CN109251244A - A kind of identification is derived from the TCR of EBV memebrane protein LMP1 antigen - Google Patents

A kind of identification is derived from the TCR of EBV memebrane protein LMP1 antigen Download PDF

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CN109251244A
CN109251244A CN201710571907.7A CN201710571907A CN109251244A CN 109251244 A CN109251244 A CN 109251244A CN 201710571907 A CN201710571907 A CN 201710571907A CN 109251244 A CN109251244 A CN 109251244A
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李懿
陈安安
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Guangzhou Institute of Biomedicine and Health of CAS
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Abstract

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

Description

A kind of identification is derived from the TCR of EBV memebrane protein LMP1 antigen
Technical field
The present invention relates to that can identify the TCR from LMP1 antigen small peptide, the invention further relates to transduce above-mentioned TCR to obtain LMP1 specificity T cell and they prevent and treat LMP1 related disease in purposes.
Background technique
EBV is a kind of generally existing nerpes vinrus hominis of global range.Studies have shown that is more than in 95% adult human body Containing the antibody for being directed to this virus, this also means that them in a certain stage by this virus infection mistake.It is most of to be felt The internal all one's life of the people contaminated can all have EBV, generally seldom go wrong.However, in some cases, EBV and some cancers The generation of disease is related, including Burkitt lymphoma (Burkitt ' s lymphoma), Hodgkin lymphoma (Hodgkin Lymphoma), lymphocytic hyperplasia disease (PTLD) or nasopharyngeal carcinoma etc. after EBV positive graft.For example, LMP1 belongs to the incubation period film of EBV Albumen can express (Raab-Traub N.Epstein-Barr virus in the by most nasopharyngeal carcinoma cells pathogenesis of NPC[J].Semin Cancer Biol,2002,12(6):431-441.).LMP1 gives birth in the cell It is degraded to micromolecule polypeptide after, and forms compound in conjunction with MHC (main histocompatibility complex) molecule, is presented to Cell surface.ILWRLGATI is the small peptide derived from LMP1 antigen, is a kind of target of LMP1 treating correlative diseases.For upper The treatment of disease is stated, the methods of chemotherapy and radiation treatment can be used, but can all damage to the normal cell of itself.
T cell adoptive immunotherapy is that will there is the reaction-ive T cell of specificity to be transferred in patient body target cell antigen, It is set to play a role for target cell.T cell receptor (TCR) is a kind of memebrane protein on T cell surface, can be identified corresponding The antigen small peptide of target cell surface.In immune system, pass through the TCR and the main histocompatbility of small peptide-of antigen small peptide specificity The combination of complex (pMHC compound) causes T cell and antigen presenting cell (APC) is directly physically contacted, then T cell And other cell membrane surface molecules of both APC just interact, and cause a series of subsequent cell signal transmitting and its His physiological reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, this field skill Art personnel are dedicated to isolating the TCR for having specificity to LMP1 antigen small peptide, and the TCR transduceed T cell to obtain pair LMP1 antigen small peptide has the T cell of specificity, so that them be made to play a role in cellular immunotherapy.
Summary of the invention
The purpose of the present invention is to provide a kind of T cell receptors for identifying LMP1 antigen small peptide.
The first aspect of the present invention, provides a kind of T cell receptor (TCR), and the TCR can be with ILWRLGATI-HLA A0201 compound combines.
In another preferred example, the TCR includes TCR α chain variable domain and TCR β chain variable domain, the TCR α chain variable domain CDR3 amino acid sequence be CAVGSNFGNEKLTF (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domain Amino acid sequence be CSARRLGTEAFF (SEQ ID NO:15).
In another preferred example, 3 complementary determining regions (CDR) of the TCR α chain variable domain are as follows:
αCDR1-DSVNN(SEQ ID NO:10)
αCDR2-IPSGT(SEQ ID NO:11)
αCDR3-CAVGSNFGNEKLTF(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-CSARRLGTEAFF(SEQ ID NO:15)。
In another preferred example, the TCR includes TCR α chain variable domain and TCR β chain variable domain, the TCR α chain variable domain To have the amino acid sequence of at least 90% sequence identity with SEQ ID NO:1；And/or the TCR β chain variable domain be with SEQ ID NO:5 has the amino acid sequence of at least 90% sequence identity.
In another preferred example, the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.
In another preferred example, the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5.
In another preferred example, the TCR is α β heterodimer, and it includes TCR α chain constant region TRAC*01 and TCR β Chain constant region TRBC1*01 or TRBC2*01.
In another preferred example, the α chain amino acid sequence of the TCR is the β chain ammonia of the SEQ ID NO:3 and/or TCR Base acid sequence is SEQ ID NO:7.
In another preferred example, the TCR is soluble.
In another preferred example, the TCR is single-stranded.
In another preferred example, the TCR is formed by connecting with β chain variable domain by peptide catenation sequence by α chain variable domain.
In another preferred example, the TCR is in α chain variable region amino acid the 11st, 13,19,21,53,76,89,91 or There is one or more dash forward in 94 and/or α chain J gene small peptide amino acid inverse the 3rd, 5th reciprocal or 7th reciprocal Become；And/or the TCR is in β chain variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th and/or β chain J In gene small peptide amino acid inverse the 2nd, 4th reciprocal or 6th reciprocal there is one or more to be mutated, wherein amino acid position Number is set by the Position Number listed in IMGT (international immunogenetics information system).
In another preferred example, the TCR includes (a) all or part of TCR α chain in addition to transmembrane domain；And (b) all or part of TCR β chain in addition to transmembrane domain；
And (a) and (b) respectively contains functional variable domain, or includes functional variable domain and the TCR At least part of chain constant domain.
In another preferred example, cysteine residues form artificial disulfide bond between α the and β chain constant domain of the TCR.
In another preferred example, the cysteine residues of artificial disulfide bond are formed in the TCR instead of selected from following One or more groups of sites:
The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；With
The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1.
In another preferred example, the α chain amino acid sequence of the TCR is the β chain of the SEQ ID NO:26 and/or TCR Amino acid sequence is SEQ ID NO:28.
In another preferred example, artificial interchain disulfide bond is contained between the α chain variable region of the TCR and β chain constant region.
In another preferred example, which is characterized in that the cysteine residues of artificial interchain disulfide bond are formed in the TCR Instead of selected from following one or more groups of sites:
The 46th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1；
The 47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1；
The 46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1；Or
The 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1.
In another preferred example, the TCR is comprising α chain variable domain and β chain variable domain and in addition to transmembrane domain All or part of β chain constant domain, but it does not contain α chain constant domain, the α chain variable domain and β chain of the TCR forms heterogeneous dimerization Body.
In another preferred example, the α chain of the TCR and/or the end C- or N- of β chain are combined with conjugate.
In another preferred example, the conjugate in conjunction with the T cell receptor is detectable marker, therapeutic agent, PK are repaired The combination of decorations part or any of these substances.Preferably, the therapeutic agent is anti-CD 3 antibodies.
The second aspect of the present invention provides a kind of multivalent TCR complex, and it includes at least two TCR molecules, and its In at least one TCR molecule be first aspect present invention described in TCR.
The third aspect of the present invention, provides a kind of nucleic acid molecules, and the nucleic acid molecules include to encode first party of the present invention The nucleic acid sequence of TCR molecule or its complementary series described in face.
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chain variable domain: 2。
In another preferred example, the nucleic acid molecules include the nucleotide sequence SEQ ID of coding TCR β chain variable domain NO:6.
In another preferred example, the nucleic acid molecules include coding TCR α chain nucleotide sequence SEQ ID NO:4 and/or Nucleotide sequence SEQ ID NO:8 comprising encoding TCR β chain.
The fourth aspect of the present invention, provides a kind of carrier, and the carrier contains core described in third aspect present invention Acid molecule；Preferably, the carrier is viral vectors；It is highly preferred that the carrier is slow virus carrier.
The fifth aspect of the present invention provides a kind of isolated host cell, contains the present invention in the host cell Nucleic acid molecules described in the third aspect present invention of external source are integrated in carrier described in fourth aspect or genome.
The sixth aspect of the present invention provides a kind of cell, nucleic acid described in the cell transduction third aspect present invention Carrier described in molecule or fourth aspect present invention；Preferably, the cell is T cell or stem cell.
The seventh aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains pharmaceutically acceptable load TCR described in body and first aspect present invention, TCR compound described in second aspect of the present invention, third aspect present invention institute Cell described in carrier described in the nucleic acid molecules stated, fourth aspect present invention or sixth aspect present invention.
The eighth aspect of the present invention provides T cell receptor described in first aspect present invention or second aspect of the present invention Nucleic acid molecules described in the TCR compound, third aspect present invention, carrier or this hair described in fourth aspect present invention The purposes of cell described in bright 6th aspect, is used to prepare the drug for the treatment of tumour or autoimmune disease.
The ninth aspect of the present invention provides a kind of method for treating disease, including suitable to object in need for the treatment of application TCR compound, third of the present invention described in T cell receptor described in the first aspect present invention of amount or second aspect of the present invention Nucleic acid molecules described in aspect, cell or this hair described in carrier or sixth aspect present invention described in fourth aspect present invention Pharmaceutical composition described in bright 7th aspect；
Preferably, the disease is tumour, and the preferably described tumour includes Burkitt lymphoma (Burkitt ' s Lymphoma), lymphocytic hyperplasia disease (PTLD) or nasopharynx after Hodgkin lymphoma (Hodgkin lymphoma), EBV positive graft Cancer etc..
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively that TCR α chain variable domain amino acid sequence, TCR α chain are variable Domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, the TCR α chain amino acid sequence with leader sequence And the TCR α chain nucleotide sequence with leader sequence.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively that TCR β chain variable domain amino acid sequence, TCR β chain are variable Domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, the TCR β chain amino acid sequence with leader sequence And the TCR β chain nucleotide sequence with leader sequence.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chain.
Fig. 5 a and Fig. 5 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chain.
Fig. 6 is the glue figure of the sTCR obtained after purification.Leftmost side swimming lane is molecular weight marker (marker), intermediate Swimming lane is non-reduced glue, and rightmost side swimming lane is to go back virgin rubber.
Fig. 7 is BIAcore dynamics figure of the sTCR of the present invention in conjunction with ILWRLGATI-HLA A0201 compound Spectrum.
Fig. 8 shows that the T cell for the TCR of the present invention that transduces has activation well anti-the target cell for loading its special small peptide It answers, the target cell of target cell and the non-specific small peptide of load to unsupported corresponding small peptide does not have activating reaction.
Fig. 9 shows LMP1 CD8+T cell to the target cell of load LMP1PX224129-137ILWRLGATI small peptide The lethal effect of LCL-A02/A11 is obvious；The target cell LCL-A02/A11 for not loading small peptide or load non-specificity small peptide is killed Wound effect is unobvious.
Specific embodiment
The present inventor after extensive and in-depth study, have found with LMP1 antigen small peptide ILWRLGATI (SEQ ID NO: 9) TCR that can be specifically bound, the antigen small peptide ILWRLGATI can form compound with HLA A0201 and be in together It is delivered to cell surface.Carrier the present invention also provides the nucleic acid molecules for encoding the TCR and comprising the nucleic acid molecules.Separately Outside, the present invention also provides the cells for the TCR of the present invention that transduces.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, for The presentation of antigen has specificity, and different individuals has different MHC, can present small peptide different in a kind of proteantigen to respectively From APC cell surface.The MHC of the mankind is commonly referred to as HLA gene or HLA complex.
T cell receptor (TCR) is the unique of specific antigen peptide of the presentation on main histocompatibility complex (MHC) Receptor.In immune system, T cell is caused by the combination of the TCR and pMHC compound of antigentic specificity and antigen presentation is thin Born of the same parents (APC) are directly physically contacted, and then other cell membrane surface molecules of both T cell and APC just interact, this A series of subsequent cell signal transmitting and other physiological reactions are just caused, so that the T cell of different antigentic specificities Immunological effect is played to its target cell.
TCR be as α chain/β chain or γ chain/δ chain in the form of heterodimer existing for cell membrane surface glycoprotein.? TCR heterodimer is made of α and β chain in 95% T cell, and 5% T cell has the TCR being made of γ and δ chain.It The right heterogeneous dimerization TCR of α β has α chain and β chain, and α chain and β chain constitute the subunit of α β heterodimeric TCR.In a broad sense, α and β are each Chain includes variable region, bonding pad and constant region, and β chain usually contains short variable region also between variable region and bonding pad, but should Variable region is often regarded as a part of bonding pad.Each variable region includes 3 be entrenched in frame structure (framework regions) A CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compound, wherein CDR3 by Variable region and bonding pad recombinate, referred to as hypervariable region.α the and β chain of TCR generally regards that each there are two " structural domains " can be changed as Domain and constant domain, variable domain are made of the variable region connected and bonding pad.The sequence of TCR constant domain can be in international immune genetic It learns and is found in the public database of information system (IMGT), if the constant domain sequence of TCR molecule alpha chain is " TRAC*01 ", TCR divides The constant domain sequence of sub- β chain is " TRBC1*01 " or " TRBC2*01 ".In addition, α the and β chain of TCR also includes transmembrane region and cytoplasm Area, cytoplasmic region are very short.
In the present invention, term " polypeptide of the present invention ", " TCR of the invention ", " T cell receptor of the invention " is interchangeable makes With.
Native interchain disulfide bond and artificial interchain disulfide bond
Natural TCR membrane-proximal region C α and C β interchain exist one group of disulfide bond, the present invention in referred to as " two sulphur of native interchain Key ".In the present invention, by what is be artificially introduced, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond claim For " artificial interchain disulfide bond ".
For convenience of the position of description disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequence in the present invention Position Number by from N-terminal to C-terminal sequence successively carry out Position Number, in TRBC1*01 or TRBC2*01, by from N-terminal to The 60th amino acid of the sequence of C-terminal successively is P (proline), then can describe it as TRBC1*01 or TRBC2*01 in the present invention The Pro60 of exons 1 can also be stated that the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1, for another example It is Q (glutamine) by the 61st amino acid of the sequence from N-terminal to C-terminal successively in TRBC1*01 or TRBC2*01, then it is of the invention In can describe it as the Gln61 of TRBC1*01 or TRBC2*01 exons 1, can also be stated that TRBC1*01 or TRBC2* 61st amino acids of 01 exons 1, other and so on.In the present invention, the amino acid sequence of variable region TRAV and TRBV Position Number, according to the Position Number listed in IMGT.Such as some amino acid in TRAV, the Position Number listed in IMGT is 46, then the 46th amino acids of TRAV, other and so on are described it as in the present invention.In the present invention, the sequence of other amino acid Column position number has specified otherwise, then presses specified otherwise.
Detailed description of the invention
TCR molecule
In antigen processing pathways, antigen is degraded in the cell, is then carried by MHC molecule to cell surface.T is thin Born of the same parents' receptor can identify the peptide-MHC compound of Antigen Presenting Cell surface.Therefore, the first aspect of the present invention provides one kind It can be in conjunction with the TCR molecule of ILWRLGATI-HLA A0201 compound.Preferably, the TCR molecule is separation or purifying 's.α the and β chain of the TCR respectively has 3 complementary determining regions (CDR).
It is preferably carried out in mode at of the invention one, the α chain of the TCR includes with following amino acid sequence CDR:
αCDR1-DSVNN(SEQ ID NO:10)
αCDR2-IPSGT(SEQ ID NO:11)
αCDR3-CAVGSNFGNEKLTF(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-CSARRLGTEAFF(SEQ ID NO:15)。
The CDR region amino acid sequence of aforementioned present invention can be embedded into chimeric to prepare in any suitable frame structure TCR.As long as frame structure is compatible with the CDR region of TCR of the invention, those skilled in the art's disclosed CDR region according to the present invention It can design or synthesize the TCR molecule with corresponding function.Therefore, TCR molecule of the present invention refers to comprising above-mentioned α and/or β The TCR molecule of chain CDR region sequence and any suitable frame structure.TCR α chain variable domain of the present invention is to have with SEQ ID NO:1 There are at least 90%, preferably 95%, the more preferably amino acid sequence of 98% sequence identity；And/or TCR β chain of the present invention can Variable domain is to have at least 90%, preferably 95% with SEQ ID NO:5, the amino acid sequence of more preferably 98% sequence identity Column.
In a preference of the invention, TCR molecule of the invention is the heterodimer being made of α and β chain.Specifically Ground, on the one hand the α chain of the heterogeneous dimerization TCR molecule includes variable domain and constant domain, the α chain variable domain amino acid sequence packet CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:11) and CDR3 (SEQ ID NO:12) containing above-mentioned α chain.Preferably, The TCR molecule includes α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domain of the TCR molecule Amino acid sequence is SEQ ID NO:1.On the other hand, the β chain of the heterogeneous dimerization TCR molecule includes variable domain and constant domain, The β chain variable domain amino acid sequence include above-mentioned β chain CDR1 (SEQ ID NO:13), CDR2 (SEQ ID NO:14) and CDR3(SEQ ID NO:15).Preferably, the TCR molecule includes β chain variable domain amino acid sequence SEQ ID NO:5.It is more excellent Selection of land, the β chain variable domain amino acid sequence of the TCR molecule are SEQ ID NO:5.
In a preference of the invention, TCR molecule of the invention is the portion by some or all of α chain and/or β chain The single chain TCR molecules for dividing or all forming.Description in relation to single chain TCR molecules can be with bibliography Chung et al (1994) Proc.Natl.Acad.Sci.USA 91,12654-12658.According to document, those skilled in the art can be easily Building includes the single chain TCR molecules in the area CDRs of the present invention.Specifically, the single chain TCR molecules include V α, V β and C β, preferably According to the sequential connection from N-terminal to C-terminal.
CDR1 (SEQ ID NO:10) of the α chain variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned α chain, CDR2 (SEQ ID NO:11) and CDR3 (SEQ ID NO:12).Preferably, the single chain TCR molecules include α chain variable domain ammonia Base acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO: 1.The β chain variable domain amino acid sequence of the single chain TCR molecules includes CDR1 (SEQ ID NO:13), the CDR2 of above-mentioned β chain (SEQ ID NO:14) and CDR3 (SEQ ID NO:15).Preferably, the single chain TCR molecules include β chain variable domain amino acid Sequence SEQ ID NO:5.It is highly preferred that the β chain variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:5.
In a preference of the invention, the constant domain of TCR molecule of the invention is the constant domain of people.Art technology Personnel know or can be obtained by consulting the public database of pertinent texts or IMGT (international immunogenetics information system) Obtain the constant domain amino acid sequence of people.For example, the constant domain sequence of TCR molecule alpha chain of the present invention can be " TRAC*01 ", TCR divides The constant domain sequence of sub- β chain can be " TRBC1*01 " or " TRBC2*01 ".The amino acid sequence provided in the TRAC*01 of IMGT The 53rd be Arg, indicate herein are as follows: the Arg53 of TRAC*01 exons 1, other and so on.Preferably, TCR of the present invention The amino acid sequence of molecule alpha chain is that the amino acid sequence of SEQ ID NO:3 and/or β chain is SEQ ID NO:7.
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.As immunoglobulin (antibody) is made The same for antigen recognition molecule, at this moment TCR can also need to obtain soluble TCR points by development and application in diagnosing and treating Son.Soluble TCR molecule does not include its transmembrane region.STCR has very extensive purposes, it cannot be only used for research TCR With the interaction of pMHC, it is also possible to make the diagnostic tool of detection infection or the marker as autoimmunity disease.Similarly, may be used Dissolubility TCR can be used to for therapeutic agent (such as cytotoxin compounds or immunostimulating compound) to be transported to presentation specificity The cell of antigen, in addition, sTCR can also with other molecules (e.g., anti-CD 3 antibodies) in conjunction with redirecting T cell, from And make the cell of its targeting presentation specific antigen.The present invention also obtains the solubility for having specificity to LMP1 antigen small peptide TCR。
To obtain sTCR, on the one hand, TCR of the present invention can be to be introduced between the residue of itself α and β chain constant domain The TCR of artificial disulfide bond.Cysteine residues form artificial interchain disulfide bond between α the and β chain constant domain of the TCR.Half Guang Histidine residue can be substituted in other amino acid residues of appropriate site in natural TCR to form artificial interchain disulfide bond.For example, Replace the Thr48 of TRAC*01 exons 1 and replaces the cysteine residues of the Ser57 of TRBC1*01 or TRBC2*01 exons 1 To form disulfide bond.It introduces cysteine residues and may also is that TRAC*01 exons 1 with other sites for forming disulfide bond The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1；The Tyr10 and TRBC1*01 of TRAC*01 exons 1 or The Ser17 of TRBC2*01 exons 1；Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1 Asp59；The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；TRAC*01 exons 1 Arg53 and TRBC1*01 or TRBC2*01 exons 1 Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1 or The Ala19 of TRBC2*01 exons 1；Or Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1 Glu20.I.e. cysteine residues are instead of any group of site in above-mentioned α and β chain constant domain.It can be in TCR constant domain of the present invention One or more C-terminals truncate most 50 or most 30 or most 15 or most 10 or most 8 or less Amino acid can also be by the way that day will be formed so that it does not include cysteine residues to achieve the purpose that lack natural disulphide bonds The cysteine residues of right disulfide bond sport another amino acid to reach above-mentioned purpose.
As described above, TCR of the invention may be embodied in the artificial disulfide bond introduced between the residue of itself α and β chain constant domain. It should be noted that the artificial disulfide bond with or without introducing described above between constant domain, TCR of the invention can be constant containing TRAC Domain sequence and TRBC1 or TRBC2 constant domain sequence.The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequence of TCR can It is connected by the natural disulphide bonds being present in TCR.
To obtain sTCR, on the other hand, TCR of the present invention further includes the TCR to mutate in its hydrophobic core region, The mutation of these hydrophobic core regions is preferably capable making the stability-enhanced mutation of sTCR of the present invention, such as in publication number Described in patent document for WO2014/206304.Such TCR can mutate in its following hydrophobic core position of variable domain: (α and/or β chain) variable region amino acid the 11st, 13,19,21,53,76,89,91,94 and/or α chain J gene (TRAJ) small peptide Amino acid position the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptide amino acid position reciprocal is 2nd, 4,6 reciprocal, wherein ammonia The Position Number of base acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).Those skilled in the art Member knows above-mentioned international immunogenetics information system, and the amino acid residue that different TCR can be obtained according to the database exists Position Number in IMGT.
The TCR that hydrophobic core region mutates in the present invention can be by α and the β chain of a flexible peptide chain link TCR can Variable domain and the solvable single-stranded TCR of stability constituted.It should be noted that in the present invention flexible peptide chain can be any suitable connection TCR α and The peptide chain of β chain variable domain.
In addition, patent document PCT/CN2016/077680 is also disclosed in the α chain variable region of TCR for stability Introducing artificial interchain disulfide bond between β chain constant region can be such that the stability of TCR significantly improves.Therefore, height parent of the invention Artificial interchain disulfide bond can also be contained between the α chain variable region and β chain constant region of power TCR.Specifically, in the α of the TCR The cysteine residues of artificial interchain disulfide bond are formed between chain variable region and β chain constant region instead of the 46th ammonia of TRAV 60th amino acids of base acid and TRBC1*01 or TRBC2*01 exons 1；The 47th amino acids and TRBC1*01 of TRAV or 61 amino acids of TRBC2*01 exons 1；The of the 46th amino acids of TRAV and TRBC1*01 or TRBC2*01 exons 1 61 amino acids；Or TRAV the 47th amino acids and TRBC1*01 or TRBC2*01 exons 1 the 60th amino acids.It is preferred that Ground, such TCR may include all or part of TCR α chain of (I) in addition to its transmembrane domain, and (II) removes its cross-film knot All or part of TCR β chain other than structure domain, wherein (I) and (II) variable domain comprising TCR chain and at least part is constant Domain, α chain and β chain form heterodimer.It is highly preferred that such TCR may include α chain variable domain and β chain variable domain and All or part of β chain constant domain in addition to transmembrane domain, but it does not contain α chain constant domain, the α chain variable domain of the TCR Heterodimer is formed with β chain.
TCR of the invention can also be provided in the form of multivalence complex.Multivalent TCR complex of the invention include two, Three, four or more TCR of the present invention are combined and the polymer that is formed, can such as be generated with four dimerization domains of p53 The compound that the tetramer or multiple TCR of the present invention are formed in conjunction with another molecule.TCR compound of the invention can be used for body Outer or tracking in vivo or targeting present the cell of specific antigen, it can also be used to which generating has other multivalence TCR of such application multiple Close the intermediate of object.
TCR of the invention can be used alone, can also with conjugate with covalent or other modes in conjunction with, preferably with covalently side Formula combines.The conjugate includes that detectable marker (for diagnostic purpose, presents wherein the TCR is used to detect The presence of the cell of ILWRLGATI-HLA A0201 compound), therapeutic agent, PK (protein kinase) modified part or it is any more than The combination of these substances combines or coupling.
Detectable marker for diagnostic purposes includes but is not limited to: fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.
Can in conjunction with TCR of the present invention or coupling therapeutic agent include but is not limited to: 1. radionuclides (Koppe etc., 2005, (Cancer metastasis reviews) 24,539 is commented in metastasis of cancer)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc Segment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；5. antibody ScFv segment (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold medal (Lapotko etc., 2005, cancer communicates (Cancer letters) 239,36 to nano particle/nanometer rods；Huang etc., 2006, beauty Chemical Society 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- diaphorase (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
In addition, TCR of the invention can also be comprising derived from the heterozygosis TCR more than a kind of species sequence.For example, grinding Studying carefully display Muridae TCR can more effectively express in human T-cell than people TCR.Therefore, TCR of the present invention may include people's variable domain With the constant domain of mouse.The defect of this method is possible to cause immune response.Therefore, when it is used for the treatment of adoptive T cell There should be regulation scheme to carry out immunosupress, to allow to express the implantation of the T cell of Muridae.
It should be understood that amino acid name herein is indicated using international single English alphabet or three English alphabets, amino The corresponding relationship of the single English alphabet and three English alphabets of sour title is as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser (S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
Nucleic acid molecules
The second aspect of the present invention provides the nucleic acid molecules of coding first aspect present invention TCR molecule or part thereof, institute Stating part can be one or more CDR, the variable domain and α chain and/or β chain of α and/or β chain.
The nucleotide sequence for encoding first aspect present invention TCR molecule alpha chain CDR region is as follows:
αCDR1-(SEQ ID NO:16)
αCDR2-(SEQ ID NO:17)
αCDR3-(SEQ ID NO:18)
The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR region is as follows:
βCDR1-(SEQ ID NO:19)
βCDR2-(SEQ ID NO:20)
βCDR3-(SEQ ID NO:21)
Therefore, the nucleotide sequence for encoding the nucleic acid molecules of the present invention of TCR α chain of the present invention includes SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, and/or the nucleotide sequence of nucleic acid molecules of the present invention of coding TCR β chain of the present invention includes SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
The nucleotide sequence of nucleic acid molecules of the present invention can be it is single-stranded or double-stranded, the nucleic acid molecules can be RNA or DNA, and may include or not include introne.Preferably, the nucleotide sequence of nucleic acid molecules of the present invention does not include introne But polypeptide of the present invention can be encoded, such as encodes the nucleotide sequence packet of the nucleic acid molecules of the present invention of TCR α chain variable domain of the present invention The nucleotide sequence for including the nucleic acid molecules of the present invention of SEQ ID NO:2 and/or coding TCR β chain variable domain of the present invention includes SEQ ID NO:6.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 present invention nucleic acid sequence shown in the drawings or degeneracy.With One of example in the present invention illustrates that " variant of degeneracy " refer to that coding has the protein sequence of SEQ ID NO:1, But the differentiated nucleic acid sequence of sequence with SEQ ID NO:2.
Nucleotide sequence can be through codon optimization.Different cells is above different in the utilization of specific codon , the codon in sequence can be changed to increase expression quantity according to the type of cell.Mammalian cell and various other The codon usage table of biology is well known to those skilled in the art.
Nucleic acid molecules full length sequence or its segment of the invention usually can with but be not limited to PCR amplification method, recombination method or Artificial synthesized method obtains.At present, it is already possible to obtain encoding completely by chemical synthesis TCR of the present invention (or its segment, Or derivatives thereof) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or Such as carrier) and cell in.DNA can be coding strand or noncoding strand.
Carrier
It, can in vivo or body the invention further relates to the carrier comprising nucleic acid molecules of the invention, including expression vector The construct of outer expression.Common carrier includes bacterial plasmid, bacteriophage and animals and plants virus.
Viral delivery systems include but is not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, Retroviral vector, slow virus carrier, baculovirus vector.
Preferably, nucleotide of the invention can be transferred in cell by carrier, such as in T cell, so that the cell table Up to the TCR of LMP1 antigentic specificity.Ideally, which can should express to continual high levels in T cell.
Cell
The invention further relates to the genetically engineered host cell of carrier or coded sequence of the invention.The host Contain in carrier or chromosome of the invention in cell and is integrated with nucleic acid molecules of the invention.Host cell is selected from: prokaryotic cell And eukaryocyte, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..
In addition, the invention also includes the isolated cell for expressing TCR of the invention, especially T cell.The T cell can spread out It is born from the T cell separated from subject, or can be the mixed cellularity group separated from subject, such as periphery hemolymph is thin A part of born of the same parents (PBL) group.Such as, which can be isolated from peripheral blood mononuclear cells (PBMC), can be CD4+T helper cell Or CD8+Cytotoxic T cell.The cell can be in CD4+T helper cell/CD8+In the mixing group of cytotoxic T cell.Generally Ground, the cell can be activated with antibody (e.g., the antibody of anti-CD3 or anti-CD28), to allow them to more easily receive to turn Dye, such as transfected with the carrier of the nucleotide sequence comprising encoding TCR molecule of the present invention.
Alternatively, cell of the invention can also be or derived from stem cell, such as candidate stem cell (HSC).Gene is turned Moving to HSC not will lead in cell surface expression TCR, because stem cell surface does not express CD3 molecule.However, when stem cell point It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecule will start in thymocyte The surface expression introducing TCR molecule.
There are many method be suitable for being carried out with the DNA or RNA of coding TCR of the present invention T cell transfection (e.g., the such as Robbins, (2008)J.Immunol.180:6116-6131).The T cell for expressing TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat Rev for carrying out adoptive treatment Cancer8 (4): 299-308).
LMP1 antigen-related disease
The invention further relates to the method with LMP1 related disease is treated and/or prevented in subject comprising adoptive Shift the step of LMP1 specific T-cells are to the subject.The LMP1 specific T-cells can recognize ILWRLGATI-HLA A0201 compound.
The T cell of LMP1 specificity of the invention can be used for treating any presentation LMP1 antigen small peptide ILWRLGATI-HLA The LMP1 related disease of A0201 compound.Including but not limited to Burkitt lymphoma (Burkitt ' s lymphoma), Huo Qijin Lymphocytic hyperplasia disease (PTLD) or nasopharyngeal carcinoma etc. after lymthoma (Hodgkin lymphoma), EBV positive graft.
Treatment method
Can by separation with the patient of LMP1 antigen-related disease or the T cell of volunteer, and will be of the invention TCR is imported in above-mentioned T cell, is then fed back to the cell that these genetic engineerings are modified in patient body to treat.Therefore, The present invention provides a kind of method for treating LMP1 related disease, the T cell including the expression TCR of the present invention that will be separated, preferably Ground, the T cell derive from patient itself, are input in patient body.Generally, the T cell including (1) separation patient, (2) are with originally Invention nucleic acid molecules or the nucleic acid molecules ex vivo transduction T cell that can encode TCR molecule of the present invention, (3) modify genetic engineering T cell be input in patient body.The quantity of separation, transfection and the cell fed back can be determined by doctor.
Main advantages of the present invention are:
(1) TCR of the invention can turn simultaneously in conjunction with LMP1 antigen small peptide composite I LWRLGATI-HLA A0201 The cell for having led TCR of the present invention can have very strong lethal effect by specific activation and to target cell.
Following specific embodiment, the present invention is further explained.It should be understood that these embodiments be merely to illustrate the present invention and It is not used in and limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, Such as (Sambrook and Russel l et al., molecular cloning: laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing house) described in condition, or according to proposed by manufacturer Condition.Unless otherwise stated, otherwise percentage and number are calculated by weight.Unless otherwise stated, otherwise percentage and number It calculates by weight.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
Embodiment 1 clones PRAME specific T-cells
Utilize synthesis small peptide (Nanjing Jin Sikang Biotechnology Co., Ltd) ILWRLGATI (SEQ ID NO.9, the present invention In be named as PX224) stimulation from genotype be HLA-A02 healthy volunteer peripheral blood lymphocytes (PBL).It will be short Peptide and the HLA-A*0201 renaturation for having biotin labeling, prepare pHLA monoploid.These monoploid and the strepto- marked with PE Avidin (BD company) is combined into the tetramer of PE label, sorts the tetramer and anti-CD8-APC double positive cells.Amplification point The cell of choosing, and secondary sorting is carried out according to the above method, then monoclonal is carried out with limiting dilution assay.
Since entire experiment flow takes a long time, influence factor is extremely more, and experiment is more complicated, and the performance of cell at all can not Prediction, even across screening layer by layer with stringent detection, the success rate for obtaining corresponding T cell monoclonal is also very low.One As in the case of merely through several batches experiment be difficult obtain have desired activities TCR.
In the present invention, by inventor's in-depth study and a large amount of experiment, double sun of the condition of satisfaction have been finally obtained Property monoclonal cell.Monoclonal cell tetramer staining, the double positive colonies screened are as shown in Figure 3.
Even if successfully screening obtains T cell monoclonal, TCR obtained from this also not necessarily can satisfy requirement, It is very poor with the affinity of corresponding epitope after renaturation or renaturation because the TCR in many cases, obtained can not be succeeded, very It can not extremely combine.It needs further to combine active verifying.
Embodiment 2 obtains the building of the tcr gene and carrier of LMP1 specific T-cell clones
Use Quick-RNATMMiniPrep (ZYMO research) extracting embodiment 1 in screen PX224 specificity, The total serum IgE of the restrictive T cell clone of HLA-A02.The synthesis of cDNA expands examination using the SMART RACE cDNA of clontech Agent box, the primer of use are the C-terminal conserved regions designed in mankind's tcr gene.It is enterprising that sequence is cloned into carrier T (TAKARA) Row sequencing.Through being sequenced, the α chain and β chain-ordering structure 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 chain variable domain amino acid sequence, TCR α chain variable domain nucleotide sequence, TCR α chain amino Acid sequence and TCR α chain nucleotide sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c and Fig. 2 d are respectively TCR β chain variable domain amino acid sequence, TCR β chain variable domain nucleotide sequence, TCR β chain amino acid sequence and TCR β chain nucleotide sequence.
Identified, α chain includes the CDR with following amino acid sequence:
αCDR1-DSVNN(SEQ ID NO:10)
αCDR2-IPSGT(SEQ ID NO:11)
αCDR3-CAVGSNFGNEKLTF(SEQ ID NO:12)
β chain includes the CDR with following amino acid sequence:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-CSARRLGTEAFF(SEQ ID NO:15)。
The full-length gene of TCR α chain and β chain is cloned into Lentiviral respectively by overlapping (overlap) PCR pLenti(addgene).Specifically: with overlap PCR by the area the V gene of TCR α chain and TCR β chain respectively with mouse TCR α chain It is attached to obtain TCR α -2A-TCR β segment with the conserved region area C of TCR β chain.By Lentiviral and TCR α -2A-TCR β digestion connects to obtain pLenti-LMP1TRA-2A-TRB-IRES-NGFR plasmid.It is used as control, while also building expression The slow virus carrier pLenti-eGFP of eGFP.Pseudovirus is packed with 293T/17 again later.
Expression, refolding and the purifying of the 3 solvable TCR of LMP1 antigen small peptide specificity of embodiment
To obtain soluble TCR molecule, α the and β chain of TCR molecule of the invention can only include its variable domain and portion respectively Point constant domain, and a cysteine residues are introduced in the constant domain of α and β chain respectively to form artificial interchain disulfide bond, The position of introducing cysteine residues is respectively the Ser57 of the Thr48 and TRBC2*01 exons 1 of TRAC*01 exons 1；Its α The amino acid sequence and nucleotide sequence of chain are distinguished as shown in figures 4 a and 4b, the amino acid sequence and nucleotide sequence of β chain Respectively as shown in figure 5 a and 5b, the cysteine residues of introducing with overstriking and underline alphabetical indicate.Pass through " molecular cloning Laboratory manual " (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) Described in standard method the objective gene sequence of above-mentioned TCR α and β chain is inserted respectively into expression vector pET28a after synthesizing + (Novagene), the cloning site of upstream and downstream are NcoI and NotI respectively.Insert Fragment is errorless by sequencing confirmation.
The expression vector of TCR α and β chain is converted by chemical transformation respectively and enters expression bacterium BL21 (DE3), bacterium It is grown with LB culture solution, in OD600It is induced when=0.6 with final concentration 0.5mM IPTG, the packet formed after α the and β chain expression of TCR Contain body to extract by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, forgives Body is finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT) (DTT), 10mM ethylenediamine tetra-acetic acid (EDTA), 20mM Tris (pH 8.1) in.
Dissolved TCR α and β chain are quickly mixed in 5M urea, 0.4M arginine, 20mM Tris with the mass ratio of 1:1 (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Solution is placed in dialysis (4 DEG C) in the deionized water of 10 times of volumes afterwards, changes deionized water into buffer (20mM after 12 hours Tris, pH 8.0) continue at 4 DEG C of dialysis 12 hours.Solution after the completion of dialysis after 0.45 μM of membrane filtration, by yin from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains the successful α and β dimer of renaturation TCR is confirmed by SDS-PAGE glue.TCR then pass through gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) it is further purified.TCR purity after purification is greater than 90% by SDS-PAGE measurement, and concentration is by BCA method It determines.The SDS-PAGE glue figure for the sTCR that the present invention obtains is as shown in Figure 6.
Embodiment 4 combines characterization
BIAcore analysis
This example demonstrated soluble TCR molecules of the present invention can be special with ILWRLGATI-HLA A0201 compound The opposite sex combines.
Using BIAcore T200 real-time analyzer detect TCR molecule obtained in embodiment 3 and embodiment 5 with The combination activity of ILWRLGATI-HLA A0201 compound.It is slow that coupling is added in the antibody (GenScript) of anti-Streptavidin Antibody, is then flowed through the CM5 chip activated in advance with EDC and NHS, made by fliud flushing (10mM sodium-acetate buffer, pH 4.77) Antibody is fixed on chip surface, finally closes unreacted activating surface with the hydrochloric acid solution of ethanol amine, completes coupling process, even Connection horizontal about 15,000RU.
The Streptavidin of low concentration is set to flow through the chip surface of coated antibody, then by ILWRLGATI-HLA A0201 compound flows through sense channel, and another channel is as reference channel, then by the biotin of 0.05mM with the stream of 10 μ L/min Speed flows through chip 2min, closes the remaining binding site of Streptavidin.
The preparation process of above-mentioned ILWRLGATI-HLA A0201 compound is as follows:
A. it purifies
The E.col i bacterium solution for collecting 100ml inducing expression heavy chain or light chain uses 10ml after 4 DEG C of 8000g are centrifuged 10min PBS washing thalline is primary, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) later Thallus is resuspended in concussion, and rotates in room temperature and be incubated for 20min, and later in 4 DEG C, 6000g is centrifuged 15min, discards supernatant, collection is forgiven Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated for 5min；Add 30ml The BugBuster of 10 times of dilution is mixed, and 4 DEG C of 6000g are centrifuged 15min；It discards supernatant, 30ml is added to dilute 10 times of BugBuster Inclusion body is resuspended, mixes, 4 DEG C of 6000g are centrifuged 15min, are repeated twice, and add 30ml 20mM Tris-HCl pH 8.0 that packet is resuspended Contain body, mix, 4 DEG C of 6000g are centrifuged 15min, finally dissolve inclusion body, SDS-PAGE detection with 20mM Tris-HCl 8M urea Inclusion body purity, BCA kit survey concentration.
B. renaturation
The small peptide ILWRLGATI (Beijing SBS Genetech gene technology Co., Ltd) of synthesis is dissolved in DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain 8M urea, 20mM Tris pH 8.0,10mM DTT dissolve, and are added before renaturation 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA are further denaturalized.Renaturation is added with 25mg/L (final concentration) in ILWRLGATI peptide Buffer (0.4M L-arginine, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced form Glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, Heavy chain is added in three times, and 8h/ times), renaturation carries out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detection renaturation success.
C. it is purified after renaturation
Make dialysis with the 20mM Tris pH 8.0 of 10 volumes to replace renaturation buffer, at least replacement buffer comes twice Sufficiently reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volume).Instrument (the general electricity of GE is purified using Akta Gas company), the 0-400mM NaCl linear gradient liquid that 20mM Tris pH 8.0 is prepared elutes albumen, and pMHC is about in 250mM It is eluted at NaCl, collects all peak components, SDS-PAGE detects purity.
D. biotinylation
It with Mill ipore super filter tube by the pMHC molecular concentration of purifying, while being 20mM Tris pH by buffer exchange 8.0, biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D- is then added Biotin, 100 μ g/ml BirA enzymes (GST-BirA), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detects biotinylation Completely.
E. the compound after purifying biological element
PMHC molecular concentration after being marked biotinylation with Mi llipore super filter tube is to 1ml, using Gel filtration The pMHC for analysing purifying biological element purifies instrument (GE General Electric Co. Limited) using Akta, is pre-equilibrated with filtered PBS HiPrepTM16/60S200HR column (GE General Electric Co. Limited), load 1ml concentrated biotinylation pMHC molecule, is then used PBS is with the elution of 1ml/min flow velocity.Biotinylated pMHC molecule occurs in about 55ml as unimodal elution.Merging contains egg The component of white matter is concentrated with Mill ipore super filter tube, and BCA method (Thermo) measures protein concentration, and protease is added and inhibits The packing of biotinylated pMHC molecule is stored in -80 DEG C by agent cocktail (Roche).
Using BIAcore Evaluation software computational dynamics parameter, obtain the TCR molecule of solubility of the invention with The kinetic profile that ILWRLGATI-HLA A0201 compound combines is as shown in Figure 7.Map shows that the present invention obtains solvable Property TCR molecule can be in conjunction with ILWRLGATI-HLA A0201 compound.Meanwhile the present invention also is had detected using the above method The combination of soluble TCR molecule and other several irrelevant antigen small peptides and HLA compound is active, as the result is shown TCR of the present invention Molecule and other irrelevant antigens are without combination.
5 LMP1 specific t-cell receptor slow virus of embodiment packaging transfects LMP1TCR with primary T cells
(a) (Express-In-mediated transient is transiently transfected by the quick mediation of 293T/17 cell Transfection slow virus) is prepared
Utilize the slow virus of gene of the third generation slow virus packaging system packaging containing TCR needed for encoding.It is situated between using quick Lead transient transfection (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)) with 4 kinds of plasmids, (one kind containing pLenti-LMP1TRA-2A-TRB-IRES-NGFR described in embodiment 2 is slow Viral vectors, and 3 kinds of plasmids containing other components necessary to building infectiousness but non-replicating lentiviral particle) transfection 293T/17 cell.
To be transfected, the 0th day kind cell, in 15 cm dishes, kind upper 1.7 × 107A 293T/17 cell makes thin Born of the same parents are evenly distributed on culture dish, and convergence degree is slightly above 50%.1st day transfected plasmids pack pLenti-LMP1 TRA-2A- TRB-IRES-NGFR and pLenti-eGFP pseudovirus, by the above expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV It is mixed with pMD.2G, the dosage of a 15 cm diameter plates is as follows: 22.5 micrograms: 15 micrograms: 15 micrograms: 7.5 micrograms.Transfection The ratio of reagent PEI-MAX and plasmid is 2:1, and the usage amount of each plate is 114.75 micrograms.Concrete operations are as follows: expression matter 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 liquor；Corresponding amount PEI is taken to mix with 1800 microlitres of OPTI-MEM culture mediums Even, being stored at room temperature 5 minutes becomes PEI mixed liquor.DNA mixed liquor and PEI mixed liquor are mixed and are being stored at room temperature 30 Minute, then 3150 microlitres of OPTI-MEM culture mediums are added, it is added to has been converted into 11.25 milliliters of OPTI-MEM after mixing 293T/17 cell in, shake gently culture dish, make culture medium be uniformly mixed, 37 DEG C/5%CO2Lower culture.It is small to transfect 5-7 When, remove transfection media, change into containing 10% fetal calf serum DMEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt)) complete medium, 37 DEG C/5%CO2Lower culture.Training of the collection containing wrapped slow virus in 3rd and the 4th day Support base supernatant.For the slow virus of harvest packaging, collected culture supernatant 3000g is centrifuged 15 minutes removal cell fragments, It filters through 0.45 micron filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB), finally uses again The concentration tube (Merck Mi Libo (Merck Mill ipore)) of 50KD interception, catalog number (Cat.No.) UFC905096) it is concentrated, it removes Most of supernatant is removed, 1 milliliter is finally concentrated to, is frozen for -80 DEG C after equal part packing.Pseudovirus sample is taken to carry out virus titer survey Fixed, step is referring to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit specification.It is used as control, while also packet turns The pseudovirus of pLenti-eGFP.
(b) with the lentiviruses transduction primary T cells containing LMP1 specific t-cell receptor gene
CD8 is separated to from the blood of healthy volunteer+T cell, then with the lentiviruses transduction packed.It is thin to count these Born of the same parents, in 48 orifice plates, containing 30IU/ml IL-2 containing 10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT with 1 × 10 in 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture mediums)6It is a thin Born of the same parents/milliliter (0.5 milliliter/hole) with 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 kit is measured, it is added in the ratio of MOI=10 dense The slow virus of the PRAME specific t-cell receptor gene of contracting, 32 DEG C, 900g centrifugation infection 1 hour.It is removed after infection slow Cell is resuspended with 1640 culture mediums containing 30IU/ml IL-2 containing 10%FBS in virus infection liquid, and 37 DEG C/5%CO2Lower training It supports 3 days.Transduction counted cell, diluting cells to 0.5 × 10 after 3 days6A cells/ml.The cell of counting in every two days, replacement Or the fresh culture containing 30IU/ml IL-2 is added, maintain cell 0.5 × 106-1×106A cells/ml.From the 3rd It begins through flow cytometry cell, for function test (for example, the ELISPOT of IFN-γ release since the 5th day With non-radioactive cell toxicity detection).Since the 10th day or when cell slows down division and size becomes smaller, stored frozen etc. Point cell, at least 4 × 106A cell/pipe (1 × 107A cells/ml, 90%FBS/10%DMSO).
6 cell-stimulating functional verification of embodiment
ELISPOT scheme
Following tests is carried out to prove the activation that target cell specificity reacts of T cell of TCR- transduction.It utilizes Readout of the IFN-γ yield of ELISPOT testing inspection as t cell activation.
Reagent
Test medium: 10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), (Ji Bu can by RPMI1640 Company (Gibco), catalog number (Cat.No.) C11875500bt)
Washing buffer: 0.01M PBS/0.05% polysorbas20
PBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT)
96 orifice plate of PVDF ELISPOT (Merck Mi Libo (Merck Mil lipore), catalog number (Cat.No.) MSIPS4510)
People's IFN-γ ELISPOT PVDF- enzyme reagent kit (BD) (captures equipped with required every other reagent and detection is anti- Body, Streptavidin-alkaline phosphatase and BCIP/NBT solution)
Method
Target cell preparation
The target cell of the present embodiment is the B-lymphoblastoid cell lines (LCL) of Epstein-Barr viral (EBV) conversion. B95-8 cell induces culture medium supernatant of the production containing EBV, 4 DEG C/600g centrifugation 10 through myristoyl acetic acid phorbol ester (TPA) Minute removal impurity, then crosses 0.22 micron filter, and equal part dispenses -70 DEG C of preservations.It is HLA-A11/A02/A24 from genotype The peripheral blood lymphocytes (PBL) of the healthy volunteer of (including homozygote and heterozygote), taking 10 milliliters of concentration is 2 × 107/ milli The PBL suspension risen is added after cyclosporin in 25 square centimeters of culture bottle in 37 DEG C/CO2It is small that 1 is incubated in incubator When, quick-thawing portion EBV, by 1/10 dilution be added in above-mentioned cell, gently shake up and culture bottle is uprightly placed in 37 DEG C/ CO2It is cultivated in incubator.10 milliliters of culture mediums of addition continue to cultivate after culture 12 days, and further expansion culture is gone forward side by side after about 30 days Row flow cytometer detection, wherein CD19+CD23hiCD58+For B-lymphoblastoid cell lines (LCL).This ELISPOT is tested with HLA- A02LCL is target cell.
Effector cell's preparation
Effector cell's (T cell) of this test is to express LMP1 specificity TCR through flow cytometry in embodiment 3 CD8+T cell, and with the CD8 of same volunteer+T is as negative control effector cell.With AntiCD3 McAb/CD28 coating pearl (T cell expansion Increase object, lifetechnologies) stimulation T cell, with lentiviruses transduction (the foundation implementation for carrying LMP1 specificity TCR gene Example 3), in the 1640 culture mediums amplification containing 10%FBS containing 30IU/ml IL-2 until 9-12 days after transduction, then by these Cell is placed in test medium, and 300g room temperature is centrifuged 10 minutes and is washed.Then by cell with 2 × required final concentration is resuspended In test medium.Same processing negative control effector cell.
ELISPOT
According to the specification that manufacturer provides, prepare orifice plate as described below: with 10 milliliters of sterile PBS of every block of plate by 1:200 It dilutes anti-human IFN-γ and captures antibody, 100 microlitres of dilution is then captured into antibody etc. point, each hole is added.Orifice plate is incubated at 4 DEG C Overnight.After incubation, orifice plate is washed to remove extra capture antibody.The RPMI 1640 that 10%FBS is contained in 100 microlitres/hole is added Culture medium simultaneously incubates orifice plate 2 hours at room temperature to close orifice plate.Then culture medium is washed away from orifice plate, by flicking on paper The washing buffer of any remnants is removed with ELISPOT orifice plate is patted.
LMP1 CD8+(T cell of TCR transduction of the present invention, effector cell are also named as T cell in the present invention “IL14CD8+TCELL”)、CD8+T cell (negative control effector cell) and LCL-A02/A11 (target cell) are according to 3 institute of embodiment Preparation is stated, and corresponding small peptide is added in corresponding experimental group, wherein PX224 is LMP1PX224129-137ILWRLGATI small peptide, Remaining is non-LMP1 TCR specific bond small peptide.
Then all components of test are added by ELISPOT orifice plate using following sequence:
130 microlitres of target cells, 77000 cells/mls (obtain about 10000 target cell/holes in total).
50 microlitres of effector cells's (1000 LMP1 TCR positive T cells).
20 microlitre 10-5Other small peptide solution (final concentration of 10 of the LMP1PX224129-137ILWRLGATI/ of mol/L-6 Mol/L).
All holes prepare addition in triplicate.
Then orifice plate (37 DEG C/5%CO2) second day overnight is incubated, culture medium is abandoned, is washed orifice plate 2 times with distilled water, then use Washing buffer is washed 3 times, is patted on paper handkerchief to remove remaining washing buffer.Then dilute with the PBS containing 10%FBS Detection primary antibody is released, each hole is added by 100 microlitres/hole.It incubates orifice plate 2 hours, then is washed 3 times with washing buffer at room temperature, Orifice plate is patted on paper handkerchief to remove excessive washing buffer.
Streptavidin-alkaline phosphatase is diluted by 1:100 with the PBS containing 10%FBS, by 100 microlitres of diluted chains Mould Avidin-alkaline phosphatase is added each hole and incubates orifice plate 1 hour at room temperature.Then 3 PBS are washed with washing buffer Washing 2 times, pats orifice plate on paper handkerchief to remove excessive washing buffer and PBS.Kit is added after washing to provide 100 microlitres/hole of BCIP/NBT solution develop.It is protected from light during development with masking foil covering orifice plate, stands 5-15 minutes. The spot of conventional detection development orifice plate during this period, determines the Best Times for terminating reaction.Remove BCIP/NBT solution and with pair It steams water and rinses orifice plate to stop developing reaction, then drying removes orifice plate bottom, be dried at room temperature for orifice plate until each hole It is completely dried, recycles immunodotting plate count meter (CTL, Celltech Ltd. (Cellular Technology Limited the)) spot that counterdie is formed in counting orifice.
As a result
The T cell (as described above) for examining LMP1 TCR transduction is tested to load LMP1PX224129- by ELISPOT The IFN-γ release that the target cell of 137ILWRLGATI small peptide and the target cell of non-specific small peptide react.Utilize graphpad Prism6 draws the ELSPOT amount of speckle observed in each hole.
Experimental results are shown in figure 8, individual LMP1 CD8+T cell (effector cell) or LCL cell (target cell) addition Corresponding small peptide release IFN-γ is seldom.
LMP1 CD8+T cell (effector cell) can with addition PX224 LCL-A02/A11 cell react release compared with More IFN-γ.
LMP1 CD8+T cell (effector cell) discharges very the IFN-γ for the LCL-A02/A11 cell for adding other small peptides It is few.
CD8+T cell (negative control effector cell) discharges very the IFN-γ of the LCL-A02/A11 cell of addition PX224 It is few.
To sum up, the T cell of transduction TCR of the present invention has good activating reaction to the target cell for loading its special small peptide, The target cell of target cell and the non-specific small peptide of load to unsupported corresponding small peptide does not have activating reaction.
7 cell killing functional verification of embodiment
The test is the colorimetric alternate test of 51Cr release cell toxicity test, quantitative determines the cream discharged after cell cracking Acidohydrogenase (LDH).The LDH of release in the medium is detected using the enzyme reaction of coupling in 30 minutes, LDH can in enzyme reaction A kind of tetrazolium salts (INT) are made to be converted into red formazan (formazan).The amount of the red product of generation and the cell number of cracking It is directly proportional.Collection 490nm visible light extinction Value Data can be collected with 96 hole read plates of standard.
Material
CytoToxNon-radioactive cell toxicity detection (Pu Luomaige company, G1780) contains substrate mixture, test Buffer, cracked solution and stop buffer.
Test medium: 5%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), 1% penicillin/streptomycin (Ji Bu can company, catalogue Number 15070-063).
Micropore round bottom tissue culturing plates with 96 hole (Nucor Corporation (Nunc), catalog number (Cat.No.) 163320)
96 hole immuno plate Maxisorb (Nucor Corporation (Nunc), catalog number (Cat.No.) 442404)
Method
Target cell preparation
Target cell LCL used in the test is as the target cell preparation method in aforementioned ELISPOT scheme.It is trained in test Support in base and prepare target cell: target cell concentration is adjusted to 3 × 105A/milliliter, every hole take 50 microlitres to obtain 1.5 × 104A cell/ Hole.
Effector cell's preparation
Effector cell's (T cell) of this test is to express LMP1 specificity TCR through flow cytometry in embodiment 3 CD8+T cell.Effector cell and target cell ratio use 10:1,5:1,2.5:1,1.25:1 (to be diluted to 3 × 10 if 10:16 A/milliliter, every hole take 50 microlitres to obtain 1.5 × 105A cells/well).
The preparation of small peptide solution
LMP1PX224129-137ILWRLGATI (or other non-specific small peptides) small peptide with containing 5%FBS without phenol red 1640 culture medium of RPMI is diluted to 10-5Working solution in range, ultimate density 10 after making it be added to experimental group-6M。
(a) effector cell is detected by the small peptide that target cell loads various concentration and kills ability
Test prepares
All components of test are added by micropore round bottom tissue culturing plates with 96 hole using following sequence:
Each hole is added in -50ul target cell (preparation as described above)
Each hole is added in -50ul effector cell (preparation as described above)
Each hole is added in -12ul small peptide solution
- 8ul culture fills into hole (do not load small peptide experimental group and directly mend 20ul culture medium).
Prepare control group as described below:
Small peptide experimental group is not loaded: containing 50ul effector cell and 50ul target cell.
The spontaneous release of effector cell: only 50ul effector cell.
The spontaneous release of target cell: only 50ul target cell.
Target cell maximum release: only 50ul target cell.
Reagent culture medium control: only 120ul culture medium.
All holes prepare in triplicate, and final volume is 120ul (inadequate is supplied with culture medium).
37 DEG C incubate 24 hours.Before collecting all hole supernatants, by target cell maximum release control wells in -70 DEG C of placement cells About 30 minutes, then melt 15 minutes at 37 DEG C, so that target cell all cracks.
It is centrifuged plate 4 minutes in 250g.The 50ul supernatant in each hole of test panel is transferred to 96 hole immuno plates The corresponding aperture of Maxisorb plate.Using test buffer (12ml) reconstituted substrate mixture, then plus 50ul is to each hole of plate.It is flat Plate close the lid after at shady place incubation at room temperature 30 minutes.Each hole of plate is added to terminate reaction in 50ul stop bath.It is added The absorbance of record 490nm is counted after stop bath in 1 hour.
Calculated result
Culture medium is deducted from all absorbance values of release group from experimental group, the spontaneous release group of target cell and effector cell The absorbance value of background.
It brings the corrected value obtained among the above into following formula, calculates each effect target than generated cytotoxicity Percentage.
Cytotoxicity=100 % × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximum-target cell from Hair)
As a result
By the non-radioactive cell toxicity detection T cell (as described above) for examining LMP1 TCR transduction to load The LDH release that the target cell of LMP1PX224129-137ILWRLGATI small peptide and non-specific small peptide reacts.It utilizes Graphpad prism6 draws 490nm visible light light absorption value in each hole.
Experimental data statistical result as shown in figure 9, be in load LMP1PX224129-137ILWRLGATI small peptide concentration 10-6When M effect target ratio is 10:1 and 5:1, LMP1 CD8+T cell is obvious to the lethal effect of target cell LCL-A02/A11；To not The target cell LCL-A02/A11 lethal effect for loading small peptide or loading non-specificity small peptide is unobvious.Homologous CD8+T cell is to negative The LCL-A02/A11 lethal effect for carrying LMP1PX224129-137ILWRLGATI small peptide is unobvious.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>a kind of identification is derived from the TCR of EBV memebrane protein LMP1 antigen
<130> P2017-1290
<160> 29
<170> PatentIn version 3.5
<210> 1
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223>TCR α chain variable domain
<400> 1
Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu Gly
1 5 10 15
Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu
20 25 30
Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr
35 40 45
Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val
50 55 60
Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr
65 70 75 80
Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn Phe Gly Asn Glu
85 90 95
Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile Ile Pro
100 105 110
<210> 2
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223>TCR α chain variable domain
<400> 2
ggaatacaag tggagcagag tcctccagac ctgattctcc aggagggagc caattccacg 60
ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 120
ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 180
gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 240
gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 300
gggactggaa caagactcac catcataccc 330
<210> 3
<211> 251
<212> PRT
<213> artificial sequence
<220>
<223>TCR α chain
<400> 3
Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu Gly
1 5 10 15
Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu
20 25 30
Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr
35 40 45
Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val
50 55 60
Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr
65 70 75 80
Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn Phe Gly Asn Glu
85 90 95
Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile Ile Pro Asn Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
195 200 205
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn
210 215 220
Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe
225 230 235 240
Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 4
<211> 753
<212> DNA
<213> artificial sequence
<220>
<223>TCR α chain
<400> 4
ggaatacaag tggagcagag tcctccagac ctgattctcc aggagggagc caattccacg 60
ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 120
ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 180
gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 240
gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 300
gggactggaa caagactcac catcataccc aatatccaga accctgaccc tgccgtgtac 360
cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 420
caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 480
gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 540
tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 600
ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 660
aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 720
aatctgctca tgacgctgcg gctgtggtcc agc 753
<210> 5
<211> 114
<212> PRT
<213> artificial sequence
<220>
<223>TCR β chain variable domain
<400> 5
Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly
1 5 10 15
Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr
20 25 30
Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala
35 40 45
Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys
50 55 60
Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
65 70 75 80
Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala
85 90 95
Arg Arg Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr
100 105 110
Val Val
<210> 6
<211> 342
<212> DNA
<213> artificial sequence
<220>
<223>TCR β chain variable domain
<400> 6
ggtgctgtcg tctctcaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60
atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120
aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180
ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240
gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctcg gaggctcggc 300
actgaagctt tctttggaca aggcaccaga ctcacagttg ta 342
<210> 7
<211> 291
<212> PRT
<213> artificial sequence
<220>
<223>TCR β chain
<400> 7
Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly
1 5 10 15
Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr
20 25 30
Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala
35 40 45
Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys
50 55 60
Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
65 70 75 80
Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala
85 90 95
Arg Arg Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr
100 105 110
Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe
115 120 125
Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val
130 135 140
Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp
145 150 155 160
Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro
165 170 175
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser
180 185 190
Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe
195 200 205
Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
210 215 220
Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
225 230 235 240
Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val
245 250 255
Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu
260 265 270
Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg
275 280 285
Lys Asp Phe
290
<210> 8
<211> 873
<212> DNA
<213> artificial sequence
<220>
<223>TCR β chain
<400> 8
ggtgctgtcg tctctcaaca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60
atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120
aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180
ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240
gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctcg gaggctcggc 300
actgaagctt tctttggaca aggcaccaga ctcacagttg tagaggacct gaacaaggtg 360
ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420
gccacactgg tgtgcctggc cacaggcttc ttccccgacc acgtggagct gagctggtgg 480
gtgaatggga aggaggtgca cagtggggtc agcacggacc cgcagcccct caaggagcag 540
cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 600
tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660
gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720
ggtagagcag actgtggctt tacctcggtg tcctaccagc aaggggtcct gtctgccacc 780
atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt cagcgccctt 840
gtgttgatgg ccatggtcaa gagaaaggat ttc 873
<210> 9
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223>antigen small peptide
<400> 9
Ile Leu Trp Arg Leu Gly Ala Thr Ile
1 5
<210> 10
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> α CDR1
<400> 10
Asp Ser Val Asn Asn
1 5
<210> 11
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> α CDR2
<400> 11
Ile Pro Ser Gly Thr
1 5
<210> 12
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> α CDR3
<400> 12
Cys Ala Val Gly Ser Asn Phe Gly Asn Glu Lys Leu Thr Phe
1 5 10
<210> 13
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> β CDR1
<400> 13
Asp Phe Gln Ala Thr Thr
1 5
<210> 14
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> β CDR2
<400> 14
Ser Asn Glu Gly Ser Lys Ala
1 5
<210> 15
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> β CDR3
<400> 15
Cys Ser Ala Arg Arg Leu Gly Thr Glu Ala Phe Phe
1 5 10
<210> 16
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> α CDR1
<400> 16
gactctgtga acaat 15
<210> 17
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> α CDR2
<400> 17
attccctcag ggaca 15
<210> 18
<211> 42
<212> DNA
<213> artificial sequence
<220>
<223> α CDR3
<400> 18
tgtgctgtgg gatctaactt tggaaatgag aaattaacct tt 42
<210> 19
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> β CDR1
<400> 19
gactttcagg ccacaact 18
<210> 20
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> β CDR2
<400> 20
tccaatgagg gctccaaggc c 21
<210> 21
<211> 36
<212> DNA
<213> artificial sequence
<220>
<223> β CDR3
<400> 21
tgcagtgctc ggaggctcgg cactgaagct ttcttt 36
<210> 22
<211> 271
<212> PRT
<213> artificial sequence
<220>
<223>with the TCR α chain of leader sequence
<400> 22
Met Lys Arg Ile Leu Gly Ala Leu Leu Gly Leu Leu Ser Ala Gln Val
1 5 10 15
Cys Cys Val Arg Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile
20 25 30
Leu Gln Glu Gly Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser
35 40 45
Val Asn Asn Leu Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile
50 55 60
Asn Leu Phe Tyr Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser
65 70 75 80
Ala Thr Thr Val Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser
85 90 95
Ser Gln Thr Thr Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn
100 105 110
Phe Gly Asn Glu Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile
115 120 125
Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp
130 135 140
Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser
145 150 155 160
Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp
165 170 175
Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
180 185 190
Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn
195 200 205
Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
210 215 220
Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu
225 230 235 240
Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys
245 250 255
Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265 270
<210> 23
<211> 813
<212> DNA
<213> artificial sequence
<220>
<223>with the TCR α chain of leader sequence
<400> 23
atgaagagga tattgggagc tctgctgggg ctcttgagtg cccaggtttg ctgtgtgaga 60
ggaatacaag tggagcagag tcctccagac ctgattctcc aggagggagc caattccacg 120
ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 180
ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 240
gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 300
gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 360
gggactggaa caagactcac catcataccc aatatccaga accctgaccc tgccgtgtac 420
cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480
caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540
gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600
tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660
ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720
aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780
aatctgctca tgacgctgcg gctgtggtcc agc 813
<210> 24
<211> 305
<212> PRT
<213> artificial sequence
<220>
<223>with the TCR β chain of leader sequence
<400> 24
Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ser Gly Leu Gly Ala
1 5 10 15
Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly Thr Ser
20 25 30
Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe
35 40 45
Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser
50 55 60
Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys
65 70 75 80
Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr
85 90 95
Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Arg
100 105 110
Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val
115 120 125
Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
130 135 140
Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu
145 150 155 160
Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
165 170 175
Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys
180 185 190
Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu
195 200 205
Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
210 215 220
Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
225 230 235 240
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
245 250 255
Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser
260 265 270
Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala
275 280 285
Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp
290 295 300
Phe
305
<210> 25
<211> 915
<212> DNA
<213> artificial sequence
<220>
<223>with the TCR β chain of leader sequence
<400> 25
atgctgctgc ttctgctgct tctggggcca ggctccgggc ttggtgctgt cgtctctcaa 60
catccgagct gggttatctg taagagtgga acctctgtga agatcgagtg ccgttccctg 120
gactttcagg ccacaactat gttttggtat cgtcagttcc cgaaacagag tctcatgctg 180
atggcaactt ccaatgaggg ctccaaggcc acatacgagc aaggcgtcga gaaggacaag 240
tttctcatca accatgcaag cctgaccttg tccactctga cagtgaccag tgcccatcct 300
gaagacagca gcttctacat ctgcagtgct cggaggctcg gcactgaagc tttctttgga 360
caaggcacca gactcacagt tgtagaggac ctgaacaagg tgttcccacc cgaggtcgct 420
gtgtttgagc catcagaagc agagatctcc cacacccaaa aggccacact ggtgtgcctg 480
gccacaggct tcttccccga ccacgtggag ctgagctggt gggtgaatgg gaaggaggtg 540
cacagtgggg tcagcacgga cccgcagccc ctcaaggagc agcccgccct caatgactcc 600
agatactgcc tgagcagccg cctgagggtc tcggccacct tctggcagaa cccccgcaac 660
cacttccgct gtcaagtcca gttctacggg ctctcggaga atgacgagtg gacccaggat 720
agggccaaac ccgtcaccca gatcgtcagc gccgaggcct ggggtagagc agactgtggc 780
tttacctcgg tgtcctacca gcaaggggtc ctgtctgcca ccatcctcta tgagatcctg 840
ctagggaagg ccaccctgta tgctgtgctg gtcagcgccc ttgtgttgat ggccatggtc 900
aagagaaagg atttc 915
<210> 26
<211> 196
<212> PRT
<213> artificial sequence
<220>
<223>sTCR α chain
<400> 26
Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu Gly
1 5 10 15
Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu
20 25 30
Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr
35 40 45
Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val
50 55 60
Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr
65 70 75 80
Asp Ser Gly Val Tyr Phe Cys Ala Val Gly Ser Asn Phe Gly Asn Glu
85 90 95
Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile Ile Pro Tyr Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr
195
<210> 27
<211> 588
<212> DNA
<213> artificial sequence
<220>
<223>sTCR α chain
<400> 27
ggtattcagg ttgaacagag tcctccagac ctgattctcc aggagggagc caattccacg 60
ctgcggtgca atttttctga ctctgtgaac aatttgcagt ggtttcatca aaacccttgg 120
ggacagctca tcaacctgtt ttacattccc tcagggacaa aacagaatgg aagattaagc 180
gccacgactg tcgctacgga acgctacagc ttattgtaca tttcctcttc ccagaccaca 240
gactcaggcg tttatttctg tgctgtggga tctaactttg gaaatgagaa attaaccttt 300
gggactggaa caagactcac catcataccc tatatccaga atccggaccc ggccgtttat 360
cagctgcgtg atagcaaaag cagcgataaa agcgtgtgcc tgttcaccga ttttgatagc 420
cagaccaacg tgagccagag caaagatagc gatgtgtaca tcaccgataa atgcgtgctg 480
gatatgcgca gcatggattt caaaagcaat agcgcggttg cgtggagcaa caaaagcgat 540
tttgcgtgcg cgaacgcgtt taacaacagc atcatcccgg aagatacg 588
<210> 28
<211> 244
<212> PRT
<213> artificial sequence
<220>
<223>sTCR β chain
<400> 28
Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly
1 5 10 15
Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr
20 25 30
Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala
35 40 45
Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys
50 55 60
Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr
65 70 75 80
Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala
85 90 95
Arg Arg Leu Gly Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr
100 105 110
Val Val Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe
115 120 125
Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val
130 135 140
Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp
145 150 155 160
Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro
165 170 175
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser
180 185 190
Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe
195 200 205
Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
210 215 220
Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
225 230 235 240
Gly Arg Ala Asp
<210> 29
<211> 732
<212> DNA
<213> artificial sequence
<220>
<223>sTCR β chain
<400> 29
ggtgcagttg tttctcagca tccgagctgg gttatctgta agagtggaac ctctgtgaag 60
atcgagtgcc gttccctgga ctttcaggcc acaactatgt tttggtatcg tcagttcccg 120
aaacagagtc tcatgctgat ggcaacttcc aatgagggct ccaaggccac atacgagcaa 180
ggcgtcgaga aggacaagtt tctcatcaac catgcaagcc tgaccttgtc cactctgaca 240
gtgaccagtg cccatcctga agacagcagc ttctacatct gcagtgctcg gaggctcggc 300
actgaagctt tctttggaca aggcaccaga ctcacagttg tagaagatct gaaaaatgtg 360
tttccgccgg aagtcgcggt gttcgaaccg tcggaagccg aaattagcca tacccagaaa 420
gcaacgctgg tgtgcctggc taccggcttt tatccggatc atgtggaact gtcctggtgg 480
gttaacggca aagaagtgca ctcaggtgtt tgtacggatc cgcagccgct gaaagaacaa 540
ccggcactga atgactcgcg ttatgctctg agttcccgtc tgcgcgttag cgccaccttc 600
tggcaggatc cgcgtaacca ctttcgctgt caggtccaat tctacggcct gtccgaaaat 660
gatgaatgga cccaggaccg tgcaaaaccg gtcacgcaaa tcgtgtcagc agaagcttgg 720
ggtcgtgcag at 732

Claims (10)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be with ILWRLGATI-HLAA0201 compound knot It closes；Preferably, the TCR includes TCR α chain variable domain and TCR β chain variable domain, which is characterized in that the TCR α chain variable domain CDR3 amino acid sequence be CAVGSNFGNEKLTF (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domain Amino acid sequence be CSARRLGTEAFF (SEQ ID NO:15)；
It is highly preferred that 3 complementary determining regions (CDR) of the TCR α chain variable domain are as follows:
αCDR1-DSVNN(SEQ ID NO:10)
αCDR2-IPSGT(SEQ ID NO:11)
αCDR3-CAVGSNFGNEKLTF(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domain are as follows:
βCDR1-DFQATT(SEQ ID NO:13)
βCDR2-SNEGSKA(SEQ ID NO:14)
βCDR3-CSARRLGTEAFF(SEQ ID NO:15)。
2. TCR as described in claim 1, which is characterized in that described it includes TCR α chain variable domain and TCR β chain variable domain TCR α chain variable domain is the amino acid sequence for having at least 90% sequence identity with SEQ ID NO:1；And/or the TCR β chain Variable domain is the amino acid sequence for having at least 90% sequence identity with SEQ ID NO:5.
3. TCR as described in claim 1, which is characterized in that the α chain of the TCR and/or the end C- or N- of β chain are combined with Conjugate；Preferably, the conjugate in conjunction with the T cell receptor is detectable marker, therapeutic agent, PK modified part or appoints The combination of what these substance；Preferably, the therapeutic agent is anti-CD 3 antibodies.
4. a kind of multivalent TCR complex, which is characterized in that contain at least two TCR molecule, and at least one TCR therein Molecule is TCR described in any one of the claims.
5. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules include to encode TCR described in any of the above-described claim points The nucleic acid sequence or its complementary series of son；
Preferably, the nucleic acid molecules include the nucleotide sequence SEQ ID NO:2 of coding TCR α chain variable domain；And/or
The nucleic acid molecules include the nucleotide sequence SEQ ID NO:6 of coding TCR β chain variable domain.
6. a kind of carrier, which is characterized in that the carrier contains nucleic acid molecules described in claim 5；Preferably, described Carrier is viral vectors；It is highly preferred that the carrier is slow virus carrier.
7. a kind of isolated host cell, which is characterized in that contain the carrier described in claim 6 in the host cell Or nucleic acid molecules described in the claim 5 of external source are integrated in chromosome.
8. a kind of cell, which is characterized in that institute in nucleic acid molecules described in the cell transduction claim 5 or claim 6 State carrier；Preferably, the cell is T cell or stem cell.
9. a kind of pharmaceutical composition, which is characterized in that the composition contains pharmaceutically acceptable carrier and claim TCR described in any one of 1-3, TCR compound described in claim 4, nucleic acid molecules described in claim 5 or power Benefit requires cell described in 8.
10. TCR compound or right described in T cell receptor of any of claims 1-3 or claim 4 It is required that the purposes of cell described in 8, which is characterized in that be used to prepare the drug for the treatment of tumour or autoimmune disease.
CN201710571907.7A 2017-07-13 2017-07-13 TCR (T cell receptor) for recognizing LMP1 antigen derived from EBV (Epstein-Barr Virus) membrane protein Active CN109251244B (en)

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CN114891109A (en) * 2022-05-20 2022-08-12 华东师范大学 Method for renaturation of trastuzumab single-chain antibody and single-chain antibody with sequence homology thereof

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