Gln Gly Glu Ala Phe Phe
1 5 10
<210> 16
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223> α CDR1
<400> 16
aacagtgctt ttcaatac 18
<210> 17
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223> α CDR2
<400> 17
acatactcca gtggtaac 18
<210> 18
<211> 42
<212> DNA
<213>Artificial sequence
<220>
<223> α CDR3
<400> 18
tgtgcaatga gggcaattca gggagcccag aagctggtat tt 42
<210> 19
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223> β CDR1
<400> 19
cctagacacg acact 15
<210> 20
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223> β CDR2
<400> 20
ttttatgaaa agatgcag 18
<210> 21
<211> 36
<212> DNA
<213>Artificial sequence
<220>
<223> β CDR3
<400> 21
tgtgccagca gccgcggaca gggtgaagct ttcttt 36
<210> 22
<211> 275
<212> PRT
<213>Artificial sequence
<220>
<223>TCR α chains with targeting sequencing
<400> 22
Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro
20 25 30
Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser
35 40 45
Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys
50 55 60
Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp
65 70 75 80
Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu
85 90 95
Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala
100 105 110
Met Arg Ala Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr
115 120 125
Arg Leu Thr Ile Asn Pro 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>TCR α chains with targeting sequencing
<400> 23
atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60
agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120
gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180
tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240
ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300
tcacagccca gtgattcagc cacctacctc tgtgcaatga gggcaattca gggagcccag 360
aagctggtat ttggccaagg aaccaggctg actatcaacc caaatatcca 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> 317
<212> PRT
<213>Artificial sequence
<220>
<223>TCR β chains with targeting sequencing
<400> 24
Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu
1 5 10 15
Cys Arg Val Met Leu Cys Leu Leu Gly Ala Gly Ser Val Ala Ala Gly
20 25 30
Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala
35 40 45
Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr
50 55 60
Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu
65 70 75 80
Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln
85 90 95
Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu
100 105 110
Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly Gln Gly Glu
115 120 125
Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val Glu Asp Leu Asn
130 135 140
Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu
145 150 155 160
Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe
165 170 175
Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val
180 185 190
His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala
195 200 205
Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala
210 215 220
Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe
225 230 235 240
Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro
245 250 255
Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly
260 265 270
Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu
275 280 285
Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser
290 295 300
Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe
305 310 315
<210> 25
<211> 951
<212> DNA
<213>Artificial sequence
<220>
<223>TCR β chains with targeting sequencing
<400> 25
atgcttagtc ctgacctgcc tgactctgcc tggaacacca ggctcctctg ccgtgtcatg 60
ctttgtctcc tgggagcagg ttcagtggct gctggagtca tccagtcccc aagacatctg 120
atcaaagaaa agagggaaac agccactctg aaatgctatc ctatccctag acacgacact 180
gtctactggt accagcaggg tccaggtcag gacccccagt tcctcatttc gttttatgaa 240
aagatgcaga gcgataaagg aagcatccct gatcgattct cagctcaaca gttcagtgac 300
tatcattctg aactgaacat gagctccttg gagctggggg actcagccct gtacttctgt 360
gccagcagcc gcggacaggg tgaagctttc tttggacaag gcaccagact cacagttgta 420
gaggacctga acaaggtgtt cccacccgag gtcgctgtgt ttgagccatc agaagcagag 480
atctcccaca cccaaaaggc cacactggtg tgcctggcca caggcttctt ccccgaccac 540
gtggagctga gctggtgggt gaatgggaag gaggtgcaca gtggggtcag cacggacccg 600
cagcccctca aggagcagcc cgccctcaat gactccagat actgcctgag cagccgcctg 660
agggtctcgg ccaccttctg gcagaacccc cgcaaccact tccgctgtca agtccagttc 720
tacgggctct cggagaatga cgagtggacc caggataggg ccaaacccgt cacccagatc 780
gtcagcgccg aggcctgggg tagagcagac tgtggcttta cctcggtgtc ctaccagcaa 840
ggggtcctgt ctgccaccat cctctatgag atcctgctag ggaaggccac cctgtatgct 900
gtgctggtca gcgcccttgt gttgatggcc atggtcaaga gaaaggattt c 951
<210> 26
<211> 198
<212> PRT
<213>Artificial sequence
<220>
<223>STCR α chains
<400> 26
Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr
20 25 30
Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met
35 40 45
Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Met Arg Ala Ile Gln Gly
85 90 95
Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg Leu Thr Ile Asn Pro
100 105 110
Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys
115 120 125
Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr
130 135 140
Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys
145 150 155 160
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala
165 170 175
Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser
180 185 190
Ile Ile Pro Glu Asp Thr
195
<210> 27
<211> 594
<212> DNA
<213>Artificial sequence
<220>
<223>STCR α chains
<400> 27
cagaaagaag tggaacagga ccctggacca ctcagtgttc cagagggagc cattgtttct 60
ctcaactgca cttacagcaa cagtgctttt caatacttca tgtggtacag acagtattcc 120
agaaaaggcc ctgagttgct gatgtacaca tactccagtg gtaacaaaga agatggaagg 180
tttacagcac aggtcgataa atccagcaag tatatctcct tgttcatcag agactcacag 240
cccagtgatt cagccaccta cctctgtgca atgagggcaa ttcagggagc ccagaagctg 300
gtatttggcc aaggaaccag gctgactatc aacccatata tccagaatcc ggacccggcc 360
gtttatcagc tgcgtgatag caaaagcagc gataaaagcg tgtgcctgtt caccgatttt 420
gatagccaga ccaacgtgag ccagagcaaa gatagcgatg tgtacatcac cgataaatgc 480
gtgctggata tgcgcagcat ggatttcaaa agcaatagcg cggttgcgtg gagcaacaaa 540
agcgattttg cgtgcgcgaa cgcgtttaac aacagcatca tcccggaaga tacg 594
<210> 28
<211> 240
<212> PRT
<213>Artificial sequence
<220>
<223>STCR β chains
<400> 28
Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu
1 5 10 15
Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr
20 25 30
Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe
35 40 45
Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser
50 55 60
Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu
65 70 75 80
Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Arg Gly Gln
85 90 95
Gly Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val Glu Asp
100 105 110
Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu
115 120 125
Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr
130 135 140
Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys
145 150 155 160
Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln
165 170 175
Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val
180 185 190
Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys Gln Val
195 200 205
Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala
210 215 220
Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp
225 230 235 240
<210> 29
<211> 720
<212> DNA
<213>Artificial sequence
<220>
<223>STCR β chains
<400> 29
gcaggtgtta ttcagtcccc aagacatctg atcaaagaaa agagggaaac agccactctg 60
aaatgctatc ctatccctag acacgacact gtctactggt accagcaggg tccaggtcag 120
gacccccagt tcctcatttc gttttatgaa aagatgcaga gcgataaagg aagcatccct 180
gatcgattct cagctcaaca gttcagtgac tatcattctg aactgaacat gagctccttg 240
gagctggggg actcagccct gtacttctgt gccagcagcc gcggacaggg tgaagctttc 300
tttggacaag gcaccagact cacagttgta gaagatctga aaaatgtgtt tccgccggaa 360
gtcgcggtgt tcgaaccgtc ggaagccgaa attagccata cccagaaagc aacgctggtg 420
tgcctggcta ccggctttta tccggatcat gtggaactgt cctggtgggt taacggcaaa 480
gaagtgcact caggtgtttg tacggatccg cagccgctga aagaacaacc ggcactgaat 540
gactcgcgtt atgctctgag ttcccgtctg cgcgttagcg ccaccttctg gcaggatccg 600
cgtaaccact ttcgctgtca ggtccaattc tacggcctgt ccgaaaatga tgaatggacc 660
caggaccgtg caaaaccggt cacgcaaatc gtgtcagcag aagcttgggg tcgtgcagat 720

Claims (10)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be with QLEERTWLL-HLAA0201 compound knots It closes；Preferably, the TCR includes TCR α chains variable domains and TCR β chain variable domains, which is characterized in that the TCR α chain variable domains CDR3 amino acid sequence be CAMRAIQGAQKLVF (SEQ ID NO.12)；And/or the CDR3 of the TCR β chain variable domains Amino acid sequence be CASSRGQGEAFF (SEQ ID NO.15)；
It is highly preferred that 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
αCDR1-NSAFQY (SEQ ID NO.10)
αCDR2-TYSSGN (SEQ ID NO.11)
αCDR3-CAMRAIQGAQKLVF (SEQ ID NO.12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
βCDR1-PRHDT (SEQ ID NO.13)
βCDR2-FYEKMQ (SEQ ID NO.14)
βCDR3-CASSRGQGEAFF (SEQ ID NO.15)。
2. TCR as described in claim 1, which is characterized in that described it includes TCR α chains variable domains and TCR β chain variable domains TCR α chain variable domains are the amino acid sequence for having at least 90% sequence identity with SEQ ID NO.1；And/or the TCR β chains Variable domain be and SEQ ID NO：5 amino acid sequences at least 90% sequence identity.
3. TCR as described in claim 1, which is characterized in that the α chains of the TCR and/or the ends C- or N- of β chains are combined with Conjugate；Preferably, the conjugate combined with the T cell receptor is detectable marker, therapeutic agent, PK modified parts 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 include at least two TCR molecules, and at least one TCR therein Molecule is the 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 TCR points encoded described in any of the above-described claim The nucleic acid sequence or its complementary series of son；
Preferably, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chain variable domains：2；And/or
The nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR β chain variable domains：6.
6. a kind of carrier, which is characterized in that the carrier contains the 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 host cell of separation, which is characterized in that contain the carrier described in claim 6 in the host cell Or the 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 the nucleic acid molecules or claim 6 described in the cell transduction claim 5 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 The TCR compounds described in TCR, claim 4 described in any one of 1-3, the nucleic acid molecules described in claim 5 or power Profit requires the cell described in 8.
10. the TCR compounds or right described in T cell receptor or claim 4 described in any one of claim 1-3 It is required that the purposes of the cell described in 8, which is characterized in that be used to prepare the drug for the treatment of tumour or autoimmune disease.
CN201710214669.4A 2017-04-01 2017-04-01 TCR for recognizing PASD1 antigen short peptide Active CN108659114B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022166904A1 (en) * 2021-02-03 2022-08-11 香雪生命科学技术（广东）有限公司 T-cell receptor for identifying hpv

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010038027A1 (en) * 2008-10-02 2010-04-08 Cancer Research Technology Limited Immunogenic peptides and uses thereof
WO2013169858A1 (en) * 2012-05-08 2013-11-14 The Broad Institute, Inc. Diagnostic and treatment methods in patients having or at risk of developing resistance to cancer therapy
WO2016187508A2 (en) * 2015-05-20 2016-11-24 The Broad Institute Inc. Shared neoantigens

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010038027A1 (en) * 2008-10-02 2010-04-08 Cancer Research Technology Limited Immunogenic peptides and uses thereof
WO2013169858A1 (en) * 2012-05-08 2013-11-14 The Broad Institute, Inc. Diagnostic and treatment methods in patients having or at risk of developing resistance to cancer therapy
WO2016187508A2 (en) * 2015-05-20 2016-11-24 The Broad Institute Inc. Shared neoantigens

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022166904A1 (en) * 2021-02-03 2022-08-11 香雪生命科学技术（广东）有限公司 T-cell receptor for identifying hpv

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