CN106699874B - Identify the T cell receptor of PRAME antigen small peptide - Google Patents

Abstract

The present invention provides a kind of T cell receptor (TCR) that can specifically bind the small peptide PYLGQMINL derived from PRAME antigen, the antigen small peptide PYLGQMINL can with HLA A2402 formed compound and together be presented to cell surface.The present invention also provides encode the nucleic acid molecules of the TCR and include the carrier of the nucleic acid molecules.In addition, the cell the present invention also provides the TCR of the present invention that transduces.

Description

Identify the T cell receptor of PRAME antigen small peptide

Technical field

The present invention relates to that can identify the TCR from PRAME antigen small peptide, the invention further relates to transduce above-mentioned TCR to obtain PRAME specificity T cell and their purposes in PRAME relevant diseases are prevented and treated.

Background technology

PRAME is melanoma specific antigen (preferentially expressed antigen of Melanoma, PRAME), 88% it is initial and 95% transfer melanoma in have expression (Ikeda H, et al.Immunity,1997,6(2):199-208), normal skin tissue and benign melanocyte are not expressed then.PRAME is in cell Micromolecule polypeptide is degraded to after interior generation, and compound is combined to form with MHC (main histocompatibility complex) molecule, by It is delivered to cell surface.PYLGQMINL is the small peptide derived from PRAME antigen, is a kind of target of PRAME treating correlative diseases. Except melanoma, PRAME is also expressed in kinds of tumors, swollen including squamous cell lung carcinoma, breast cancer, clear-cell carcinoma, incidence Knurl, Huo Jiejin lymphomas, sarcoma and medulloblastoma etc. (van't Veer LJ, et al.Nature, 2002,415 (6871):530-536；Boon K,et al.Oncogene,2003,22(48):7687-7694) in addition, it is in leukaemia PRAME also has notable expression, acute lymphoblastic leukemia 17%~42%, acute myeloblastic leukemia 30%~64% (SteinbachD,et al.Cancer Genet Cytogene,2002,138(1):89-91).For controlling for above-mentioned disease The methods for the treatment of, chemotherapy and radiation treatment may be employed, but the normal cell of itself can all be damaged.

T cell adoptive immunotherapy is to be transferred to the reaction-ive T cell for having specificity to target cell antigen in patient body, It is made to play a role for target cell.T cell receptor (TCR) is a kind of memebrane protein on T cell surface, can be identified corresponding The antigen small peptide of target cell surface.In immune system, pass through TCR and the small peptide-main histocompatbility of antigen small peptide specificity 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, and cause a series of subsequent cell signals transmission and its His physiological reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, this field skill Art personnel are directed to isolating the TCR for having specificity to PRAME antigen small peptide and TCR's T cell are transduceed to obtain pair PRAME antigen small peptide has the T cell of specificity, 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 T cell receptors for identifying PRAME antigen small peptide.

The first aspect of the present invention, provides a kind of T cell receptor (TCR), and the TCR can be with PYLGQMINL-HLA A2402 compounds combine.

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 VVRGNNARLM (SEQ ID NO:12)；And/or the ammonia of the CDR3 of the TCR β chain variable domains Base acid sequence is ASSGSAAGANVLT (SEQ ID NO:15).

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

αCDR1-NSASQS(SEQ ID NO:10)

αCDR2-VYSSGN(SEQ ID NO:11)

αCDR3-VVRGNNARLM(SEQ ID NO:12)；And/or

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

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSGSAAGANVLT(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 For with SEQ ID NO:1 has the amino acid sequence of at least 90% sequence thereto；And/or the TCR β chains variable domain be with SEQ ID NO：5 have the amino acid sequence of at 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 soluble.

In another preference, the TCR is single-stranded.

In another preference, the TCR is to be formed by connecting by α chains variable domain with β chains variable domain by peptide catenation sequence.

In another preference, the TCR is in α chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or 94 and/or α chain J gene small peptides amino acid are 3rd reciprocal, it is one or more prominent to have in 5th reciprocal or 7th reciprocal Become；And/or the TCR is in β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th and/or β chains J Gene small peptide amino acid is 2nd reciprocal, has one or more mutation, wherein amino acid position in 4th reciprocal or 6th reciprocal Number 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 (a) in addition to transmembrane domain；And (b) all or part of TCR β chains in addition to transmembrane domain；

And and (b) respectively contains functional variable domain (a), or includes 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 of artificial disulfide bond are formed in the TCR instead of selected from following One or more groups of sites：

The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

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

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

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

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

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

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

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

In another 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, which is characterized in that the cysteine residues of artificial interchain disulfide bond are formed in the TCR Instead of selected from following one or more groups of sites：

The 46th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；

The 47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1s；

The 46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s；Or

The 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s.

In another preference, the TCR includes α chains variable domain and β chains variable domain and in addition to transmembrane domain All or part of β chains constant domain, but it does not contain α chain constant domains, α chains variable domain and the β chains of the TCR form heterogeneous dimerization Body.

In another 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 that is combined with the T cell receptor is detectable, therapeutic agent, PK are repaiied Decorations part or the combination of these any substances.Preferably, the therapeutic agent is anti-CD 3 antibodies.

The second aspect of the present invention provides a kind of multivalent TCR complex, it includes at least two TCR molecules, and its In at least one TCR molecules be the TCR described in first aspect present invention.

The third aspect of the present invention provides a kind of nucleic acid molecules, and the nucleic acid molecules, which include, encodes first party of the present invention The nucleotide sequence or its complementary series of TCR molecules described in face.

In another preference, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chain variable domains： 2 or SEQ ID NO:33.

In another preference, the nucleic acid molecules include the nucleotide sequence SEQ ID of coding TCR β chain variable domains NO：6 or SEQ ID NO:35.

In another preference, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chains：4 and/or Include the nucleotide sequence SEQ ID NO of coding TCR β chains：8.

The fourth aspect of the present invention, provides a kind of carrier, and the carrier contains the core described in third aspect present invention Acid molecule；Preferably, the carrier is viral vectors；It is highly preferred that the carrier is slow virus carrier.

The fifth aspect of the present invention provides a kind of separated host cell, contains the present invention in the host cell The nucleic acid molecules described in the third aspect present invention of external source are integrated in carrier or genome described in fourth aspect.

The sixth aspect of the present invention provides a kind of cell, the nucleic acid described in the cell transduction third aspect present invention Carrier described in molecule or fourth aspect present invention；Preferably, the cell is T cell or stem cell.

The seventh aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains pharmaceutically acceptable load The TCR compounds described in TCR, second aspect of the present invention, third aspect present invention institute described in body and first aspect present invention Carrier described in the nucleic acid molecules stated, fourth aspect present invention or the cell described in sixth aspect present invention.

The eighth aspect of the present invention provides T cell receptor or second aspect of the present invention described in first aspect present invention Nucleic acid molecules described in the TCR compounds, third aspect present invention, carrier or this hair described in fourth aspect present invention The purposes of cell described in bright 6th aspect is used to prepare the drug for the treatment of tumour or autoimmune disease.

The ninth aspect of the present invention provides a kind of method for treating disease, including giving object application in need for the treatment of suitable The TCR compounds described in T cell receptor or second aspect of the 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 cell or this hair described in sixth aspect present invention Pharmaceutical composition described in bright 7th aspect；

Preferably, the disease is tumour, and preferably described tumour includes melanoma and other tumours such as stomach Cancer, lung cancer (e.g., squamous cell lung carcinoma), cancer of the esophagus, carcinoma of urinary bladder, head and neck neoplasm (e.g., head and neck squamous cell carcinoma), prostate Cancer, breast cancer, colon cancer, oophoroma, clear-cell carcinoma, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukaemia etc..

It is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

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

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

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

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

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

Fig. 6 is the glue figure of the sTCR obtained after purification.For leftmost side swimming lane to go back virgin rubber, intermediate swimming lane is molecular weight It marks (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 obtained after purification.Left side swimming lane is molecular weight marker (marker), right Breathing arm road is non-reduced glue.

Figure 12 is the BIAcore dynamics figures that sTCR of the present invention is combined with PYLGQMINL-HLA A2402 compounds Spectrum.

Figure 13 is the BIAcore power that soluble single-chain T CR of the present invention is combined with PYLGQMINL-HLA A2402 compounds Learn collection of illustrative plates.

Figure 14 is the activation experiment result of the effector cell of transduction TCR of the present invention.

Specific embodiment

The present inventor has found and PRAME antigen small peptide PYLGQMINL (SEQ ID by in-depth study extensively NO:9) TCR that can be specifically bound, the antigen small peptide PYLGQMINL can with HLA A2402 formed compound and together with by It is presented to cell surface.The present invention also provides encode the nucleic acid molecules of the TCR and include the carrier of the nucleic acid molecules. In addition, the cell the present invention also provides the TCR of the present invention that transduces.

Term

MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, for The presentation of antigen has specificity, and different individuals has different MHC, can present small peptide different in a kind of proteantigen to respectively From APC cell surfaces.The MHC of the mankind is commonly referred to as HLA genes or HLA complexs.

T cell receptor (TCR) is the unique of specific antigen peptide of the presentation in main histocompatibility complex (MHC) Receptor.In immune system, trigger T cell and antigen presentation thin 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 subsequent cell signals transmission and other physiological reactions are just caused, so that the T cell of different antigentic specificities Immunological effect is played to its target cell.

TCR is the glycoprotein of the cell membrane surface as existing for α chains/β chains or γ chains/δ chains in the form of heterodimer. TCR heterodimers are made of α and β chains in 95% T cell, and 5% T cell has the TCR being made of γ and δ chains.My god The heterogeneous dimerization TCR of right α β have α chains and β chains, and α chains and β chains form 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 contain short variable region usually also between variable region and bonding pad, but should Variable region is often regarded as a part for bonding pad.Each variable region includes 3 be entrenched in frame structure (framework regions) A CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compounds, wherein CDR3 by Variable region and bonding pad recombinate, and are referred to as hypervariable region.α the and β chains of TCR generally regard that each there are two " structural domain " is i.e. variable as Domain and constant domain, variable domain are made of the variable region connected and bonding pad.The sequence of TCR constant domains can be in international immune genetic It learns and is found in the public database of information system (IMGT), if the constant domain sequence of TCR molecule alpha chains is " TRAC*01 ", TCR divides The constant domain sequence of sub- β chains is " TRBC1*01 " or " TRBC2*01 ".In addition, α the and β chains of TCR also include transmembrane region and kytoplasm Area, cytoplasmic region are very short.

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

Native interchain disulfide bond and artificial interchain disulfide bond

There is one group of disulfide bond in membrane-proximal region C α and the C β interchains of natural TCR, referred to as " two sulphur of native interchain 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 For " artificial interchain disulfide bond ".

For convenience of the position of description disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequences in the present invention Position Number by from N-terminal to C-terminal order successively carry out Position Number, in TRBC1*01 or TRBC2*01, by from N-terminal to The 60th amino acid of the order of C-terminal successively is P (proline), then can describe it as TRBC1*01 or TRBC2*01 in the present invention The Pro60 of exons 1 can also be stated that the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s, for another example Be Q (glutamine) by the 61st amino acid of 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 and so on.In the present invention, the amino acid sequence of variable region TRAV and TRBV Position Number, according to the Position Number listed in IMGT.Such as some amino acid in TRAV, the Position Number listed in IMGT is 46, then the 46th amino acids of TRAV are described it as in the present invention, other and so on.In the present invention, the sequence of other amino acid Column position number has specified otherwise, then by specified otherwise.

Detailed description of the invention

TCR molecules

In antigen processing pathways, antigen is degraded in the cell, is then carried by MHC molecule to cell surface.T is thin Born of the same parents' receptor can identify the peptide-MHC compounds of Antigen Presenting Cell surface.Therefore, the first aspect of the present invention provides one kind The TCR molecules of PYLGQMINL-HLA A2402 compounds can be combined.Preferably, the TCR molecules are separated or purify 's.α the and β chains of the TCR respectively have 3 complementary determining regions (CDR).

One in the present invention is preferably carried out in mode, and the α chains of the TCR are included with following amino acid sequence CDR：

αCDR1-NSASQS(SEQ ID NO:10)

αCDR2-VYSSGN(SEQ ID NO:11)

αCDR3-VVRGNNARLM(SEQ ID NO:12)；And/or

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

βCDR1-SEHNR(SEQ I D NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSGSAAGANVLT(SEQ ID NO:15)。

The CDR region amino acid sequence of the invention described above can be embedded into chimeric to prepare in any suitable frame structure TCR.As long as frame structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art's disclosed CDR region according to the present invention It 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 chains variable domain of the present invention be and SEQ ID NO:1 tool There is the amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence thereto；And/or TCR β chains of the present invention can Variable domain be and SEQ ID NO：5 have at least 90%, preferably 95%, the amino acid sequence of more preferably 98% sequence thereto Row.

In the preference of the present invention, TCR molecules of the invention are the heterodimers being made of α and β chains.Specifically Ground, 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：And CDR3 (SEQ ID NO 11):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, The β chains variable domain amino acid sequence includes CDR1 (the SEQ ID NO of 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 the preference of the present invention, TCR molecules of the invention are part or all of and/or β chains the portions by α chains The single chain TCR molecules for dividing or all forming.Description in relation to 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 Structure includes the single chain TCR molecules in 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.

The α chains variable domain amino acid sequence of the single chain TCR molecules includes CDR1 (the SEQ ID NO of above-mentioned α chains：10)、 CDR2(SEQ ID NO：And CDR3 (SEQ ID NO 11):12).Preferably, the single chain TCR molecules include α chain variable domain ammonia Base acid sequence SEQ ID NO:1.It is highly preferred that the α chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO: 1.The β chains variable domain amino acid sequence of the single chain TCR molecules includes CDR1 (the SEQ ID NO of above-mentioned β chains：13)、CDR2 (SEQ ID NO：And CDR3 (SEQ ID NO 14)：15).Preferably, the single chain TCR molecules include β chain variable domain amino acids Sequence SEQ ID NO:5.It is highly preferred that the β chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:5.

In the preference of the present invention, the constant domain of TCR molecules of the invention is the constant domain of people.Art technology Personnel know or can be obtained by consulting the public database of pertinent texts or IMGT (international immunogenetics information system) Obtain the constant domain amino acid sequence of people.For example, the constant domain sequence of TCR molecule alphas chain of the present invention can be " TRAC*01 ", TCR points The constant domain sequence of sub- β chains can be " TRBC1*01 " or " TRBC2*01 ".The amino acid sequence provided in the TRAC*01 of IMGT The 53rd for Arg, be expressed as herein：The Arg53 of TRAC*01 exons 1s, other and so on.Preferably, TCR of the present invention The amino acid sequence of molecule alpha chain is SEQ ID NO:The amino acid sequence of 3 and/or β chains is SEQ ID NO:7.

Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.As immunoglobulin (antibody) is made The same for antigen recognizing molecule, at this moment TCR can also need to obtain soluble TCR points by development and application in diagnose and treat Son.Soluble TCR molecules do not include its transmembrane region.STCR has very extensive purposes, it cannot be only used for research TCR With the interaction of pMHC, it is also possible to make the diagnostic tool of detection infection or the marker as autoimmunity disease.Similarly, may be used Dissolubility TCR can be used to therapeutic agent (such as cytotoxin compounds or immunostimulating compound) being transported to presentation specificity The cell of antigen, in addition, sTCR can also with other molecules (e.g., anti-CD 3 antibodies) with reference to redirecting T cell, from And make the cell of its targeting presentation specific antigen.The present invention also obtains the solubility for having specificity to PRAME antigen small peptide TCR。

To obtain sTCR, on the one hand, TCR of the present invention can be 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, Substitute the cysteine residues of the Thr48 of TRAC*01 exons 1s and the Ser57 of substitution TRBC1*01 or TRBC2*01 exons 1s To form disulfide bond.Cysteine residues are introduced to can also be 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 are instead of any group of site in above-mentioned α and β chain constant domains.It can be in TCR constant domains of the present invention One or more C-terminals truncate most 50 or most 30 or most 15 or most 10 or most 8 or less Amino acid so that its not including cysteine residues come achieve the purpose that lack natural disulphide bonds, also can be by the way that day will be formed The cysteine residues of right disulfide bond sport another amino acid to reach above-mentioned purpose.

As described above, the TCR of the present invention may be embodied in the artificial disulfide bond introduced between the residue of itself α and β chain constant domain. It should be noted that the artificial disulfide bond with or without introducing described above between constant domain, it is constant that TCR of the invention can contain TRAC Domain sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences of TCR can It is connected by the natural disulphide bonds being present in TCR.

To obtain sTCR, on the other hand, TCR of the present invention is additionally included in the TCR that its hydrophobic core region is undergone mutation, The mutation of these hydrophobic core regions is preferably capable the stability-enhanced mutation for making 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 genes (TRAJ) small peptide Amino acid position the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptides amino acid position reciprocal is 2nd, 4,6 reciprocal, wherein ammonia The Position Number of base acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).People in the art Member knows above-mentioned international immunogenetics information system, and the amino acid residue that different TCR can be obtained according to the database exists Position Number in IMGT.

The TCR that hydrophobic core region is undergone mutation in the present invention can be by α and the β chain of a flexible peptide chain link TCR can Variable domain and the solvable single-stranded TCR of stability formed.It should be noted that in the present invention flexible peptide chain can be any suitable connection TCR α with The peptide chain of β chain variable domains.Single chain soluble TCR, α the chain variable domain amino acid sequence such as built in the embodiment of the present invention 4 For SEQ ID NO:32, the nucleotides sequence of coding is classified as SEQ ID NO:33；β 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.It specifically, can in the α chains of the TCR Become between area and β chain constant regions formed the cysteine residues of artificial interchain disulfide bond instead of：The 46th amino acids of TRAV With the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；The 47th amino acids and TRBC1*01 of TRAV or 61 amino acids of TRBC2*01 exons 1s；The 46th amino acids of TRAV and the of TRBC1*01 or TRBC2*01 exons 1s 61 amino acids；Or the 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s.It is preferred that Ground, such TCR can include all or part of TCR α chains and (II) of (I) in addition to its transmembrane domain and remove 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 include α chains variable domain and β chains variable domain and All or part of β chains constant domain in addition to transmembrane domain, but it does not contain α chain constant domains, the α chain variable domains of the TCR Heterodimer is formed with β chains.

The present invention TCR can also multivalence complex form provide.The present invention multivalent TCR complex include two, Three, the four or more TCR of the present invention polymers that are combined and are formed, can such as be generated with four dimerization domains of p53 The tetramer or multiple TCR of the present invention and another molecule with reference to and the compound that is formed.The TCR compounds of the present invention can be used for body Outer or tracking in vivo or targeting present the cell of specific antigen, it can also be used to generate other multivalence TCR with such application and answer Close the intermediate of object.

The TCR of the present invention can be used alone, and can also be combined with conjugate with covalent or other modes, preferably with covalently side Formula combines.The conjugate includes detectable and (for diagnostic purpose, is presented wherein the TCR is used to detect The presence of the cell of PYLGQMINL-HLA A2402 compounds), therapeutic agent, PK (protein kinase) modified parts or it is any more than The combination of these substances combines or coupling.

Detectable for diagnostic purposes includes but not limited to：Fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.

The therapeutic agent that can be combined or be coupled with TCR of the present invention includes but not limited to：1. radionuclide (Koppe etc., 2005, metastasis of cancer comment (Cancer metastasis reviews) 24,539)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc Segment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；5. antibody ScFv segments (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold medal 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, the TCR of the present invention can also be comprising the heterozygosis TCR being derived from more than a kind of species sequence.For example, it grinds Study carefully display Muridae TCR can more effectively to express than people TCR in human T-cell.Therefore, TCR of the present invention can include people's variable domain With the constant domain of mouse.The defects 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 to express the implantation of the T cell of Muridae.

It is to be understood that amino acid name is represented using international single English alphabet or three English alphabets herein, amino The correspondence of the single English alphabet and three English alphabets of sour title is as follows：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys (C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser (S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。

Nucleic acid molecules

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

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

αCDR1-aacagtgcttctcagtct(SEQ ID NO:16)

αCDR2-gtatactccagtggtaat(SEQ ID NO:17)

αCDR3-gtggtgagggggaacaatgccagactcatg(SEQ ID NO:18)

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

βCDR1-tctgaacacaaccgc(SEQ ID NO:19)

βCDR2-ttccagaatgaagctcaa(SEQ ID NO:20)

βCDR3-gccagcagtgggagcgctgctggggccaacgtcctgact(SEQ ID NO:21)

Therefore, encoding the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chains of the present invention includes SEQ ID NO:16、SEQ ID NO:17 and SEQ ID NO:18 and/or the nucleotide sequences of nucleic acid molecules of the present invention of coding TCR β chains of the present invention 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, such as the nucleotide sequence bag of the nucleic acid molecules of the present invention of coding TCR α chain variable domains of the present invention Include SEQ ID NO:2 and/or the nucleotide sequences of nucleic acid molecules of the present invention of coding TCR β chain variable domains of the present invention include SEQ ID NO:6.Alternatively, the nucleotide sequence of the nucleic acid molecules of the present invention of coding TCR α chain variable domains of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequences of nucleic acid molecules of the present invention of coding 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.It is alternatively, of the invention The nucleotides sequence of nucleic acid molecules is classified as SEQ ID NO:31.

It is to 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 attached nucleotide sequence shown in figure 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 the codon in sequence be changed to increase expression quantity according to the type of cell.Mammalian cell and various other The codon usage table of biology is well known to those skilled in the art.

The present invention nucleic acid molecules full length sequence or its segment usually can with but be not limited to PCR amplification method, recombination method or Artificial synthesized method obtains.At present, it is already possible to completely by chemical synthesis come obtain encoding TCR of the present invention (or its segment, Or derivatives thereof) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or Such as carrier) and cell in.DNA can be coding strand or noncoding strand.

Carrier

It, can in vivo or body including expression vector the invention further relates to the carrier for the nucleic acid molecules for including the present invention The construct of outer expression.Common carrier includes bacterial plasmid, bacteriophage and animals and plants virus.

Viral delivery systems include but not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, Retroviral vector, slow virus carrier, baculovirus vector.

Preferably, the nucleotide of the present invention can be transferred in cell by carrier, such as in T cell so that the cell table Up to the TCR of PRAME antigen specificity.Ideally, which can should express continual high levels in T cell.

Cell

The invention further relates to the host cells generated with the carrier or coded sequence of the present invention through genetic engineering.The host The nucleic acid molecules of the present invention are integrated in carrier or chromosome containing the present invention in cell.Host cell is selected from：Prokaryotic cell And eukaryocyte, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..

In addition, the separated cell of the TCR present invention additionally comprises the expression present invention, particularly T cell.The T cell can spread out It is born from from the separated T cell of subject or can be the separated mixed cellularity group from subject, such as periphery hemolymph is thin The part of born of the same parents (PBL) group.Such as, which can be isolated from peripheral blood mononuclear cells (PBMC), can be CD4⁺Helper cell Or CD8⁺Cytotoxic T cell.The cell can be in CD4⁺Helper cell/CD8⁺In the mixing group of cytotoxic T cell.Generally Ground, the cell can use antibody (e.g., the antibody of anti-CD3 or anti-CD28) to activate, to allow them to easily receive to turn Dye, such as transfected with the carrier comprising the nucleotide sequence for encoding TCR molecules of the present invention.

Alternatively, cell of the invention can also be or 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 It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecules will start in thymocyte The surface expression introducing TCR molecules.

There are many method be suitable for the DNA or RNA of coding TCR of the present invention carry out 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).

PRAME antigen relevant disease

The invention further relates to being treated in subject and/or preventing the method with PRAME relevant diseases, including adoptive Shift the step of PRAME specific T-cells are to the subject.The PRAME specific T-cells can recognize that PYLGQMINL-HLA A2402 compounds.

The T cell of the PRAME specificity of the present invention can be used for treating any presentation PRAME antigen small peptide PYLGQMINL- The PRAME relevant diseases of HLA A2402 compounds.Including but not limited to tumour, such as melanoma and other entity tumors are such as Stomach cancer, lung cancer, cancer of the esophagus, carcinoma of urinary bladder, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, oophoroma etc..

Therapy

Can by separation suffer from the patient of PRAME antigen relevant disease or the T cell of volunteer, and by the present invention TCR is imported in above-mentioned T cell, then feeds back in patient body to treat by the cell that these genetic engineerings are modified.Therefore, The present invention provides a kind of method for treating PRAME relevant diseases, including by the T cell of separated expression TCR of the present invention, preferably Ground, the T cell in itself, are input in patient body from patient.Usually, the T cell of (1) separation patient is included, (2) are with originally Invention nucleic acid molecules or the nucleic acid molecules ex vivo transduction T cell that can encode TCR molecules of the present invention, (3) modify genetic engineering T cell be input in patient body.The quantity of separation, transfection and the cell fed back can be determined by doctor.

Main advantages of the present invention are：

(1) TCR of the invention can be combined with PRAME antigen small peptide compound PYLGQMINL-HLA A2402, be turned simultaneously The cell for having led TCR of the present invention can have very strong lethal effect by specific activation and to target cell.

Following specific embodiment, the present invention is further explained.It is to be understood that these embodiments be merely to illustrate the present invention and It is not used in and limits the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to normal condition, Such as (Sambrook and Russell et al., molecular cloning：Laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing houses) described in condition or according to the condition proposed by manufacturer.Unless In addition illustrate, otherwise percentage and number are calculated by weight.Unless otherwise stated, otherwise percentage and number are calculated by weight. Experiment material and reagent used can obtain unless otherwise instructed from commercially available channel in following embodiment.

Embodiment 1 clones PRAME antigen small peptide specific T-cells

Using synthesizing small peptide PYLGQMINL (SEQ ID NO.9；Beijing SBS Genetech gene technology Co., Ltd) stimulate come from In the peripheral blood lymphocytes (PBL) for the healthy volunteer that genotype is HLA-A2402.By PYLGQMINL small peptides with carrying life The HLA-A2402 renaturation of object element mark, prepares pHLA monoploid.These monoploid and Streptavidin (the BD public affairs with PE marks Department) 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 carries out monoclonal with limiting dilution assay.Monoclonal cell tetramer staining, screening The double positive colonies arrived are as shown in Figure 3.

Embodiment 2 obtains the tcr gene of PRAME 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 PYLGQMINL specificity, HLA-A2402 restricted T cell clone.The synthesis of cDNA is using clontech's SMART RACE cDNA amplification kits, the primer of use are designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned It is sequenced on to carrier T (TAKARA).It should be noted that the sequence is complementary series, 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 nucleotide 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, have targeting sequencing TCR β chain amino acid sequences with And the TCR β chain nucleotide sequences with targeting sequencing.

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

αCDR1-NSASQS(SEQ ID NO:10)

αCDR2-VYSSGN(SEQ ID NO:11)

αCDR3-VVRGNNARLM(SEQ ID NO:12)

β chains include the CDR with following amino acid sequence：

βCDR1-SEHNR(SEQ ID NO:13)

βCDR2-FQNEAQ(SEQ ID NO:14)

βCDR3-ASSGSAAGANVLT(SEQ ID NO:15)

The full-length gene of TCR α chains and β chains is cloned into Lentiviral respectively by being overlapped (overlap) PCR pLenti(addgene).Specially：The full-length gene of TCR α chains and TCR β chains is attached to obtain TCR with overlap PCR α -2A-TCR β segments.Lentiviral and TCR α -2A-TCR β digestions are connected to obtain pLenti-TRA-2A-TRB- IRES-NGFR plasmids.It is used as control, while the also slow virus carrier pLenti-eGFP of structure expression eGFP.It uses again afterwards 293T/17 packs pseudovirus.

Expression, refolding and the purifying of the solvable TCR of embodiment 3PRAME antigens small peptide specificity

To obtain soluble TCR molecules, α the and β chains of TCR molecules of the invention can only include its variable domain and portion respectively Divide constant domain, and introduce a cysteine residues in the constant domain of α and β chains respectively to form artificial interchain disulfide bond, The position for introducing cysteine residues is respectively the Ser57 of the Thr48 and TRBC2*01 exons 1s of TRAC*01 exons 1s；Its α The amino acid sequence of chain is distinguished as shown in figures 4 a and 4b with nucleotide sequence, the amino acid sequence and nucleotide sequence of β chains Respectively as shown in figure 5 a and 5b, the cysteine residues of introducing with overstriking and underline alphabetical represent.Pass through《Molecular cloning Laboratory manual》(Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) Described in standard method the objective gene sequence of above-mentioned TCR α and β chains are inserted respectively into expression vector pET28a after synthesis + (Novagene), the cloning site of upstream and downstream is NcoI and NotI respectively.Insert Fragment confirms errorless by sequencing.

The expression vector of TCR α and β chains is converted by chemical transformation into expression bacterium BL21 (DE3), bacterium respectively It is grown with LB culture solutions, in OD₆₀₀It is induced when=0.6 with final concentration 0.5mM IPTG, the bag formed after α the and β chains expression of TCR Contain body to extract by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, 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.

Dissolved TCR α and β chains are with 1：1 mass ratio is quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Solution is placed in the deionized water of 10 times of volumes dialysis (4 DEG C) afterwards, 12 it is small when after change deionized water into buffer solution (20mM Tris, pH 8.0) continue at 4 DEG C dialysis 12 it is small when.Solution after the completion of dialysis after 0.45 μM of membrane filtration, by the moon from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains the successful α and β dimers of renaturation TCR is confirmed by SDS-PAGE glue.TCR then by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) it is further purified.TCR purity after purification is measured by SDS-PAGE is more than 90%, and concentration is by BCA methods It determines.The SDS-PAGE glue figures for the sTCR that the present invention obtains are as shown in Figure 6.

The generation of the soluble single-chain T CR of 4 PRAME antigen small peptide specificity of embodiment

According to patent document WO2014/206304, using the method for rite-directed mutagenesis by TCR α and β in embodiment 2 The variable domain of chain has been built into the soluble single-chain T CR molecules of a stabilization with flexible small peptide (linker) connection.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 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 target gene through I double digestion of Nco I and Not, it is connected with by the pET28a carriers of I double digestion of Nco I and Not. Connection product converts and to E.coli DH5 α, is coated with the LB tablets containing kanamycins, 37 DEG C of inversion overnight incubations, positive gram of picking Grand progress PCR screenings, are sequenced positive recombinant, determine that sequence correctly extracts recombinant plasmid transformed to E.coli afterwards BL21 (DE3), for expressing.

Expression, renaturation and the purifying of the soluble single-chain T CR of 5 PRAME antigen small peptide specificity of embodiment

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 culture to OD600 be 0.6-0.8, add in IPTG to final concentration of 0.5mM, 37 DEG C continue to cultivate 4h.5000rpm centrifugation 15min harvest cell pellets, are cracked with Bugbuster Master Mix (Merck) Cell pellet, 6000rpm centrifugation 15min recycling inclusion bodys, then 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.

In the single-stranded TCR inclusion body proteins dissolved to 5mg, 2.5mL buffer solutions (6M Gua-HCl, 50mM Tris- is added in HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration of 10mM, 37 DEG C of processing 30min.With injection Device is to 125mL renaturation buffers (100mM Tris-HCl pH 8.1,0.4M L-arginines, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) in treated single-stranded TCR, 4 DEG C of stirrings are added dropwise Then renaturation solution is packed into the cellulose membrane bag filter that interception is 4kDa by 10min, bag filter is placed in the water of 1L precoolings, 4 DEG C It is slowly stirred overnight.17 it is small when after, dialyzate changes into the buffer solutions (20mM Tris-HCl pH 8.0) of 1L precoolings, 4 DEG C after Then continuous dialysis 8h changes dialyzate into identical fresh buffer and continues dialysed overnight.17 it is small when after, sample through 0.45 μm filter Membrane filtration, by anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgas, with 20mM Tris-HCl The 0-1M NaCl linear gradient elution liquid purifying proteins that pH 8.0 is prepared, the elution fraction of collection carry out SDS-PAGE analyses, bag It is further carried out after component concentration containing single-stranded TCR with solvent resistant column (Superdex 75 10/300, GE Healthcare) Purifying, target components also carry 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 for the soluble single-chain T CR that the present invention obtains are as shown in figure 11.

Embodiment 6 combines characterization

BIAcore is analyzed

It can be special with PYLGQMINL-HLA A2402 compounds this example demonstrated soluble TCR molecules of the present invention The opposite sex combines.

Detected using BIAcore T200 real-time analyzers the TCR molecules that are obtained in embodiment 3 and embodiment 5 with The combination activity of PYLGQMINL-HLA A2402 compounds.It is slow that the antibody (GenScript) of anti-Streptavidin is added in into coupling Then antibody is flowed through the CM5 chips activated in advance with EDC and NHS, made by fliud flushing (10mM sodium-acetate buffers, pH 4.77) Antibody is fixed on chip surface, finally closes unreacted activating surface with the hydrochloric acid solution of ethanolamine, completes coupling process, even Connection horizontal about 15,000RU.

The Streptavidin of low concentration is made to flow through the chip surface of coated antibody, then by PYLGQMINL-HLA A2402 compounds flow through sense channel, and another passage is as reference channel, then by the biotin of 0.05mM with the stream of 10 μ L/min Speed flows through chip 2min, closes the remaining binding site of Streptavidin.

The preparation process of above-mentioned PYLGQMINL-HLA A2402 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 centrifuge 10min PBS washing thallines are once, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) afterwards Thalline is resuspended in concussion, and is rotated in room temperature and be incubated 20min, after 4 DEG C, 6000g centrifugation 15min, supernatant discarding, collection forgives Body.

Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Add 30ml The BugBuster of 10 times of dilution, mixing, 4 DEG C of 6000g centrifuge 15min；Supernatant discarding, the BugBuster that 30ml is added to dilute 10 times Inclusion body, mixing is resuspended, 4 DEG C of 6000g centrifuge 15min, are repeated twice, and add 30ml 20mM Tris-HCl pH 8.0 that bag is resuspended Contain body, mixing, 4 DEG C of 6000g centrifuge 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 PYLGQMINL (Beijing SBS Genetech gene technology Co., Ltd) of synthesis is dissolved in DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain is dissolved with 8M urea, 20mM Tris pH 8.0,10mM DTT, is added in before renaturation 3M guanidine hydrochlorides, 10mM sodium acetates, 10mM EDTA are further denatured.PYLGQMINL peptides are added in into renaturation with 25mg/L (final concentration) Buffer solution (0.4M L-arginines, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced forms Glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, Heavy chain adds in three times, 8h/ times), renaturation carries out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detections renaturation success.

C. purified after renaturation

Make dialysis with the 20mM Tris pH 8.0 of 10 volumes to replace renaturation buffer, at least replace 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 gradients liquid elution albumen that 20mM Tris pH 8.0 are prepared, pMHC is about in 250mM It is eluted at NaCl, collects all peak components, SDS-PAGE detection purity.

D. biotinylation

With Millipore super filter tubes by the pMHC molecular concentrations of purifying, while it is 20mM Tris pH by buffer exchange 8.0, then add in 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), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detections biotinylation Completely.

E. the compound after purifying biological elementization

PMHC molecular concentrations after biotinylation is marked with Millipore super filter tubes are to 1ml, using gel permeation chromatography The pMHC of purifying biological elementization, using Akta purifying instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibrations HiPrep^TM 16/60S200HR columns (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated loading 1ml, then with PBS with 1ml/ Min flow velocitys elute.Biotinylated pMHC molecules occur in about 55ml as unimodal elution.Merge the group containing protein Point, it is concentrated with Millipore super filter tubes, BCA methods (Thermo) measure protein concentration, add in protease inhibitors cocktail (Roche) packing of biotinylated pMHC molecules is stored in -80 DEG C.

Using BIAcore Evaluation software computational dynamics parameters, obtain the TCR molecules of solubility of the invention with And the kinetic profile point that the soluble single-chain T CR molecules of the invention built are combined with PYLGQMINL-HLA A2402 compounds Not as shown in Figure 12 and Figure 13.Collection of illustrative plates shows that the soluble TCR molecules and soluble single-chain T CR molecules that the present invention obtains are all It can be combined with PYLGQMINL-HLA A2402 compounds.Meanwhile also solubility of the invention is had detected using the above method TCR molecules and other several irrelevant antigen small peptides and the combination of HLA compounds are active, the results show TCR molecules of the present invention and its His irrelevant antigen is without combination.

Embodiment 7PRAME antigen small peptide specificity TCRs slow virus is packed to be transfected with primary T cells

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

Utilize slow virus of the third generation slow virus packaging system packaging containing the gene of TCR needed for coding.It is situated between using quick Lead transient transfection (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)), with a kind of 4 kinds of plasmids (slow virus containing pLenti-TRA-2A-TRB-IRES-NGFR described in embodiment 2 Carrier and 3 kinds of plasmids containing other components necessary to structure infectiousness but non-replicating lentiviral particle) transfection 293T Cell.

(b) with the lentiviruses transduction primary T cells of the T cell receptor gene containing PRAME antigen small peptide specificity

It is separated to CD8+T cells from the blood of healthy volunteer, then with the lentiviruses transduction packed.It is thin to count these Born of the same parents, in 48 orifice plates, containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10%FBS (Ji Bu can company (Gibco), Catalog number (Cat.No.) C10010500BT) 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture mediums in 1 × 10⁶ A cells/ml (0.5 milliliter/hole) and AntiCD3 McAb/CD28 antibody-coating globule (T cell amplified matter, the life pre-washed Technologies, catalog number (Cat.No.) 11452D) be incubated overnight stimulates altogether, cell：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 PRAME antigen small peptide specific t-cell receptor gene of contracting, 32 DEG C, when 900g centrifugations infection 1 is small.Infection finishes Slow-virus infection liquid is removed afterwards, with the culture of 1640 containing the 10%FBS base weight containing 50IU/ml IL-2 and 10ng/ml IL-7 Outstanding cell, 37 DEG C/5%CO₂Lower culture 3 days.Next day carries out the second wheel infection in the same way.Second of transduction counts after 3 days Cell, diluting cells to 0.5 × 10⁶A cells/ml.It counts a cell within every two days, replaces or add in and contain 50IU/ml The fresh culture of IL-2 and 10ng/ml IL-7 maintains cell 0.5 × 10⁶-1×10⁶A cells/ml.It was opened from the 3rd day Begin through flow cytometry cell, for function test (for example, the ELISPOT of IFN-γ release and non-since the 5th day Radioactivity cytotoxicity detects).Since the 10th day or when cell slows down division and size becomes smaller, stored frozen decile is thin Born of the same parents, at least 4 × 10⁶A cell/pipe (1 × 10⁷A cells/ml, 90%FBS/10%DMSO).

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

ELISPOT schemes

Following experiment is carried out to prove activating reaction of the T cell to target cell specificity of TCR- transductions.Utilize ELISPOT Readout of the IFN-γ yield of testing inspection as t cell activation.

Reagent

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

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

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

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

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

Method

It is prepared by target cell

Target cell used is LCL cells in this experiment.Target cell is prepared in assay medium：Target cell concentration is adjusted to 2.0×10⁵A/milliliter takes 100 microlitres per hole so as to obtain 2.0 × 10⁴A cells/well.

It is prepared by effector cell

Effector cell's (T cell) of this experiment is to express PRAME antigen of the present invention through flow cytometry in embodiment 7 The CD8+T cells of small peptide specificity TCR, and with the CD8+T of same volunteer's untransfected TCR of the present invention as a control group.With anti- CD3/CD28 coating pearls (T cell amplified matter, life technologies) stimulate T cell, special with PRAME antigen small peptide is carried The lentiviruses transduction (according to embodiment 7) of different in nature tcr gene is containing 10% containing 50IU/ml IL-2 and 10ng/ml IL-7 FBS 1640 culture mediums amplification until transduction after 9-12 days, then these cells are placed in test medium, 300g room temperature from The heart is washed for 10 minutes.Then by cell with 2 × required final concentration is resuspended in test medium.Similary processing negative control Effector cell.

It is prepared by small peptide solution

Corresponding small peptide is added in corresponding target cell (LCL) 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, prepare orifice plate as described below：1 is pressed with 10 milliliters of sterile PBS of every block of plate：200 It dilutes anti-human IFN-γ and captures antibody, 100 microlitres of dilution then is captured antibody etc. point adds in each hole.Orifice plate is incubated at 4 DEG C Overnight.After incubation, orifice plate is washed to remove extra capture antibody.Add in the RPMI 1640 that 10%FBS is contained in 100 microlitres/hole Culture medium, and incubate at room temperature orifice plate 2 it is small when to close orifice plate.Then culture medium is washed away from orifice plate, by light on paper Bullet and ELISPOT orifice plates are patted to remove the lavation buffer solution of any remnants.

Then all components of experiment are added in by ELISPOT orifice plates using following order：

100 microlitres of target cell 2*10⁵A cells/ml (obtains about 2*10 in total⁴A target cell/hole).

100 microlitres of effector cell (1*10⁴A control effector cell/hole and PRAME TCR positive T cells/hole).

All holes prepare addition in duplicate.

Then (37 DEG C/5%CO overnight of orifice plate is incubated₂) second day, culture medium is abandoned, orifice plate is washed 2 times with distilled water, then uses Lavation buffer solution washs 3 times, pats to remove remaining lavation buffer solution on paper handkerchief.Then pressed with the PBS containing 10%FBS 1：200 dilution detection antibody, each hole is added in by 100 microlitres/hole.When incubation orifice plate 2 is small 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 diluted chains Mould Avidin-alkaline phosphatase adds in each hole and when incubation orifice plate 1 is small 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.Kit is added in after washing to provide 100 microlitres/hole of BCIP/NBT solution develop.It is protected from light during development with masking foil covering orifice plate, stands 5-15 minutes. The spot of conventional detection development orifice plate during this period determines to terminate the Best Times of reaction.Remove BCIP/NBT solution and with pair It steams water and rinses 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 (as described above) for examining TCR transductions of the present invention is tested to loading PRAME antigen small peptide by ELISPOT The IFN-γ release that the target cell of PYLGQMINL reacts.Draw what is observed in each hole using graphpad prism6 ELSPOT amount of speckle.

Experimental result is as shown in figure 14, and the T cell for the TCR of the present invention that transduces has very the target cell for loading its special small peptide Good activating reaction, and the T cell for the TCR of the present invention that do not transduce is substantially without activating reaction.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited It encloses.

Sequence table

<110>Guangzhou Xiangxue Pharmaceutical Co

<120>Identify the T cell receptor of PRAME antigen small peptide

<130> P2016-2163

<150> CN201610188918.2

<151> 2016-03-29

<160> 37

<170> PatentIn version 3.5

<210> 1

<211> 110

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chain variable domains

<400> 1

Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val Pro Glu Gly

1 5 10 15

Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Ser Ala Ser Gln Ser

20 25 30

Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pro Lys Leu Leu Met

35 40 45

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

50 55 60

Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Arg Asp Ser Lys Leu

65 70 75 80

Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Arg Gly Asn Asn Ala Arg

85 90 95

Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn

100 105 110

<210> 2

<211> 330

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chain variable domains

<400> 2

cggaaggagg tggagcagga tcctggaccc ttcaatgttc cagagggagc cactgtcgct 60

ttcaactgta cttacagcaa cagtgcttct cagtctttct tctggtacag acaggattgc 120

aggaaagaac ctaagttgct gatgtccgta tactccagtg gtaatgaaga tggaaggttt 180

acagcacagc tcaatagagc cagccagtat atttccctgc tcatcagaga ctccaagctc 240

agtgattcag ccacttacct ctgtgtggtg agggggaaca atgccagact catgtttgga 300

gatggaactc agctggtggt gaagcccaat 330

<210> 3

<211> 250

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chains

<400> 3

Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val Pro Glu Gly

1 5 10 15

Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Ser Ala Ser Gln Ser

20 25 30

Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pro Lys Leu Leu Met

35 40 45

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

50 55 60

Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Arg Asp Ser Lys Leu

65 70 75 80

Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Arg Gly Asn Asn Ala Arg

85 90 95

Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn

165 170 175

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250

<210> 4

<211> 750

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chains

<400> 4

cggaaggagg tggagcagga tcctggaccc ttcaatgttc cagagggagc cactgtcgct 60

ttcaactgta cttacagcaa cagtgcttct cagtctttct tctggtacag acaggattgc 120

aggaaagaac ctaagttgct gatgtccgta tactccagtg gtaatgaaga tggaaggttt 180

acagcacagc tcaatagagc cagccagtat atttccctgc tcatcagaga ctccaagctc 240

agtgattcag ccacttacct ctgtgtggtg agggggaaca atgccagact catgtttgga 300

gatggaactc agctggtggt gaagcccaat atccagaacc ctgaccctgc cgtgtaccag 360

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 480

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 660

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 720

ctgctcatga cgctgcggct gtggtccagc 750

<210> 5

<211> 115

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chain variable domains

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gly

85 90 95

Ser Ala Ala Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser Arg Leu

100 105 110

Thr Val Leu

115

<210> 6

<211> 345

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chain variable domains

<400> 6

gatactggag tctcccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcagtgggag cgctgctggg 300

gccaacgtcc tgactttcgg ggccggcagc aggctgaccg tgctg 345

<210> 7

<211> 294

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chains

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gly

85 90 95

Ser Ala Ala Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser Arg Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Arg Lys Asp Ser Arg Gly

290

<210> 8

<211> 882

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chains

<400> 8

gatactggag tctcccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcagtgggag cgctgctggg 300

gccaacgtcc tgactttcgg ggccggcagc aggctgaccg tgctggagga cctgaaaaac 360

gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 420

aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 480

tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 540

cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 600

ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 660

aatgacgagt ggacccagga tagggccaaa cctgtcaccc agatcgtcag cgccgaggcc 720

tggggtagag cagactgtgg cttcacctcc gagtcttacc agcaaggggt cctgtctgcc 780

accatcctct atgagatctt gctagggaag gccaccttgt atgccgtgct ggtcagtgcc 840

ctcgtgctga tggccatggt caagagaaag gattccagag gc 882

<210> 9

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>Antigen small peptide

<400> 9

Pro Tyr Leu Gly Gln Met Ile Asn Leu

1 5

<210> 10

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR1

<400> 10

Asn Ser Ala Ser Gln Ser

1 5

<210> 11

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR2

<400> 11

Val Tyr Ser Ser Gly Asn

1 5

<210> 12

<211> 10

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR3

<400> 12

Val Val Arg Gly Asn Asn Ala Arg Leu Met

1 5 10

<210> 13

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR1

<400> 13

Ser Glu His Asn Arg

1 5

<210> 14

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR2

<400> 14

Phe Gln Asn Glu Ala Gln

1 5

<210> 15

<211> 13

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR3

<400> 15

Ala Ser Ser Gly Ser Ala Ala Gly Ala Asn Val Leu Thr

1 5 10

<210> 16

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR1

<400> 16

aacagtgctt ctcagtct 18

<210> 17

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR2

<400> 17

gtatactcca gtggtaat 18

<210> 18

<211> 30

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR3

<400> 18

gtggtgaggg ggaacaatgc cagactcatg 30

<210> 19

<211> 15

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR1

<400> 19

tctgaacaca accgc 15

<210> 20

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR2

<400> 20

ttccagaatg aagctcaa 18

<210> 21

<211> 39

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR3

<400> 21

gccagcagtg ggagcgctgc tggggccaac gtcctgact 39

<210> 22

<211> 271

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chains with targeting sequencing

<400> 22

Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser

1 5 10 15

Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe

20 25 30

Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn

35 40 45

Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu

50 55 60

Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg

65 70 75 80

Phe Thr Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile

85 90 95

Arg Asp Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Arg

100 105 110

Gly Asn Asn Ala Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 23

<211> 813

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chains with targeting sequencing

<400> 23

atgatatcct tgagagtttt actggtgatc ctgtggcttc agttaagctg ggtttggagc 60

caacggaagg aggtggagca ggatcctgga cccttcaatg ttccagaggg agccactgtc 120

gctttcaact gtacttacag caacagtgct tctcagtctt tcttctggta cagacaggat 180

tgcaggaaag aacctaagtt gctgatgtcc gtatactcca gtggtaatga agatggaagg 240

tttacagcac agctcaatag agccagccag tatatttccc tgctcatcag agactccaag 300

ctcagtgatt cagccactta cctctgtgtg gtgaggggga acaatgccag actcatgttt 360

ggagatggaa ctcagctggt ggtgaagccc aatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agc 813

<210> 24

<211> 313

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chains with targeting sequencing

<400> 24

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

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

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Gly Ser Ala Ala Gly Ala Asn Val Leu Thr Phe Gly Ala Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Met Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 25

<211> 939

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chains with targeting sequencing

<400> 25

atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60

actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120

aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180

ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240

agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300

acagagcagg gggactcggc catgtatctc tgtgccagca gtgggagcgc tgctggggcc 360

aacgtcctga ctttcggggc cggcagcagg ctgaccgtgc tggaggacct gaaaaacgtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 939

<210> 26

<211> 203

<212> PRT

<213>Artificial sequence

<220>

<223>STCR α chains

<400> 26

Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val Pro Glu Gly

1 5 10 15

Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn Ser Ala Ser Gln Ser

20 25 30

Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu Pro Lys Leu Leu Met

35 40 45

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

50 55 60

Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile Arg Asp Ser Lys Leu

65 70 75 80

Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Arg Gly Asn Asn Ala Arg

85 90 95

Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro Asn Ile Gln

100 105 110

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

115 120 125

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

130 135 140

Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp

145 150 155 160

Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn

165 170 175

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro

180 185 190

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 27

<211> 609

<212> DNA

<213>Artificial sequence

<220>

<223>STCR α chains

<400> 27

cgcaaagaag tggaacagga tcctggaccc ttcaatgttc cagagggagc cactgtcgct 60

ttcaactgta cttacagcaa cagtgcttct cagtctttct tctggtacag acaggattgc 120

aggaaagaac ctaagttgct gatgtccgta tactccagtg gtaatgaaga tggaaggttt 180

acagcacagc tcaatagagc cagccagtat atttccctgc tcatcagaga ctccaagctc 240

agtgattcag ccacttacct ctgtgtggtg agggggaaca atgccagact catgtttgga 300

gatggaactc agctggtggt gaagcccaat atccagaacc ctgaccctgc cgtgtaccag 360

ctgagagact ctaagtcgag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 420

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaatg tgtgctagac 480

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 540

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 600

gaaagttcc 609

<210> 28

<211> 245

<212> PRT

<213>Artificial sequence

<220>

<223>STCR β chains

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gly

85 90 95

Ser Ala Ala Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser Arg Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Trp Gly Arg Ala Asp

245

<210> 29

<211> 735

<212> DNA

<213>Artificial sequence

<220>

<223>STCR β chains

<400> 29

gataccggcg tgagccagga ccccagacac aagatcacaa agaggggaca gaatgtaact 60

ttcaggtgtg atccaatttc tgaacacaac cgcctttatt ggtaccgaca gaccctgggg 120

cagggcccag agtttctgac ttacttccag aatgaagctc aactagaaaa atcaaggctg 180

ctcagtgatc ggttctctgc agagaggcct aagggatctt tctccacctt ggagatccag 240

cgcacagagc agggggactc ggccatgtat ctctgtgcca gcagtgggag cgctgctggg 300

gccaacgtcc tgactttcgg ggccggcagc aggctgaccg tgctggagga cctgaaaaac 360

gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 420

aaggccacac tggtgtgcct ggccaccggt ttctaccccg accacgtgga gctgagctgg 480

tgggtgaatg ggaaggaggt gcacagtggg gtctgcacag acccgcagcc cctcaaggag 540

cagcccgccc tcaatgactc cagatacgct ctgagcagcc gcctgagggt ctcggccacc 600

ttctggcagg acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 660

aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 720

tggggtagag cagac 735

<210> 30

<211> 249

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR

<400> 30

Ala Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val Pro Glu

1 5 10 15

Gly Glu Thr Val Ser Ile Asn Cys Thr Tyr Ser Asn Ser Ala Ser Gln

20 25 30

Ser Phe Phe Trp Tyr Arg Gln Asp Ser Arg Lys Glu Pro Lys Leu Leu

35 40 45

Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Glu Ile Arg Asp Val Lys

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Arg Gly Asn Asn Ala

85 90 95

Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Lys Pro Gly Gly

100 105 110

Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

115 120 125

Ser Glu Gly Gly Thr Gly Asp Thr Gly Val Ser Gln Asp Pro Arg His

130 135 140

Lys Ser Val Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile

145 150 155 160

Ser Glu His Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Pro Gly Gln Gly

165 170 175

Pro Glu Phe Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser

180 185 190

Arg Leu Leu Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe

195 200 205

Ser Thr Leu Glu Ile Gln Arg Val Glu Gln Gly Asp Ser Ala Met Tyr

210 215 220

Leu Cys Ala Ser Ser Gly Ser Ala Ala Gly Ala Asn Val Leu Thr Phe

225 230 235 240

Gly Ala Gly Ser Arg Leu Thr Val Leu

245

<210> 31

<211> 747

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR

<400> 31

gctcgtaaag aagttgaaca ggatccgggc ccgtttaacg tcccggaagg tgaaaccgtg 60

tctattaact gcacgtattc aaattcggcc agccagagct ttttctggta ccgtcaagat 120

tcgcgcaaag aaccgaaact gctgatgagc gtctatagct ctggcaacga agacggtcgc 180

tttaccgcac agctgaatcg tgctagccaa tatatttctc tggaaatccg tgatgtgaaa 240

ccgagtgact ccgcgacgta cctgtgtgtg gttcgtggca acaatgcccg cctgatgttc 300

ggcgatggta cccagctgac ggttaaaccg ggcggtggct cggaaggtgg cggtagcgaa 360

ggcggtggct ctgaaggtgg cggtagtgaa ggcggtaccg gtgatacggg tgtctcccag 420

gacccgcgcc ataaatcagt taaacgtggt cagaacgtca cctttcgctg cgacccgatt 480

agcgaacaca atcgtctgta ttggtaccgt cagaccccgg gtcaaggtcc ggaatttctg 540

acgtatttcc agaacgaagc acaactggaa aaatctcgtc tgctgtccga tcgcttttca 600

gctgaacgtc cgaaaggctc attctcgacc ctggaaatcc agcgcgttga acagggtgac 660

tctgcgatgt acctgtgtgc aagttccggt agtgcagcag gtgcaaatgt gctgaccttc 720

ggcgctggtt cccgtctgac ggttctg 747

<210> 32

<211> 110

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR α chains

<400> 32

Ala Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe Asn Val Pro Glu

1 5 10 15

Gly Glu Thr Val Ser Ile Asn Cys Thr Tyr Ser Asn Ser Ala Ser Gln

20 25 30

Ser Phe Phe Trp Tyr Arg Gln Asp Ser Arg Lys Glu Pro Lys Leu Leu

35 40 45

Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Glu Ile Arg Asp Val Lys

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Arg Gly Asn Asn Ala

85 90 95

Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Thr Val Lys Pro

100 105 110

<210> 33

<211> 330

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR α chains

<400> 33

gctcgtaaag aagttgaaca ggatccgggc ccgtttaacg tcccggaagg tgaaaccgtg 60

tctattaact gcacgtattc aaattcggcc agccagagct ttttctggta ccgtcaagat 120

tcgcgcaaag aaccgaaact gctgatgagc gtctatagct ctggcaacga agacggtcgc 180

tttaccgcac agctgaatcg tgctagccaa tatatttctc tggaaatccg tgatgtgaaa 240

ccgagtgact ccgcgacgta cctgtgtgtg gttcgtggca acaatgcccg cctgatgttc 300

ggcgatggta cccagctgac ggttaaaccg 330

<210> 34

<211> 115

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR β chains

<400> 34

Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ser Val Lys Arg Gly

1 5 10 15

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

20 25 30

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

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Val Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gly

85 90 95

Ser Ala Ala Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser Arg Leu

100 105 110

Thr Val Leu

115

<210> 35

<211> 345

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR β chains

<400> 35

gatacgggtg tctcccagga cccgcgccat aaatcagtta aacgtggtca gaacgtcacc 60

tttcgctgcg acccgattag cgaacacaat cgtctgtatt ggtaccgtca gaccccgggt 120

caaggtccgg aatttctgac gtatttccag aacgaagcac aactggaaaa atctcgtctg 180

ctgtccgatc gcttttcagc tgaacgtccg aaaggctcat tctcgaccct ggaaatccag 240

cgcgttgaac agggtgactc tgcgatgtac ctgtgtgcaa gttccggtag tgcagcaggt 300

gcaaatgtgc tgaccttcgg cgctggttcc cgtctgacgg ttctg 345

<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

ggcggtggct cggaaggtgg cggtagcgaa ggcggtggct ctgaaggtgg cggtagtgaa 60

ggcggtaccg gt 72

Claims (36)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be with PYLGQMINL-HLA A2402 compound knots It closes；

And 3 complementary determining regions (CDR) of the TCR α chain variable domains are：

αCDR1-NSASQS (SEQ ID NO:10)

αCDR2-VYSSGN (SEQ ID NO:11)

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

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

βCDR1-SEHNR (SEQ ID NO:13)

βCDR2-FQNEAQ (SEQ ID NO:14)

βCDR3-ASSGSAAGANVLT (SEQ ID NO:15)。

2. TCR as described in claim 1, which is characterized in that described it includes TCR α chains variable domains and TCR β chain variable domains TCR α chains variable domain be and SEQ ID NO:1 has the amino acid sequence of at least 90% sequence thereto；And/or the TCR β chains Variable domain be and SEQ ID NO：5 have the amino acid sequence of at least 90% sequence thereto.

3. TCR as described in claim 1, which is characterized in that the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1。

4. TCR as described in claim 1, which is characterized in that the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5。

5. TCR as described in claim 1, which is characterized in that the TCR is α β heterodimers, and it includes TCR α chains are constant Area TRAC*01 and TCR β chain constant regions TRBC1*01 or TRBC2*01.

6. TCR as described in claim 5, which is characterized in that the α chain amino acid sequences of the TCR are SEQ ID NO：3 And/or the β chain amino acid sequences of the TCR are SEQ ID NO:7.

7. the TCR as described in any in claim 1-4, which is characterized in that the TCR is soluble.

8. TCR as claimed in claim 7, which is characterized in that the TCR is single-stranded.

9. TCR as claimed in claim 8, which is characterized in that the TCR is to be connected by α chains variable domain with β chain variable domains by peptide Sequence is connect to be formed by connecting.

10. TCR as claimed in claim 9, which is characterized in that the TCR α chains variable region amino acid the 11st, 13,19,21, 53rd, in 76,89,91 or the 94th and/or α chain J gene small peptides amino acid inverses the 3rd, 5th reciprocal or inverse the 7th It is mutated with one or more；And/or the TCR in β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or 94th and/or β chain J gene small peptides amino acid is 2nd reciprocal, has one or more in 4th reciprocal or 6th reciprocal Mutation, wherein amino acid position number is by the Position Number listed in IMGT (international immunogenetics information system).

11. TCR as claimed in claim 10, which is characterized in that the α chains variable domain amino acid sequence of the TCR is SEQ ID NO:The β chains variable domain amino acid sequence of the 32 and/or TCR is SEQ ID NO:34.

12. TCR as claimed in claim 11, which is characterized in that the amino acid sequence of the TCR is SEQ ID NO:30.

13. TCR as claimed in claim 7, which is characterized in that the TCR include (a) whole in addition to transmembrane domain or Part TCR α chains；And all or part of TCR β chains of (b) in addition to transmembrane domain；

And and (b) respectively contains functional variable domain (a)

14. TCR as claimed in claim 13, which is characterized in that (a) and (b) each also includes the TCR chains constant domain At least a portion.

15. the TCR as described in claim 13 or 14, which is characterized in that α and β chain of the cysteine residues in the TCR is constant Artificial disulfide bond is formed between domain.

16. TCR as claimed in claim 15, which is characterized in that the cysteine that artificial disulfide bond is formed in the TCR is residual Base is 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.

17. TCR as claimed in claim 16, which is characterized in that the α chain amino acid sequences of the TCR are SEQ ID NO:26 And/or the β chain amino acid sequences of the TCR are SEQ ID NO:28.

18. TCR as claimed in claim 13, which is characterized in that contain between the α chains variable region of the TCR and β chain constant regions Artificial interchain disulfide bond.

19. TCR as claimed in claim 18, which is characterized in that half Guang ammonia of artificial interchain disulfide bond is formed in the TCR Sour residue is instead of selected from following one or more groups of sites：

The 46th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；

The 47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1s；

The 46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s；Or

The 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s.

20. the TCR as described in claim 18 or 19, which is characterized in that the TCR include α chains variable domain and β chains variable domain with And all or part of β chains constant domain in addition to transmembrane domain, but it does not contain α chain constant domains, the α chains of the TCR are variable Domain forms heterodimer with β chains.

21. TCR as described in claim 1, which is characterized in that the α chains of the TCR and/or C- the or N- ends of β chains are combined with Conjugate.

22. TCR as claimed in claim 21, which is characterized in that the conjugate combined with the TCR for detectable, Therapeutic agent, PK modified parts or combination thereof.

23. TCR as claimed in claim 22, which is characterized in that the therapeutic agent is anti-CD 3 antibodies.

24. a kind of multivalent TCR complex, which is characterized in that comprising at least two TCR molecules, and at least one TCR therein TCR of the molecule any one of claim 1-23.

25. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules are included any one of coding claim 1-23 The nucleotide sequence of TCR or its complementary series.

26. nucleic acid molecules as claimed in claim 25, which is characterized in that it includes the nucleotides sequences of coding TCR α chain variable domains Arrange SEQ ID NO：2 or SEQ ID NO:33.

27. the nucleic acid molecules as described in claim 25 or 26, which is characterized in that it includes the nucleosides of coding TCR β chain variable domains Acid sequence SEQ ID NO：6 or SEQ ID NO:35.

28. nucleic acid molecules as claimed in claim 25, which is characterized in that it includes the nucleotide sequence SEQ of coding TCR α chains ID NO：4 and/or include coding TCR β chains nucleotide sequence SEQ ID NO：8.

29. a kind of carrier, which is characterized in that the carrier contains any nucleic acid molecules in claim 25-28.

30. carrier as claimed in claim 29, which is characterized in that the carrier is viral vectors.

31. carrier as claimed in claim 30, which is characterized in that the carrier is slow virus carrier.

32. a kind of separated host cell, which is characterized in that containing described in claim 29-31 in the host cell Carrier or chromosome in be integrated with any nucleic acid molecules in the claim 25-28 of external source.

33. a kind of cell, which is characterized in that the cell have in transduction claim 25-28 any nucleic acid molecules or Any carrier in claim 29-31.

34. cell as claimed in claim 33, which is characterized in that the cell is T cell or stem cell.

35. a kind of pharmaceutical composition, which is characterized in that the composition contains pharmaceutically acceptable carrier and claim The TCR compounds described in TCR, claim 24 any one of 1-23, any core in claim 25-28 Any cell in acid molecule or claim 33-34.

36. the multivalent TCR complex or right described in TCR or claim 24 any one of claim 1-23 It is required that the purposes of the cell described in any in 33-34, which is characterized in that be used to prepare the drug for the treatment of tumour.