CN106749620A - Recognize the φt cell receptor of MAGE A1 antigen small peptides - Google Patents

Abstract

The invention provides a kind of φt cell receptor (TCR) that can specifically bind the small peptide KVLEYVIKV derived from MAGE A1 antigens, the antigen small peptide KVLEYVIKV can with HLA A0201 formed compound and together be presented to cell surface.Carrier present invention also offers the nucleic acid molecules for encoding the TCR and comprising the nucleic acid molecules.In addition, present invention also offers the cell of the TCR of the present invention that transduces.

Description

Recognize the φt cell receptor of MAGE-A1 antigen small peptides

Technical field

The present invention relates to be capable of identify that the TCR from MAGE-A1 antigen small peptides, the invention further relates to transduce, above-mentioned TCR comes The specific T cells of MAGE-A1 of acquisition, and their purposes in MAGE-A1 relevant diseases are prevented and treated.

Background technology

MAGE-A1 is degraded to micromolecule polypeptide as a kind of autochthonous tumor antigen after generating in the cell, and with MHC (main histocompatibility complex) molecule combines to form compound, is presented to cell surface.Research display, KVLEYVIKV It is the small peptide derived from MAGE-A1.MAGE-A1 albumen has expression, including melanoma, Yi Jiqi in kinds of tumors type His solid tumor such as stomach cancer, lung cancer, cancer of the esophagus, carcinoma of urinary bladder, SCCHN etc..For the treatment of above-mentioned disease, can To use the methods such as chemotherapy and radiation treatment, but the normal cell of itself can all be caused damage.

T cell adoptive immunotherapy is that will there is specific reaction-ive T cell to be transferred in patient body to target cell antigen, 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 specific TCR of antigen small peptide and small peptide-main histocompatbility The combination of complex (pMHC compounds) triggers T cell to be directly physically contacted with antigen presenting cell (APC), then T cell And other cell membrane surface molecules of both APC just interact, 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 has specific TCR to MAGE-A1 antigen small peptides, and TCR T cells of transduceing are obtained There is specific T cell to MAGE-A1 antigen small peptides, 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 MAGE-A1 antigen small peptides.

A kind of the first aspect of the present invention, there is provided φt cell receptor (TCR), the TCR can be with KVLEYVIKV-HLA A0201 compounds are combined.

In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chain variable domains CDR3 amino acid sequence be AFPSGGGADGLT (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domains Amino acid sequence is ASSVEGYPSYEQY (SEQ ID NO:15).

In another preference, 3 complementary determining regions (CDR) of the TCR α chain variable domains are：

αCDR1-SSNFYA (SEQ ID NO:10)

αCDR2-MTLNGDE (SEQ ID NO:11)

αCDR3-AFPSGGGADGLT (SEQ ID NO:12)；And/or

3 complementary determining regions of the TCR β chain variable domains are：

βCDR1-SGDLS (SEQ ID NO:13)

βCDR2-YYNGEE (SEQ ID NO:14)

βCDR3-ASSVEGYPSYEQY (SEQ ID NO:15)。

In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chain variable domains It is and SEQ ID NO:1 amino acid sequence with least 90% sequence thereto；And/or the TCR β chain variable domains be with 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 chain constant region TRAC*01 and TCR β Chain constant region TRBC1*01 or TRBC2*01.

In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO：The β chain ammonia of the 3 and/or TCR Base acid sequence is SEQ ID NO:7.

In another 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 chains J With one or more mutation, wherein amino acid 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) and (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 Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；With

The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.

In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO:The β chains of the 26 and/or TCR 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 the 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 the 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 NO of the nucleic acid molecules comprising coding TCR α chain variable domains： 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 Nucleotide sequence SEQ ID NO comprising coding TCR β chains：8.

The 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 vectors；It is highly preferred that described carrier is slow virus carrier.

A kind of the fifth aspect of the present invention, there is provided host cell of separation, contains the present invention in described host cell The nucleic acid molecules described in the third aspect present invention of external source are integrated with carrier or genome described in fourth aspect.

A kind of the 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.

The 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 described in the nucleic acid molecules stated, fourth aspect present invention or the cell described in sixth aspect present invention.

The eighth aspect of the present invention, there is provided φt cell receptor or second aspect present invention described in first aspect present invention Nucleic acid molecules, carrier or this hair described in fourth aspect present invention described in described TCR compounds, third aspect present invention The purposes of the cell described in bright 6th aspect, the medicine for preparing treatment tumour or autoimmune disease.

A kind of the ninth aspect of the present invention, there is provided method for treating disease, including fitted to needing the object for the treatment of to apply 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 The carrier described in nucleic acid molecules, fourth aspect present invention described in aspect or the cell or this hair described in sixth aspect present invention Pharmaceutical composition described in bright 7th aspect；

Preferably, described disease is tumour, and preferably described tumour includes melanoma, and other entity tumors are such as Stomach cancer, lung cancer, cancer of the esophagus, carcinoma of urinary bladder, SCCHN, prostate cancer, breast cancer, colon cancer, oophoroma etc..

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) 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.

Brief description of the drawings

It is variable that Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively TCR α chains variable domain amino acid sequence, TCR α chains Domain nucleotide sequence, TCR α chain amino acid sequences, TCR α chains nucleotide sequence, the TCR α chain amino acid sequences with targeting sequencing And the TCR α chain nucleotide sequences with targeting sequencing.

It is variable that Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chains variable domain amino acid sequence, TCR β chains Domain nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, the TCR β chain amino acid sequences with targeting sequencing And the TCR β chain nucleotide sequences with targeting sequencing.

Fig. 3 is the CD8 of monoclonal cell⁺And the double positive staining results of the tetramer-PE.

Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chains.

Fig. 5 a and Fig. 5 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chains.

Fig. 6 is the glue figure of the sTCR 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.Left side swimming lane is molecular weight marker (marker), right Breathing arm road is non-reduced glue.

Figure 12 is the ForteBio dynamics that sTCR of the present invention is combined with KVLEYVIKV-HLA A0201 compounds Collection of illustrative plates.

Figure 13 is that the BIAcore that soluble single-chain T CR of the present invention is combined with KVLEYVIKV--HLA A0201 compounds is moved Mechanics collection of illustrative plates.

Figure 14 be transduce TCR of the present invention effector cell activation experiment result.

Figure 15 be transduce TCR of the present invention effector cell killing experiments result.

Specific embodiment

The present inventor have found and MAGE-A1 antigen small peptide KVLEYVIKV (SEQ ID by in-depth study extensively NO:9) TCR that can be specifically bound, the antigen small peptide KVLEYVIKV can with HLA A0201 formed compound and together with by It is presented to cell surface.Carrier present invention also offers the nucleic acid molecules for encoding the TCR and comprising the nucleic acid molecules. In addition, present invention also offers the cell of the TCR of the present invention that transduces.

Term

MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, its for The presentation of antigen has specificity, and different individualities has different MHC, can present different small peptides 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, trigger T cell thin with antigen presentation by the combination of the TCR and pMHC compounds of antigentic specificity Born of the same parents (APC) are directly physically contacted, and then other cell membrane surface molecules of both T cell and APC just interact, this A series of 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 The right heterogeneous dimerization TCR of α β have α chains and β chains, and α chains and β chains constitute the subunit of α β heterodimerics TCR.In a broad sense, α and β are each Chain includes variable region, bonding pad and constant region, and β chains 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 are made up of the variable region for connecting and bonding pad.The sequence of TCR constant domains can be in international immune genetic Found in the public database for learning information system (IMGT), such as the constant domain sequence of TCR molecule alpha chains is " TRAC*01 ", TCR points The constant domain sequence of sub- β chains is " TRBC1*01 " or " TRBC2*01 ".Additionally, α the and β chains of TCR also include transmembrane region and kytoplasm Area, cytoplasmic region is very short.

In the present invention, term " polypeptide of the present invention ", " TCR of the invention ", " φt cell receptor of the invention " is interchangeable makes With.

Native interchain disulfide bond and artificial interchain disulfide bond

There is one group of disulfide bond in membrane-proximal region C α and C the β interchain of natural TCR, " the sulphur of native interchain two is referred to as in the present invention Key ".In the present invention, by what is be artificially introduced, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond claim It is " 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, the first aspect of the present invention provides one kind The TCR molecules of KVLEYVIKV-HLA A0201 compounds can be combined.Preferably, the TCR molecules are to separate or purify 's.α the and β chains of the TCR respectively have 3 complementary determining regions (CDR).

In being preferably carried out mode at one of the invention, the α chains of the TCR are included with following amino acid sequence CDR：

αCDR1-SSNFYA (SEQ ID NO:10)

αCDR2-MTLNGDE (SEQ ID NO:11)

αCDR3-AFPSGGGADGLT (SEQ ID NO:12)；And/or

3 complementary determining regions of the TCR β chain variable domains are：

βCDR1-SGDLS (SEQ ID NO:13)

βCDR2-YYNGEE (SEQ ID NO:14)

βCDR3-ASSVEGYPSYEQY (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 TCR of the 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 refer to comprising above-mentioned α and/or β The TCR molecules of chain CDR region sequence and any suitable frame structure.TCR α chain variable domains of the present invention are and SEQ ID NO:1 tool There are at least 90%, preferably 95%, the more preferably 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 invention, TCR molecules of the invention are the heterodimers being made up of α and β chains.Specifically Ground, the α chains of the one side heterogeneous dimerization TCR molecules include 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 molecules 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 invention, TCR molecules of the 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 invention, the constant domain of TCR molecules of the invention is the constant domain of people.Art technology Personnel know or can be obtained by consulting the public database of pertinent texts or IMGT (international immunogenetics information system) Obtain the constant domain amino acid sequence of people.For example, the constant domain sequence of TCR molecule alphas chain of the present invention can be " TRAC*01 ", TCR points The constant domain sequence of sub- β chains can be " TRBC1*01 " or " TRBC2*01 ".The amino acid sequence be given in the TRAC*01 of IMGT The 53rd be Arg, be expressed as herein：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 (antibody) is made For antigen recognizing molecule is the same, TCR can also be developed and be applied to diagnose and treat, and at this moment need to obtain soluble TCR points 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 its targeting is presented the cell of specific antigen.The present invention also obtain have to MAGE-A1 antigen small peptides it is specific solvable 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, Replace the cysteine residues of the Thr48 of TRAC*01 exons 1s and the Ser57 of substitution TRBC1*01 or TRBC2*01 exons 1s To form disulfide bond.Cysteine residues are introduced to be can also be to form other sites of disulfide bond：TRAC*01 exons 1s The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1s；The Tyr10 and TRBC1*01 of TRAC*01 exons 1s or The Ser17 of TRBC2*01 exons 1s；Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s Asp59；The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 exons 1s Arg53 and TRBC1*01 or TRBC2*01 exons 1s Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1s or The Ala19 of TRBC2*01 exons 1s；Or Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s Glu20.I.e. cysteine residues instead of above-mentioned α with any 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 its do not reached including cysteine residues missing natural disulphide bonds purpose, also can be by the way that day will be formed The cysteine residues of right disulfide bond sport another amino acid to reach above-mentioned purpose.

As described above, the artificial disulfide bond that TCR of the invention is introduced between may be embodied in the residue of itself α and β chain constant domain. It should be noted that the artificial disulfide bond with or without introducing mentioned above between constant domain, TCR of the 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 Connected by the natural disulphide bonds 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 its following hydrophobic core position of variable domain： (α and/or β chains) variable region amino acid the 11st, 13,19,21,53,76,89,91,94, and/or α chain J gene (TRAJ) small peptide Amino acid position is reciprocal 3rd, 5,7, and/or β chain J gene (TRBJ) small peptides amino acid position is reciprocal 2nd, 4,6, wherein ammonia The Position Number of base acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).People in the art Member knows above-mentioned international immunogenetics information system, and the amino acid residue that 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 α and the 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 in the present invention flexible peptide chain can be any suitable connection TCR α and 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 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 can significantly improve 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 and TRBC1*01 of TRAV 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) removes 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 is 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.

TCR of the invention can also multivalence complex form provide.Multivalent TCR complex of the invention comprising two, Three, the four or more TCR of the present invention polymers that are combined and are formed, can such as 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.TCR compounds of the 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 to answer The intermediate of compound.

TCR of the invention can be used alone, and can also 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 KVLEYVIKV-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 agent can produce detectable product Enzyme.

The therapeutic agent that can be combined or be coupled with TCR of the present invention 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. (Gillies etc., 1992, NAS's proceeding such as cell factor such as IL-2 (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 are (for example, DT- diaphorases (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..

In addition, TCR of the invention can also be comprising derived from the heterozygosis TCR more than a kind of species sequence.For example, grinding Studying carefully display Muridae TCR can more effectively express in human T-cell than people TCR.Therefore, TCR of the present invention can include people's variable domain With the constant domain of mouse.The defect of this method is possible to trigger 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 amino acid name is represented using international single English alphabet or three English alphabets herein, 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

The second aspect of the present invention provides the nucleic acid molecules of coding first aspect present invention TCR molecules or part thereof, institute It can be one or more CDR, the variable domain of α and/or β chains, and α chains and/or β chains to state part.

The nucleotide sequence for encoding first aspect present invention TCR molecule alpha chain CDR regions is as follows：

αCDR1-tccagcaatttttatgcc (SEQ ID NO:16)

αCDR2-atgactttaaatggggatgaa (SEQ ID NO:17)

αCDR3-gccttcccttcaggaggaggtgctgacggactcacc(SEQ ID NO:18)

The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR regions is as follows：

βCDR1-tctggagacctctct (SEQ ID NO:19)

βCDR2-tattataatggagaagag (SEQ ID NO:20)

βCDR3-gccagcagcgtagaaggctacccctcctacgagcagtac(SEQ ID NO:21)

Therefore, the nucleotide sequence for encoding the nucleic acid molecules of the present invention of TCR α chains of the present invention includes SEQ ID NO:16、SEQ ID NO:17 and SEQ ID NO:18, and/or encode the nucleotide sequence of the nucleic acid molecules of the present invention of TCR β chains of the present invention and include 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 bag of the nucleic acid molecules of the present invention of TCR α chain variable domains of the present invention Include 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, the nucleotide sequence for encoding 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 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:35.More Preferably, the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:4 and/or SEQ ID NO:8.Or, the present invention The nucleotides sequence of nucleic acid molecules is classified as SEQ ID NO:31.

It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.Therefore, 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 One of example in the present invention illustrates that " variant of degeneracy " refer to that coding has SEQ ID NO:1 protein sequence, But with SEQ ID NO:The 2 differentiated nucleotide sequence of sequence.

Nucleotide sequence can be through codon optimization.Different cells is above different in the utilization of specific codon , can change the codon in sequence to increase expression quantity 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.

Nucleic acid molecules full length sequence of the invention 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 the carrier comprising nucleic acid molecules of the invention, including expression vector, i.e., 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, can be transferred to nucleotides of the invention in cell by carrier, such as in T cell so that the cell table Up to the TCR of MAGE-A1 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 through genetic engineering with carrier of the invention or coded sequence.The host Containing being integrated with nucleic acid molecules of the invention in carrier of the invention or chromosome 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 cell of the separation for expressing TCR of the invention, particularly T cell.The T cell can spread out The T cell for from subject separate is conigenous, or can be the mixed cellularity group for from subject separate, 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 comprising the nucleotide sequence for encoding 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 Moving to HSC will not cause in cell surface expression TCR, because stem cell surface does not express CD3 molecules.However, when stem cell point Turn to and migrate during 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., the such as Robbins, (2008)J.Immunol.180:6116-6131).The T cell of expression TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat Rev for carrying out adoptive treatment Cancer8(4)：299-308).

MAGE-A1 antigen-related diseases

The invention further relates to being treated in subject and/or preventing the method with MAGE-A1 relevant diseases, it includes adopting Property transfer MAGE-A1 specific T-cells to the subject the step of.The MAGE-A1 specific T-cells can recognize that KVLEYVIKV- HLA A0201 compounds.

The specific T cells of MAGE-A1 of the invention can be used to treat any presentation MAGE-A1 antigen small peptides The MAGE-A1 relevant diseases of KVLEYVIKV-HLA A0201 compounds.Including but not limited to tumour, such as melanoma, Yi Jiqi His entity tumor for example stomach cancer, lung cancer, cancer of the esophagus, carcinoma of urinary bladder, SCCHN, prostate cancer, breast cancer, colon cancer, Oophoroma etc..

Treatment method

Can be by separating with the patient with MAGE-A1 antigen-related diseases or the T cell of volunteer, and by the present invention TCR import in above-mentioned T cell, then the cell that these genetic engineerings are modified is fed back in patient body to be treated.Cause This, is the invention provides a kind of method for treating MAGE-A1 relevant diseases, including the T of the expression TCR of the present invention that will be separate thin Born of the same parents, it is preferable that the T cell in itself, is input in patient body from patient.Usually, including (1) separate patient T cell, (2) with nucleic acid molecules of the present invention or the nucleic acid molecules ex vivo transduction T cell of TCR molecules of the present invention can be encoded, (3) are by gene work The T cell of journey 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 MAGE-A1 antigen small peptide compound KVLEYVIKV-HLA A0201, while 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 limitation 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 is calculated by weight.Unless otherwise indicated, otherwise percentage and number is calculated by weight. Experiment material used and reagent can be obtained from commercially available channel unless otherwise instructed in following examples.

Embodiment 1 clones MAGE-A1 antigen small peptide specific T-cells

Using synthesizing small peptide KVLEYVIKV (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 KVLEYVIKV 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 sorting is expanded, And secondary sorting is carried out as stated above, then 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 MAGE-A1 antigen small peptide specific T-cell clones and the structure of carrier

Use Quick-RNA^TMThe antigen small peptide screened in MiniPrep (ZYMO research) extracting embodiments 1 The total serum IgE of KVLEYVIKV specificity, HLA-A0201 restricted T cell clone.The synthesis of cDNA is using clontech's SMART RACE cDNA amplification kits, the primer of use is designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned It is sequenced on to carrier T (TAKARA).It should be noted that the sequence is complementary series, not comprising introne.Through sequencing, 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 chains variable domain amino acid sequence, TCR α chain variable domains nucleotide sequence, TCR α chain amino acid sequences, TCR α chains nucleotide sequence, the TCR α chain amino acid sequences with targeting sequencing and the TCR α chain nucleotides with targeting sequencing Sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chains variable domain amino acid sequence, TCR β chain variable domains Nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, the TCR β chain amino acid sequences with targeting sequencing with And the TCR β chain nucleotide sequences with targeting sequencing.

Identified, α chains include the CDR with following amino acid sequence：

αCDR1-SSNFYA (SEQ ID NO:10)

αCDR2-MTLNGDE (SEQ ID NO:11)

αCDR3-AFPSGGGADGLT (SEQ ID NO:12)

β chains include the CDR with following amino acid sequence：

βCDR1-SGDLS (SEQ ID NO:13)

βCDR2-YYNGEE (SEQ ID NO:14)

βCDR3-ASSVEGYPSYEQY (SEQ ID NO:15)

The full-length gene of TCR α chains and β chains is cloned into Lentiviral respectively by overlapping (overlap) PCR pLenti(addgene).Specially：The full-length gene of TCR α chains and TCR β chains is attached with overlap PCR obtains 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.Used as control, while the also slow virus carrier pLenti-eGFP of construction expression eGFP.Use again afterwards 293T/17 packs pseudovirus.

Expression, refolding and the purifying of the solvable TCR of the MAGE-A1 antigens small peptide of embodiment 3 specificity

To obtain solvable TCR molecules, α the and β chains of TCR molecules of the invention can only include its variable domain and portion respectively 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 alphabetical expression.By ＜ ＜ molecules Cloning experimentation room handbook ＞ ＞ (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and The objective gene sequence of above-mentioned TCR α and β chains is inserted respectively into expression and carried by the standard method described in Russell) after synthesis Body pET28a+ (Novagene), the cloning site of upstream and downstream is respectively NcoI and NotI.Insert Fragment confirms nothing by sequencing By mistake.

The expression vector of TCR α and β chains is converted by chemical transformation respectively and enters expression bacterium BL21 (DE3), bacterium Grown with LB nutrient solutions, in OD₆₀₀Induced with final concentration 0.5mM IPTG when=0.6, the bag formed after α the and β chains expression of TCR Contain body to be extracted by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, forgive Body is finally dissolved in 6M guanidine hydrochlorides, 10mM dithiothreitol (DTT)s (DTT), 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris (pH 8.1) in.

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 Solution is placed in dialysis (4 DEG C) in 10 times of deionized waters of volume afterwards, deionized water is changed into buffer solution (20mM after 12 hours Tris, pH 8.0) continue at 4 DEG C dialyse 12 hours.After solution after the completion of dialysis is through 0.45 μM of membrane filtration, by the moon from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains the successful α and β dimers of renaturation TCR is confirmed by SDS-PAGE glue.TCR then by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) it is further purified.TCR purity after purification is determined by SDS-PAGE and is more than 90%, and concentration is by BCA methods It is determined that.The SDS-PAGE glue figures of the sTCR that the present invention is obtained are as shown in Figure 6.

The generation of the specific soluble single-chain T CR of MAGE-A1 antigen small peptides of embodiment 4

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 soluble single-chain T CR molecule for the stabilization connected with flexible small peptide (linker).This is single-stranded Amino acid sequence and the nucleotide sequence difference of TCR molecules are as shown in figs. 7 a and 7b.The amino acid sequence of its α chain variable domain and Nucleotide sequence difference is as figures 8 a and 8 b show；The amino acid sequence and nucleotide sequence of its β chain variable domain are respectively such as Fig. 9 a With shown in Fig. 9 b；Amino acid sequence and the nucleotide sequence difference of its linker sequence are as as-shown-in figures 10 a and 10b.

By genes of interest through Nco I and the double digestions of Not I, connect with the pET28a carriers by Nco I and the double digestions of Not I Connect.Connection product is converted to E.coli DH5 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 recombinant plasmid transformed to E.coli afterwards BL21 (DE3), for expressing.

The expression of the specific soluble single-chain T CR of MAGE-A1 antigen small peptides of embodiment 5, renaturation and purifying

BL21 (DE 3) bacterium colony containing recombinant plasmid pET28a- template strands prepared in embodiment 4 is all inoculated in In LB culture mediums containing kanamycins, 37 DEG C of cultures to OD600 are 0.6-0.8, add IPTG to final concentration of 0.5mM, 37 DEG C continue cultivate 4h.5000rpm is centrifuged 15min harvesting sediments, is cracked with Bugbuster Master Mix (Merck) Cell pellet, 6000rpm centrifugations 15min reclaims inclusion body, then is 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 removal insoluble matter, 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 that 5mg dissolves, 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 for the treatment of 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 treatment, 4 DEG C of stirrings 10min, it is the cellulose membrane bag filter of 4kDa that renaturation solution then is loaded into interception, and bag filter is placed in the water of 1L precoolings, 4 DEG C It is slowly stirred overnight.After 17 hours into, dialyzate changes the buffer solution (20mM Tris-HCl pH 8.0) of 1L precoolings, 4 DEG C after Continuous dialysis 8h, then changes dialyzate into identical fresh buffer and continues dialysed overnight.After 17 hours, sample is through 0.45 μm of filter 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 component concentration 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

Can be special with KVLEYVIKV-HLA A0201 compounds this example demonstrated soluble TCR molecules of the present invention The opposite sex is combined.

Detected using ForteBio (Octet QKe System) and BIAcore (T200) real-time analyzer respectively and implemented The TCR molecules and the binding activity of KVLEYVIKV-HLA A0201 compounds obtained in example 3 and embodiment 5.Anti- strepto- is affine The antibody (GenScript) of element adds coupling buffer (10mM sodium-acetate buffers, pH 4.77), then flows through antibody pre- The CM5 chips for first being activated with EDC and NHS, make antibody be fixed on chip surface, are finally closed not with the hydrochloric acid solution of monoethanolamine The activating surface of reaction, completes coupling process, and coupling level is about 15,000RU.

The Streptavidin of low concentration is set to flow through the chip surface of coated antibody, then by KVLEYVIKV-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, closes the remaining binding site of Streptavidin.

The preparation process of above-mentioned KVLEYVIKV-HLA A0201 compounds is as follows：

A. purify

The E.coli bacterium solutions of 100ml induced expressions heavy chain or light chain are collected, 10ml is used after 4 DEG C of 8000g are centrifuged 10min PBS washing thallines are once, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) afterwards Shake resuspended thalline, and 20min be incubated in room temperature rotation, 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 is incubated 5min；Plus 30ml The BugBuster of 10 times of dilution, mixes, and 4 DEG C of 6000g are centrifuged 15min；Supernatant discarded, plus 30ml dilutes 10 times of BugBuster 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 KVLEYVIKV (Beijing SBS Genetech gene technology Co., Ltd) for synthesizing is dissolved in 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, 10mM EDTA are further denatured.KVLEYVIKV 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 GSSG, 5mM reduced forms Glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, Heavy chain is added in three times, 8h/ times), renaturation is carried out at least 3 days to completion at 4 DEG C, and can SDS-PAGE detections renaturation success.

C. purified after renaturation

Make dialysis to change renaturation buffer with the 20mM Tris pH 8.0 of 10 volumes, at least change buffer solution and come twice Fully reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Instrument (the general electricity of GE is purified using Akta Gas company), the 0-400mM NaCl linear gradient liquid wash-out proteins that 20mM Tris pH 8.0 are prepared, pMHC is about in 250mM Eluted 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 biotinylation is marked with Millipore super filter tubes to 1ml, using gel permeation chromatography The pMHC of purifying biological elementization, purifies instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibrations HiPrep using Akta^TM 16/60S200HR posts (GE General Electric Co. Limited), loading 1ml concentrated biotinylation pMHC molecules, then with PBS with 1ml/ Min flow velocitys are eluted.Biotinylated pMHC molecules occur in about 55ml as unimodal wash-out.Merge the group containing protein Point, being concentrated with Millipore super filter tubes, BCA methods (Thermo) determine protein concentration, add protease inhibitors cocktail (Roche) packing of biotinylated pMHC molecules is stored in -80 DEG C.

Data Analysis 7.1 and BIAcore Evaluation software computational dynamics parameters are utilized respectively, are obtained The soluble single-chain T CR molecules that soluble TCR molecules of the invention and the present invention build are answered with KVLEYVIKV-HLA A0201 The kinetic profile difference that compound is combined is as shown in Figure 12 and Figure 13.Collection of illustrative plates shows, the soluble TCR molecules that the present invention is obtained with And soluble single-chain T CR molecules can be combined with KVLEYVIKV-HLA A0201 compounds.Meanwhile, also using the above method The TCR molecules and other several irrelevant antigen small peptides and the binding activity of HLA compounds of solubility of the invention are have detected, is as a result shown Show TCR molecules of the present invention with other irrelevant antigens without combination.

The MAGE A1 antigen small peptide specificity TCRs slow virus of embodiment 7 is packed and primary T cells transfection

A () transiently transfects (Express-In-mediated transient by the quick mediation of 293T cells Transfection slow virus) is prepared

Using third generation slow virus packaging system slow virus of the packaging containing the gene of TCR needed for coding.Using quick Jie Lead transient transfection (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)), with 4 kinds of plasmid (one kind containing pLenti-RHAMMTRA-2A-TRB-IRES-NGFR described in embodiment 2 Slow virus carrier, and 3 kinds of plasmids containing structure infectiousness but other components necessary to non-replicating lentiviral particle) turn Dye 293T cells.

To be transfected, the 0th day kind cell, in 15 cm dishes, plants upper 1.7 × 10⁷Individual 293T cells, make cell equal Even to be distributed on culture dish, degree of converging is slightly above 50%.1st day transfected plasmids, pack pLenti-TRA-2A-TRB-IRES- NGFR and pLenti-eGFP pseudovirus, above expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV and pMD.2G are mixed Even, the consumption of a 15 cm diameter plates is as follows：22.5 micrograms：15 micrograms:15 micrograms：7.5 micrograms.Transfection reagent PEI- The ratio of MAX and plasmid is 2:The usage amount of 1, each plate PEI-MAX is 120 micrograms.Concrete operations are：Expression plasmid with Packaging plasmid mixes in adding 1800 microlitres of OPTI-MEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) 31985-070) culture medium Uniformly, being stored at room temperature 5 minutes turns into DNA mixed liquors；Respective amount PEI is taken to be well mixed with 1800 microlitres of OPTI-MEM culture mediums, Being stored at room temperature 5 minutes turns into PEI mixed liquors.DNA mixed liquors and PEI mixed liquors are mixed and 30 points are being stored at room temperature Clock, then add 3150 microlitres of OPTI-MEM culture mediums, it is well mixed after be added to and have been converted to 11.25 milliliters of OPTI-MEM's In 293T cells, culture dish is gently rocked, be well mixed culture medium, cultivated under 37 DEG C/5%CO2.Transfection 5-7 hours, removal Transfection media, changes the DMEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt) containing 10% hyclone into) Complete medium, 37 DEG C/5%CO₂Lower culture.Collect the culture medium supernatant containing wrapped slow virus within 3rd and the 4th day.To receive The slow virus of packaging is obtained, collected culture supernatant 3000g is centrifuged 15 minutes removal cell fragments, then through 0.22 micron of mistake Filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB) is filtered, finally with the concentration of 50KD interceptions Pipe (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) UFC905096) is concentrated, and removes most of supernatant, finally 1 milliliter is concentrated to, -80 DEG C freeze after decile packing.Taking pseudovirus sample carries out virus titer measure, and step is with reference to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit specification.Used as control, while also bag turns the pseudovirus of pLenti-eGFP.

B () is with the lentiviruses transduction primary T cells containing the MAGE specific φt cell receptor genes of A1 antigen small peptides

It is separated to CD8+T cells from the blood of healthy volunteer, then with the lentiviruses transduction packed.Count these thin Born of the same parents, in 48 orifice plates, containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10%FBS (Ji Bu can company (Gibco), Catalog number (Cat.No.) C10010500BT) 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture mediums in 1 × 10⁶ Individual cells/ml (0.5 milliliter/hole) and the AntiCD3 McAb for pre-washing/CD28 antibody-coating globule (T cell amplified matter, life Technologies, catalog number (Cat.No.) 11452D) overnight incubation stimulation, cell altogether：Pearl=3：1.

After stimulating overnight, according to the virus titer that p24ELISA kits are measured, added in the ratio of MOI=10 dense The slow virus of the MAGE A1 antigen small peptide specific t-cell receptor genes of contracting, 32 DEG C, 900g centrifugations infection 1 hour.Infect Slow-virus infection liquid is removed after finishing, with 1640 culture mediums containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 Re-suspended cell, 37 DEG C/5%CO₂Lower culture 3 days.Next day carries out the second wheel infection in the same way.Second transduction is counted after 3 days Number cell, diluting cells to 0.5 × 10⁶Individual cells/ml.Count a cell within every two days, replace or addition contains 50IU/ml The fresh culture of IL-2 and 10ng/ml IL-7, maintains cell 0.5 × 10⁶-1×10⁶Individual cells/ml.Opened from the 3rd day Begin by flow cytometry cell, for function test (for example, the ELISPOT of IFN-γ release and non-since the 5th day Radioactivity cytotoxicity is detected).Since the 10th day or when cell slows down division and size diminishes, stored frozen decile is thin Born of the same parents, at least 4 × 10⁶Individual cell/pipe (1 × 10⁷Individual cells/ml, 90%FBS/10%DMSO).

Embodiment 8 transduce TCR of the present invention T cell activation experiment

ELISPOT schemes

Tests below is carried out to prove the activating reaction of the T cell to target cell specificity of TCR transductions.Using ELISPOT The IFN-γ yield of testing inspection as t cell activation readout.

Reagent

Test medium：10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), (Ji Bu can for RPMI1640 Company (Gibco), catalog number (Cat.No.) C11875500bt)

Lavation buffer solution (PBST)：0.01M PBS/0.05% polysorbas20s

PBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT)

The orifice plates of PVDF ELISPOT 96 (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) MSIPS4510)

People's IFN-γ ELISPOT PVDF- enzyme reagent kits (BD) (catches and detection is anti-equipped with required every other reagent Body, Streptavidin-alkaline phosphatase and BCIP/NBT solution)

Method

It is prepared by target cell

Target cell used is T2 cells in this experiment.Target cell is prepared in assay medium：Target cell concentration is adjusted to 2.0×10⁵Individual/milliliter, 100 microlitres are taken per hole so as to obtain 2.0 × 10⁴Individual cells/well.

It is prepared by effector cell

Effector cell's (T cell) of this experiment is that through flow cytometry to express MAGE A1 of the present invention in embodiment 7 anti- The CD8+T cells of former small peptide specificity TCR, and with the CD8+T of same volunteer's untransfected TCR of the present invention as a control group.With AntiCD3 McAb/CD28 is coated with pearl (T cell amplified matter, life technologies) stimulates T cell, and with carrying, MAGE A1 antigens are short The lentiviruses transduction (according to embodiment 7) of peptide specific tcr gene, in containing containing 50IU/ml IL-2 and 10ng/ml IL-7 Until 9-12 days after transduction, then be placed in these cells in test medium, 300g is normal by the 1640 culture mediums amplification of 10%FBS Temperature centrifugation is washed for 10 minutes.Then cell is resuspended in test medium with 2 × required final concentration.Same treatment is negative Control effector cell.

It is prepared by small peptide solution

Correspondence small peptide is added in corresponding target cell (T2) experimental group, makes final concentration of 1 μ of the small peptide in ELISPOT orifice plates g/ml。

ELISPOT

According to the specification that manufacturer provides, preparation orifice plate as described below：1 is pressed with 10 milliliters of aseptic PBS of every block of plate：200 Dilute anti-human IFN-γ and catch antibody, 100 microlitres of dilution then is caught into antibody etc. point adds each hole.Orifice plate is incubated at 4 DEG C Overnight.After incubation, wash orifice plate to remove unnecessary seizure antibody.The RPMI 1640 for adding 100 microlitres/hole to contain 10%FBS Culture medium, and orifice plate is incubated at room temperature 2 hours to close orifice plate.Then culture medium is washed away from orifice plate, by light on paper Bullet and pat ELISPOT orifice plates with remove it is any remnants lavation buffer solution.

Then all components that will be tested using following order add ELISPOT orifice plates：

100 microlitres of target cell 2*10⁵Individual cells/ml (obtains about 2*10 altogether⁴Individual target cell/hole).

100 microlitres of effector cell (1*10⁴Individual control effector cell/hole and MAGE A1 TCR positive T cells/hole).

All holes prepare addition in duplicate.

Then overnight (37 DEG C/5%CO of orifice plate is incubated₂) second day, culture medium is abandoned, orifice plate is washed with distilled water 2 times, then use Lavation buffer solution is washed 3 times, pats to remove the lavation buffer solution of remnants on paper handkerchief.Then pressed with the PBS containing 10%FBS 1：200 dilution detection antibodies, each hole is added by 100 microlitres/hole.Incubate orifice plate 2 hours at room temperature, then washed with lavation buffer solution 3 times, orifice plate is patted on paper handkerchief to remove excessive lavation buffer solution.

1 is pressed with the PBS containing 10%FBS：100 dilution Streptavidin-alkaline phosphatases, by 100 microlitres of chains of dilution Mould Avidin-alkaline phosphatase adds each hole and incubates orifice plate 1 hour at room temperature.Then 4 PBS are washed with lavation buffer solution Washing 2 times, pats orifice plate to remove excessive lavation buffer solution and PBS on paper handkerchief.Washing adds kit to provide after finishing 100 microlitres/hole of BCIP/NBT solution developed.Orifice plate lucifuge is covered with masking foil during developing, 5-15 minutes is stood. The spot of conventional detection development orifice plate, determines the Best Times of terminating reaction during this period.Removal BCIP/NBT solution and with pair Steam water to rinse orifice plate to stop developing reaction, dry, then remove orifice plate bottom, be dried at room temperature for orifice plate until each hole It is completely dried, recycles immunodotting plate count meter (CTL, Celltech Ltd. (Cellular Technology Limited the)) spot that counterdie is formed in counting orifice.

As a result

The T cell for testing inspection (as described above) TCR transductions of the present invention by ELISPOT is short to load MAGE A1 antigens The IFN-γ release that the target cell of peptide GLSNLTHVL reacts.Using what is observed in each hole of graphpad prism6 draftings ELSPOT amount of speckle.

As shown in figure 14, the T cell of the TCR of the present invention that transduces has very experimental result to the target cell for loading its special small peptide Good activating reaction, and the T cell of the TCR of the present invention that do not transduce does not have activating reaction substantially.

Embodiment 9 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.The experiment is the colorimetric alternate test that 51Cr discharges cell toxicity test, quantitative determination cell cracking The lactic dehydrogenase (LDH) for discharging afterwards.Release LDH in the medium is detected using the enzyme reaction of coupling in 30 minutes, in enzyme LDH can make a kind of tetrazolium salts (INT) be converted into red formazans (formazan) in reaction.The amount of the red product of generation with split The cell number of solution is directly proportional.Collection 490nm visible ray extinction Value Datas can be collected with 96 hole read plates of standard.

Those skilled in the art know the method for detecting cell function using the release experiment of LDH.The present embodiment effect is thin Born of the same parents' (T cell) are the CD8+T for expressing MAGE A1 antigens small peptide specificity TCR of the present invention in embodiment 7 through flow cytometry Cell, target cell system is 293T and U266B1.According to nanostring results, wherein, U266B1 expression MAGE A1 antigens, 293T does not express MAGE A1 antigens substantially, in this, as control.

Prepare LDH flat boards first.Test the 1st day, each component that will be tested in the following order adds flat board：Culture keynote Whole effector cell is to 2X10⁶Individual cells/ml, culture medium adjusts each target cell system to 5X10⁵Individual cells/ml.After well mixed Take 100 μ L target cell systems 5X10⁵Individual cells/ml (i.e. 50,000 cells/wells), 100 μ L effector cells 2X10⁶Individual cell/milli Liter (i.e. 200,000 cells/well) is added in corresponding aperture, and sets three multiple holes.Effector cell's idiomorphic pore is set simultaneously, and target is thin Born of the same parents' idiomorphic pore, target cell largest hole, volume correction control wells and culture medium ground control hole.Be 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.Experimental result is as schemed Shown in 15, curve NC-293T is reaction of the effector cell of untransfected TCR of the present invention to 293T cell lines in figure；Curve NC- U266B1 is reaction of the effector cell of untransfected TCR of the present invention to U266B1 cell lines；Curve TCR-293T is this hair of transfection Reaction of the effector cell of bright TCR to 293T cell lines；Curve TCR-U266B1 is the effector cell couple for transfecting TCR of the present invention The reaction of U266B1 cell lines.From the results, it was seen that the effector cell of transduction TCR of the present invention is thin to expressing the target of related antigen Born of the same parents have lethal effect, and the target cell to not expressing related antigen does not have lethal effect substantially.

The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, those skilled in the art can Made various changes or modifications with to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Sequence table

<110>Guangzhou Xiangxue Pharmaceutical Co

<120>Recognize the φt cell receptor of MAGE-A1 antigen small peptides

<130> P2017-0068

<150> CN201610190650.6

<151> 2016-03-29

<160> 37

<170> PatentIn version 3.5

<210> 1

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chain variable domains

<400> 1

Ile Leu Asn Val Glu Gln Ser Pro Gln Ser Leu His Val Gln Glu Gly

1 5 10 15

Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro Ser Ser Asn Phe Tyr Ala

20 25 30

Leu His Trp Tyr Arg Trp Glu Thr Ala Lys Ser Pro Glu Ala Leu Phe

35 40 45

Val Met Thr Leu Asn Gly Asp Glu Lys Lys Lys Gly Arg Ile Ser Ala

50 55 60

Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr Leu Tyr Ile Lys Gly Ser

65 70 75 80

Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Phe Pro Ser Gly Gly

85 90 95

Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu Ile Ile Gln

100 105 110

Pro Tyr

<210> 2

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chain variable domains

<400> 2

atactgaacg tggaacaaag tcctcagtca ctgcatgttc aggagggaga cagcaccaat 60

ttcacctgca gcttcccttc cagcaatttt tatgccttac actggtacag atgggaaact 120

gcaaaaagcc ccgaggcctt gtttgtaatg actttaaatg gggatgaaaa gaagaaagga 180

cgaataagtg ccactcttaa taccaaggag ggttacagct atttgtacat caaaggatcc 240

cagcctgaag actcagccac atacctctgt gccttccctt caggaggagg tgctgacgga 300

ctcacctttg gcaaagggac tcatctaatc atccagccct at 342

<210> 3

<211> 254

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chains

<400> 3

Ile Leu Asn Val Glu Gln Ser Pro Gln Ser Leu His Val Gln Glu Gly

1 5 10 15

Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro Ser Ser Asn Phe Tyr Ala

20 25 30

Leu His Trp Tyr Arg Trp Glu Thr Ala Lys Ser Pro Glu Ala Leu Phe

35 40 45

Val Met Thr Leu Asn Gly Asp Glu Lys Lys Lys Gly Arg Ile Ser Ala

50 55 60

Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr Leu Tyr Ile Lys Gly Ser

65 70 75 80

Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Phe Pro Ser Gly Gly

85 90 95

Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu Ile Ile Gln

100 105 110

Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

195 200 205

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

210 215 220

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

225 230 235 240

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250

<210> 4

<211> 762

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chains

<400> 4

atactgaacg tggaacaaag tcctcagtca ctgcatgttc aggagggaga cagcaccaat 60

ttcacctgca gcttcccttc cagcaatttt tatgccttac actggtacag atgggaaact 120

gcaaaaagcc ccgaggcctt gtttgtaatg actttaaatg gggatgaaaa gaagaaagga 180

cgaataagtg ccactcttaa taccaaggag ggttacagct atttgtacat caaaggatcc 240

cagcctgaag actcagccac atacctctgt gccttccctt caggaggagg tgctgacgga 300

ctcacctttg gcaaagggac tcatctaatc atccagccct atatccagaa ccctgaccct 360

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 420

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 660

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 720

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 762

<210> 5

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chain variable domains

<400> 5

Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr Ala Thr Gly

1 5 10 15

Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val

20 25 30

Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu Ile Gln

35 40 45

Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu Glu Arg Phe

50 55 60

Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn Leu Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Val Glu

85 90 95

Gly Tyr Pro Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr

<210> 6

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chain variable domains

<400> 6

gattctggag tcacacaaac cccaaagcac ctgatcacag caactggaca gcgagtgacg 60

ctgagatgct cccctaggtc tggagacctc tctgtgtact ggtaccaaca gagcctggac 120

cagggcctcc agttcctcat tcagtattat aatggagaag agagagcaaa aggaaacatt 180

cttgaacgat tctccgcaca acagttccct gacttgcact ctgaactaaa cctgagctct 240

ctggagctgg gggactcagc tttgtatttc tgtgccagca gcgtagaagg ctacccctcc 300

tacgagcagt acttcgggcc gggcaccagg ctcacggtca ca 342

<210> 7

<211> 293

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chains

<400> 7

Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr Ala Thr Gly

1 5 10 15

Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val

20 25 30

Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu Ile Gln

35 40 45

Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu Glu Arg Phe

50 55 60

Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn Leu Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Val Glu

85 90 95

Gly Tyr Pro Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

115 120 125

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

130 135 140

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

145 150 155 160

Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro

165 170 175

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser

180 185 190

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe

195 200 205

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

210 215 220

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

225 230 235 240

Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val

245 250 255

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

260 265 270

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

275 280 285

Lys Asp Ser Arg Gly

290

<210> 8

<211> 879

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chains

<400> 8

gattctggag tcacacaaac cccaaagcac ctgatcacag caactggaca gcgagtgacg 60

ctgagatgct cccctaggtc tggagacctc tctgtgtact ggtaccaaca gagcctggac 120

cagggcctcc agttcctcat tcagtattat aatggagaag agagagcaaa aggaaacatt 180

cttgaacgat tctccgcaca acagttccct gacttgcact ctgaactaaa cctgagctct 240

ctggagctgg gggactcagc tttgtatttc tgtgccagca gcgtagaagg ctacccctcc 300

tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 360

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 480

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 540

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 600

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660

gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 720

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 780

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 840

gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 879

<210> 9

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>Antigen small peptide

<400> 9

Lys Val Leu Glu Tyr Val Ile Lys Val

1 5

<210> 10

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR1

<400> 10

Ser Ser Asn Phe Tyr Ala

1 5

<210> 11

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR2

<400> 11

Met Thr Leu Asn Gly Asp Glu

1 5

<210> 12

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR3

<400> 12

Ala Phe Pro Ser Gly Gly Gly Ala Asp Gly Leu Thr

1 5 10

<210> 13

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR1

<400> 13

Ser Gly Asp Leu Ser

1 5

<210> 14

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR2

<400> 14

Tyr Tyr Asn Gly Glu Glu

1 5

<210> 15

<211> 13

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR3

<400> 15

Ala Ser Ser Val Glu Gly Tyr Pro Ser Tyr Glu Gln Tyr

1 5 10

<210> 16

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR1

<400> 16

tccagcaatt tttatgcc 18

<210> 17

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR2

<400> 17

atgactttaa atggggatga a 21

<210> 18

<211> 36

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR3

<400> 18

gccttccctt caggaggagg tgctgacgga ctcacc 36

<210> 19

<211> 15

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR1

<400> 19

tctggagacc tctct 15

<210> 20

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR2

<400> 20

tattataatg gagaagag 18

<210> 21

<211> 39

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR3

<400> 21

gccagcagcg tagaaggcta cccctcctac gagcagtac 39

<210> 22

<211> 276

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chains with targeting sequencing

<400> 22

Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His

1 5 10 15

Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser

20 25 30

Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro

35 40 45

Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys

50 55 60

Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys

65 70 75 80

Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr

85 90 95

Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys

100 105 110

Ala Phe Pro Ser Gly Gly Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly

115 120 125

Thr His Leu Ile Ile Gln Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val

130 135 140

Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe

145 150 155 160

Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp

165 170 175

Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe

180 185 190

Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys

195 200 205

Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro

210 215 220

Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu

225 230 235 240

Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg

245 250 255

Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg

260 265 270

Leu Trp Ser Ser

275

<210> 23

<211> 828

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chains with targeting sequencing

<400> 23

atggagaaga atcctttggc agccccatta ctaatcctct ggtttcatct tgactgcgtg 60

agcagcatac tgaacgtgga acaaagtcct cagtcactgc atgttcagga gggagacagc 120

accaatttca cctgcagctt cccttccagc aatttttatg ccttacactg gtacagatgg 180

gaaactgcaa aaagccccga ggccttgttt gtaatgactt taaatgggga tgaaaagaag 240

aaaggacgaa taagtgccac tcttaatacc aaggagggtt acagctattt gtacatcaaa 300

ggatcccagc ctgaagactc agccacatac ctctgtgcct tcccttcagg aggaggtgct 360

gacggactca cctttggcaa agggactcat ctaatcatcc agccctatat ccagaaccct 420

gaccctgccg tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc 480

accgattttg attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca 540

gacaaaactg tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg 600

agcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac 660

accttcttcc ccagcccaga aagttcctgt gatgtcaagc tggtcgagaa aagctttgaa 720

acagatacga acctaaactt tcaaaacctg tcagtgattg ggttccgaat cctcctcctg 780

aaagtggccg ggtttaatct gctcatgacg ctgcggctgt ggtccagc 828

<210> 24

<211> 312

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chains with targeting sequencing

<400> 24

Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala

1 5 10 15

Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr

20 25 30

Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp

35 40 45

Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe

50 55 60

Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu

65 70 75 80

Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn

85 90 95

Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser

100 105 110

Ser Val Glu Gly Tyr Pro Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 25

<211> 936

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chains with targeting sequencing

<400> 25

atgggcttca ggctcctctg ctgtgtggcc ttttgtctcc tgggagcagg cccagtggat 60

tctggagtca cacaaacccc aaagcacctg atcacagcaa ctggacagcg agtgacgctg 120

agatgctccc ctaggtctgg agacctctct gtgtactggt accaacagag cctggaccag 180

ggcctccagt tcctcattca gtattataat ggagaagaga gagcaaaagg aaacattctt 240

gaacgattct ccgcacaaca gttccctgac ttgcactctg aactaaacct gagctctctg 300

gagctggggg actcagcttt gtatttctgt gccagcagcg tagaaggcta cccctcctac 360

gagcagtact tcgggccggg caccaggctc acggtcacag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210> 26

<211> 207

<212> PRT

<213>Artificial sequence

<220>

<223>STCR α chains

<400> 26

Ile Leu Asn Val Glu Gln Ser Pro Gln Ser Leu His Val Gln Glu Gly

1 5 10 15

Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro Ser Ser Asn Phe Tyr Ala

20 25 30

Leu His Trp Tyr Arg Trp Glu Thr Ala Lys Ser Pro Glu Ala Leu Phe

35 40 45

Val Met Thr Leu Asn Gly Asp Glu Lys Lys Lys Gly Arg Ile Ser Ala

50 55 60

Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr Leu Tyr Ile Lys Gly Ser

65 70 75 80

Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Phe Pro Ser Gly Gly

85 90 95

Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu Ile Ile Gln

100 105 110

Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 27

<211> 621

<212> DNA

<213>Artificial sequence

<220>

<223>STCR α chains

<400> 27

attctgaacg tggaacaaag tcctcagtca ctgcatgttc aggagggaga cagcaccaat 60

ttcacctgca gcttcccttc cagcaatttt tatgccttac actggtacag atgggaaact 120

gcaaaaagcc ccgaggcctt gtttgtaatg actttaaatg gggatgaaaa gaagaaagga 180

cgaataagtg ccactcttaa taccaaggag ggttacagct atttgtacat caaaggatcc 240

cagcctgaag actcagccac atacctctgt gccttccctt caggaggagg tgctgacgga 300

ctcacctttg gcaaagggac tcatctaatc atccagccct atatccagaa ccctgaccct 360

gccgtgtacc agctgagaga ctctaagtcg agtgacaagt ctgtctgcct attcaccgat 420

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 480

tgtgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 540

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 600

ttccccagcc cagaaagttc c 621

<210> 28

<211> 244

<212> PRT

<213>Artificial sequence

<220>

<223>STCR β chains

<400> 28

Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr Ala Thr Gly

1 5 10 15

Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val

20 25 30

Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu Ile Gln

35 40 45

Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu Glu Arg Phe

50 55 60

Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn Leu Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Val Glu

85 90 95

Gly Tyr Pro Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

115 120 125

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

130 135 140

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

145 150 155 160

Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro

165 170 175

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser

180 185 190

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe

195 200 205

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

210 215 220

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

225 230 235 240

Gly Arg Ala Asp

<210> 29

<211> 732

<212> DNA

<213>Artificial sequence

<220>

<223>STCR β chains

<400> 29

gatagcggcg tgacccaaac cccaaagcac ctgatcacag caactggaca gcgagtgacg 60

ctgagatgct cccctaggtc tggagacctc tctgtgtact ggtaccaaca gagcctggac 120

cagggcctcc agttcctcat tcagtattat aatggagaag agagagcaaa aggaaacatt 180

cttgaacgat tctccgcaca acagttccct gacttgcact ctgaactaaa cctgagctct 240

ctggagctgg gggactcagc tttgtatttc tgtgccagca gcgtagaagg ctacccctcc 300

tacgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 360

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 420

gccacactgg tgtgcctggc caccggtttc taccccgacc acgtggagct gagctggtgg 480

gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 540

cccgccctca atgactccag atacgctctg agcagccgcc tgagggtctc ggccaccttc 600

tggcaggacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 660

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 720

ggtagagcag ac 732

<210> 30

<211> 252

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR

<400> 30

Ala Ile Leu Asn Val Glu Gln Ser Pro Gln Ser Leu His Val Gln Glu

1 5 10 15

Gly Asp Ser Val Asn Ile Thr Cys Ser Phe Pro Ser Ser Asn Phe Tyr

20 25 30

Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys Ser Pro Glu Ala Leu

35 40 45

Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys Lys Gly Arg Ile Ser

50 55 60

Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr Leu Tyr Ile Lys Arg

65 70 75 80

Val Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Phe Pro Ser Gly

85 90 95

Gly Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu Met Ile

100 105 110

Gln Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

115 120 125

Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Asp Ser Gly Val Thr Gln

130 135 140

Thr Pro Lys His Leu Ser Val Ala Thr Gly Gln Arg Val Thr Leu Arg

145 150 155 160

Cys Ser Pro Arg Ser Gly Asp Leu Ser Val Tyr Trp Tyr Gln Gln Ser

165 170 175

Leu Asp Gln Gly Leu Gln Phe Leu Ile Gln Tyr Tyr Asn Gly Glu Glu

180 185 190

Arg Ala Lys Gly Asn Ile Pro Glu Arg Phe Ser Ala Gln Gln Phe Pro

195 200 205

Asp Leu His Ser Glu Leu Asn Ile Ser Ser Val Glu Pro Gly Asp Ser

210 215 220

Ala Leu Tyr Phe Cys Ala Ser Ser Val Glu Gly Tyr Pro Ser Tyr Glu

225 230 235 240

Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu

245 250

<210> 31

<211> 756

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR

<400> 31

gctattctta atgttgaaca gagcccgcaa tctctgcatg tgcaggaagg tgatagtgtt 60

aacatcacct gctcctttcc gagctctaat ttctatgcgc tgcactggta ccgctgggaa 120

accgcgaaaa gcccggaagc cctgtttgtc atgacgctga acggtgacga gaaaaagaaa 180

ggccgcattt cagccaccct gaatacgaaa gaaggttatt cgtacctgta tatcaaacgt 240

gttcagccgg aagatagcgc aacctatctg tgtgcttttc cgtctggcgg tggcgcagac 300

ggtctgacct tcggtaaagg cacgcatctg atgattcaac cgggtggcgg ttcagaaggc 360

ggtggctcgg aaggtggcgg tagcgaaggc ggtggctctg aaggtggcac cggtgattca 420

ggcgtgaccc agacgccgaa acacctgtct gtcgcgaccg gtcaacgtgt gacgctgcgt 480

tgcagtccgc gttccggtga tctgtcagtt tactggtatc agcaatcgct ggaccagggc 540

ctgcaattcc tgattcagta ttacaacggt gaagaacgcg caaaaggcaa tatcccggaa 600

cgttttagtg ctcagcaatt cccggatctg cattccgaac tgaatatcag ttccgttgaa 660

ccgggtgaca gtgcgctgta tttttgtgcc tcatcggtcg aaggctaccc gagctatgaa 720

cagtacttcg gtccgggcac ccgtctgacg gttctg 756

<210> 32

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR α chains

<400> 32

Ala Ile Leu Asn Val Glu Gln Ser Pro Gln Ser Leu His Val Gln Glu

1 5 10 15

Gly Asp Ser Val Asn Ile Thr Cys Ser Phe Pro Ser Ser Asn Phe Tyr

20 25 30

Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys Ser Pro Glu Ala Leu

35 40 45

Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys Lys Gly Arg Ile Ser

50 55 60

Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr Leu Tyr Ile Lys Arg

65 70 75 80

Val Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Phe Pro Ser Gly

85 90 95

Gly Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His Leu Met Ile

100 105 110

Gln Pro

<210> 33

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR α chains

<400> 33

gctattctta atgttgaaca gagcccgcaa tctctgcatg tgcaggaagg tgatagtgtt 60

aacatcacct gctcctttcc gagctctaat ttctatgcgc tgcactggta ccgctgggaa 120

accgcgaaaa gcccggaagc cctgtttgtc atgacgctga acggtgacga gaaaaagaaa 180

ggccgcattt cagccaccct gaatacgaaa gaaggttatt cgtacctgta tatcaaacgt 240

gttcagccgg aagatagcgc aacctatctg tgtgcttttc cgtctggcgg tggcgcagac 300

ggtctgacct tcggtaaagg cacgcatctg atgattcaac cg 342

<210> 34

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR β chains

<400> 34

Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ser Val Ala Thr Gly

1 5 10 15

Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val

20 25 30

Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu Ile Gln

35 40 45

Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Pro Glu Arg Phe

50 55 60

Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn Ile Ser Ser

65 70 75 80

Val Glu Pro Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Val Glu

85 90 95

Gly Tyr Pro Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Leu

<210> 35

<211> 342

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR β chains

<400> 35

gattcaggcg tgacccagac gccgaaacac ctgtctgtcg cgaccggtca acgtgtgacg 60

ctgcgttgca gtccgcgttc cggtgatctg tcagtttact ggtatcagca atcgctggac 120

cagggcctgc aattcctgat tcagtattac aacggtgaag aacgcgcaaa aggcaatatc 180

ccggaacgtt ttagtgctca gcaattcccg gatctgcatt ccgaactgaa tatcagttcc 240

gttgaaccgg gtgacagtgc gctgtatttt tgtgcctcat cggtcgaagg ctacccgagc 300

tatgaacagt acttcggtcc gggcacccgt ctgacggttc tg 342

<210> 36

<211> 24

<212> PRT

<213>Artificial sequence

<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

<220>

<223>Single-stranded TCR catenation sequences

<400> 37

ggtggcggtt cagaaggcgg tggctcggaa ggtggcggta gcgaaggcgg tggctctgaa 60

ggtggcaccg gt 72

Claims (10)

1. a kind of φt cell receptor (TCR), it is characterised in that the TCR can be with KVLEYVIKV-HLAA0201 compound knots 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 AFPSGGGADGLT (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domains Amino acid sequence is ASSVEGYPSYEQY (SEQ ID NO:15)；

It is highly preferred that 3 complementary determining regions (CDR) of the TCR α chain variable domains are：

αCDR1-SSNFYA(SEQ ID NO:10)

αCDR2-MTLNGDE(SEQ ID NO:11)

αCDR3-AFPSGGGADGLT(SEQ ID NO:12)；And/or

3 complementary determining regions of the TCR β chain variable domains are：

βCDR1-SGDLS(SEQ ID NO:13)

βCDR2-YYNGEE(SEQ ID NO:14)

βCDR3-ASSVEGYPSYEQY(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 points 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 the nucleic acid molecules described in claim 5；Preferably, it is described Carrier is viral vectors；It is highly preferred that described carrier is slow virus carrier.

7. a kind of host cell of separation, it is characterised in that contain the carrier described in claim 6 in described host cell Or the nucleic acid molecules described in the claim 5 of external source are integrated with chromosome.

8. a kind of cell, it is characterised in that institute in the nucleic acid molecules or claim 6 described in the cell transduction claim 5 State carrier；Preferably, the cell is T cell or stem cell.

9. a kind of pharmaceutical composition, it is characterised in that the composition contains pharmaceutically acceptable carrier and claim The TCR compounds described in TCR, claim 4 any one of 1-3, the nucleic acid molecules described in claim 5 or power Profit requires the cell described in 8.

10. the TCR compounds or right described in the φt cell receptor or claim 4 any one of claim 1-3 It is required that the purposes of the cell described in 8, it is characterised in that the medicine for preparing treatment tumour or autoimmune disease.