CN106632660A - T cell receptor (TCR) capable of identifying NY-ESO-1 antigen short-peptides - Google Patents

T cell receptor (TCR) capable of identifying NY-ESO-1 antigen short-peptides Download PDF

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CN106632660A
CN106632660A CN201610958444.5A CN201610958444A CN106632660A CN 106632660 A CN106632660 A CN 106632660A CN 201610958444 A CN201610958444 A CN 201610958444A CN 106632660 A CN106632660 A CN 106632660A
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CN106632660B (en
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李懿
相瑞瑞
吴万里
林燕梅
李思韵
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Xiangxue Life Science Technology (Guangdong) Co.,Ltd.
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GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
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Abstract

The invention provides a T cell receptor (TCR) capable of being specifically combined with short-peptide SLLMWITQC derived from a NY-ESO-1 antigen. The antigen short-peptide SLLMWITQC and HLA A0201 can form a compound and are presented to the surfaces of cells together. The invention further provides nucleic acid molecules for encoding the TCR and a carrier comprising the nucleic acid molecules. In addition, the invention further provides cells for transducing the TCR.

Description

The TCR of identification NY-ESO-1 antigen small peptides
Technical field
The present invention relates to the TCR from NY-ESO-1 antigen small peptides is capable of identify that, the invention further relates to the above-mentioned TCR that transduces comes Acquisition NY-ESO-1 specificity T cell, and they prevention and treatment NY-ESO-1 relevant diseases in purposes.
Background technology
NY-ESO-1 belongs to tumor-testis antigen (Cancer-Testis Antigen, CTA) family, can be in testis, ovum Express in the tumor tissues of nest tissue and number of different types, and do not express in other normal structures, be a species specificity Stronger tumour antigen.NY-ESO-1 is a kind of endogenous antigen, and after generating in the cell micromolecule polypeptide is degraded to, and with MHC (main histocompatibility complex) molecule combines to form compound, is presented to cell surface.SLLMWITQC be derived from The small peptide of NY-ESO-1 antigens, is a kind of target of NY-ESO-1 treating correlative diseases.Research shows that NY-ESO-1 is various swollen There is expression in tumor tissue, in neuroblastoma (Rodolfo M, et al., Cancer Res, 2003,63 (20):6948- 6955), sarcoma (Jungbhth A A et al., Int J Cancer, 200l, 94 (2):252-256), malignant mela noma (Barrow C,et al.,Clin Cancer Res,2006,12(3Pt 1):There is very high expression in 764-771), while Prostate cancer, carcinoma of urinary bladder, breast cancer, Huppert's disease, hepatocellular carcinoma, oral squamous cell carcinomas (Ries J, et al., Anticancer RES,2009,29(12):5125-5130) and the cancer of the esophagus (Fujita S, Clin Cancer Res, 2004,10(19):Also there is higher expression in 6551-6558).For the treatment of above-mentioned disease, can be using chemotherapy and radiation Property the method such as treatment, but all the normal cell of itself can be caused damage.
T cell adoptive immunotherapy is to proceed to the reaction-ive T cell to target cell antigen with specificity in patient body, Make it be directed to target cell to play a role.φt cell receptor (TCR) is a kind of memebrane protein on T cell surface, and it is capable of identify that accordingly The antigen small peptide of target cells.In immune system, by the TCR and small peptide-main histocompatbility of antigen small peptide specificity The combination of complex (pMHC compounds) causes T cell and the direct physical contact of antigen presenting cell (APC), then T cell And other cell membrane surface molecules of both APC just interact, cause a series of follow-up cell signals transmission and its His physiological reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, this area skill Art personnel are devoted to isolating the TCR for having specificity to NY-ESO-1 antigens small peptide, and the TCR is transduceed T cell to obtain There must be the T cell of specificity to NY-ESO-1 antigens small peptide, so that they play a role in cellular immunotherapy.
The content of the invention
It is an object of the invention to provide a kind of φt cell receptor of identification NY-ESO-1 antigen small peptides.
A kind of a first aspect of the present invention, there is provided φt cell receptor (TCR), the TCR can be with SLLMWITQC-HLA A0201 compounds are combined.
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, and the TCR α chains can The amino acid sequence of the CDR3 of variable domain is ATDANGKII (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domains Amino acid sequence be ASSLGSNEQY (SEQ ID NO:15).
In another preference, 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
α CDR1- TSINN(SEQ ID NO:10)
α CDR2- IRSNERE(SEQ ID NO:11)
α CDR3- ATDANGKII(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
β CDR1- SGHDY(SEQ ID NO:13)
β CDR2- FNNNVP(SEQ ID NO:14)
β CDR3- ASSLGSNEQY(SEQ ID NO:15)。
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, and the TCR α chains can Variable domain is and SEQ ID NO:1 amino acid sequence with least 90% sequence thereto；And/or the TCR β chain variable domains It is and SEQ ID NO：5 amino acid sequences with least 90% sequence thereto.
In another preference, the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.
In another preference, the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5.
In another preference, the TCR is α β heterodimers, and it includes TCR α chains constant regions TRAC*01 and TCR β chains constant region TRBC1*01 or TRBC2*01.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO：3 and/or TCR β chain ammonia Base acid sequence is SEQ ID NO:7.
In another preference, the TCR is solvable.
In another preference, the TCR is single-stranded.
In another preference, the TCR is formed by connecting by peptide catenation sequence with β chains variable domain by α chains variable domain.
In another preference, the TCR is in α chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or It is prominent with one or more in 94, and/or α chain J gene small peptides amino acid inverse the 3rd, 5th reciprocal or inverse the 7th Become；And/or the TCR is in β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th, and/or β chain J With one or more mutation, wherein amino acid position in gene small peptide amino acid inverse the 2nd, 4th reciprocal or inverse the 6th Numbering is put by the Position Number listed in IMGT (international immunogenetics information system).
In another preference, the α chains variable domain amino acid sequence of the TCR includes SEQ ID NO:32 and/or described The β chains variable domain amino acid sequence of TCR includes SEQ ID NO:34.
In another preference, the amino acid sequence of the TCR is SEQ ID NO:30.
In another preference, the TCR includes all or part of TCR α chains of (a) in addition to membrane spaning domain；And The all or part of TCR β chains of (b) in addition to membrane spaning domain；
And (a) with (b) each self-contained functional variable domain, or comprising functional variable domain and the TCR At least a portion of chain constant domain.
In another preference, cysteine residues form artificial disulfide bond between α the and β chain constant domains of the TCR.
In another preference, the cysteine residues that artificial disulfide bond is formed in the TCR instead of selected from following One or more groups of sites：
The Ser57 of the Thr48 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser77 of the Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser17 of the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Asp59 of the Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Glu15 of the Ser15 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser54 of the Arg53 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ala19 of the Pro89 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；With
The Glu20 of the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO:26 and/or TCR β chains Amino acid sequence is SEQ ID NO:28.
In another preference, artificial interchain disulfide bond is contained between the α chains variable region of the TCR and β chain constant regions.
In another preference, it is characterised 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：
46th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；
47th amino acids and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；
46th amino acids and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；Or
47th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV.
In another preference, the TCR is comprising α chains variable domain and β chains variable domain and in addition to membrane spaning domain All or part of β chains constant domain, but it does not contain α chain constant domains, α chains variable domain and the β chains of the TCR form heterogeneous dimerization Body.
In another preference, the α chains of the TCR and/or C- the or N- ends of β chains are combined with conjugate.
In another preference, the conjugate combined with the φt cell receptor is detectable, therapeutic agent, PK are repaiied Decorations part or the combination of any these materials.Preferably, the therapeutic agent is anti-CD 3 antibodies.
A kind of a second aspect of the present invention, there is provided multivalent TCR complex, it includes at least two TCR molecules, and its At least one of TCR molecules be first aspect present invention described in TCR.
A kind of a third aspect of the present invention, there is provided nucleic acid molecules, the nucleic acid molecules include coding first party of the present invention The nucleotide sequence or its complementary series of the TCR molecules described in face.
In another preference, nucleotide sequence SEQ ID of the nucleic acid molecules comprising coding TCR α chain variable domains NO：2 or SEQ ID NO:33.
In another preference, described nucleotide sequence SEQ ID of the nucleic acid molecules comprising coding TCR β chain variable domains NO：6 or SEQ ID NO:35.
In another preference, nucleotide sequence SEQ ID NO of the nucleic acid molecules comprising coding TCR α chains：4 and/ Or the nucleotide sequence SEQ ID NO comprising coding TCR β chains：8.
A fourth aspect of the present invention, there is provided a kind of carrier, described carrier contains the core described in third aspect present invention Acid molecule；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
A fifth aspect of the present invention, there is provided a kind of detached host cell, containing the present invention in described host cell The nucleic acid molecules being integrated with carrier or genome described in fourth aspect described in the third aspect present invention of external source.
A kind of a sixth aspect of the present invention, there is provided cell, the nucleic acid described in the cell transduction third aspect present invention Carrier described in molecule or fourth aspect present invention；Preferably, the cell is T cell or stem cell.
A seventh aspect of the present invention, there is provided a kind of pharmaceutical composition, the composition contains pharmaceutically acceptable load The TCR compounds described in TCR, second aspect present invention, third aspect present invention institute described in body and first aspect present invention Carrier or the cell described in sixth aspect present invention described in the nucleic acid molecules stated, fourth aspect present invention.
A eighth aspect of the present invention, there is provided the φt cell receptor or second aspect present invention described in first aspect present invention Nucleic acid molecules, the carrier described in fourth aspect present invention described in described TCR compounds, third aspect present invention or this The purposes of the cell described in bright 6th aspect, for preparing the medicine for the treatment of tumour or autoimmune disease.
A kind of a ninth aspect of the present invention, there is provided method for treating disease, applies suitable including to object in need for the treatment of The TCR compounds described in φt cell receptor or second aspect present invention, the present invention the 3rd described in the first aspect present invention of amount Nucleic acid molecules, the carrier described in fourth aspect present invention or the cell described in sixth aspect present invention described in aspect or this Pharmaceutical composition described in bright 7th aspect；
Preferably, described disease be tumour, preferably described tumour include neuroblastoma, sarcoma, melanoma, Prostate cancer, carcinoma of urinary bladder, breast cancer, Huppert's disease, hepatocellular carcinoma, oral squamous cell carcinomas, the cancer of the esophagus and cancer of the stomach, lung cancer, head Neck squamous cell cancer, colon cancer, oophoroma etc..
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and have in below (eg embodiment) Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively TCR α chain variable domain amino acid sequences, TCR α chains can Variable domain nucleotide sequence, TCR α chain amino acid sequences, TCR α chain nucleotide sequences, the TCR α chain amino with targeting sequencing Acid sequence and the TCR α chain nucleotide sequences with targeting sequencing.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chain variable domain amino acid sequences, TCR β chains can Variable domain nucleotide sequence, TCR β chain amino acid sequences, TCR β chain nucleotide sequences, the TCR β chain amino with targeting sequencing Acid sequence and the TCR β chain nucleotide sequences with targeting sequencing.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chains.
Fig. 5 a and Fig. 5 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chains.
Fig. 6 is the glue figure of the sTCR for obtaining after purification.To go back virgin rubber, middle swimming lane is molecular weight to leftmost side swimming lane Mark (marker), rightmost side swimming lane is non-reduced glue.
Fig. 7 a and Fig. 7 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR.
Fig. 8 a and Fig. 8 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR α chains.
Fig. 9 a and Fig. 9 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR β chains.
Figure 10 a and Figure 10 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR catenation sequences (linker).
Figure 11 is the glue figure of the soluble single-chain T CR for obtaining after purification.
Figure 12 is the BIAcore dynamics figures that sTCR of the present invention is combined with SLLMWITQC-HLA A0201 compounds Spectrum.
Figure 13 is that the BIAcore that soluble single-chain T CR of the present invention is combined with SLLMWITQC--HLA A0201 compounds is moved Mechanics collection of illustrative plates.
Figure 14 shows that the cell of the TCR of the present invention that transduces has lethal effect to the target cell for expressing related antigen, and right Do not express the target cell of related antigen does not have lethal effect substantially.
Specific embodiment
The present inventor have found and NY-ESO-1 antigen small peptide SLLMWITQC (SEQ ID through extensively in-depth study NO:9) TCR that can be specifically bound, the antigen small peptide SLLMWITQC can with HLA A0201 formed compound and together with by It is presented to cell surface.Present invention also offers encoding the nucleic acid molecules and the carrier comprising the nucleic acid molecules of the TCR. In addition, present invention also offers the cell of transduction TCR of the present invention.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, its for The presentation of antigen has specificity, and different individualities has different MHC, can present small peptides different in a kind of proteantigen to respectively From APC cell surfaces.The MHC of the mankind is commonly referred to HLA genes or HLA complexs.
φt cell receptor (TCR), is the unique of specific antigen peptide of the presentation in main histocompatibility complex (MHC) Acceptor.In immune system, cause T cell thin with antigen presentation by the combination of the TCR and pMHC compounds of antigentic specificity The direct physical contact of born of the same parents (APC), then other cell membrane surface molecules of both T cell and APC just interact, this A series of follow-up cell signal transmission and other physiological reactions are just caused, so that the T cell of different antigentic specificities Immunological effect is played to its target cell.
TCR is the glycoprotein of the surface of cell membrane existed in heterodimer form by α chains/β chains or γ chains/δ chains. TCR heterodimers are made up of α and β chains in 95% T cell, and 5% T cell has the TCR being made up of γ and δ chains.My god So heterogeneous dimerization TCR of α β has α chains and β chains, and α chains and β chains 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 chains generally contain short variable region also between variable region and bonding pad, but should Variable region is often regarded as a part for bonding pad.Each variable region includes 3 be entrenched in frame structure (framework regions) Individual CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compounds, wherein CDR3 by Variable region and bonding pad restructuring are formed, and are referred to as hypervariable region.α the and β chains of TCR are typically regarded as respectively has two " domains " i.e. variable Domain and constant domain, variable domain is made up of the variable region and bonding pad that connect.The sequence of TCR constant domains can be in international immune genetic Find in the public database for learning information system (IMGT), such as the constant domain sequence of TCR molecule alpha chains is " TRAC*01 ", TCR divides The constant domain sequence of sub- β chains is " TRBC1*01 " or " TRBC2*01 ".Additionally, α the and β chains of TCR are also comprising transmembrane region and kytoplasm Area, cytoplasmic region is very short.
In the present invention, term " polypeptide of the present invention ", the TCR of the present invention " ", " φt cell receptor of the present invention " is interchangeable makes With.
Native interchain disulfide bond and artificial interchain disulfide bond
There is one group of disulfide bond in the membrane-proximal region C α and C β interchain of natural TCR, " the sulphur of native interchain two is referred to as in the present invention Key ".In the present invention, will be manually-injected, 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 for describing disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequences in the present invention Position Number carry out Position Number by the order from N-terminal to C-terminal successively, in such as TRBC1*01 or TRBC2*01, by from N-terminal to The 60th amino acid of C-terminal order successively is P (proline), then can describe it as TRBC1*01 or TRBC2*01 in the present invention The Pro60 of exons 1, can also be stated that the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s, and for example Be Q (glutamine) by the 61st amino acid of the order from N-terminal to C-terminal successively in TRBC1*01 or TRBC2*01, then it is of the invention In can describe it as the Gln61 of TRBC1*01 or TRBC2*01 exons 1s, can also be stated that TRBC1*01 or TRBC2* 61st amino acids of 01 exons 1, other are by that analogy.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 certain amino acid in TRAV, the Position Number listed in IMGT is 46, then the amino acids of TRAV the 46th are described it as in the present invention, other are by that analogy.In the present invention, the sequence of other amino acid Column position numbering has specified otherwise, then by specified otherwise.
Detailed description of the invention
TCR molecules
In antigen processing pathways, antigen is degraded in the cell, is then carried to cell surface by MHC molecule.T is thin Born of the same parents' acceptor is capable of identify that the peptide-MHC compounds of Antigen Presenting Cell surface.Therefore, a first aspect of the present invention provides one kind Can be with reference to the TCR molecules of SLLMWITQC-HLA A0201 compounds.Preferably, the TCR molecules are detached or purifying 's.α the and β chains of the TCR respectively have 3 complementary determining regions (CDR).
One in the present invention is preferably carried out in mode, and the α chains of the TCR are included with following amino acid sequence CDR：
α CDR1- TSINN(SEQ ID NO:10)
α CDR2- IRSNERE(SEQ ID NO:11)
α CDR3- ATDANGKII(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
β CDR1- SGHDY(SEQ ID NO:13)
β CDR2- FNNNVP(SEQ ID NO:14)
β CDR3- ASSLGSNEQY(SEQ ID NO:15)。
The CDR region amino acid sequence of the invention described above 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 the TCR of the present invention, those skilled in the art are according to CDR region disclosed by the invention Can just design or synthesize the TCR molecules with corresponding function.Therefore, TCR molecules of the present invention are referred to comprising above-mentioned α and/or β The TCR molecules of chain CDR region sequence and any suitable frame structure.TCR α chain variable domains of the present invention are and SEQ ID NO:1 tool Have at least 90%, preferably 95%, more preferably the amino acid sequence of 98% sequence thereto；And/or TCR β chains of the present invention can Variable domain is and SEQ ID NO：5 have at least 90%, preferably 95%, the amino acid sequence of more preferably 98% sequence thereto Row.
In a preference of the present invention, the TCR molecules of the present invention are the heterodimers being made up of α and β chains.Specifically Ground, the α chains of the one side heterogeneous dimerization TCR molecule are comprising variable domain and constant domain, the α chains variable domain amino acid sequence bag CDR1 (SEQ ID NO containing above-mentioned α chains：10)、CDR2(SEQ ID NO：11) with CDR3 (SEQ ID NO:12).Preferably, The TCR molecules include α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domains of the TCR molecules Amino acid sequence is SEQ ID NO:1.On the other hand, the β chains of the heterogeneous dimerization TCR molecule include variable domain and constant domain, CDR1 (SEQ ID NO of the β chains variable domain amino acid sequence comprising above-mentioned β chains:13)、CDR2(SEQ ID NO：14) and CDR3(SEQ ID NO:15).Preferably, the TCR molecules include β chain variable domain amino acid sequence SEQ ID NO:5.It is more excellent Selection of land, the β chains variable domain amino acid sequence of the TCR molecules is SEQ ID NO:5.
In a preference of the present invention, the TCR molecules of the present invention are by part or all of and/or β chains the portion of α chains The single chain TCR molecules for dividing or all constituting.Description about single chain TCR molecules may be referred to document Chung et al (1994) Proc.Natl.Acad.Sci.USA 91,12654-12658.According to document, those skilled in the art can be easily Build the single chain TCR molecules comprising CDRs areas of the present invention.Specifically, the single chain TCR molecules include V α, V β and C β, preferably According to being linked in sequence from N-terminal to C-terminal.
CDR1 (SEQ ID NO of the α chains variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned α chains：10)、 CDR2(SEQ ID NO：11) with 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 chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO: 1.CDR1 (SEQ ID NO of the β chains variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned β chains：13)、CDR2 (SEQ ID NO：14) with CDR3 (SEQ ID NO：15).Preferably, the single chain TCR molecules include β chain variable domain amino acids Sequence SEQ ID NO:5.It is highly preferred that the β chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:5.
In a preference of the present invention, the constant domain of the TCR molecules of the present invention is the constant domain of people.Art technology Personnel know or can be obtained by consulting the public database of pertinent texts or IMGT (international immunogenetics information system) Obtain the constant domain amino acid sequence of people.For example, the constant domain sequence of TCR molecule alphas chain of the present invention can be " TRAC*01 ", and TCR divides The constant domain sequence of sub- β chains can be " TRBC1*01 " or " TRBC2*01 ".The amino acid sequence be given in the TRAC*01 of IMGT The 53rd be Arg, here is expressed as：The Arg53 of TRAC*01 exons 1s, other are by that analogy.Preferably, TCR of the present invention The amino acid sequence of molecule alpha chain is SEQ ID NO:3, and/or the amino acid sequence of β chains is SEQ ID NO:7.
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.As immunoglobulin (Ig) (antibody) is made The same for antigen recognizing molecule, TCR can also be developed and be applied to diagnose and treat, and at this moment need to obtain soluble TCR point Son.Soluble TCR molecules do not include its transmembrane region.STCR has very extensive purposes, and it cannot be only used for studying TCR With the interaction of pMHC, it is also possible to make the diagnostic tool or the mark as autoimmunity disease of detection infection.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 combine to redirect T cell with other molecules (e.g., anti-CD 3 antibodies), from And make its targeting present the cell of specific antigen.The present invention also obtain has the solvable of specificity to NY-ESO-1 antigens small peptide Property TCR.
To obtain sTCR, on the one hand, TCR of the present invention can be introduced between the residue of itself α and β chain constant domain The TCR of artificial disulfide bond.Cysteine residues form artificial interchain disulfide bond between α the and β chain constant domains of the TCR.Half Guang Histidine residue can be substituted in other amino acid residues of appropriate site in natural TCR to form artificial interchain disulfide bond.For example, The cysteine residues of the Thr48 for replacing TRAC*01 exons 1s and the Ser57 for replacing TRBC1*01 or TRBC2*01 exons 1s To form disulfide bond.Introduce cysteine residues to can also be with other sites for forming disulfide bond：TRAC*01 exons 1s The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1s；The Tyr10 and TRBC1*01 of TRAC*01 exons 1s or The Ser17 of TRBC2*01 exons 1s；The Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s Asp59；The Glu15 of the Ser15 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 exons 1s Arg53 and TRBC1*01 or TRBC2*01 exons 1s Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1s or The Ala19 of TRBC2*01 exons 1s；Or the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s Glu20.I.e. cysteine residues instead of above-mentioned α with arbitrary group of site in β chain constant domains.Can be in TCR constant domains of the present invention One or more C-terminals truncate most 50 or most 30 or most 15 or most 10 or most 8 or less Amino acid so that it is not including cysteine residues reaching the purpose of disappearance natural disulphide bonds, also can be by the way that day will be formed So the cysteine residues of disulfide bond sport another amino acid to reach above-mentioned purpose.
As described above, the TCR of the present invention may be embodied in the artificial disulfide bond introduced between the residue of itself α and β chain constant domain. It should be noted that with or without the artificial disulfide bond of introducing mentioned above between constant domain, the TCR of the present invention can be constant containing TRAC Domain sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences of TCR can Natural disulphide bonds connection in by being present in TCR.
To obtain sTCR, on the other hand, TCR of the present invention is additionally included in the TCR that its hydrophobic core region is undergone mutation, The mutation in 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 undergo mutation in the hydrophobic core position of its following variable domain： (α and/or β chains) variable region amino acid the 11st, 13,19,21,53,76,89,91,94, and/or α chain J genes (TRAJ) small peptides Amino acid position is reciprocal 3rd, 5,7, and/or β chain J genes (TRBJ) small peptides amino acid position is reciprocal 2nd, 4,6, wherein ammonia The Position Number of base acid sequence is by the Position Number listed in international immunogenetics information system (IMGT).People in the art Member knows above-mentioned international immunogenetics information system, and the amino acid residue that can obtain different TCR according to the database exists Position Number in IMGT.
The TCR that hydrophobic core region is undergone mutation in the present invention can be by the α and β chain of a flexible peptide chain connection TCR can Variable domain and the solvable single-stranded TCR of stability that constitutes.It should be noted that flexible peptide chain can be any suitable connection TCR α in the present invention With the peptide chain of β chain variable domains.The single chain soluble TCR built such as in the embodiment of the present invention 4, its α chain variable domain amino acid sequence It is classified as SEQ ID NO:32, the nucleotides sequence of coding is classified as SEQ ID NO:33；β chains variable domain amino acid sequence is SEQ ID NO:34, the nucleotides sequence of coding is classified as SEQ ID NO:35.
In addition, for stability, patent document 201510260322.4 is also disclosed in the α chains variable region of TCR and β Artificial interchain disulfide bond is introduced between chain constant region significantly improves can the stability of TCR.Therefore, high-affinity of the invention Artificial interchain disulfide bond can also be contained between the α chains variable region of TCR and β chain constant regions.Specifically, can in the α chains of the TCR The cysteine residues that artificial interchain disulfide bond is formed between change area and β chain constant regions instead of：46th amino acids of TRAV With the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；47th amino acids of TRAV and TRBC1*01 or 61 amino acids of TRBC2*01 exons 1s；46th amino acids of TRAV and the of TRBC1*01 or TRBC2*01 exons 1s 61 amino acids；Or the 47th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV.It is preferred that Ground, such TCR can include all or part of TCR α chains of (I) in addition to its membrane spaning domain, and (II) except its cross-film knot All or part of TCR β chains beyond structure domain, wherein (I) and (II) variable domain comprising TCR chains and at least a portion are constant Domain, α chains form heterodimer with β chains.It is highly preferred that such TCR can comprising α chains variable domain and β chains variable domain and All or part of β chains constant domain in addition to membrane spaning domain, but it does not contain α chain constant domains, the α chain variable domains of the TCR Heterodimer is formed with β chains.
The present invention TCR can also multivalence complex form provide.The present invention multivalent TCR complex comprising two, Three, the four or more TCR of the present invention polymers that combine and formed, such as can be produced with four dimerization domains of p53 The tetramer, or the compound that multiple TCR of the present invention are combined and formed with another molecule.The TCR compounds of the present invention can be used for body Cell that is outer or following the trail of or target presentation specific antigen in vivo, it can also be used to produce other multivalence TCR with such application and answer The intermediate of compound.
The TCR of the present invention can be used alone, and also can be combined with covalent or other modes with conjugate, preferably with covalently side Formula is combined.The conjugate includes that detectable (is diagnostic purpose, wherein the TCR is used to detect presenting The presence of the cell of SLLMWITQC-HLA A0201 compounds), therapeutic agent, PK (protein kinase) modification part or it is any more than The combination of these materials is combined or is coupled.
Detectable for diagnostic purposes is included but is not limited to：Fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (CT technology) contrast preparation can produce detectable product Enzyme.
The therapeutic agent that can be combined with TCR of the present invention or be coupled is included but is not limited to：1. radionuclide (Koppe etc., 2005, metastasis of cancer comment (Cancer metastasis reviews) 24,539)；2. biological 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, NAS's 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 Fragment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；5. antibody ScFv fragments (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold Nano particle/nanometer rods (Lapotko etc., 2005, cancer communication (Cancer letters) 239,36；Huang etc., 2006, it is beautiful Chemical Society of state magazine (Journal of the American Chemical Society) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,11631)；9. magnetic nanosphere；10. pro-drug activation enzymes (for example, DT- diaphorases (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
In addition, the TCR of the present invention can also be comprising derived from more than a kind of heterozygosis TCR of species sequence.For example, grind Studying carefully display Muridae TCR can more effectively express in human T-cell than people TCR.Therefore, TCR of the present invention can include people's variable domain With the constant domain of mouse.The defect of this method is possible to cause immune response.Therefore, when it is used for adoptive T cell treatment There should be regulation scheme to carry out immunosupress, to allow the implantation of the T cell for expressing Muridae.
It should be understood that herein amino acid name is represented using international single English alphabet or three English alphabets, amino Single English alphabet of sour title is as follows with the corresponding relation of three English alphabets：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
A second aspect of the present invention provides the nucleic acid molecules of coding first aspect present invention TCR molecule or part thereof, institute It can be one or more CDR, the variable domain of α and/or β chains, and α chains and/or β chains to state part.
The nucleotide sequence of coding first aspect present invention TCR molecule alpha chain CDR region is as follows：
α CDR1- actagtataaacaat(SEQ ID NO:16)
α CDR2- atacgttcaaatgaaagagag(SEQ ID NO:17)
α CDR3- gctacggacgcaaacggcaagatcatc (SEQ ID NO:18)
The nucleotide sequence of coding first aspect present invention TCR molecule β chain CDR regions is as follows：
β CDR1- tcaggacacgactac(SEQ ID NO:19)
β CDR2- tttaacaacaacgttccg(SEQ ID NO:20)
β CDR3- gccagcagtttagggagcaacgagcagtac (SEQ ID NO:21)
Therefore, encoding the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chains of the present invention includes SEQ ID NO:16、 SEQ ID NO:17 and SEQ ID NO:18, and/or encode the nucleotide sequence of the nucleic acid molecules of the present invention of TCR β chains of the present invention Including SEQ ID NO:19、SEQ ID NO:20 and SEQ ID NO:21.
The nucleotide sequence of nucleic acid molecules of the present invention can be it is single-stranded or double-stranded, the nucleic acid molecules can be RNA or DNA, and can include or not comprising 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, for example, encodes the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chain variable domains of the present invention Including SEQ ID NO:2 and/or the nucleotide sequence of the nucleic acid molecules of the present invention for encoding TCR β chain variable domains of the present invention include SEQ ID NO:6.Or, encoding the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chain variable domains of the present invention includes SEQ ID NO:33 and/or encode TCR β chain variable domains of the present invention nucleic acid molecules of the present invention nucleotide sequence including SEQ ID NO:35.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.Or Person, the nucleotides sequence of nucleic acid molecules of the present invention is classified as SEQ ID NO:31.
It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.Therefore, compile The nucleotide sequence of code book invention TCR can variant identical with the nucleotide sequence shown in accompanying drawing of the present invention or degeneracy.With Illustrating, " variant of degeneracy " refers to that coding has SEQ ID NO to one of example in the present invention:1 protein sequence, But with SEQ ID NO:The 2 differentiated nucleotide sequence of sequence.
Nucleotide sequence can be Jing codon optimizations.Different cells is different in the utilization of concrete codon , can change the codon in sequence to increase expression according to the type of cell.Mammalian cell and various other Biological codon usage table be well known to a person skilled in the art.
The present invention nucleic acid molecules full length sequence or its fragment generally can with but be not limited to PCR TRAPs, recombination method or Artificial synthesized method is obtained.At present, it is already possible to obtain encoding completely by chemical synthesis TCR of the present invention (or its fragment, Or derivatives thereof) DNA sequence dna.Then can by the DNA sequence dna introduce various existing DNA moleculars as known in the art (or Such as carrier) and cell in.DNA can be coding strand or noncoding strand.
Carrier
The invention further relates to include the carrier of the nucleic acid molecules of the present invention, including expression vector that is, can in vivo or body The construct of outer expression.Conventional 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, carrier can be transferred to the nucleotides of the present invention in cell, such as in T cell so that the cell table Up to the TCR of NY-ESO-1 antigentic specificities.Ideally, the carrier should in T cell continual high levels earth's surface Reach.
Cell
The invention further relates to the host cell produced with the carrier or coded sequence Jing genetic engineerings of the present invention.The host The nucleic acid molecules of the present invention are integrated with carrier or chromosome containing the present invention in cell.Host cell is selected from：Prokaryotic And eukaryotic, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..
In addition, present invention additionally comprises the detached cell of the TCR of the expression present invention, particularly T cell.The T cell can spread out T cell detached from experimenter is conigenous, or can be the detached mixed cellularity group from experimenter, such as periphery hemolymph is thin The part of born of the same parents (PBL) group.Such as, the cell can be isolated from PMBC (PBMC), can be CD4+Helper cell Or CD8+Cytotoxic T cell.The cell can be in CD4+Helper cell/CD8+In the mixing group of cytotoxic T cell.Typically Ground, the cell can be activated with antibody (e.g., the antibody of anti-CD3 or anti-CD28), to allow them to easily receive to turn Dye, for example, transfected with the carrier of the nucleotide sequence comprising coding TCR molecules 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 Move to HSC to be not result in cell surface expression TCR, because stem cell surface does not express CD3 molecules.However, when stem cell point Turn to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecules will start in thymocyte The surface expression introducing TCR molecules.
Have many methods be suitable for being carried out with the DNA or RNA that encode TCR of the present invention T cell transfection (e.g., Robbins etc., (2008)J.Immunol.180:6116-6131).The T cell of expression TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that carry out adoptive treatment many appropriate methods (e.g., Rosenberg etc., (2008) Nat Rev Cancer8(4)：299-308).
NY-ESO-1 antigen-related diseases
The invention further relates to the method with NY-ESO-1 relevant diseases is treated and/or prevented in experimenter, it included The step of property shifts NY-ESO-1 specific T-cells to the experimenter.The NY-ESO-1 specific T-cells can recognize that SLLMWITQC-HLA A0201 compounds.
The T cell of the NY-ESO-1 specificity of the present invention can be used to treat any presentation NY-ESO-1 antigen small peptides The NY-ESO-1 relevant diseases of SLLMWITQC-HLA A0201 compounds, including but not limited to tumour, preferably described tumour bag Include neuroblastoma, sarcoma, melanoma, prostate cancer, carcinoma of urinary bladder, breast cancer, Huppert's disease, hepatocellular carcinoma, mouth Chamber squamous carcinoma, the cancer of the esophagus and cancer of the stomach, lung cancer, SCCHN, colon cancer, oophoroma etc..
Treatment method
Can pass through separate with NY-ESO-1 antigen-related diseases patient or the T cell of volunteer, and by this Bright TCR is imported in above-mentioned T cell, subsequently feeds back in patient body to be treated by the cell of these genetic engineering modifications. Therefore, the invention provides a kind of method for treating NY-ESO-1 relevant diseases, including by the T of detached expression TCR of the present invention Cell, it is preferable that the T cell derives from patient itself, is input in patient body.Usually, it is thin including the T of (1) separation patient Born of the same parents, (2) are with nucleic acid molecules of the present invention or can encode the nucleic acid molecules ex vivo transduction T cell of TCR molecules of the present invention, and (3) are by base Because the T cell of engineering modification is input in patient body.The quantity of the cell for separate, transfecting and feeding back can be determined by doctor.
Main advantages of the present invention are：
(1) TCR of the invention can be combined with NY-ESO-1 antigen small peptide compound SLLMWITQC-HLA A0201, together When transduceed TCR of the present invention cell can by specific activation and to target cell have very strong lethal effect.
Following specific embodiment, is expanded on further the present invention.It should be understood that these embodiments be merely to illustrate the present invention and It is not used in restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, Such as (Sambrook and Russell et al., molecular cloning：Laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing houses) described in condition, or according to the condition proposed by manufacturer.Unless Explanation in addition, otherwise percentage and number are calculated by weight.Unless otherwise indicated, otherwise percentage and number are calculated by weight. Experiment material used and reagent can be obtained if no special instructions from commercially available channel in following examples.
Embodiment 1 clones NY-ESO-1 antigen small peptide specific T-cells
Using synthesis small peptide SLLMWITQC (SEQ ID NO.:9；Beijing SBS Genetech gene technology Co., Ltd) stimulate and From in the PBLC (PBL) of the healthy volunteer that genotype is HLA-A0201.By SLLMWITQC small peptides with carry The HLA-A0201 renaturation of biotin labeling, prepares pHLA monoploid.These monoploid and the Streptavidin (BD marked with PE Company) tetramer of PE marks is combined into, sort the tetramer and anti-CD8-APC double positive cells.The cell of amplification sorting, And secondary sorting is carried out as stated above, subsequently carry out monoclonal with limiting dilution assay.Monoclonal cell tetramer staining, sieve The double positive colonies chosen are as shown in Figure 3.
Embodiment 2 obtains the tcr gene of NY-ESO-1 antigen small peptide specific T-cell clones and the structure of carrier
Use Quick-RNATMThe antigen small peptide screened in MiniPrep (ZYMO research) extracting embodiments 1 The total serum IgE of SLLMWITQC specificity, HLA-A0201 restricted T cell clone.The synthesis of cDNA is using clontech's SMART RACE cDNA amplification kits, the primer of employing is designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned It is sequenced to carrier T (TAKARA).It should be noted that the sequence is complementary series, not comprising introne.Jing is sequenced, this pair of sun Property clonal expression TCR α chains and β chain-orderings structure respectively as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e With Fig. 1 f be respectively TCR α chain variable domain amino acid sequences, TCR α chain variable domain nucleotide sequences, TCR α chain amino acid sequences, TCR α chain nucleotide sequences, the TCR α chain amino acid sequences with targeting sequencing and the TCR α chain cores with targeting sequencing Nucleotide sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chain variable domain amino acid sequences, TCR β chains Variable domain nucleotide sequence, TCR β chain amino acid sequences, TCR β chain nucleotide sequences, the TCR β chain ammonia with targeting sequencing Base acid sequence and the TCR β chain nucleotide sequences with targeting sequencing.
Identified, α chains include the CDR with following amino acid sequence：
α CDR1- TSINN(SEQ ID NO:10)
α CDR2- IRSNERE(SEQ ID NO:11)
α CDR3- ATDANGKII(SEQ ID NO:12)
β chains include the CDR with following amino acid sequence：
β CDR1-SGHDY(SEQ ID NO:13)
β CDR2-FNNNVP(SEQ ID NO:14)
β CDR3-ASSLGSNEQY(SEQ ID NO:15)
Respectively the full-length gene of TCR α chains and β chains is cloned into Lentiviral by overlapping (overlap) PCR pLenti(addgene).Specially：The full-length gene of TCR α chains and TCR β chains is attached with overlap PCR is obtained TCR α -2A-TCR β fragments.Lentiviral and TCR α -2A-TCR β digestions connection are obtained into pLenti-TRA-2A- TRB-IRES-NGFR plasmids.Use as control, while the also slow virus carrier pLenti-eGFP of construction expression eGFP.Afterwards again Pseudovirus is packed with 293T/17.
The expression of the NY-ESO-1 antigens small peptide of the embodiment 3 solvable TCR of specificity, refolding and purifying
To obtain solvable TCR molecules, α the and β chains of the TCR molecules of the present invention can respectively only comprising its variable domain and portion Divide in constant domain, and the constant domain of α and β chains and introduce a cysteine residues respectively to form artificial interchain disulfide bond, The position of introducing cysteine residues is respectively the Ser57 of the Thr48 and TRBC2*01 exons 1s of TRAC*01 exons 1s；Its α The amino acid sequence of chain is distinguished as shown in figures 4 a and 4b with nucleotide sequence, the amino acid sequence and nucleotide sequence of its β chain Respectively as shown in figure 5 a and 5b, introducing cysteine residues are with overstriking and underline letter representation.Pass through《Molecular cloning Laboratory manual》(Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) Described in standard method the objective gene sequence ECDC of above-mentioned TCR α and β chains is inserted respectively into expression vector into after PET28a+ (Novagene), the cloning site of upstream and downstream is respectively NcoI and NotI.Insert Fragment confirms errorless through sequencing.
The expression vector of TCR α and β chains is entered into expression bacterium BL21 (DE3) by chemical transformation conversion respectively, carefully Bacterium is grown with LB nutrient solutions, in OD600Induced with final concentration 0.5mM IPTG when=0.6, formed after α the and β chains expression of TCR Inclusion body is extracted by BugBuster Mix (Novagene), and the repeated multiple times washing of Jing BugBuster solution, bag Contain body and be finally dissolved in 6M guanidine hydrochlorides, 10mM dithiothreitol (DTT)s (DTT), 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris In (pH 8.1).
TCR α and β chains after dissolving are with 1：1 mass ratio is quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Afterwards solution is placed in into dialysis (4 DEG C) in the deionized water of 10 times of volumes, deionized water is changed into buffer solution (20mM after 12 hours Tris, pH 8.0) continue at 4 DEG C dialyse 12 hours.Solution after the completion of dialysis Jing after 0.45 μM of membrane filtration, by the moon from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.It is dimeric that eluting peak contains renaturation successful α and β TCR is confirmed by SDS-PAGE glue.TCR subsequently passes through gel permeation chromatography (HiPrep 16/60, Sephacryl S-100 HR, GE Healthcare) it is further purified.TCR purity after purification is determined through SDS-PAGE and is more than 90%, and concentration is by BCA Method determines.The SDS-PAGE glue figures of the sTCR that the present invention is obtained are as shown in Figure 6.
The generation of the soluble single-chain T CR of the NY-ESO-1 antigens small peptide of embodiment 4 specificity
According to patent document WO2014/206304, using the method for rite-directed mutagenesis by TCR α and β in embodiment 2 The variable domain of chain has been built into a stable soluble single-chain T CR molecule connected with flexible small peptide (linker).This is single-stranded The amino acid sequence of TCR molecules and nucleotide sequence are respectively as shown in figs. 7 a and 7b.The amino acid sequence of its α chain variable domain and Nucleotide sequence is respectively as figures 8 a and 8 b show；The amino acid sequence and nucleotide sequence of its β chain variable domain is respectively such as Fig. 9 a With shown in Fig. 9 b；The amino acid sequence of its linker sequence and nucleotide sequence are respectively as as-shown-in figures 10 a and 10b.
By genes of interest Jing Nco I and the double digestions of Not I, connect with the pET28a carriers through Nco I and the double digestions of Not I Connect.Connection product is converted to E.coliDH5 α, and LB flat board of the coating containing kanamycins, 37 DEG C of inversion overnight incubations, picking is positive Clone into performing PCR screening, positive recombinant is sequenced, determine that sequence correctly extracts afterwards recombinant plasmid transformed to E.coli BL21 (DE3), for expressing.
The expression of soluble single-chain T CR of the NY-ESO-1 antigens small peptide of embodiment 5 specificity, renaturation and purifying
The BL21 containing recombinant plasmid pET28a- template strands (DE 3) bacterium colony prepared in embodiment 4 is all inoculated in In LB culture mediums containing kanamycins, it is 0.6-0.8, addition IPTG to final concentration of 0.5mM, 37 that 37 DEG C are cultivated to OD600 DEG C continue cultivate 4h.5000rpm is centrifuged 15min harvesting sediments, is cracked with Bugbuster Master Mix (Merck) Cell pellet, 6000rpm centrifugation 15min reclaim inclusion body, then are washed to remove cell with Bugbuster (Merck) Fragment and membrane component, 6000rpm centrifugation 15min, collect inclusion body.By solubilization of inclusion bodies in buffer solution (20mM Tris-HCl PH 8.0,8M urea) in, high speed centrifugation removes insoluble matter, and supernatant is standby in -80 DEG C of preservations with being dispensed after BCA standard measures With.
To in the single-stranded TCR inclusion body proteins of 5mg dissolvings, 2.5mL buffer solutions (6M Gua-HCl, 50mM Tris- is added HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration of 10mM, 37 DEG C of process 30min.With injection Device is to 125mL renaturation buffers (100mM Tris-HCl pH 8.1,0.4M L-arginines, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) the middle single-stranded TCR being added dropwise after above-mentioned process, 4 DEG C of stirrings 10min, then loads cellulose membrane bag filter of the interception for 4kDa by renaturation solution, and bag filter is placed in the water of 1L precoolings, 4 DEG C It is slowly stirred overnight.After 17 hours, change dislysate the buffer solution (20mM Tris-HCl pH 8.0) of 1L precoolings into, 4 DEG C after Continuous dialysis 8h, then changes dislysate into identical fresh buffer and continues dialysed overnight.After 17 hours, 0.45 μm of filter of sample Jing Membrane filtration, by anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgas, uses 20mM Tris-HCl The 0-1M NaCl linear gradient elution liquid purifying proteins that pH 8.0 is prepared, the elution fraction of collection carries out SDS-PAGE analyses, wraps Further carried out with solvent resistant column (Superdex 7510/300, GE Healthcare) after the concentration of component containing single-stranded TCR Purifying, target components are also carried out SDS-PAGE analyses.
Elution fraction for BIAcore analyses further tests its purity using gel filtration.Condition is：Chromatographic column Agilent Bio SEC-3 (7.8 × 300mm of 300A, φ), mobile phase be 150mM phosphate buffers, flow velocity 0.5mL/min, 25 DEG C of column temperature, ultraviolet detection wavelength 214nm.
The SDS-PAGE glue figures of the soluble single-chain T CR that the present invention is obtained are as shown in figure 11.
Embodiment 6 is combined and characterized
BIAcore is analyzed
This example demonstrated the TCR molecules of the present invention of solubility can be special with SLLMWITQC-HLA A0201 compounds The opposite sex is combined.
Detected using BIAcore T200 real-time analyzers the TCR molecules that obtain in embodiment 3 and embodiment 5 with The binding activity of SLLMWITQC-HLA A0201 compounds.The antibody (GenScript) of anti-Streptavidin is added to be coupled and is delayed Liquid (10mM sodium-acetate buffers, pH 4.77) is rushed, then antibody flow through the CM5 chips for being activated with EDC and NHS in advance, made Antibody is fixed on chip surface, finally closes unreacted activating surface with the hydrochloric acid solution of monoethanolamine, completes coupling process, even Connection level is about 15,000RU.
The Streptavidin for making low concentration flows through the chip surface of coated antibody, then by SLLMWITQC-HLA A0201 compounds flow through sense channel, another passage as reference channel, then by the biotin of 0.05mM with the stream of 10 μ L/min Speed flows through chip 2min, the remaining binding site of closing Streptavidin.
The preparation process of above-mentioned SLLMWITQC-HLA A0201 compounds is as follows：
A. purify
The E.coli bacterium solutions of 100ml abduction deliverings heavy chain or light chain are collected, after 4 DEG C of 8000g centrifugation 10min 10ml is used PBS washing thallines are once, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) afterwards Shake resuspended thalline, and in room temperature rotation incubation 20min, after 4 DEG C, 6000g centrifugation 15min, supernatant discarded, collection is forgiven Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation incubation 5min；Plus 30ml The BugBuster of 10 times of dilution, mixes, and 4 DEG C of 6000g are centrifuged 15min；Supernatant discarded, plus the BugBuster of 10 times of 30ml dilutions Resuspended inclusion body, mixes, and 4 DEG C of 6000g are centrifuged 15min, are repeated twice, plus the resuspended bags of 30ml 20mM Tris-HCl pH 8.0 Contain body, mix, 4 DEG C of 6000g are centrifuged 15min, finally dissolve inclusion body, SDS-PAGE detections with 20mM Tris-HCl 8M urea Inclusion body purity, BCA kits survey concentration.
B. renaturation
The small peptide SLLMWITQC (Beijing SBS Genetech gene technology Co., Ltd) of synthesis is dissolved in into DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain is dissolved with 8M urea, 20mM Tris pH 8.0,10mM DTT, is added before renaturation 3M guanidine hydrochlorides, 10mM sodium acetates, the further denaturation of 10mM EDTA.SLLMWITQC peptides are added into renaturation with 25mg/L (final concentration) Buffer solution (0.4M L-arginines, 100mM Tris pH 8.3,2mM EDTA, 0.5mM GSSGs, 5mM reduced forms Glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, Heavy chain is added in three times, 8h/ time), renaturation carries out at least 3 days at 4 DEG C to completing, and can SDS-PAGE detections renaturation success.
C. purify after renaturation
Renaturation buffer is changed as dialysis with the 20mM Tris pH 8.0 of 10 volumes, buffer solution is at least changed and is come twice Fully reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Instrument (the general electricity of GE is purified using Akta Gas company), the 0-400mM NaCl linear gradient liquid wash-out proteins that 20mM Tris pH 8.0 are prepared, pMHC is about in 250mM Elute at NaCl, collect all peak components, SDS-PAGE detection purity.
D. biotinylation
The pMHC molecular concentrations that will be purified with Millipore super filter tubes, while being 20mM Tris pH by buffer exchange 8.0, it is subsequently adding biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D- Biotin, 100 μ g/ml BirA enzymes (GST-BirA), overnight, whether SDS-PAGE detections biotinylation for incubation at room temperature mixture Completely.
E. the compound after purifying biological elementization
PMHC molecular concentrations after with Millipore super filter tubes biotinylation is marked to 1ml, using gel permeation chromatography The pMHC of purifying biological elementization, using Akta purifying instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibration HiPrepTM 16/60 S200 HR posts (GE General Electric Co. Limited), load the concentrated biotinylation pMHC molecules of 1ml, then with PBS with 1ml/min flow velocitys are eluted.Biotinylated pMHC molecules occur in about 55ml as unimodal wash-out.Merge containing protein Component, is concentrated with Millipore super filter tubes, and BCA methods (Thermo) determine protein concentration, adds protease inhibitors The packing of biotinylated pMHC molecules is stored in -80 DEG C by cocktail (Roche).
Using BIAcore Evaluation software computational dynamics parameters, obtain the TCR molecules of solubility of the invention with And the kinetic profile point that the soluble single-chain T CR molecules of present invention structure are combined with SLLMWITQC-HLA A0201 compounds Not as shown in Figure 12 and Figure 13.Collection of illustrative plates shows that the soluble TCR molecules for obtaining of the invention and soluble single-chain T CR molecules are all Can be combined with SLLMWITQC-HLA A0201 compounds.Meanwhile, also have detected solubility of the invention using said method The binding activity of TCR molecules and other several irrelevant antigen small peptides and HLA compounds, as a result show TCR molecules of the present invention and its His irrelevant antigen is without combination.
Embodiment 7 transduce TCR of the present invention cell killing experiments
The present embodiment determines the release of LDH by non-radioactive cell toxicity test, so as to verify transduction TCR of the present invention Cell killing ability.
Those skilled in the art know the method for detecting cell function using the release experiment of LDH.The present embodiment LDH is tested With the PBL cell Jing slow-virus transfection TCR being separated to from the blood of healthy volunteer as effector cell.Target cell system is A375 (3525), MEL624 (1605), NCI-H1650 (2) and MEL526 (4) cell.According to nanostring results, wherein, A375 (3525) and MEL624 (1605) expression NY-ESO-1 antigens.NCI-H1650 (2) and MEL526 (4) do not express NY- substantially ESO-1 antigens, in this, as control.
Prepare LDH flat boards first.Test the 1st day, in the following order each component of test is added into flat board：Culture keynote Whole effector cell is to 2 X 106Individual cells/ml, culture medium adjusts each target cell system to 5 X 105Individual cells/ml.Mixing is equal The X 10 of 100 μ L target cell systems 5 is taken after even5Individual cells/ml (i.e. 50,000 cells/well), the X 10 of 100 μ L effector cells 26 Individual cells/ml (i.e. 200,000 cells/well) is added in corresponding aperture, and arranges three multiple holes.Effector cell is set simultaneously certainly Hair engaging aperture, target cell idiomorphic pore, target cell largest hole, volume correction control wells and culture medium ground control hole.It is incubated overnight (37 DEG C, 5%CO2).Test the 2nd day, detection colour developing records light absorption value with ELIASA (Bioteck) after terminating reaction in 490nm.It is real Test result as shown in figure 14, the cell of the TCR of the present invention that transduces has lethal effect to the target cell for expressing related antigen, and to not The target cell of expression related antigen does not have lethal effect substantially.
The all documents referred in the present invention are all incorporated as in this application reference, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content for having read the present invention, those skilled in the art can To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.
- 1435 sequence tables
<110>Guangzhou Xiangxue Pharmaceutical Co
<120>The TCR of identification NY-ESO-1 antigen small peptides
<130> P2016-1815
<150> CN201510751225.5
<151> 2015-11-04
<160> 37
<170> PatentIn version 3.5
<210> 1
<211> 110
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>TCR α chain variable domains
<400> 1
Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly
1 5 10 15
Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
20 25 30
Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu
35 40 45
Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr
50 55 60
Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser Arg
65 70 75 80
Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys
85 90 95
Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn
100 105 110
<210> 2
<211> 330
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>TCR α chain variable domains
<400> 2
agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60
atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120
agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180
ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240
gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 300
aaagggacac gacttcatat tctccccaat 330
<210> 3
<211> 250
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>TCR α chains
<400> 3
Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly
1 5 10 15
Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
20 25 30
Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu
35 40 45
Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr
50 55 60
Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser Arg
65 70 75 80
Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys
85 90 95
Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn Ile Gln
100 105 110
Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp
115 120 125
Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser
130 135 140
Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp
145 150 155 160
Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn
165 170 175
Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro
180 185 190
Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu
195 200 205
Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu
210 215 220
Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn
225 230 235 240
Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250
<210> 4
<211> 750
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>TCR α chains
<400> 4
agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60
atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120
agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180
ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240
gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 300
aaagggacac gacttcatat tctccccaat atccagaacc ctgaccctgc cgtgtaccag 360
ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420
acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 480
atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540
gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600
gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 660
tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 720
ctgctcatga cgctgcggct gtggtccagc 750
<210> 5
<211> 112
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>TCR β chain variable domains
<400> 5
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr
100 105 110
<210> 6
<211> 336
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>TCR β chain variable domains
<400> 6
gatgctggag ttatccagtc accccggcac gaggtgacag agatgggaca agaagtgact 60
ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120
cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180
cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240
ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttagg gagcaacgag 300
cagtacttcg ggccgggcac caggctcacg gtcaca 336
<210> 7
<211> 291
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>TCR β chains
<400> 7
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr
100 105 110
Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
115 120 125
Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu
130 135 140
Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
145 150 155 160
Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys
165 170 175
Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu
180 185 190
Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
195 200 205
Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
210 215 220
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
225 230 235 240
Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser
245 250 255
Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala
260 265 270
Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp
275 280 285
Ser Arg Gly
290
<210> 8
<211> 873
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>TCR β chains
<400> 8
gatgctggag ttatccagtc accccggcac gaggtgacag agatgggaca agaagtgact 60
ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120
cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180
cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240
ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttagg gagcaacgag 300
cagtacttcg ggccgggcac caggctcacg gtcacagagg acctgaaaaa cgtgttccca 360
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420
ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 480
gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 540
ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 600
aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660
tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 720
gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 780
tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 840
atggccatgg tcaagagaaa ggattccaga ggc 873
<210> 9
<211> 9
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>Antigen small peptide
<400> 9
Ser Leu Leu Met Trp Ile Thr Gln Cys
1 5
<210> 10
<211> 5
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223> α CDR1
<400> 10
Thr Ser Ile Asn Asn
1 5
<210> 11
<211> 7
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223> α CDR2
<400> 11
Ile Arg Ser Asn Glu Arg Glu
1 5
<210> 12
<211> 9
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223> α CDR3
<400> 12
Ala Thr Asp Ala Asn Gly Lys Ile Ile
1 5
<210> 13
<211> 5
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223> β CDR1
<400> 13
Ser Gly His Asp Tyr
1 5
<210> 14
<211> 6
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223> β CDR2
<400> 14
Phe Asn Asn Asn Val Pro
1 5
<210> 15
<211> 10
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223> β CDR3
<400> 15
Ala Ser Ser Leu Gly Ser Asn Glu Gln Tyr
1 5 10
<210> 16
<211> 15
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223> α CDR1
<400> 16
actagtataa acaat 15
<210> 17
<211> 21
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223> α CDR2
<400> 17
atacgttcaa atgaaagaga g 21
<210> 18
<211> 27
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223> α CDR3
<400> 18
gctacggacg caaacggcaa gatcatc 27
<210> 19
<211> 15
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223> β CDR1
<400> 19
tcaggacacg actac 15
<210> 20
<211> 18
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223> β CDR2
<400> 20
tttaacaaca acgttccg 18
<210> 21
<211> 30
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223> β CDR3
<400> 21
gccagcagtt tagggagcaa cgagcagtac 30
<210> 22
<211> 270
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>TCR α chains with targeting sequencing
<400> 22
Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu
1 5 10 15
Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser
20 25 30
Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser
35 40 45
Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val
50 55 60
His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg
65 70 75 80
Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile
85 90 95
Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp
100 105 110
Ala Asn Gly Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu
115 120 125
Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser
130 135 140
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln
145 150 155 160
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys
165 170 175
Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val
180 185 190
Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn
195 200 205
Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys
210 215 220
Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn
225 230 235 240
Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val
245 250 255
Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265 270
<210> 23
<211> 810
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>TCR α chains with targeting sequencing
<400> 23
atggaaactc tcctgggagt gtctttggtg attctatggc ttcaactggc tagggtgaac 60
agtcaacagg gagaagagga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 120
atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 180
agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 240
ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 300
gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 360
aaagggacac gacttcatat tctccccaat atccagaacc ctgaccctgc cgtgtaccag 420
ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480
acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540
atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600
gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660
gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720
tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780
ctgctcatga cgctgcggct gtggtccagc 810
<210> 24
<211> 310
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>TCR β chains with targeting sequencing
<400> 24
Met Asp Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala
1 5 10 15
Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr
20 25 30
Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His
35 40 45
Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu
50 55 60
Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro
65 70 75 80
Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu
85 90 95
Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala
100 105 110
Ser Ser Leu Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu
115 120 125
Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
130 135 140
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
145 150 155 160
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
165 170 175
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
180 185 190
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
195 200 205
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
210 215 220
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
225 230 235 240
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
245 250 255
Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly
260 265 270
Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
275 280 285
Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
290 295 300
Arg Lys Asp Ser Arg Gly
305 310
<210> 25
<211> 930
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>TCR β chains with targeting sequencing
<400> 25
atggactcct ggaccctctg ctgtgtgtcc ctttgcatcc tggtagcaaa gcacacagat 60
gctggagtta tccagtcacc ccggcacgag gtgacagaga tgggacaaga agtgactctg 120
agatgtaaac caatttcagg acacgactac cttttctggt acagacagac catgatgcgg 180
ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240
gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300
tcagaaccca gggactcagc tgtgtacttc tgtgccagca gtttagggag caacgagcag 360
tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 420
gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480
gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540
aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600
aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660
ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720
acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780
gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840
gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900
gccatggtca agagaaagga ttccagaggc 930
<210> 26
<211> 203
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>STCR α chains
<400> 26
Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly
1 5 10 15
Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
20 25 30
Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu
35 40 45
Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr
50 55 60
Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser Arg
65 70 75 80
Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys
85 90 95
Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Asn Ile Gln
100 105 110
Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp
115 120 125
Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser
130 135 140
Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp
145 150 155 160
Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn
165 170 175
Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro
180 185 190
Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200
<210> 27
<211> 609
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>STCR α chains
<400> 27
agccagcagg gcgaagaaga tcctcaggcc ttgagcatcc aggagggtga aaatgccacc 60
atgaactgca gttacaaaac tagtataaac aatttacagt ggtatagaca aaattcaggt 120
agaggccttg tccacctaat tttaatacgt tcaaatgaaa gagagaaaca cagtggaaga 180
ttaagagtca cgcttgacac ttccaagaaa agcagttcct tgttgatcac ggcttcccgg 240
gcagcagaca ctgcttctta cttctgtgct acggacgcaa acggcaagat catctttgga 300
aaagggacac gacttcatat tctccccaat atccagaacc ctgaccctgc cgtgtaccag 360
ctgagagact ctaagtcgag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420
acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaatg tgtgctagac 480
atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540
gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600
gaaagttcc 609
<210> 28
<211> 242
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>STCR β chains
<400> 28
Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr
100 105 110
Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
115 120 125
Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu
130 135 140
Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
145 150 155 160
Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys
165 170 175
Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu
180 185 190
Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys
195 200 205
Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
210 215 220
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
225 230 235 240
Ala Asp
<210> 29
<211> 726
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>STCR β chains
<400> 29
gatgcgggcg tgattcagtc accccggcac gaggtgacag agatgggaca agaagtgact 60
ctgagatgta aaccaatttc aggacacgac taccttttct ggtacagaca gaccatgatg 120
cggggactgg agttgctcat ttactttaac aacaacgttc cgatagatga ttcagggatg 180
cccgaggatc gattctcagc taagatgcct aatgcatcat tctccactct gaagatccag 240
ccctcagaac ccagggactc agctgtgtac ttctgtgcca gcagtttagg gagcaacgag 300
cagtacttcg ggccgggcac caggctcacg gtcacagagg acctgaaaaa cgtgttccca 360
cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 420
ctggtgtgcc tggccaccgg tttctacccc gaccacgtgg agctgagctg gtgggtgaat 480
gggaaggagg tgcacagtgg ggtctgcaca gacccgcagc ccctcaagga gcagcccgcc 540
ctcaatgact ccagatacgc tctgagcagc cgcctgaggg tctcggccac cttctggcag 600
gacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 660
tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 720
gcagac 726
<210> 30
<211> 245
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR
<400> 30
Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly
1 5 10 15
Glu Asn Val Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
20 25 30
Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu
35 40 45
Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr
50 55 60
Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Val Arg
65 70 75 80
Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys
85 90 95
Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro Gly Gly Gly
100 105 110
Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
115 120 125
Glu Gly Gly Thr Gly Asp Ala Gly Val Thr Gln Ser Pro Arg His Glu
130 135 140
Ser Val Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser
145 150 155 160
Gly His Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Pro Lys Arg Gly Leu
165 170 175
Glu Leu Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly
180 185 190
Met Pro Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser
195 200 205
Thr Leu Lys Ile Gln Pro Val Glu Pro Arg Asp Ser Ala Val Tyr Phe
210 215 220
Cys Ala Ser Ser Leu Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr
225 230 235 240
Arg Leu Thr Val Thr
245
<210> 31
<211> 735
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR
<400> 31
tctcaacaag gtgaagaaga tccgcaggcc ctgagtattc aagaaggtga aaatgtgacc 60
atcaactgct cttacaaaac gagtatcaac aatctgcagt ggtaccgtca aaattctggc 120
cgcggtctgg ttcatctgat tctgatccgt tccaacgaac gcgaaaaaca ctcaggccgt 180
ctgcgcgtta ccctggatac cagcaaaaaa tcttctagtc tggaaatcac cgcagtccgt 240
ccggcagata cggcaagcta tttttgtgca accgacgcta atggtaaaat tatcttcggc 300
aaaggtaccc gcctgcatat tctgccgggc ggtggctccg aaggtggcgg ttcagaaggc 360
ggtggctcgg aaggtggcgg tagcgaaggc ggtaccggtg atgcgggtgt cacgcagtct 420
ccgcgtcatg aaagtgtgga aatgggccaa gaagttacgc tgcgctgcaa accgatcagc 480
ggtcacgact acctgttttg gtaccgtcag accccgaaac gcggcctgga actgctgatc 540
tacttcaaca ataacgttcc gattgatgac tcgggtatgc cggaagatcg ttttagcgcg 600
aaaatgccga atgcctcgtt cagcacgctg aaaattcagc cggtcgaacc gcgtgactcc 660
gcagtgtatt tttgtgcttc ctcactgggc agtaacgaac agtacttcgg cccgggtacc 720
cgtctgaccg tgacg 735
<210> 32
<211> 109
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR α chains
<400> 32
Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly
1 5 10 15
Glu Asn Val Thr Ile Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu
20 25 30
Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu
35 40 45
Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr
50 55 60
Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Glu Ile Thr Ala Val Arg
65 70 75 80
Pro Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Asn Gly Lys
85 90 95
Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro
100 105
<210> 33
<211> 327
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR α chains
<400> 33
tctcaacaag gtgaagaaga tccgcaggcc ctgagtattc aagaaggtga aaatgtgacc 60
atcaactgct cttacaaaac gagtatcaac aatctgcagt ggtaccgtca aaattctggc 120
cgcggtctgg ttcatctgat tctgatccgt tccaacgaac gcgaaaaaca ctcaggccgt 180
ctgcgcgtta ccctggatac cagcaaaaaa tcttctagtc tggaaatcac cgcagtccgt 240
ccggcagata cggcaagcta tttttgtgca accgacgcta atggtaaaat tatcttcggc 300
aaaggtaccc gcctgcatat tctgccg 327
<210> 34
<211> 112
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR β chains
<400> 34
Asp Ala Gly Val Thr Gln Ser Pro Arg His Glu Ser Val Glu Met Gly
1 5 10 15
Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu
20 25 30
Phe Trp Tyr Arg Gln Thr Pro Lys Arg Gly Leu Glu Leu Leu Ile Tyr
35 40 45
Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg
50 55 60
Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln
65 70 75 80
Pro Val Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Gly Ser Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr
100 105 110
<210> 35
<211> 336
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR β chains
<400> 35
gatgcgggtg tcacgcagtc tccgcgtcat gaaagtgtgg aaatgggcca agaagttacg 60
ctgcgctgca aaccgatcag cggtcacgac tacctgtttt ggtaccgtca gaccccgaaa 120
cgcggcctgg aactgctgat ctacttcaac aataacgttc cgattgatga ctcgggtatg 180
ccggaagatc gttttagcgc gaaaatgccg aatgcctcgt tcagcacgct gaaaattcag 240
ccggtcgaac cgcgtgactc cgcagtgtat ttttgtgctt cctcactggg cagtaacgaa 300
cagtacttcg gcccgggtac ccgtctgacc gtgacg 336
<210> 36
<211> 24
<212> PRT
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR catenation sequences
<400> 36
Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly
1 5 10 15
Gly Gly Ser Glu Gly Gly Thr Gly
20
<210> 37
<211> 72
<212> DNA
<213>Artificial sequence (artificial)
<220>
<223>Single-stranded TCR catenation sequences
<400> 37
ggcggtggct ccgaaggtgg cggttcagaa ggcggtggct cggaaggtgg cggtagcgaa 60
ggcggtaccg gt 72

Claims (10)

1. a kind of φt cell receptor (TCR), it is characterised in that the TCR can be tied with SLLMWITQC-HLAA0201 compounds Close；Preferably, described TCR includes TCR α chains variable domains and TCR β chain variable domains, it is characterised in that the TCR α chain variable domains CDR3 amino acid sequence be ATDANGKII (SEQ ID NO:12)；And/or the amino of the CDR3 of the TCR β chain variable domains Acid sequence is ASSLGSNEQY (SEQ ID NO:15)；
It is highly preferred that 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
α CDR1-TSINN(SEQ ID NO:10)
α CDR2-IRSNERE(SEQ ID NO:11)
α CDR3-ATDANGKII(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
β CDR1-SGHDY(SEQ ID NO:13)
β CDR2-FNNNVP(SEQ ID NO:14)
β CDR3-ASSLGSNEQY(SEQ ID NO:15)。
2. TCR as claimed in claim 1, it is characterised in that it includes TCR α chains variable domains and TCR β chain variable domains, described TCR α chain variable domains are and SEQ ID NO:1 amino acid sequence with least 90% sequence thereto；And/or the TCR β chains Variable domain is and SEQ ID NO：5 amino acid sequences with least 90% sequence thereto.
3. TCR as claimed in claim 1, it is characterised in that the α chains of the TCR and/or C- the or N- ends of β chains are combined with Conjugate；Preferably, the conjugate for being combined with the φt cell receptor is detectable, therapeutic agent, PK modifications part or appoints The combination of what these material；Preferably, the therapeutic agent is anti-CD 3 antibodies.
4. a kind of multivalent TCR complex, it is characterised in that comprising at least two TCR molecules, and at least one TCR therein Molecule is the TCR any one of the claims.
5. a kind of nucleic acid molecules, it is characterised in that the nucleic acid molecules include TCR point described in any of the above-described claim of coding The nucleotide sequence or its complementary series of son；
Preferably, nucleotide sequence SEQ ID NO of the described nucleic acid molecules comprising coding TCR α chain variable domains：2 or SEQ ID NO:33；And/or
Nucleotide sequence SEQ ID NO of the described nucleic acid molecules comprising coding TCR β chain variable domains：6 or SEQ ID NO:35.
6. a kind of carrier, it is characterised in that described carrier contains arbitrary described nucleic acid molecules in claim 5；Preferably, Described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
7. a kind of detached host cell, it is characterised in that containing the carrier described in claim 6 in described host cell Or it is integrated with arbitrary described nucleic acid molecules in the claim 5 of external source in chromosome.
8. a kind of cell, it is characterised in that arbitrary described nucleic acid molecules or claim in the cell transduction claim 5 Carrier described in 6；Preferably, the cell is T cell or stem cell.
9. a kind of pharmaceutical composition, it is characterised in that the composition contains pharmaceutically acceptable carrier and claim Arbitrary described nucleic acid point in TCR compounds, claim 5 described in TCR, claim 4 any one of 1-3 Cell described in son or claim 8.
10. the TCR compounds or right described in the φt cell receptor or claim 4 any one of claim 1-3 Require the purposes of the cell described in 8, it is characterised in that for preparing the medicine for the treatment of tumour or autoimmune disease.
CN201610958444.5A 2015-11-04 2016-11-03 TCR for recognizing NY-ESO-1 antigen short peptide Active CN106632660B (en)

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CN108948184A (en) * 2017-05-22 2018-12-07 广东香雪精准医疗技术有限公司 A kind of identification is derived from the T cell receptor of PRAME antigen small peptide
CN109251244B (en) * 2017-07-13 2021-10-26 中国科学院广州生物医药与健康研究院 TCR (T cell receptor) for recognizing LMP1 antigen derived from EBV (Epstein-Barr Virus) membrane protein
CN109251244A (en) * 2017-07-13 2019-01-22 中国科学院广州生物医药与健康研究院 A kind of identification is derived from the TCR of EBV memebrane protein LMP1 antigen
CN111133104A (en) * 2017-08-11 2020-05-08 特里比奥迪卡有限责任公司 Methods of generating epitopes that bind to recognition molecules by templated assembly
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CN111819194B (en) * 2018-02-26 2024-06-04 基因医疗免疫疗法有限责任公司 NYESO T Cell Receptor (TCR)
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CN112300268A (en) * 2019-08-02 2021-02-02 广东香雪精准医疗技术有限公司 High affinity T cell receptors recognizing NY-ESO-1 antigens
CN112442118A (en) * 2019-08-30 2021-03-05 深圳普瑞金生物药业有限公司 TCR and application thereof
CN112442118B (en) * 2019-08-30 2023-02-14 深圳普瑞金生物药业股份有限公司 TCR and application thereof
WO2022002014A1 (en) * 2020-07-01 2022-01-06 华夏英泰（北京）生物技术有限公司 T cell antigen receptor, multimeric complex thereof and preparation method and use thereof
WO2022170509A1 (en) 2021-02-09 2022-08-18 深圳普瑞金生物药业有限公司 Tcr and application thereof

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