CN106519019B - Identify the TCR of PRAME antigen - Google Patents

Abstract

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

Description

Identify the TCR of PRAME antigen

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 thin Micromolecule polypeptide is degraded to, and combined to form compound with MHC (main histocompatibility complex) molecule after intracellular generation, It is presented to cell surface.GLSNLTHVL is the small peptide derived from PRAME antigen, is one kind of PRAME treating correlative diseases Target.In addition to melanoma, PRAME is also expressed in kinds of tumors, including squamous cell lung carcinoma, breast cancer, clear-cell carcinoma, head Tumor colli, 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, its PRAME also has notable expression, acute lymphoblastic leukemia 17%~42%, acute myeloblastic leukemia in leukaemia 30%~64% (SteinbachD, et al.Cancer Genet Cytogene, 2002,138 (1):89-91).For upper The methods of stating the treatment of disease, chemotherapy and radiation treatment may be used, but the normal cell of itself can all be damaged.

T cell adoptive immunotherapy is that the reaction-ive T cell for having specificity to target cell antigen is transferred to patient body It is interior, 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 identify phase The antigen small peptide for the target cell surface answered.In immune system, pass through TCR and small peptide-main tissue phase of antigen small peptide specificity The combination of capacitive complex (pMHC compounds) causes T cell to be directly physically contacted with antigen presenting cell (APC), then T Other cell membrane surface molecules of both cell and APC just interact, and a series of subsequent cell signals is caused to transmit With other physiological reactions so that the T cell of different antigentic specificity plays immunological effect to its target cell.Therefore, ability Field technique personnel are dedicated to isolating the TCR that has specificity to NY-ESO-1 antigens small peptide and the TCR transduce T cell There is specific T cell to NY-ESO-1 antigens small peptide to obtain, so as to which them be made to play work in cellular immunotherapy With.

Invention content

The purpose of the present invention is 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 GLSNLTHVL-HLA A0201 compounds combine.

In another preferred example, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chains are variable The amino acid sequence of the CDR3 in domain is AEIPSTGANNLF (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domains Amino acid sequence be ASSQGGTQY (SEQ ID NO:15).

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

αCDR1-DSSSTY(SEQ ID NO:10)

αCDR2-IFSNMDM(SEQ ID NO:11)

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

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

βCDR1-SGHDN(SEQ ID NO:13)

βCDR2-FVKESK(SEQ ID NO:14)

βCDR3-ASSQGGTQY(SEQ ID NO:15)。

In another preferred example, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chains are variable Domain is and SEQ ID NO:1 has the amino acid sequence of at least 90% sequence identity；And/or the TCR β chain variable domains are With SEQ ID NO：5 have the amino acid sequence of at least 90% sequence identity.

In another preferred example, the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.

In another preferred example, the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5.

In another preferred example, the TCR is α β heterodimers, and it includes TCR α chain constant region TRAC*01 and TCR β Chain constant region TRBC1*01 or TRBC2*01.

In another preferred example, the α chain amino acid sequences of the TCR are SEQ ID NO：The β chains of the 3 and/or TCR Amino acid sequence is SEQ ID NO:7.

In another preferred example, the TCR is soluble.

In another preferred example, the TCR is single-stranded.

In another preferred example, the TCR be by α chains variable domain and β chains variable domain by peptide catenation sequence connect and Into.

In another preferred example, the TCR in α chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or 94th and/or α chain J gene small peptides amino acid is 3rd reciprocal, has one or more in 5th reciprocal or 7th reciprocal Mutation；And/or the TCR is in β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th and/or β Chain J gene small peptides amino acid is 2nd reciprocal, has one or more mutation, wherein amino in 4th reciprocal or 6th reciprocal Sour Position Number is by the Position Number listed in IMGT (international immunogenetics information system).

In another preferred example, 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 preferred example, the amino acid sequence of the TCR is SEQ ID NO:30.

In another preferred example, 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 (a) and (b) respectively contains functional variable domain, or include functional variable domain and the TCR At least part of chain constant domain.

In another preferred example, cysteine residues form artificial two sulphur between α the and β chain constant domains of the TCR Key.

In another preferred example, the cysteine residues of artificial disulfide bond are formed in the TCR instead of selected from following One or more groups of sites：

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

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

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

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

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

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

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

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

In another preferred example, the α chain amino acid sequences of the TCR are SEQ ID NO:The β chains of the 26 and/or TCR Amino acid sequence is SEQ ID NO:28.

In another preferred example, artificial interchain disulfide bond is contained between the α chains variable region of the TCR and β chain constant regions.

In another preferred example, which is characterized in that the cysteine that artificial interchain disulfide bond is formed in the TCR is residual Base 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.

In another preferred example, 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 preferred example, the α chains of the TCR and/or C- the or N- ends of β chains are combined with conjugate.

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

The second aspect of the present invention provides a kind of multivalent TCR complex, it includes at least two TCR molecules, and At least one TCR molecules therein are 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 nucleic acid sequence or its complementary series of TCR molecules described in face.

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

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

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

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

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

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

The eighth aspect of the present invention provides the T cell receptor described in first aspect present invention or second party of the present invention The nucleic acid molecules described in TCR compounds, third aspect present invention described in face, the carrier described in fourth aspect present invention or sheet The purposes of the cell described in the 6th aspect is invented, 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 described in the first aspect present invention of amount, third of the present invention The carrier described in nucleic acid molecules, fourth aspect present invention described in aspect or the cell described in sixth aspect present invention or sheet Invent the pharmaceutical composition described in the 7th aspect；

Preferably, the disease is tumour, and preferably described tumour includes melanoma, squamous cell lung carcinoma, mammary gland Cancer, clear-cell carcinoma, head and neck neoplasm, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukaemia (including but it is unlimited In, acute lymphoblastic leukemia, acute myeloblastic leukemia), melanoma and other entity tumors such as gastric cancer, lung Cancer, cancer of the esophagus, carcinoma of urinary bladder, head and neck squamous cell carcinoma, prostate cancer, breast cancer, colon cancer, oophoroma etc..

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) 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, Not repeated them here.

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 can Variable domain nucleotide sequence, TCR α chain amino acid sequences, TCR α chains nucleotide sequence, the TCR α chain amino acids with targeting sequencing Sequence and the TCR α chain nucleotide sequences with targeting sequencing.

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

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

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

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

Fig. 6 is the glue figure of the sTCR obtained after purification.Leftmost side swimming lane is molecular weight to go back virgin rubber, intermediate swimming lane 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 BIAcore dynamics that sTCR of the present invention is combined with GLSNLTHVL-HLA A0201 compounds Collection of illustrative plates.

Figure 12 be transduce TCR of the present invention T cell activation experiment result.

Specific embodiment

The present inventor after extensive and in-depth study, has found and PRAME antigen small peptide GLSNLTHVL (SEQ ID NO:9) TCR that can be specifically bound, the antigen small peptide GLSNLTHVL can form compound and together with HLA A0201 It is presented to cell surface.The present invention also provides encode the nucleic acid molecules of the TCR and include the load of the nucleic acid molecules Body.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 and arrive Respective APC cell surfaces.The MHC of the mankind is commonly referred to as HLA genes or HLA complexs.

T cell receptor (TCR) is the unique of specific antigen peptide of the presentation in main histocompatibility complex (MHC) Receptor.In immune system, T cell is caused by the combination of the TCR and pMHC compounds of antigentic specificity and antigen presentation is thin Born of the same parents (APC) are directly physically contacted, and then other cell membrane surface molecules of both T cell and APC just interact, This just causes a series of subsequent cell signals transmission and other physiological reactions, so that the T of different antigentic specificities is thin Born of the same parents play immunological effect 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. The heterogeneous dimerization TCR of natural 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 Each chains of β include variable region, bonding pad and constant region, and β chains contain short variable region usually also between variable region and bonding pad, But the variable region is often regarded as a part for bonding pad.Each variable region includes and is entrenched in frame structure (framework regions) In 3 CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compounds, wherein CDR3 is recombinated by variable region and bonding pad, is referred to as hypervariable region.α the and β chains of TCR generally regard that each there are two " structures as Domain " i.e. variable 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 state It is found in the public database of border immunogenetics information system (IMGT), as the constant domain sequence of TCR molecule alpha chains is " TRAC*01 ", the constant domain sequence of TCR molecule β chains is " TRBC1*01 " or " TRBC2*01 ".In addition, α the and β chains of TCR are also Comprising transmembrane region and cytoplasmic region, cytoplasmic region is very short.

In the present invention, term " polypeptide of the present invention ", " TCR of the invention ", " T cell receptor of the invention " are interchangeable It uses.

Native interchain disulfide bond and artificial interchain disulfide bond

In the membrane-proximal region C α of natural TCR, there are one group of disulfide bond with C β interchains, are known as " two sulphur of native interchain in of the invention Key ".In the present invention, by what is be artificially introduced, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond claim For " artificial interchain disulfide bond ".

For convenience of the position of description disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequences in the present invention Position Number by from N-terminal to C-terminal sequence successively carry out Position Number, in TRBC1*01 or TRBC2*01, by from N-terminal To the 60th amino acid of the sequence of C-terminal successively be P (proline), then the present invention in can describe it as TRBC1*01 or The Pro60 of TRBC2*01 exons 1s can also be stated that the 60th bit amino of TRBC1*01 or TRBC2*01 exons 1s Acid is Q (glutamine) by the 61st amino acid of the sequence from N-terminal to C-terminal successively for another example in TRBC1*01 or TRBC2*01, The Gln61 of TRBC1*01 or TRBC2*01 exons 1s can be then described it as in the present invention, can also be stated that TRBC1* 61st amino acids of 01 or TRBC2*01 exons 1s, other and so on.In the present invention, the ammonia of variable region TRAV and TRBV The Position Number of base acid sequence, according to the Position Number listed in IMGT.Such as some amino acid in TRAV, listed in IMGT Position Number for 46, then describe it as the 46th amino acids of TRAV in the present invention, other and so on.In the present invention, The Sequence position numbers of his amino acid have 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 Cell receptor can identify the peptide-MHC compounds of Antigen Presenting Cell surface.Therefore, the first aspect of the present invention provides one Kind can combine the TCR molecules of GLSNLTHVL-HLA A0201 compounds.Preferably, the TCR molecules are separation or pure Change.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-DSSSTY(SEQ ID NO:10)

αCDR2-IFSNMDM(SEQ ID NO:11)

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

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

βCDR1-SGHDN(SEQ ID NO:13)

βCDR2-FVKESK(SEQ ID NO:14)

βCDR3-ASSQGGTQY(SEQ ID NO:15)。

The CDR region amino acid sequence of the invention described above can be embedded into embedding to prepare in any suitable frame structure Close TCR.As long as frame structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art are according to CDR disclosed by the invention Area 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 the TCR molecules of β chain CDR region sequences and any suitable frame structure.TCR α chains variable domain of the present invention be and SEQ ID NO: 1 has at least 90%, preferably 95%, the more preferably amino acid sequence of 98% sequence identity；And/or TCR β of the present invention Chain variable domain be and SEQ ID NO：5 have at least 90%, preferably 95%, the more preferably amino of 98% sequence identity Acid sequence.

In the preference of the present invention, TCR molecules of the invention are the heterodimers being made of α and β chains.Tool Body, on the one hand the α chains of the heterogeneous dimerization TCR molecules include variable domain and constant domain, the α chains variable domain amino acid sequence Row include CDR1 (the SEQ ID NO of above-mentioned α chains：10)、CDR2(SEQ ID NO：And CDR3 (SEQ ID NO 11):12).It is preferred that Ground, the TCR molecules include α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chains of the TCR molecules can Domain 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 perseverance Localization, the β chains variable domain amino acid sequence include CDR1 (the SEQ ID NO of above-mentioned β chains:13)、 CDR2(SEQ ID NO： And CDR3 (SEQ ID NO 14):15).Preferably, the TCR molecules include β chain variable domain amino acid sequence SEQ ID NO: 5.It is highly preferred that 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 the part or all of and/or β chains by α chains The single chain TCR molecules partly or entirely formed.Description in relation to single chain TCR molecules can be with bibliography Chung et al (1994)Proc.Natl.Acad.Sci.USA 91,12654-12658.According to document, those skilled in the art's energy Enough easily structures include 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 end 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 domains Amino acid 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.This field skill Art personnel know or can by consult the public database of pertinent texts or IMGT (international immunogenetics information system) come 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 ", The constant domain sequence of TCR molecule β chains can be " TRBC1*01 " or " TRBC2*01 ".The amino provided in the TRAC*01 of IMGT The 53rd of acid sequence is Arg, is expressed as herein：The Arg53 of TRAC*01 exons 1s, other and so on.Preferably, originally The amino acid sequence for inventing TCR molecule alpha chains 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.Such as immunoglobulin (antibody) The same as antigen recognition molecule, at this moment TCR can also need to obtain soluble TCR by development and application in diagnose and treat Molecule.Soluble TCR molecules do not include its transmembrane region.STCR has very extensive purposes, it cannot be only used for studying The interaction of TCR and pMHC, it is also possible to make the diagnostic tool of detection infection or the marker as autoimmunity disease.It is similar Ground, sTCR can be used to therapeutic agent (such as cytotoxin compounds or immunostimulating compound) being transported to presentation The cell of specific antigen, in addition, sTCR can also be with other molecules (e.g., anti-CD 3 antibodies) with reference to redirecting T Cell, so as to which its targeting be made to present the cell of specific antigen.The present invention is also obtained has specificity to PRAME antigen small peptide STCR.

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.Example Such as, the cysteine of the Ser57 of the Thr48 of substitution TRAC*01 exons 1s and substitution TRBC1*01 or TRBC2*01 exons 1s Residue forms disulfide bond.Cysteine residues are introduced to can also be to form other sites of disulfide bond：It is aobvious outside TRAC*01 The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of son 1；The Tyr10 and TRBC1* of TRAC*01 exons 1s The Ser17 of 01 or 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；It is aobvious outside TRAC*01 The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of son 1；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 30 or 15 or 10 or 8 or more most most most most Few amino acid, so that it does not achieve the purpose that lack natural disulphide bonds including cysteine residues, it also can be by that will be formed The cysteine residues of natural disulphide bonds sport another amino acid to reach above-mentioned purpose.

As described above, the TCR of the present invention may be embodied in artificial two sulphur introduced between the residue of itself α and β chain constant domain Key.It should be noted that the artificial disulfide bond with or without introducing described above between constant domain, TCR of the invention can contain TRAC Constant domain sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC constant domains sequence of TCR and TRBC1 or TRBC2 constant domain sequences Row can be 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 mutates, 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 mutate in its following hydrophobic core position of variable domain： (α and/or β chains) variable region amino acid the 11st, 13,19,21,53,76,89,91,94 and/or α chain J genes (TRAJ) are short Peptide ammino acid position the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptides amino acid position reciprocal is 2nd, 4,6 reciprocal, The Position Number of middle amino acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).This field Technical staff knows above-mentioned international immunogenetics information system, and the amino acid that different TCR can be obtained according to the database is residual Position Number of the base in IMGT.

The TCR that hydrophobic core region mutates in the present invention can be by α and the β chain of a flexible peptide chain link TCR can Variable domain and the solvable single-stranded TCR of stability formed.It should be noted that flexible peptide chain can be any suitable connection TCR α in the present invention With the peptide chain of β chain variable domains.Single chain soluble TCR, α the chain variable domain amino acid sequence such as built in the embodiment of the present invention 4 It is classified as SEQ ID NO:32, the nucleotides sequence of coding is classified as SEQ ID NO:33；β chains variable domain amino acid sequence is SEQ ID NO:34, the nucleotides sequence of coding is classified as SEQ ID NO:35.

In addition, for stability, patent document 201510260322.4 also disclose the α chains variable region of TCR with Introducing artificial interchain disulfide bond between β chain constant regions can significantly improve the stability of TCR.Therefore, height of the invention is affine Artificial interchain disulfide bond can also be contained between the α chains variable region of power TCR and β chain constant regions.Specifically, in the α of the TCR Formed between chain variable region and β chain constant regions the cysteine residues of artificial interchain disulfide bond instead of：The 46th ammonia of TRAV 60th amino acids of base acid and TRBC1*01 or TRBC2*01 exons 1s；The 47th amino acids and TRBC1*01 of TRAV or 61 amino acids of TRBC2*01 exons 1s；The 46th amino acids of TRAV and TRBC1*01 or TRBC2*01 exons 1s 61st amino acids；Or the 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s. Preferably, such TCR can include (I) all or part of TCR α chains in addition to its transmembrane domain and (II) except its across All or part of TCR β chains other than spanning domain, wherein (I) and (II) includes the variable domain and at least part of TCR chains Constant domain, α chains form heterodimer with β chains.It is highly preferred that such TCR can include α chains variable domain and β chain variable domains 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 can Variable domain forms heterodimer 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 covalent Mode combines.The conjugate includes detectable marker and (for diagnostic purpose, is presented wherein the TCR is used to detect The presence of the cell of GLSNLTHVL-HLA A0201 compounds), therapeutic agent, PK (protein kinase) modified parts or it is any more than The combination of these substances combines or coupling.

Detectable marker for diagnostic purposes includes but not limited to：Fluorescence or luminous marker, radioactive label Object, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable production The enzyme of object.

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 (for example, DT- diaphorases (DTD) or Biphenyl base 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, have Researches show that Muridae TCR can be expressed more effectively in human T-cell than people TCR.Therefore, TCR of the present invention may include that people is variable Domain and the constant domain of mouse.The defects of this method is possible to cause immune response.Therefore, it is used for adoptive T cell treatment at it When should have regulation scheme to carry out immunosupress, with allow express Muridae T cell implantation.

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

Nucleic acid molecules

The second aspect of the present invention provides the nucleic acid molecules of coding first aspect present invention TCR molecules or part thereof, institute It can be 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-gacagctcctccacctac(SEQ ID NO:16)

αCDR2-attttttcaaatatggacatg(SEQ ID NO:17)

αCDR3-gcagagatcccctcaactggggcaaacaacctcttc(SEQ ID NO:18)

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

βCDR1-tctggacatgataat(SEQ ID NO:19)

βCDR2-tttgtgaaagagtctaaa(SEQ ID NO:20)

βCDR3-gccagcagccaaggggggacccagtac(SEQ ID NO:21)

Therefore, the nucleotide sequence for encoding the nucleic acid molecules of the present invention of TCR α chains of the present invention includes SEQ ID NO: 16、 SEQ ID NO:17 and SEQ ID NO:18 and/or coding TCR β chains of the present invention nucleic acid molecules of the present invention nucleotide sequence Including SEQ ID NO:19、SEQ ID NO:20 and SEQ ID NO:21.

The nucleotide sequence of nucleic acid molecules of the present invention can be it is single-stranded or double-stranded, the nucleic acid molecules can be RNA or DNA, and can include or not comprising introne.Preferably, the nucleotide sequence of nucleic acid molecules of the present invention does not include introne But polypeptide of the present invention can be encoded, such as the nucleotide sequence of the nucleic acid molecules of the present invention of coding TCR α chain variable domains of the present invention Including 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.It is highly preferred that the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:4 and/or SEQ ID NO: 8.Or Person, the nucleotides sequence of nucleic acid molecules of the present invention are classified as SEQ ID NO:31.

It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.Therefore, it compiles The nucleic acid sequence of code book invention TCR can variant identical with the attached nucleic acid 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 albumen sequence Row, but with SEQ ID NO:The 2 differentiated nucleic acid 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 it is a variety of its The codon usage table of allogene 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 obtain encoding TCR of the present invention (or its piece by chemical synthesis completely Section, or derivatives thereof) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art In (or such as carrier) and cell.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, such as in T cell by carrier so that the cell table Up to the TCR of PRAME antigen specificity.Ideally, which can should express to continual high levels in T cell.

Cell

The invention further relates to the present invention carrier or the genetically engineered host cell of coded sequence.The place The nucleic acid molecules of the present invention are integrated in carrier or chromosome containing the present invention in chief cell.Host cell is selected from：Protokaryon is thin Born of the same parents and eukaryocyte, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..

In addition, the cell of the separation the invention also includes the TCR of the expression present invention, particularly T cell.The T cell can spread out It is born from the T cell detached from subject or can be the mixed cellularity group detached from subject, such as periphery hemolymph The part of cell (PBL) group.Such as, which can be isolated from peripheral blood mononuclear cells (PBMC), can be CD4⁺Assist T Cell or CD8⁺Cytotoxic T cell.The cell can be in CD4⁺T helper cell/CD8⁺In the mixing group of cytotoxic T cell. Usually, which can use antibody (e.g., the antibody of anti-CD3 or anti-CD28) to activate, to allow them to be easier to connect It is transfected, such as is 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 to express TCR in cell surface, because stem cell surface does not express CD3 molecules.However, when stem cell point It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecules will start in thymocyte The surface expression introducing TCR molecules.

It is suitable for carrying out T cell transfection (e.g., Robbins with the DNA or RNA for encoding TCR of the present invention there are many method Wait, (2008) J.Immunol.180:6116-6131).The T cell of expression TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat for carrying out adoptive treatment Rev 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 adopting Property transfer PRAME specific T-cells to the subject the step of.The PRAME specific T-cells can recognize that GLSNLTHVL-HLA A0201 compounds.

The T cell of the PRAME specificity of the present invention can be used for treating any presentation PRAME antigen small peptide GLSNLTHVL- The PRAME relevant diseases of HLA A0201 compounds, such as tumour.It is thin that the tumour includes but not limited to melanoma, lung squamous Born of the same parents' cancer, breast cancer, clear-cell carcinoma, head and neck neoplasm, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukaemia (packet Include but be not limited to, acute lymphoblastic leukemia, acute myeloblastic leukemia), gastric cancer, lung cancer, cancer of the esophagus, carcinoma of urinary bladder, neck Portion's squamous cell carcinoma, prostate cancer, colon cancer, oophoroma etc..

Therapy

Can be suffered from by separation with the patient of PRAME antigen relevant disease or the T cell of volunteer, and will be of the invention TCR import in above-mentioned T cell, then the cell that these genetic engineerings are modified is fed back in patient body to treat.Cause This, is the present invention provides a kind of method for treating PRAME relevant diseases, including the T cell of expression TCR of the present invention that will be detached, Preferably, which derives from patient in itself, is input in patient body.Usually, include the T cell of (1) separation patient, (2) with nucleic acid molecules of the present invention or the nucleic acid molecules ex vivo transduction T cells of TCR molecules of the present invention can be encoded, (3) are by gene The T cell of engineering modification is 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 GLSNLTHVL-HLA A0201, simultaneously Transduceed TCR of the present invention cell can by specific activation and to target cell have very strong lethal effect.

Following specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.Test method without specific conditions in the following example, usually according to conventional strip Part, 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 proposed by manufacturer Condition.Unless otherwise stated, otherwise percentage and number are calculated by weight.Unless otherwise stated, otherwise percentage and number It calculates by weight.Experiment material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.

Embodiment 1 clones PRAME antigen small peptide specific T-cells

Using synthesizing small peptide GLSNLTHVL (SEQ ID NO.:9；Beijing SBS Genetech gene technology Co., Ltd) it stimulates and From in the peripheral blood lymphocytes (PBL) for the healthy volunteer that genotype is HLA-A0201.By GLSNLTHVL small peptides with carrying The HLA-A0201 renaturation of biotin labeling prepares pHLA monoploid.These monoploid and the Streptavidin (BD marked with PE Company) tetramer of PE labels 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 monoclonal is carried out with limiting dilution assay.Monoclonal cell tetramer staining, The double positive colonies screened 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 GLSNLTHVL specificity, HLA-A0201 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.By sequence gram It is grand to being sequenced in carrier T (TAKARA).It should be noted that the sequence is complementary series, not comprising introne.Through sequencing, this pair The α chains and β chain-orderings structure of the TCR of positive colony expression as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, is schemed respectively 1e and Fig. 1 f are respectively TCR α chains variable domain amino acid sequence, TCR α chain variable domains nucleotide sequence, TCR α chain amino acid sequences Row, TCR α chains nucleotide sequence, the TCR α chain amino acid sequences with targeting sequencing and the TCR α chain cores with targeting sequencing Nucleotide sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively that TCR β chains variable domain amino acid sequence, TCR β chains can Variable domain nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, the TCR β chain amino acids with targeting sequencing Sequence and the TCR β chain nucleotide sequences with targeting sequencing.

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

αCDR1-DSSSTY(SEQ ID NO:10)

αCDR2-IFSNMDM(SEQ ID NO:11)

αCDR3-AEIPSTGANNLF(SEQ ID NO:12)

β chains include the CDR with following amino acid sequence：

βCDR1-SGHDN(SEQ ID NO:13)

βCDR2-FVKESK(SEQ ID NO:14)

βCDR3-ASSQGGTQY(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 with overlap PCR TCR 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.Later Again pseudovirus is packed with 293T/17.

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 respectively only comprising its variable domain and Portion constant domain, and a cysteine residues are introduced in the constant domain of α and β chains respectively to form artificial two sulphur of interchain Key, the position for introducing cysteine residues are respectively Thr48 the and TRBC2*01 exons 1s of TRAC*01 exons 1s Ser57；The amino acid sequence of its α chain and nucleotide sequence respectively as shown in figures 4 a and 4b, the amino acid sequence of β chains with For nucleotide sequence respectively as shown in Fig. 5 a and Fig. 5 b, the cysteine residues of introducing with overstriking and underline alphabetical represent.It is logical It crosses《Molecular Cloning: A Laboratory room handbook》(Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) described in standard method by the objective gene sequence of above-mentioned TCR α and β chains after synthesis respectively Expression vector pET28a+ (Novagene) is inserted into, the cloning site of upstream and downstream is NcoI and NotI respectively.Insert Fragment passes through It is errorless to cross sequencing confirmation.

The expression vector of TCR α and β chains by chemical transformation is converted respectively and enters expression bacterium BL21 (DE3), carefully Bacterium is grown with LB culture solutions, in OD₆₀₀It is induced with final concentration 0.5mM IPTG when=0.6, is formed after α the and β chains expression of TCR Inclusion body is extracted by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, packet Contain body and be finally dissolved in 6M guanidine hydrochlorides, 10mM dithiothreitol (DTT)s (DTT), 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris In (pH 8.1).

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.It is mixed Solution is placed in dialysis (4 DEG C) in the deionized water of 10 times of volumes after conjunction, deionized water is changed into buffer solution after 12 hours (20mM Tris, pH 8.0) continues at 4 DEG C and dialyses 12 hours.Solution after the completion of dialysis after 0.45 μM of membrane filtration, It is purified by anion-exchange column (HiTrap Q HP, 5ml, GE Healthcare).Eluting peak contain the successful α of renaturation and The TCR of β dimers 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 measures big by SDS-PAGE In 90%, concentration is determined by BCA methods.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 embodiment 4PRAME antigens small peptide specificity

According to patent document WO2014/206304, using the method for rite-directed mutagenesis by TCR α and β in embodiment 2 The variable domain of chain has been built into the soluble single-chain T CR molecules of a stabilization connected with flexible small peptide (linker).This is single-stranded Amino acid sequence and the nucleotide sequence difference of TCR molecules are as shown in figs. 7 a and 7b.The amino acid sequence of its α chain variable domain And nucleotide sequence difference is as figures 8 a and 8 b show；The amino acid sequence and nucleotide sequence of its β chain variable domain are respectively as schemed Shown in 9a and 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 connect 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 embodiment 5PRAME antigens small peptide specificity

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 precipitates, are split with Bugbuster Master Mix (Merck) Cell precipitate, 6000rpm centrifugation 15min recycling inclusion bodys are solved, then is washed to remove carefully with Bugbuster (Merck) Born of the same parents' fragment and membrane component, 6000 rpm centrifugation 15min, collect inclusion body.By solubilization of inclusion bodies in buffer solution (20mM Tris- 8.0,8 M urea of HCl pH) in, high speed centrifugation removes insoluble matter, is dispensed after supernatant BCA standard measures, in -80 DEG C It saves backup.

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 note Emitter 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 is added dropwise, 4 DEG C are stirred 10min to be mixed, renaturation solution is then packed into the cellulose membrane bag filter that interception is 4kDa, bag filter is placed in the water of 1L precoolings, 4 DEG C are slowly stirred overnight.After 17 hours, by dialyzate change into 1L precooling buffer solution (20mM Tris-HCl pH 8.0), 4 DEG C continue the 8h that dialyses, then changing dialyzate into identical fresh buffer continues dialysed overnight.After 17 hours, sample warp 0.45 μm of membrane filtration by anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgas, uses 20mM The 0-1M NaCl linear gradient elution liquid purifying proteins that Tris-HCl pH 8.0 are prepared, the elution fraction of collection carry out SDS- PAGE is analyzed, and solvent resistant column (Superdex 75 10/300, GE are further used after the component concentration comprising single-stranded TCR Healthcare it) is purified, target components also carry out SDS-PAGE analyses.

Elution fraction for BIAcore analyses further tests its purity using gel filtration.Condition is：Chromatography 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 6.

Embodiment 6 combines characterization

BIAcore is analyzed

It can be with GLSNLTHVL-HLA A0201 compounds spy 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 GLSNLTHVL-HLA A0201 compounds.The antibody (GenScript) of anti-Streptavidin is added in into coupling Then antibody is flowed through the CM5 cores activated in advance with EDC and NHS by buffer solution (10mM sodium-acetate buffers, pH 4.77) Piece makes antibody be fixed on chip surface, finally closes unreacted activating surface with the hydrochloric acid solution of ethanol amine, completes coupling Process, horizontal coupling is about 15,000 RU.

The Streptavidin of low concentration is made to flow through the chip surface of coated antibody, then by GLSNLTHVL-HLA A0201 compounds flow through sense channel, and another channel is as reference channel, then by the biotin of 0.05 mM with 10 μ L/min's Flow velocity flows through chip 2min, closes the remaining binding site of Streptavidin.

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

A. it purifies

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 primary, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) later Thalline is resuspended in concussion, and is rotated in room temperature and be incubated 20min, after 4 DEG C, 6000g centrifugation 15min discard supernatant, collect packet Contain body.

Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Add 30ml The BugBuster of 10 times of dilution, mixing, 4 DEG C of 6000g centrifuge 15min；It discards supernatant, 30ml is added to dilute 10 times Inclusion body, mixing is resuspended in BugBuster, and 4 DEG C of 6000g centrifuge 15min, are repeated twice, add 30ml 20mM Tris-HCl pH 8.0 are resuspended inclusion body, mixing, and 4 DEG C of 6000g centrifuge 15min, finally dissolve inclusion body with 20mM Tris-HCl 8M urea, SDS-PAGE detects inclusion body purity, and BCA kits survey concentration.

B. renaturation

The small peptide GLSNLTHVL (Beijing SBS Genetech gene technology Co., Ltd) of synthesis is dissolved in DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain 8M urea, 20mM Tris pH 8.0,10mM DTT dissolve, and are added in before renaturation 3M guanidine hydrochlorides, 10mM sodium acetates, 10mM EDTA are further denaturalized.GLSNLTHVL peptides are added in again with 25mg/L (final concentration) (0.4M L-arginines, 100 mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM are also for property buffer solution Prototype glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add the light chain of 20mg/L and the heavy chain of 90mg/L (final concentration, heavy chain add in three times, 8h/ times), renaturation carry out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detections answer Property success.

C. it is purified after renaturation

Make dialysis to replace renaturation buffer with the 20mM Tris pH 8.0 of 10 volumes, at least replace buffer solution twice Fully reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, then load Onto HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Using Akta purifying instrument, (GE is general Electric corporation), 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, 10 mM 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 element

PMHC molecular concentrations after biotinylation is marked with Millipore super filter tubes are to 1ml, using Gel filtration The pMHC of purifying biological element is analysed, using Akta purifying instrument (GE General Electric Co. Limited), is pre-equilibrated with filtered PBS HiPrep^TM16/60S200HR columns (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated 1 ml of loading, Ran Houyong PBS is eluted with 1ml/min flow velocitys.Biotinylated pMHC molecules occur in about 55ml as unimodal elution.Merging contains egg The component of white matter is concentrated with Millipore super filter tubes, and BCA methods (Thermo) measure protein concentration, is added in protease and is inhibited The packing of biotinylated pMHC molecules is stored in -80 DEG C by agent cocktail (Roche).

Using BIAcore Evaluation software computational dynamics parameters, obtain the TCR molecules of solubility of the invention with The kinetic profile difference that GLSNLTHVL-HLA A0201 compounds combine is as shown in figure 11.Collection of illustrative plates shows that the present invention obtains Soluble TCR molecules can be combined with GLSNLTHVL-HLA A0201 compounds.Meanwhile it is also had detected using the above method The combination activity of soluble TCR molecules of the invention and other several irrelevant antigen small peptides and HLA compounds, as a result shows this TCR molecules are invented with other irrelevant antigens 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 4 kinds of plasmid (one kind containing pLenti-RHAMMTRA-2A-TRB-IRES-NGFR described in embodiment 2 Slow virus carrier and 3 kinds of plasmids containing other components necessary to structure infectiousness but non-replicating lentiviral particle) Transfect 293T cells.

To be transfected, the 0th day kind cell, in 15 cm dishes, kind upper 1.7 × 10⁷A 293T cells, make cell It is evenly distributed on culture dish, degree of converging is slightly above 50%.1st day transfected plasmids pack pLenti-RHAMMTRA-2A- TRB-IRES-NGFR and pLenti-eGFP pseudovirus, by more than expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV With pMD.2G mixings, the dosage of a 15 cm diameter plates is as follows：22.5 micrograms：15 micrograms:15 micrograms：7.5 microgram.Turn The ratio of transfection reagent PEI-MAX and plasmid is 2:The usage amount of 1, each plate PEI-MAX are 120 micrograms.Concrete operations are： Expression plasmid and packaging plasmid add in 1800 microlitres of OPTI-MEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) 31985- 070) it is uniformly mixed in culture medium, being stored at room temperature 5 minutes becomes DNA mixed liquors；Take corresponding amount PEI and 1800 microlitres of OPTI- MEM culture mediums are uniformly mixed, and being stored at room temperature 5 minutes becomes PEI mixed liquors.DNA mixed liquors and PEI mixed liquors are blended in one It rises and is being stored at room temperature 30 minutes, then add 3150 microlitres of OPTI-MEM culture mediums, be added to have been converted to after mixing In the 293T cells of 11.25 milliliters of OPTI-MEM, culture dish is gently shaken, is uniformly mixed culture medium, 37 DEG C/5%CO₂Under Culture.Transfection 5-7 hours removes transfection media, and changing the DMEM containing 10% fetal calf serum into, ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt)) complete medium, 37 DEG C/5%CO₂Lower culture.Collection in 3rd and the 4th day contains The culture medium supernatant of the slow virus of packaging.For the slow virus of harvest packaging, collected culture supernatant 3000g is centrifuged 15 Minute removal cell fragment, then through 0.22 micron filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB it) filters, finally with the concentration tube of 50KD interceptions (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) UFC905096 it) is concentrated, removes most of supernatant, be finally concentrated to 1 milliliter, frozen for -80 DEG C after decile packing.Take vacation Viral sample carries out virus titer measure, and step is with reference to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit explanation Book.It is used as control, while also packet turns the pseudovirus of pLenti-eGFP.

(b) with the primary T cells of lentiviruses transduction 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 × 10⁶A 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 stimulation altogether, cell：Pearl=3：1.

After stimulation overnight, according to the virus titer that p24ELISA kits are measured, added in the ratio of MOI=10 The slow virus of the PRAME antigen small peptide specific t-cell receptor gene of concentration, 32 DEG C, 900g centrifugations infection 1 hour.It has infected Slow-virus infection liquid is removed after finishing, with 1640 culture mediums containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 Cell is resuspended, is cultivated 3 days under 37 DEG C/5%CO2.Next day carries out the second wheel infection in the same way.Second of transduction is counted after 3 days Number cell, diluting cells to 0.5 × 10⁶A cells/ml.It counts a cell within every two days, replaces or add in and contain 50IU/ The fresh culture of ml IL-2 and 10ng/ml IL-7 maintain cell 0.5 × 10⁶-1×10⁶A cells/ml.From the 3rd It begins through flow cytometry cell, for function test (for example, what IFN- γ discharged since the 5th day ELISPOT and non-radioactive cell toxicity detection).It is cold since the 10th day or when cell slows down division and size becomes smaller Freeze storage etc. and divide cell, 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

Tests below is carried out to prove activating reaction of the T cell to target cell specificity of TCR- transductions.It utilizes Readout of the IFN-γ yield of ELISPOT testing inspections as t cell activation.

Reagent

Test medium：10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), 1640 (Ji Bu of RPMI Can company (Gibco), catalog number (Cat.No.) C11875500bt)

Washing buffer (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 Mill ipore), 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 in this experiment is T2 cells.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 of the present invention through flow cytometry in embodiment 7 to resist The CD8+T cells of former small peptide specificity TCR, and with the CD8+T of same volunteer's untransfected TCR of the present invention as a control group.With AntiCD3 McAb/CD28 coating pearls (T cell amplified matter, life technologies) stimulate T cell, with carrying PRAME antigen small peptide The lentiviruses transduction (according to embodiment 7) of specificity TCR gene, in containing containing 50IU/ml IL-2 and 10ng/ml IL-7 Then these cells are placed in test medium, 300g by the 1640 culture mediums amplification of 10%FBS until 9-12 days after transduction Room temperature is centrifuged 10 minutes and is washed.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 (T2) experimental group, makes final concentration of 1 μ of the small peptide in ELISPOT orifice plates g/ml。

ELISPOT

According to the specification that manufacturer provides, 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 that 10%FBS is contained in 100 microlitres/hole 1640 culture mediums, and orifice plate is incubated at room temperature 2 hours to close orifice plate.Then culture medium is washed away from orifice plate, by paper On flick and pat ELISPOT orifice plates to remove the washing buffer of any remnants.

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

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 It is washed 3 times with washing buffer, pats to remove remaining washing buffer on paper handkerchief.Then with containing 10%FBS's PBS presses 1：200 dilution detection antibody, each hole is added in by 100 microlitres/hole.Orifice plate is incubated at room temperature 2 hours, then use washing buffer Liquid washs 3 times, and orifice plate is patted on paper handkerchief to remove excessive washing buffer.

1 is pressed with the PBS containing 10%FBS：100 dilution Streptavidin-alkaline phosphatases, by 100 microlitres of diluted chains Mould Avidin-alkaline phosphatase adds in each hole and incubates orifice plate at room temperature 1 hour.Then it is washed 4 times with washing buffer PBS is washed 2 times, and orifice plate is patted on paper handkerchief to remove excessive washing buffer and PBS.Kit is added in after washing Develop in the 100 microlitres/hole of BCIP/NBT solution of offer.It is protected from light during development with masking foil covering orifice plate, stands 5-15 Minute.The spot of conventional detection development orifice plate during this period determines to terminate the Best Times of reaction.Remove BCIP/NBT solution And orifice plate is rinsed to stop developing reaction with distilled water, it dries, then orifice plate bottom is removed, it is straight to be dried at room temperature for orifice plate It is completely dried to each hole, recycles immunodotting plate count meter (CTL, Celltech Ltd. (Cellular Technology Limited)) counterdie is formed in counting orifice spot.

As a result

The T cell (as described above) for examining TCR transductions of the present invention is tested to loading PRAME antigen small peptides by ELISPOT The IFN-γ release that the target cell of GLSNLTHVL reacts.Draw what is observed in each hole using graphpad prism6 ELSPOT amount of speckle.

Experimental result is as shown in figure 12, and the T cell for the TCR of the present invention that transduces has 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, just as each document coverlet It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, people in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

Sequence table

<110>Guangzhou Xiangxue Pharmaceutical Co

<120>Identify the TCR of PRAME antigen

<130> P2016-1703

<150> CN201510756448.0

<151> 2015-11-06

<160> 37

<170> PatentIn version 3.5

<210> 1

<211> 113

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chain variable domains

<400> 1

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

1 5 10 15

Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

20 25 30

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

35 40 45

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu

50 55 60

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln

65 70 75 80

Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ile Pro Ser Thr Gly

85 90 95

Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg Leu Thr Val Ile Pro

100 105 110

Tyr

<210> 2

<211> 339

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chain variable domains

<400> 2

ggagaggatg tggagcagag tcttttcctg agtgtccgag agggagacag ctccgttata 60

aactgcactt acacagacag ctcctccacc tacttatact ggtataagca agaacctgga 120

gcaggtctcc agttgctgac gtatattttt tcaaatatgg acatgaaaca agaccaaaga 180

ctcactgttc tattgaataa aaaggataaa catctgtctc tgcgcattgc agacacccag 240

actggggact cagctatcta cttctgtgca gagatcccct caactggggc aaacaacctc 300

ttctttggga ctggaacgag actcaccgtt attccctat 339

<210> 3

<211> 253

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chains

<400> 3

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

1 5 10 15

Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

20 25 30

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

35 40 45

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu

50 55 60

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln

65 70 75 80

Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ile Pro Ser Thr Gly

85 90 95

Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg Leu Thr Val Ile Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250

<210> 4

<211> 759

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chains

<400> 4

ggagaggatg tggagcagag tcttttcctg agtgtccgag agggagacag ctccgttata 60

aactgcactt acacagacag ctcctccacc tacttatact ggtataagca agaacctgga 120

gcaggtctcc agttgctgac gtatattttt tcaaatatgg acatgaaaca agaccaaaga 180

ctcactgttc tattgaataa aaaggataaa catctgtctc tgcgcattgc agacacccag 240

actggggact cagctatcta cttctgtgca gagatcccct caactggggc aaacaacctc 300

ttctttggga ctggaacgag actcaccgtt attccctata tccagaaccc cgaccctgcc 360

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 420

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 480

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 660

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 720

gggtttaatc tgctcatgac gctgcggctg tggtccagc 759

<210> 5

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chain variable domains

<400> 5

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

1 5 10 15

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

20 25 30

Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe Leu Leu His

35 40 45

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

50 55 60

Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys Val Gln

65 70 75 80

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

85 90 95

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

100 105 110

<210> 6

<211> 333

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chain variable domains

<400> 6

gaagctggag ttactcagtt ccccagccac agcgtaatag agaagggcca gactgtgact 60

ctgagatgtg acccaatttc tggacatgat aatctttatt ggtatcgacg tgttatggga 120

aaagaaataa aatttctgtt acattttgtg aaagagtcta aacaggatga gtccggtatg 180

cccaacaatc gattcttagc tgaaaggact ggagggacgt attctactct gaaggtgcag 240

cctgcagaac tggaggattc tggagtttat ttctgtgcca gcagccaagg ggggacccag 300

tacttcgggc caggcacgcg gctcctggtg ctc 333

<210> 7

<211> 290

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chains

<400> 7

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

1 5 10 15

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

20 25 30

Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe Leu Leu His

35 40 45

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

50 55 60

Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys Val Gln

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser

275 280 285

Arg Gly

290

<210> 8

<211> 870

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chains

<400> 8

gaagctggag ttactcagtt ccccagccac agcgtaatag agaagggcca gactgtgact 60

ctgagatgtg acccaatttc tggacatgat aatctttatt ggtatcgacg tgttatggga 120

aaagaaataa aatttctgtt acattttgtg aaagagtcta aacaggatga gtccggtatg 180

cccaacaatc gattcttagc tgaaaggact ggagggacgt attctactct gaaggtgcag 240

cctgcagaac tggaggattc tggagtttat ttctgtgcca gcagccaagg ggggacccag 300

tacttcgggc caggcacgcg gctcctggtg ctcgaggacc tgaaaaacgt gttcccaccc 360

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 420

gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 480

aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 540

aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 600

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 660

acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 720

gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 780

gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 840

gccatggtca agagaaagga ttccagaggc 870

<210> 9

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223>Antigen small peptide

<400> 9

Gly Leu Ser Asn Leu Thr His Val Leu

1 5

<210> 10

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR1

<400> 10

Asp Ser Ser Ser Thr Tyr

1 5

<210> 11

<211> 7

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR2

<400> 11

Ile Phe Ser Asn Met Asp Met

1 5

<210> 12

<211> 12

<212> PRT

<213>Artificial sequence

<220>

<223> α CDR3

<400> 12

Ala Glu Ile Pro Ser Thr Gly Ala Asn Asn Leu Phe

1 5 10

<210> 13

<211> 5

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR1

<400> 13

Ser Gly His Asp Asn

1 5

<210> 14

<211> 6

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR2

<400> 14

Phe Val Lys Glu Ser Lys

1 5

<210> 15

<211> 9

<212> PRT

<213>Artificial sequence

<220>

<223> β CDR3

<400> 15

Ala Ser Ser Gln Gly Gly Thr Gln Tyr

1 5

<210> 16

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR1

<400> 16

gacagctcct ccacctac 18

<210> 17

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR2

<400> 17

attttttcaa atatggacat g 21

<210> 18

<211> 36

<212> DNA

<213>Artificial sequence

<220>

<223> α CDR3

<400> 18

gcagagatcc cctcaactgg ggcaaacaac ctcttc 36

<210> 19

<211> 15

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR1

<400> 19

tctggacatg ataat 15

<210> 20

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR2

<400> 20

tttgtgaaag agtctaaa 18

<210> 21

<211> 27

<212> DNA

<213>Artificial sequence

<220>

<223> β CDR3

<400> 21

gccagcagcc aaggggggac ccagtac 27

<210> 22

<211> 274

<212> PRT

<213>Artificial sequence

<220>

<223>TCR α chains with targeting sequencing

<400> 22

Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu

1 5 10 15

Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser

20 25 30

Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser

35 40 45

Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu

50 55 60

Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln

65 70 75 80

Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg

85 90 95

Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu

100 105 110

Ile Pro Ser Thr Gly Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ser Ser

<210> 23

<211> 822

<212> DNA

<213>Artificial sequence

<220>

<223>TCR α chains with targeting sequencing

<400> 23

atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60

agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120

ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180

ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240

agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300

cagactgggg actcagctat ctacttctgt gcagagatcc cctcaactgg ggcaaacaac 360

ctcttctttg ggactggaac gagactcacc gttattccct atatccagaa ccccgaccct 420

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540

actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc 660

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gc 822

<210> 24

<211> 309

<212> PRT

<213>Artificial sequence

<220>

<223>TCR β chains with targeting sequencing

<400> 24

Met Val Ser Arg Leu Leu Ser Leu Val Ser Leu Cys Leu Leu Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

Asp Asn Leu Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe

50 55 60

Leu Leu His Phe Val Lys Glu Ser Lys Gln Asp Glu Ser Gly Met Pro

65 70 75 80

Asn Asn Arg Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Lys Asp Ser Arg Gly

305

<210> 25

<211> 927

<212> DNA

<213>Artificial sequence

<220>

<223>TCR β chains with targeting sequencing

<400> 25

atggtttcca ggcttctcag tttagtgtcc ctttgtctcc tgggagcaaa gcacatagaa 60

gctggagtta ctcagttccc cagccacagc gtaatagaga agggccagac tgtgactctg 120

agatgtgacc caatttctgg acatgataat ctttattggt atcgacgtgt tatgggaaaa 180

gaaataaaat ttctgttaca ttttgtgaaa gagtctaaac aggatgagtc cggtatgccc 240

aacaatcgat tcttagctga aaggactgga gggacgtatt ctactctgaa ggtgcagcct 300

gcagaactgg aggattctgg agtttatttc tgtgccagca gccaaggggg gacccagtac 360

ttcgggccag gcacgcggct cctggtgctc gaggacctga aaaacgtgtt cccacccgag 420

gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggtg 480

tgcctggcca caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag 540

gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc cgccctcaat 600

gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660

cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720

caggataggg ccaaacctgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780

tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag 840

atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt gctgatggcc 900

atggtcaaga gaaaggattc cagaggc 927

<210> 26

<211> 206

<212> PRT

<213>Artificial sequence

<220>

<223>STCR α chains

<400> 26

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

1 5 10 15

Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

20 25 30

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

35 40 45

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu

50 55 60

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln

65 70 75 80

Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ile Pro Ser Thr Gly

85 90 95

Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg Leu Thr Val Ile Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 27

<211> 618

<212> DNA

<213>Artificial sequence

<220>

<223>STCR α chains

<400> 27

ggtgaagatg ttgaacagag tcttttcctg agtgtccgag agggagacag ctccgttata 60

aactgcactt acacagacag ctcctccacc tacttatact ggtataagca agaacctgga 120

gcaggtctcc agttgctgac gtatattttt tcaaatatgg acatgaaaca agaccaaaga 180

ctcactgttc tattgaataa aaaggataaa catctgtctc tgcgcattgc agacacccag 240

actggggact cagctatcta cttctgtgca gagatcccct caactggggc aaacaacctc 300

ttctttggga ctggaacgag actcaccgtt attccctata tccagaaccc tgaccctgcc 360

gtgtaccagc tgagagactc taagtcgagt gacaagtctg tctgcctatt caccgatttt 420

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaatgt 480

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 540

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 600

cccagcccag aaagttcc 618

<210> 28

<211> 241

<212> PRT

<213>Artificial sequence

<220>

<223>STCR β chains

<400> 28

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

1 5 10 15

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

20 25 30

Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe Leu Leu His

35 40 45

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

50 55 60

Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys Val Gln

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Asp

<210> 29

<211> 723

<212> DNA

<213>Artificial sequence

<220>

<223>STCR β chains

<400> 29

gaagcaggtg ttacccagtt ccccagccac agcgtaatag agaagggcca gactgtgact 60

ctgagatgtg acccaatttc tggacatgat aatctttatt ggtatcgacg tgttatggga 120

aaagaaataa aatttctgtt acattttgtg aaagagtcta aacaggatga gtccggtatg 180

cccaacaatc gattcttagc tgaaaggact ggagggacgt attctactct gaaggtgcag 240

cctgcagaac tggaggattc tggagtttat ttctgtgcca gcagccaagg ggggacccag 300

tacttcgggc caggcacgcg gctcctggtg ctcgaggacc tgaaaaacgt gttcccaccc 360

gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 420

gtgtgcctgg ccaccggttt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 480

aaggaggtgc acagtggggt ctgcacagac ccgcagcccc tcaaggagca gcccgccctc 540

aatgactcca gatacgctct gagcagccgc ctgagggtct cggccacctt ctggcaggac 600

ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 660

acccaggata gggccaaacc cgtcacccag atcgtcagcg ccgaggcctg gggtagagca 720

gac 723

<210> 30

<211> 247

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR

<400> 30

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

1 5 10 15

Ser Val Ser Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

20 25 30

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

35 40 45

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Ser

50 55 60

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Glu Asp Val Gln

65 70 75 80

Pro Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ile Pro Ser Thr Gly

85 90 95

Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg Leu Thr Val Ile Pro

100 105 110

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

115 120 125

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

130 135 140

Ser His Ser Ser Ile Glu Lys Gly Gln Thr Val Thr Leu Arg Cys Asp

145 150 155 160

Pro Ile Ser Gly His Asp Asn Leu Tyr Trp Tyr Arg Arg Val Pro Gly

165 170 175

Lys Glu Ile Lys Phe Leu Leu His Phe Val Lys Glu Ser Lys Gln Asp

180 185 190

Glu Ser Gly Met Pro Asn Asn Arg Phe Asn Ala Glu Arg Thr Gly Gly

195 200 205

Thr Tyr Ser Thr Leu Lys Ile Gln Pro Val Glu Pro Glu Asp Ser Gly

210 215 220

Val Tyr Phe Cys Ala Ser Ser Gln Gly Gly Thr Gln Tyr Phe Gly Pro

225 230 235 240

Gly Thr Arg Leu Thr Val Leu

245

<210> 31

<211> 741

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR

<400> 31

ggtgaagatg ttgaacagtc cctgtttctg tcagtgcgtg aaggcgattc tgttagtatt 60

aactgcacct acacggacag ctctagtacc tatctgtact ggtataaaca ggaaccgggc 120

gcgggtctgc aactgctgac gtacattttt agcaacatgg atatgaaaca ggaccaacgt 180

ctgaccgtct cgctgaataa gaaagataaa cacctgagcc tgcgcatcga agatgtgcag 240

ccgggtgact ctgcgattta cttctgtgcc gaaatcccga gtacgggcgc gaacaacctg 300

tttttcggca ccggtacgcg tctgaccgtc atcccgggcg gtggctcgga aggtggcggt 360

agcgaaggcg gtggctctga aggtggcggt agtgaaggcg gtacgggtga agccggtgtg 420

acccagtttc cgagccattc ctcaattgaa aaaggccaaa ccgttacgct gcgctgcgat 480

ccgatcagcg gtcacgacaa cctgtactgg tatcgtcgcg ttccgggcaa agaaattaaa 540

tttctgctgc atttcgtcaa agaatccaaa caggatgaaa gcggtatgcc gaacaatcgt 600

ttcaatgcag aacgcaccgg cggtacctat tccaccctga aaatccaacc ggtcgaaccg 660

gaagacagtg gcgtgtactt ttgtgcttcg agccagggcg gtacccaata tttcggcccg 720

ggtacgcgcc tgaccgtgct g 741

<210> 32

<211> 112

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR α chains

<400> 32

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

1 5 10 15

Ser Val Ser Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

20 25 30

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

35 40 45

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Ser

50 55 60

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Glu Asp Val Gln

65 70 75 80

Pro Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ile Pro Ser Thr Gly

85 90 95

Ala Asn Asn Leu Phe Phe Gly Thr Gly Thr Arg Leu Thr Val Ile Pro

100 105 110

<210> 33

<211> 336

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR α chains

<400> 33

ggtgaagatg ttgaacagtc cctgtttctg tcagtgcgtg aaggcgattc tgttagtatt 60

aactgcacct acacggacag ctctagtacc tatctgtact ggtataaaca ggaaccgggc 120

gcgggtctgc aactgctgac gtacattttt agcaacatgg atatgaaaca ggaccaacgt 180

ctgaccgtct cgctgaataa gaaagataaa cacctgagcc tgcgcatcga agatgtgcag 240

ccgggtgact ctgcgattta cttctgtgcc gaaatcccga gtacgggcgc gaacaacctg 300

tttttcggca ccggtacgcg tctgaccgtc atcccg 336

<210> 34

<211> 111

<212> PRT

<213>Artificial sequence

<220>

<223>Single-stranded TCR β chains

<400> 34

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

1 5 10 15

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

20 25 30

Tyr Trp Tyr Arg Arg Val Pro Gly Lys Glu Ile Lys Phe Leu Leu His

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 35

<211> 333

<212> DNA

<213>Artificial sequence

<220>

<223>Single-stranded TCR β chains

<400> 35

gaagccggtg tgacccagtt tccgagccat tcctcaattg aaaaaggcca aaccgttacg 60

ctgcgctgcg atccgatcag cggtcacgac aacctgtact ggtatcgtcg cgttccgggc 120

aaagaaatta aatttctgct gcatttcgtc aaagaatcca aacaggatga aagcggtatg 180

ccgaacaatc gtttcaatgc agaacgcacc ggcggtacct attccaccct gaaaatccaa 240

ccggtcgaac cggaagacag tggcgtgtac ttttgtgctt cgagccaggg cggtacccaa 300

tatttcggcc cgggtacgcg cctgaccgtg ctg 333

<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

ggcggtacgg gt 72

Claims (36)

1. a kind of T cell receptor（TCR）, which is characterized in that the TCR can be with GLSNLTHVL-HLA A0201 compound knots It closes, it includes TCR α chains variable domains and TCR β chain variable domains；Also, 3 complementary determining regions of the TCR α chain variable domains（CDR） For：

α CDR1- DSSSTY (SEQ ID NO: 10)

α CDR2- IFSNMDM (SEQ ID NO: 11)

α CDR3- AEIPSTGANNLF (SEQ ID NO: 12)；With

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

β CDR1- SGHDN (SEQ ID NO: 13)

β CDR2- FVKESK (SEQ ID NO: 14)

β CDR3- ASSQGGTQY (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 identity；And/or the TCR β chains Variable domain be and SEQ ID NO：5 have the amino acid sequence of at least 90% sequence identity.

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

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

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 marker, 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 Molecule is the TCR described in any one of the claims.

25. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules are included described in any one of coding claim 1-23 The nucleic acid 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 host cell of separation, which is characterized in that containing any in claim 29-31 in the host cell Any nucleic acid molecules in the claim 25-28 of external source are integrated in carrier or chromosome described in.

33. a kind of cell, which is characterized in that the cell transduction have the right in requirement 25-28 any nucleic acid molecules or Carrier described in any one of 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 described in any one of 1-23 or the cell described in claim 33.

36. the TCR compounds or claim described in TCR or claim 24 described in any one of claim 1-23 The purposes of cell described in 33, which is characterized in that be used to prepare the drug for the treatment of tumour.