CN106519019A - TCR for identifying PRAME antigen - Google Patents

TCR for identifying PRAME antigen Download PDF

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Publication number
CN106519019A
CN106519019A CN201610957738.6A CN201610957738A CN106519019A CN 106519019 A CN106519019 A CN 106519019A CN 201610957738 A CN201610957738 A CN 201610957738A CN 106519019 A CN106519019 A CN 106519019A
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tcr
cell
ser
seq
leu
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CN106519019B (en
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李懿
相瑞瑞
吴万里
林燕梅
李思韵
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Xiangxue Life Science Technology (Guangdong) Co.,Ltd.
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GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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  • Immunology (AREA)
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Abstract

The invention provides a T cell receptor (TCR) capable of specifically binding with a short peptide GLSNLTHVL derived from a PRAME antigen. The short peptide GLSNLTHVL can form a compound with HLA A0201 and is presented on the cell surface together with the HLA A0201. The invention further provides a nucleic acid molecule encoding the TCR and a carrier including the nucleic acid molecule. In addition, the invention further provides a cell transducing the TCR.

Description

The TCR of identification DAGE
Technical field
The present invention relates to the TCR from DAGE small peptide is capable of identify that, the invention further relates to transduce above-mentioned TCR to obtain PRAME specificitys T cell, and they prevention and treatment PRAME relevant diseases in purposes.
Background technology
PRAME is melanoma specific antigen (preferentially expressed antigen of Melanoma, PRAME), there are expression (Ikeda H, et in 88% is initial and 95% shifts melanoma al.Immunity,1997,6(2):199-208), normal skin tissue and benign melanocyte are not then expressed.PRAME is in cell Micromolecule polypeptide is degraded to after interior generation, and complex is combined to form with MHC (main histocompatibility complex) molecule, be in It is delivered to cell surface.GLSNLTHVL is the small peptide derived from DAGE, is a kind of target of PRAME treating correlative diseases. Except melanoma, PRAME is also expressed in kinds of tumors, is swollen including squamous cell lung carcinoma, breast carcinoma, renal cell carcinoma, incidence (the van't Veer LJ, et al.Nature, 2002,415 such as tumor, Huo Jiejin lymphomas, sarcoma and medulloblastoma (6871):530-536；Boon K,et al.Oncogene,2003,22(48):7687-7694) in addition, which is in leukemia PRAME also has significantly expression, acute lymphoblastic leukemia 17%～42%, acute myeloblastic leukemia 30%～64% (SteinbachD,et al.Cancer Genet Cytogene,2002,138(1):89-91).For controlling for above-mentioned disease Treat, the method such as chemotherapy and radiation treatment can be adopted, but all the normal cell of itself can be caused damage.
T cell adoptive immunotherapy is to proceed in patient body the reaction-ive T cell to target cell antigen with specificity, Make which be directed to target cell to play a role.φt cell receptor (TCR) is a kind of memebrane protein on T cell surface, and which is capable of identify that accordingly The antigen small peptide of target cells.In immune system, by the TCR of antigen small peptide specificity and small peptide-main histocompatibility The combination of complex (pMHC complex) causes T cell and the direct physical contact of antigen-presenting cell (APC), then T cell And other cell membrane surface molecules of both APC just occur to interact, cause a series of follow-up cell signals transmission and its His physiological reaction, so that the T cell of different antigenic specificities plays immunological effect to its target cell.Therefore, this area skill Art personnel are devoted to isolating the TCR for having specificity to NY-ESO-1 antigen small peptides, and the TCR is transduceed T cell to obtain There must be the T cell of specificity to NY-ESO-1 antigen small peptides, so that they play a role in cellular immunotherapy.
The content of the invention
It is an object of the invention to provide a kind of φt cell receptor of identification DAGE small peptide.
A kind of a first aspect of the present invention, there is provided φt cell receptor (TCR), the TCR can be with GLSNLTHVL-HLA A0201 complex is combined.
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chain variable domains CDR3 aminoacid sequence be AEIPSTGANNLF (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domains Aminoacid sequence is ASSQGGTQY (SEQ ID NO:15).
In another preference, 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 preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chain variable domains It is and SEQ ID NO:1 aminoacid sequence with least 90% sequence thereto；And/or the TCR β chain variable domains be with SEQ ID NO：5 aminoacid sequences with least 90% sequence thereto.
In another preference, the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.
In another preference, the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5.
In another preference, the TCR is α β heterodimers, and which includes TCR α chains constant region TRAC*01 and TCR β Chain constant region TRBC1*01 or TRBC2*01.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO：3 and/or TCR β chain ammonia Base acid sequence is SEQ ID NO:7.
In another preference, the TCR is solvable.
In another preference, the TCR is single-stranded.
In another preference, the TCR is formed by connecting by peptide catenation sequence with β chains variable domain by α chains variable domain.
In another preference, the TCR α chains variable region amino acid the 11st, 13,19,21,53,76,89,91, or It is prominent with one or more in 94, and/or α chain J gene small peptides aminoacid inverse the 3rd, 5th reciprocal or inverse the 7th Become；And/or the TCR β chains variable region amino acid the 11st, 13,19,21,53,76,89,91, or the 94th, and/or β chain J With one or more mutation, wherein aminoacid position in gene small peptide aminoacid inverse the 2nd, 4th reciprocal or inverse the 6th Numbering is put by the Position Number listed in IMGT (international immunogeneticses information system).
In another preference, the α chains variable domain amino acid sequence of the TCR includes SEQ ID NO:32 and/or described The β chains variable domain amino acid sequence of TCR includes SEQ ID NO:34.
In another preference, the aminoacid sequence of the TCR is SEQ ID NO:30.
In another preference, the TCR includes all or part of TCR α chains of (a) in addition to membrane spaning domain；And The all or part of TCR β chains of (b) in addition to membrane spaning domain；
And (a) with (b) each self-contained functional variable domain, or include functional variable domain and the TCR At least a portion of chain constant domain.
In another preference, cysteine residues form artificial disulfide bond between α the and β chain constant domains of the TCR.
In another preference, the cysteine residues that artificial disulfide bond is formed in the TCR are instead of selected from following One or more groups of sites：
The Ser57 of the Thr48 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser77 of the Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser17 of the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Asp59 of the Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Glu15 of the Ser15 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser54 of the Arg53 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ala19 of the Pro89 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；With
The Glu20 of the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO:26 and/or TCR β chains Aminoacid sequence is SEQ ID NO:28.
In another preference, between the α chains variable region and β chains constant region of the TCR, contain artificial interchain disulfide bond.
In another preference, it is characterised in that the cysteine residues of artificial interchain disulfide bond are formed in the TCR Instead of selected from following one or more groups of sites：
46th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；
47th amino acids and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；
46th amino acids and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；Or
47th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV.
In another preference, the TCR includes α chains variable domain and β chains variable domain and in addition to membrane spaning domain All or part of β chains constant domain, but it does not contain α chain constant domains, α chains variable domain and the heterogeneous dimerization of β chain formation of the TCR Body.
In another preference, C- the or N- ends of the α chains and/or β chains of the TCR are combined with conjugate.
In another preference, the conjugate combined with the φt cell receptor is detectable, therapeutic agent, PK are repaiied Decorations part or the combination of any these materials.Preferably, the therapeutic agent is anti-CD 3 antibodies.
A kind of a second aspect of the present invention, there is provided multivalent TCR complex, which includes at least two TCR molecules, and its At least one of TCR molecules be first aspect present invention described in TCR.
A kind of a third aspect of the present invention, there is provided nucleic acid molecules, the nucleic acid molecules include coding first party of the present invention The nucleotide sequence or its complementary series of the TCR molecules described in face.
In another preference, nucleotide sequence SEQ ID NO of the nucleic acid molecules comprising coding TCR α chain variable domains： 2 or SEQ ID NO:33.
In another preference, described nucleotide sequence SEQ ID of the nucleic acid molecules comprising coding TCR β chain variable domains NO：6 or SEQ ID NO:35.
In another preference, nucleotide sequence SEQ ID NO of the nucleic acid molecules comprising coding TCR α chains：4 and/or Nucleotide sequence SEQ ID NO comprising coding TCR β chains：8.
A fourth aspect of the present invention, there is provided a kind of carrier, described carrier contain the core described in third aspect present invention Acid molecule；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
A fifth aspect of the present invention, there is provided a kind of detached host cell, containing the present invention in described host cell The nucleic acid molecules being integrated with carrier or genome described in fourth aspect described in the third aspect present invention of external source.
A kind of a sixth aspect of the present invention, there is provided cell, the nucleic acid described in the cell transduction third aspect present invention Carrier described in molecule or fourth aspect present invention；Preferably, the cell is T cell or stem cell.
A seventh aspect of the present invention, there is provided a kind of pharmaceutical composition, the compositionss contain pharmaceutically acceptable load The TCR complex described in TCR, second aspect present invention, third aspect present invention institute described in body and first aspect present invention Carrier or the cell described in sixth aspect present invention described in the nucleic acid molecules stated, fourth aspect present invention.
A eighth aspect of the present invention, there is provided the φt cell receptor or second aspect present invention described in first aspect present invention Nucleic acid molecules, the carrier described in fourth aspect present invention described in described TCR complex, third aspect present invention or this The purposes of described cell in terms of bright 6th, for preparing the medicine for the treatment of tumor or autoimmune disease.
A kind of a ninth aspect of the present invention, there is provided method for treating disease, applies suitable including to object in need for the treatment of The TCR complex described in φt cell receptor or second aspect present invention, the present invention the 3rd described in the first aspect present invention of amount Nucleic acid molecules, the carrier described in fourth aspect present invention or the cell described in sixth aspect present invention described in aspect or this Described pharmaceutical composition in terms of bright 7th；
Preferably, described disease is tumor, and preferably described tumor includes melanoma, squamous cell lung carcinoma, mammary gland Cancer, renal cell carcinoma, tumor of head and neck, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukemia (include but do not limit In, acute lymphoblastic leukemia, acute myeloblastic leukemia), melanoma, and other entity tumors for example gastric cancer, pulmonary carcinoma, Esophageal carcinoma, bladder cancer, squamous cell carcinoma of the head and neck, carcinoma of prostate, breast carcinoma, colon cancer, ovarian cancer etc..
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and have in below (eg embodiment) Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
It is variable that Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively TCR α chain variable domain amino acid sequences, TCR α chains Domain nucleotide sequence, TCR α chain amino acid sequences, TCR α chain nucleotide sequences, the TCR α chain amino acid sequences with targeting sequencing And the TCR α chain nucleotide sequences with targeting sequencing.
It is variable that Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chain variable domain amino acid sequences, TCR β chains Domain nucleotide sequence, TCR β chain amino acid sequences, TCR β chain nucleotide sequences, the TCR β chain amino acid sequences with targeting sequencing And the TCR β chain nucleotide sequences with targeting sequencing.
CD8s of the Fig. 3 for monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b is respectively the aminoacid sequence and nucleotide sequence of sTCR α chains.
Fig. 5 a and Fig. 5 b is respectively the aminoacid sequence and nucleotide sequence of sTCR β chains.
Fig. 6 is the glue figure of the sTCR for obtaining after purification.To go back virgin rubber, middle swimming lane is molecular weight to leftmost side swimming lane Labelling (marker), rightmost side swimming lane are non-reduced glue.
Fig. 7 a and Fig. 7 b is respectively the aminoacid sequence and nucleotide sequence of single-stranded TCR.
Fig. 8 a and Fig. 8 b is respectively the aminoacid sequence and nucleotide sequence of single-stranded TCR α chains.
Fig. 9 a and Fig. 9 b is respectively the aminoacid sequence and nucleotide sequence of single-stranded TCR β chains.
Figure 10 a and Figure 10 b is respectively the aminoacid sequence and nucleotide sequence of single-stranded TCR catenation sequences (linker).
Figure 11 is the BIAcore kinetics figures combined with GLSNLTHVL-HLA A0201 complex by sTCR of the present invention Spectrum.
Figure 12 be transduce TCR of the present invention T cell activation experiment result.
Specific embodiment
The present inventor is have found and DAGE small peptide GLSNLTHVL (SEQ ID through extensively in-depth study NO:9) TCR that can be specifically bound, the antigen small peptide GLSNLTHVL can with HLA A0201 formed complex and together with by It is presented to cell surface.Present invention also offers the nucleic acid molecules and the carrier comprising the nucleic acid molecules of the coding TCR. In addition, present invention also offers the cell of transduction TCR of the present invention.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, its for The presentation of antigen has specificity, and different individualities has different MHC, can present different small peptides in a kind of proteantigen to each From APC cell surfaces.The MHC of the mankind is commonly referred to HLA genes or HLA complexs.
φt cell receptor (TCR), is the unique of specific antigen peptide of the presentation in main histocompatibility complex (MHC) Receptor.In immune system, cause T cell thin with antigen presentation by the combination of the TCR and pMHC complex of antigenic specificity The direct physical contact of born of the same parents (APC), then other cell membrane surface molecules of both T cell and APC just occur to interact, this A series of follow-up cell signal transmission and other physiological reactions are caused just, so that the T cell of different antigenic specificities Immunological effect is played to its target cell.
TCR is the glycoprotein of the surface of cell membrane existed with heterodimer form by α chains/β chains or γ chains/δ chains. In 95% T cell, TCR heterodimers are made up of α and β chains, and 5% T cell is with the TCR being made up of γ and δ chains.My god So there is heterogeneous dimerization TCR of α β α chains and β chains, α chains and β chains to constitute the subunit of α β heterodimeric TCR.In a broad sense, α and β are each Chain includes variable region, bonding pad and constant region, and β chains generally contain short variable region also between variable region and bonding pad, but should Variable region is often regarded as a part for bonding pad.Each variable region includes 3 be entrenched in frame structure (framework regions) Individual CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC complex, wherein CDR3 by Variable region and bonding pad restructuring are formed, and are referred to as hypervariable region.α the and β chains of TCR are typically regarded as respectively has two " domains " i.e. variable Domain and constant domain, variable domain are made up of the variable region and bonding pad that connect.The sequence of TCR constant domains can be in international immune genetic Find in the public database for learning information system (IMGT), the such as constant domain sequence of TCR molecule alpha chains is " TRAC*01 ", TCR point The constant domain sequence of sub- β chains is " TRBC1*01 " or " TRBC2*01 ".Additionally, α the and β chains of TCR also include transmembrane region and kytoplasm Area, cytoplasmic region are very short.
In the present invention, term " polypeptide of the present invention ", the TCR of the present invention " ", " φt cell receptor of the present invention " is interchangeable makes With.
Native interchain disulfide bond and artificial interchain disulfide bond
There is one group disulfide bond with C β interchains in the membrane-proximal region C α of natural TCR, in the present invention, be referred to as " two sulfur of native interchain Key ".In the present invention, will be manually-injected, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond claim For " artificial interchain disulfide bond ".
For convenience of the position of description disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 aminoacid sequences in the present invention Position Number carry out Position Number by the order from N-terminal to C-terminal successively, in such as TRBC1*01 or TRBC2*01, by from N-terminal to The 60th aminoacid of C-terminal order successively is P (proline), then can describe it as TRBC1*01 or TRBC2*01 in the present invention The Pro60 of exons 1, can also be stated that the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s, and for example In TRBC1*01 or TRBC2*01, be Q (glutamine) by the 61st aminoacid of the order from N-terminal to C-terminal successively, then it is of the invention In can describe it as the Gln61 of TRBC1*01 or TRBC2*01 exons 1s, can also be stated that TRBC1*01 or TRBC2* 61st amino acids of 01 exons 1, other are by that analogy.In the present invention, the aminoacid sequence of variable region TRAV and TRBV Position Number, according to the Position Number listed in IMGT.Such as certain aminoacid in TRAV, the Position Number listed in IMGT is 46, then the 46th amino acids of TRAV are described it as in the present invention, other are by that analogy.In the present invention, the sequence of other aminoacid Column position numbering has specified otherwise, then by specified otherwise.
Detailed description of the invention
TCR molecules
In antigen processing pathways, antigen is degraded in the cell, is then carried to cell surface by MHC molecule.T is thin Born of the same parents' receptor is capable of identify that the peptide-MHC complex of Antigen Presenting Cell surface.Therefore, a first aspect of the present invention provides one kind Can be with reference to the TCR molecules of GLSNLTHVL-HLA A0201 complex.Preferably, the TCR molecules are detached or purification 's.α the and β chains of the TCR respectively have 3 complementary determining regions (CDR).
One in the present invention is preferably carried out in mode, and the α chains of the TCR are included with following aminoacid 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 aminoacid sequence of the invention described above can be embedded into chimeric to prepare in any suitable frame structure TCR.As long as frame structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art are according to CDR region disclosed by the invention Can just design or synthesize the TCR molecules with corresponding function.Therefore, TCR molecules of the present invention are referred to comprising above-mentioned α and/or β The TCR molecules of chain CDR region sequence and any suitable frame structure.TCR α chain variable domains of the present invention are and SEQ ID NO:1 tool Have at least 90%, preferably 95%, more preferably the aminoacid sequence of 98% sequence thereto；And/or TCR β chains of the present invention can Variable domain is and SEQ ID NO：5 have at least 90%, preferably 95%, the aminoacid sequence of more preferably 98% sequence thereto Row.
In a preference of the present invention, the TCR molecules of the present invention are the heterodimers being made up of with β chains α.Specifically Ground, the α chains of the one side heterogeneous dimerization TCR molecule include variable domain and constant domain, the α chains variable domain amino acid sequence bag CDR1 containing above-mentioned α chains (SEQ ID NO：10)、CDR2(SEQ ID NO：11) with CDR3 (SEQ ID NO:12).Preferably, The TCR molecules include α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domains of the TCR molecules Aminoacid sequence is SEQ ID NO:1.On the other hand, the β chains of the heterogeneous dimerization TCR molecule include variable domain and constant domain, CDR1 (SEQ ID NO of the β chains variable domain amino acid sequence comprising above-mentioned β chains:13)、CDR2(SEQ ID NO：14) and CDR3(SEQ ID NO:15).Preferably, the TCR molecules include β chain variable domain amino acid sequence SEQ ID NO:5.It is more excellent Selection of land, the β chains variable domain amino acid sequence of the TCR molecules is SEQ ID NO:5.
In a preference of the present invention, the TCR molecules of the present invention are by the portion of the part or all of and/or β chains of α chains The single chain TCR molecules for dividing or all constituting.Description about single chain TCR molecules may be referred to document Chung et al (1994) Proc.Natl.Acad.Sci.USA 91,12654-12658.According to document, those skilled in the art can be easily Build the single chain TCR molecules comprising CDRs areas of the present invention.Specifically, the single chain TCR molecules include V α, V β and C β, preferably According to being linked in sequence from N-terminal to C-terminal.
CDR1 (SEQ ID NO of the α chains variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned α chains：10)、 CDR2(SEQ ID NO：11) with CDR3 (SEQ ID NO:12).Preferably, the single chain TCR molecules include α chain variable domain ammonia Base acid sequence SEQ ID NO:1.It is highly preferred that the α chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO: 1.CDR1 (SEQ ID NO of the β chains variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned β chains：13)、CDR2 (SEQ ID NO：14) with CDR3 (SEQ ID NO：15).Preferably, the single chain TCR molecules include β chain variable domain amino acids Sequence SEQ ID NO:5.It is highly preferred that the β chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:5.
In a preference of the present invention, the constant domain of the TCR molecules of the present invention is the constant domain of people.Art technology Personnel are known or can be obtained by the public database of access pertinent texts or IMGT (international immunogeneticses information system) Obtain the constant domain amino acid sequence of people.For example, the constant domain sequence of TCR molecule alphas chain of the present invention can be " TRAC*01 ", TCR point The constant domain sequence of sub- β chains can be " TRBC1*01 " or " TRBC2*01 ".The aminoacid sequence be given in the TRAC*01 of IMGT The 53rd be Arg, here is expressed as：The Arg53 of TRAC*01 exons 1s, other are by that analogy.Preferably, TCR of the present invention The aminoacid sequence of molecule alpha chain is SEQ ID NO:3, and/or the aminoacid sequence of β chains be SEQ ID NO:7.
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.As immunoglobulin (antibody) is made The same for antigen recognizing molecule, TCR can also be developed and be applied to diagnose and treat, and at this moment need to obtain TCR point of solubility Son.The TCR molecules of solubility do not include its transmembrane region.STCR has very extensive purposes, and it cannot be only used for studying TCR With the interaction of pMHC, it is also possible to make the diagnostic tool or the mark as autoimmune disease of detection infection.Similarly, may be used Dissolubility TCR can be used to for therapeutic agent (such as cytotoxin compounds or immunostimulating compound) to be transported to presentation specificity The cell of antigen, in addition, sTCR can also combine to redirect T cell with other molecules (e.g., anti-CD 3 antibodies), from And make its targeting present the cell of specific antigen.The present invention also obtain the solubility for having specificity to DAGE small peptide TCR。
To obtain sTCR, on the one hand, TCR of the present invention can be introduced between the residue of itself α and β chain constant domain The TCR of artificial disulfide bond.Cysteine residues form artificial interchain disulfide bond between α the and β chain constant domains of the TCR.Half Guang Histidine residue can be substituted in other amino acid residues of appropriate site in natural TCR to form artificial interchain disulfide bond.For example, The cysteine residues of the Thr48 for replacing TRAC*01 exons 1s and the Ser57 for replacing TRBC1*01 or TRBC2*01 exons 1s To form disulfide bond.Introduce cysteine residues to can also be with other sites for forming disulfide bond：TRAC*01 exons 1s The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1s；The Tyr10 and TRBC1*01 of TRAC*01 exons 1s or The Ser17 of TRBC2*01 exons 1s；The Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s Asp59；The Glu15 of the Ser15 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 exons 1s Arg53 and TRBC1*01 or TRBC2*01 exons 1s Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1s or The Ala19 of TRBC2*01 exons 1s；Or the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s Glu20.I.e. cysteine residues instead of above-mentioned α with arbitrary group of site in β chain constant domains.Can be in TCR constant domains of the present invention One or more C-terminal truncates are most 50, or most 30, or most 15, or most 10, or most 8 or less Aminoacid so which is not including cysteine residues reaching the purpose of disappearance natural disulphide bonds, also can be by day will be formed So the cysteine residues of disulfide bond sport another aminoacid to reach above-mentioned purpose.
As described above, the TCR of the present invention may be embodied in the artificial disulfide bond introduced between the residue of itself α and β chain constant domain. It should be noted that between constant domain with or without introducing mentioned above artificial disulfide bond, the present invention TCR can containing TRAC it is constant Domain sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences of TCR can By the natural disulphide bonds connection being present in TCR.
To obtain sTCR, on the other hand, TCR of the present invention is additionally included in the TCR that its hydrophobic core region is undergone mutation, The mutation in these hydrophobic core regions is preferably capable making the stability-enhanced mutation of sTCR of the present invention, such as in publication number Described in patent documentation for WO2014/206304.Such TCR can undergo mutation in the hydrophobic core position of its following variable domain： (α and/or β chains) variable region amino acid the 11st, 13,19,21,53,76,89,91,94, and/or α chain J genes (TRAJ) small peptides Amino acid position is reciprocal 3rd, 5,7, and/or β chain J genes (TRBJ) small peptides amino acid position is reciprocal 2nd, 4,6, wherein ammonia The Position Number of base acid sequence is by the Position Number listed in international immunogeneticses information system (IMGT).People in the art Member knows above-mentioned international immunogeneticses information system, and the amino acid residue that can obtain different TCR according to the data base exists Position Number in IMGT.
The TCR that hydrophobic core region is undergone mutation in the present invention can be by the α and β chain of a flexible peptide chain connection TCR can Variable domain and the solvable single-stranded TCR of stability that constitutes.It should be noted that in the present invention flexible peptide chain can be any suitable connection TCR α and The peptide chain of β chain variable domains.The single chain soluble TCR for such as building in the embodiment of the present invention 4, its α chain variable domain amino acid sequence For SEQ ID NO:32, the nucleotides sequence of coding is classified as SEQ ID NO:33；β chains variable domain amino acid sequence is SEQ ID NO: 34, the nucleotides sequence of coding is classified as SEQ ID NO:35.
In addition, for stability, patent documentation 201510260322.4 is also disclosed in the α chains variable region of TCR and β Artificial interchain disulfide bond is introduced between chain constant region significantly improves can the stability of TCR.Therefore, high-affinity of the invention Artificial interchain disulfide bond can also be contained between the α chains variable region and β chains constant region of TCR.Specifically, can in the α chains of the TCR The cysteine residues that artificial interchain disulfide bond is formed between change area and β chains constant region instead of：46th amino acids of TRAV With the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；47th amino acids of TRAV and TRBC1*01 or 61 amino acids of TRBC2*01 exons 1s；The of 46th amino acids and TRBC1*01 or TRBC2*01 exons 1s of TRAV 61 amino acids；Or the 47th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV.It is preferred that Ground, such TCR can include all or part of TCR α chains of () in addition to its membrane spaning domain, and () except its cross-film knot All or part of TCR β chains beyond structure domain, wherein () and () variable domain comprising TCR chains and at least a portion are constant Domain, α chains and β chain formation heterodimers.It is highly preferred that such TCR can include α chains variable domain and β chains variable domain and All or part of β chains constant domain in addition to membrane spaning domain, but it does not contain α chain constant domains, the α chain variable domains of the TCR With β chain formation heterodimers.
The present invention TCR can also multivalence complex form provide.The present invention multivalent TCR complex comprising two, Three, the four or more TCR of the present invention polymers that combine and formed, such as can be produced with four dimerization domains of p53 The tetramer, or the complex that multiple TCR of the present invention are combined and formed with another molecule.The TCR complex 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 produce other multivalence TCR with such application multiple The intermediate of compound.
The TCR of the present invention can be used alone, and also can be combined with covalent or other modes with conjugate, preferably with covalently side Formula is combined.The conjugate includes that detectable (is diagnostic purpose, wherein the TCR is used to detect presenting The presence of the cell of GLSNLTHVL-HLA A0201 complex), therapeutic agent, PK (protein kinase) modification part or it is any more than The combination of these materials is combined or is coupled.
Detectable for diagnostic purposes is included but is not limited to：Fluorescence or luminous marker, radioactive marker, MRI (nuclear magnetic resonance) or CT (CT technology) contrast agent can produce detectable product Enzyme.
The therapeutic agent that can be combined with TCR of the present invention or be coupled is included but is not limited to：1. radionuclide (Koppe etc., 2005, cancerometastasis comment (Cancer metastasis reviews) 24,539)；2. biological poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cytokine such as IL-2 etc. (Gillies etc., 1992, NAS's proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc Fragment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；5. antibody ScFv fragments (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold Nano-particle/nanometer rods (Lapotko etc., 2005, cancer communication (Cancer letters) 239,36；Huang etc., 2006, it is beautiful Chemical Society of state magazine (Journal of the American Chemical Society) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,11631)；9. magnetic nanosphere；10. pro-drug activation enzymes (for example, DT- diaphorases (DTD) or connection Phenyl hydrolytic enzyme-sample protein (BPHL))；11. chemotherapeutics (for example, cisplatin) or any type of nano-particle etc..
In addition, the TCR of the present invention is can also be comprising derived from more than a kind of heterozygosis TCR of species sequence.For example, grind Study carefully display Muridae TCR more effectively to express than people TCR in human T-cell.Therefore, TCR of the present invention can include people's variable domain With the constant domain of Mus.The defect of this method is possible to cause immunne response.Therefore, when which is used for adoptive T cell treatment There should be regulation scheme to carry out immunosuppressant, to allow the implantation of the T cell for expressing Muridae.
It should be understood that amino acid name adopts international single English alphabet or three English alphabets to represent herein, amino Single English alphabet of sour title is as follows with the corresponding relation of three English alphabets：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys (C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser (S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
Nucleic acid molecules
A second aspect of the present invention provides the nucleic acid molecules of coding first aspect present invention TCR molecule or part thereof, institute It can be one or more CDR, the variable domain of α and/or β chains, and α chains and/or β chains to state part.
The nucleotide sequence of coding first aspect present invention TCR molecule alpha chain CDR region is as follows：
αCDR1-gacagctcctccacctac(SEQ ID NO:16)
αCDR2-attttttcaaatatggacatg(SEQ ID NO:17)
αCDR3-gcagagatcccctcaactggggcaaacaacctcttc(SEQ ID NO:18)
The nucleotide sequence of coding 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 encode the nucleotide sequence of the nucleic acid molecules of the present invention of TCR β chains of the present invention and include SEQ ID NO:19、SEQ ID NO:20 and SEQ ID NO:21.
The nucleotide sequence of nucleic acid molecules of the present invention can be it is single-stranded or double-stranded, the nucleic acid molecules can be RNA or DNA, and can include or not include intron.Preferably, the nucleotide sequence of nucleic acid molecules of the present invention does not include intron But polypeptide of the present invention can be encoded, for example, encodes the nucleotide sequence bag of the nucleic acid molecules of the present invention of TCR α chain variable domains of the present invention Include SEQ ID NO:2 and/or the nucleotide sequence of the nucleic acid molecules of the present invention for encoding TCR β chain variable domains of the present invention include SEQ ID NO:6.Or, the nucleotide sequence for encoding the nucleic acid molecules of the present invention of TCR α chain variable domains of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecules of the present invention for encoding TCR β chain variable domains of the present invention include SEQ ID NO:35.More Preferably, the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:4 and/or SEQ ID NO:8.Or, the present invention The nucleotides sequence of nucleic acid molecules is classified as SEQ ID NO:31.
It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.Therefore, compile The nucleotide sequence of code book invention TCR can or the variant of degeneracy identical with the nucleotide sequence shown in accompanying drawing of the present invention.With Illustrating, " variant of degeneracy " refers to coding with SEQ ID NO to one of example in the present invention:1 protein sequence, But with SEQ ID NO:The 2 differentiated nucleotide sequence of sequence.
Nucleotide sequence can be the optimization of end count numeral.Different cells is different in the utilization of concrete codon , the codon in sequence can be changed increasing expression according to the type of cell.Mammalian cell and various other Biological codon usage table be well known to a person skilled in the art.
The present invention nucleic acid molecules full length sequence or its fragment generally can with but be not limited to PCR TRAP, recombination method or The method of synthetic is obtained.At present, it is already possible to obtain encoding completely by chemosynthesis TCR of the present invention (or its fragment, Or derivatives thereof) DNA sequence.Then can by the DNA sequence introduce various existing DNA moleculars as known in the art (or Such as carrier) and cell in.DNA can be coding strand or noncoding strand.
Carrier
The invention further relates to include the carrier of the nucleic acid molecules of the present invention, including expression vector that is, can in vivo or body The construct of outer expression.Conventional carrier includes bacterial plasmid, phage and animals and plants virus.
Viral delivery systems include but is not limited to adenovirus vector, adeno-associated viruses (AAV) carrier, herpesvirus vector, Retroviral vector, slow virus carrier, baculovirus vector.
Preferably, carrier can be transferred to the nucleotide of the present invention in cell, such as in T cell so that the cell table Up to the TCR of DAGE specificity.Ideally, the carrier should be expressed in T cell continual high levels.
Cell
The invention further relates to the host cell produced with the carrier or coded sequence Jing genetic engineerings of the present invention.The host The nucleic acid molecules of the present invention are integrated with carrier or chromosome containing the present invention in cell.Host cell is selected from：Prokaryotic cell And eukaryotic cell, such as escherichia coli, yeast cells, Chinese hamster ovary celI etc..
In addition, present invention additionally comprises the detached cell of the TCR of the expression present invention, particularly T cell.The T cell can be spread out It is conigenous T cell detached from experimenter, or can is the detached mixed cellularity group from experimenter, such as periphery hemolymph is thin The part of born of the same parents (PBL) group.Such as, the cell can be isolated from peripheral blood lymphocytes (PBMC), can be CD4+Helper T cell Or CD8+Cytotoxic T cell.The cell can be in CD4+Helper T cell/CD8+In the mixing group of cytotoxic T cell.Typically Ground, the cell can be activated with antibody (e.g., the antibody of anti-CD3 or anti-CD28), to allow them to easily receive to turn Dye, for example, transfected with the carrier of the nucleotide sequence comprising coding TCR molecules of the present invention.
Alternatively, cell of the invention can also be or derived from stem cell, such as hematopoietic stem cell (HSC).Gene is turned Move to HSC to be not result in cell surface expression TCR, because stem cell surface does not express CD3 molecules.However, when stem cell point Turn to when migrating to the lymphoid precursor of thymus (lymphoid precursor), the expression of CD3 molecules will start in thymocyte cell The surface expression introducing TCR molecules.
Have many methods be suitable for being carried out with the DNA or RNA that encode TCR of the present invention T cell transfection (e.g., Robbins etc., (2008)J.Immunol.180:6116-6131).The T cell of expression TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that carry out adoptive treatment many appropriate methods (e.g., Rosenberg etc., (2008) Nat Rev Cancer8(4)：299-308).
DAGE relevant disease
The invention further relates to the method with PRAME relevant diseases is treated and/or prevented in experimenter, which includes adoptive The step of transfer PRAME specific T-cells are to the experimenter.The PRAME specific T-cells can recognize that GLSNLTHVL-HLA A0201 complex.
The T cell of the PRAME specificitys of the present invention can be used to treat any presentation DAGE small peptide GLSNLTHVL- The PRAME relevant diseases of HLA A0201 complex, such as tumor.The tumor includes but is not limited to melanoma, lung squamous cell Cancer, breast carcinoma, renal cell carcinoma, tumor of head and neck, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukemia (include But be not limited to, acute lymphoblastic leukemia, acute myeloblastic leukemia), gastric cancer, pulmonary carcinoma, esophageal carcinoma, bladder cancer, incidence Squamous cell carcinoma, carcinoma of prostate, colon cancer, ovarian cancer etc..
Therapeutic Method
Can pass through to separate with the T cell with the patient or volunteer of DAGE relevant disease, and by the present invention's TCR is imported in above-mentioned T cell, subsequently feeds back in patient body to be treated by the cell of these genetic engineering modifications.Therefore, The invention provides a kind of method for treating PRAME relevant diseases, including by the T cell of TCR detached expression of the present invention, preferably Ground, the T cell derive from patient itself, are input in patient body.Usually, the T cell of patient is separated including (1), (2) are with originally Invention nucleic acid molecules can encode the nucleic acid molecules ex vivo transduction T cell of TCR molecules of the present invention, and genetic engineering is modified by (3) T cell be input in patient body.The quantity of the cell for separate, transfecting and feeding back can be determined by doctor.
Main advantages of the present invention are：
(1) TCR of the invention can be combined with DAGE small peptide complex GLSNLTHVL-HLA A0201, while turning The cell for having led TCR of the present invention can have very strong lethal effect by specific activation and to target cell.
Following specific embodiment, is expanded on further the present invention.It should be understood that these embodiments be merely to illustrate the present invention and It is not used in restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, Such as (Sambrook and Russell et al., molecular cloning：Laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing houses) described in condition, or according to the condition proposed by manufacturer.Unless Illustrate in addition, otherwise percentage ratio and number are calculated by weight.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight. In following examples, experiment material and reagent used can be obtained from commercially available channel if no special instructions.
Embodiment 1 clones DAGE small peptide specific T-cells
Using synthesis small peptide GLSNLTHVL (SEQ ID NO.:9；Beijing SBS Genetech gene technology company limited) stimulate and From in the peripheral blood lymphocyte (PBL) of the healthy volunteer that genotype is HLA-A0201.By GLSNLTHVL small peptides with carry The HLA-A0201 renaturation of biotin labeling, prepares pHLA monoploid.These monoploid and the Streptavidin (BD with PE labellings Company) tetramer of PE labellings is combined into, sort the tetramer and anti-CD8-APC double positive cells.The cell of amplification sorting, And secondary sorting is carried out as stated above, subsequently monoclonal is carried out with limiting dilution assay.Monoclonal cell tetramer staining, sieve The double positive colonies chosen are as shown in Figure 3.
Embodiment 2 obtains the tcr gene of DAGE small peptide specific T-cell clones and the structure of carrier
Use Quick-RNATMThe antigen small peptide screened in MiniPrep (ZYMO research) extracting embodiments 1 The total serum IgE of GLSNLTHVL specificitys, HLA-A0201 restricted T cell clone.The synthesis of cDNA is using clontech's SMART RACE cDNA amplification kits, the primer of employing are designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned It is sequenced to carrier T (TAKARA).It should be noted that the sequence is complementary seriess, not comprising intron.Jing is sequenced, and this pair positive Property clonal expression TCR α chains and β chain-orderings structure respectively as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e With Fig. 1 f be respectively TCR α chain variable domain amino acid sequences, TCR α chain variable domain nucleotide sequences, TCR α chain amino acid sequences, TCR α chain nucleotide sequences, the TCR α chain amino acid sequences with targeting sequencing and the TCR α chain nucleotide with targeting sequencing Sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chain variable domain amino acid sequences, TCR β chain variable domains Nucleotide sequence, TCR β chain amino acid sequences, TCR β chain nucleotide sequences, the TCR β chain amino acid sequences with targeting sequencing with And the TCR β chain nucleotide sequences with targeting sequencing.
Identified, α chains include the CDR with following aminoacid 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 aminoacid 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 into Lentiviral respectively by overlapping (overlap) PCR pLenti(addgene).Specially：The full-length gene of TCR α chains and TCR β chains is attached with overlap PCR and obtains TCR α -2A-TCR β fragments.Lentiviral and the connection of TCR α -2A-TCR β enzyme action are obtained into pLenti-TRA-2A-TRB- IRES-NGFR plasmids.Use as control, while the also slow virus carrier pLenti-eGFP of construction expression eGFP.Use again afterwards 293T/17 packs pseudoviruss.
The expression of the solvable TCR of embodiment 3PRAME antigen small peptide specificity, refolding and purification
To obtain solvable TCR molecules, α the and β chains of the TCR molecules of the present invention can only include its variable domain and portion respectively Divide in constant domain, and the constant domain of α and β chains a cysteine residues are introduced respectively to form artificial interchain disulfide bond, Introduce the Ser57 of the position respectively Thr48 and TRBC2*01 exons 1s of TRAC*01 exons 1s of cysteine residues；Its α The aminoacid sequence of chain is distinguished as shown in figures 4 a and 4b with nucleotide sequence, the aminoacid sequence and nucleotide sequence of its β chain Respectively as shown in figure 5 a and 5b, introducing cysteine residues are with overstriking and underline letter representation.Pass through《Molecular cloning Laboratory manual》(Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) Described in standard method the objective gene sequence ECDC of above-mentioned TCR α and β chains is inserted respectively into into expression vector pET28a into after + (Novagene), the cloning site of upstream and downstream is NcoI and NotI respectively.Insert Fragment confirms errorless through sequencing.
The expression vector of TCR α and β chains is entered into expression antibacterial BL21 (DE3), antibacterial by chemical transformation conversion respectively Grown with LB culture fluid, in OD600Induced with final concentration 0.5mM IPTG when=0.6, the bag formed after α the and β chains expression of TCR Contain body to be extracted by BugBuster Mix (Novagene), and the repeated multiple times washing of Jing BugBuster solution, forgive Body is finally dissolved in 6M guanidine hydrochlorides, 10mM dithiothreitol, DTTs (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris (pH 8.1) in.
TCR α and β chain after dissolving is with 1：1 mass ratio is quickly mixed in 5M carbamide, 0.4M arginine, 20mM Tris (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Solution is placed in into dialysis (4 DEG C) in the deionized water of 10 times of volumes afterwards, after 12 hours, deionized water is changed into buffer (20mM 8.0) Tris, pH continue at 4 DEG C and dialyse 12 hours.Solution after the completion of dialysis Jing after 0.45 μM of membrane filtration, by the moon from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purification.It is dimeric that eluting peak contains renaturation successful α and β TCR is confirmed by SDS-PAGE glue.TCR subsequently by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) it is further purified.TCR purity after purification is determined more than 90% through SDS-PAGE, and concentration is by BCA methods It is determined that.The SDS-PAGE glue figures of the sTCR that the present invention is obtained are as shown in Figure 6.
The generation of the soluble single-chain T CR of embodiment 4PRAME antigen small peptide specificity
According to patent documentation WO2014/206304, using the method for rite-directed mutagenesises by TCR α and β in embodiment 2 The variable domain of chain has been built into a stable soluble single-chain T CR molecule connected with flexible small peptide (linker).This is single-stranded The aminoacid sequence of TCR molecules and nucleotide sequence are respectively as shown in figs. 7 a and 7b.The aminoacid sequence of its α chain variable domain and Nucleotide sequence is respectively as figures 8 a and 8 b show；The aminoacid sequence and nucleotide sequence of its β chain variable domain is respectively such as Fig. 9 a With shown in Fig. 9 b；The aminoacid sequence of its linker sequence and nucleotide sequence are respectively as as-shown-in figures 10 a and 10b.
By I double digestion of genes of interest Jing Nco I and Not, it is connected with the pET28a carriers through I double digestion of Nco I and Not. Connection product is converted to E.coli DH5 α, LB flat board of the coating containing kanamycin, 37 DEG C of inversion overnight incubations, positive gram of picking It is grand enter performing PCR screening, positive recombinant is sequenced, determines that sequence correctly extracts recombinant plasmid transformed afterwards to E.coli BL21 (DE3), for expressing.
The expression of the soluble single-chain T CR of embodiment 5PRAME antigen small peptide specificity, renaturation and purification
The BL21 containing recombiant plasmid pET28a- template strands prepared in embodiment 4 (DE 3) bacterium colony is all inoculated in In LB culture medium containing kanamycin, it is 0.6-0.8, addition IPTG to final concentration of 0.5mM, 37 that 37 DEG C are cultivated to OD600 DEG C continue culture 4h.5000rpm is centrifuged 15min harvesting precipitate, is cracked with Bugbuster Master Mix (Merck) Cell pellet, 6000rpm centrifugation 15min reclaim inclusion body, then are washed to remove cell with Bugbuster (Merck) Fragment and membrane component, 6000rpm centrifugation 15min, collect inclusion body.By solubilization of inclusion bodies in buffer (20mM Tris-HCl PH 8.0,8M carbamide) in, high speed centrifugation removes insoluble matter, and supernatant is with subpackage is carried out after BCA standard measures, standby in -80 DEG C of preservations With.
To in the single-stranded TCR inclusion body proteins of 5mg dissolvings, 2.5mL buffer (6M Gua-HCl, 50mM Tris- is added HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration of 10mM, 37 DEG C of process 30min.With injection Device is to 125mL renaturation buffers (100mM Tris-HCl pH 8.1,0.4M L-Arginine, 5M carbamide, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) in single-stranded TCR after the above-mentioned process of Deca, 4 DEG C of stirrings Then renaturation solution is loaded cellulose membrane bag filter of the interception for 4kDa by 10min, and bag filter is placed in the water of 1L pre-coolings, 4 DEG C It is slowly stirred overnight.After 17 hours, change dialysis solution the buffer (20mM Tris-HCl pH 8.0) of 1L pre-coolings into, 4 DEG C after Continuous dialysis 8h, then changes dialysis solution into identical fresh buffer and continues dialysed overnight.After 17 hours, 0.45 μm of filter of sample Jing Membrane filtration, by anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgass, uses 20mM Tris-HCl The 0-1M NaCl linear gradient elution liquid purifying proteins that pH 8.0 is prepared, the elution fraction of collection carry out SDS-PAGE analyses, wrap Further carried out with solvent resistant column (Superdex 7510/300, GE Healthcare) after the concentration of component containing single-stranded TCR Purification, target components are also carried out SDS-PAGE analyses.
Elution fraction for BIAcore analyses further tests its purity using gel filtration.Condition is：Chromatographic column Agilent Bio SEC-3 (7.8 × 300mm of 300A, φ), mobile phase be 150mM phosphate buffers, flow velocity 0.5mL/min, 25 DEG C of column temperature, ultraviolet detection wavelength 214nm.
The SDS-PAGE glue figures of the soluble single-chain T CR that the present invention is obtained are as shown in figure 11.
Embodiment 6 is combined and is characterized
BIAcore is analyzed
This example demonstrated the TCR molecules of the present invention of solubility can be special with GLSNLTHVL-HLA A0201 complex It is anisogamy.
Detected using BIAcore T200 real-time analyzers the TCR molecules that obtain in embodiment 3 and embodiment 5 with The binding activity of GLSNLTHVL-HLA A0201 complex.The antibody (GenScript) of anti-Streptavidin is added to be coupled and is delayed (4.77), then by antibody 10mM sodium-acetate buffers, pH flow through the CM5 chips for being activated with EDC and NHS in advance, make to rush liquid Antibody is fixed on chip surface, finally closes unreacted activating surface with the hydrochloric acid solution of ethanolamine, completes coupling process, even Connection level is about 15,000RU.
The Streptavidin of low concentration is made to flow through the chip surface of coated antibody, then by GLSNLTHVL-HLA A0201 complex flows through sense channel, another passage as reference channel, then by the biotin of 0.05mM with the stream of 10 μ L/min Speed flows through chip 2min, the remaining binding site of closing Streptavidin.
The preparation process of above-mentioned GLSNLTHVL-HLA A0201 complex is as follows：
A. purification
The E.coli bacterium solutions of 100ml abduction deliverings heavy chain or light chain are collected, and 10ml are used after 4 DEG C of 8000g centrifugation 10min PBS washing thallines are once, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) afterwards Shake resuspended thalline, and in room temperature rotation incubation 20min, after 4 DEG C, 6000g centrifugation 15min, supernatant discarded, collection are forgiven Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation incubation 5min；Plus 30ml The BugBuster of 10 times of dilution, mixes, and 4 DEG C of 6000g are centrifuged 15min；Supernatant discarded, plus the BugBuster of 10 times of 30ml dilutions Resuspended inclusion body, mixes, and 4 DEG C of 6000g are centrifuged 15min, are repeated twice, plus 8.0 resuspended bags of 30ml 20mM Tris-HCl pH Contain body, mix, 4 DEG C of 6000g are centrifuged 15min, finally inclusion body, SDS-PAGE detections are dissolved with 20mM Tris-HCl 8M carbamide Inclusion body purity, BCA test kits survey concentration.
B. renaturation
The small peptide GLSNLTHVL (Beijing SBS Genetech gene technology company limited) of synthesis is dissolved in into DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain is dissolved with 8M carbamide, 20mM Tris pH 8.0,10mM DTT, is added before renaturation 3M guanidine hydrochlorides, 10mM sodium acetates, the further degeneration of 10mM EDTA.GLSNLTHVL peptides are added into renaturation with 25mg/L (final concentration) Buffer (0.4M L-Arginine, 100mM Tris pH 8.3,2mM EDTA, 0.5mM GSSGs, 5mM reduced forms Glutathion, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add the light chain and 90mg/L of 20mg/L heavy chain (final concentration, Heavy chain is added in three times, 8h/ time), renaturation carries out at least 3 days to completing at 4 DEG C, and can SDS-PAGE detections renaturation success.
C. purification after renaturation
At least changed buffer and come twice changing renaturation buffer as dialysis with the 20mM Tris pH 8.0 of 10 volumes The ionic strength of solution is reduced fully.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Using Akta purification instrument (the general electricity of GE Gas company), the 0-400mM NaCl linear gradient liquid wash-out proteins that 20mM Tris pH 8.0 are prepared, pMHC is about in 250mM Eluting at NaCl, collects all peak components, SDS-PAGE detection purity.
D. biotinylation
With Millipore super filter tubes by the pMHC molecular concentrations of purification, while being 20mM Tris pH by buffer exchange 8.0, it is subsequently adding biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D- Biotin, 100 μ g/ml BirA enzymes (GST-BirA), overnight, whether SDS-PAGE detections biotinylation for incubation at room temperature mixture Completely.
E. the complex after purifying biological elementization
With Millipore super filter tubes by the pMHC molecular concentrations after biotinylation labelling to 1ml, using gel permeation chromatography The pMHC of purifying biological elementization, using Akta purification instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibration HiPrepTM 16/60S200HR posts (GE General Electric Co. Limited), load the concentrated biotinylation pMHC molecules of 1ml, then with PBS with 1ml/ Min flow velocity eluting.Biotinylated pMHC molecules occur as unimodal eluting in about 55ml.Merge the group containing protein Point, being concentrated with Millipore super filter tubes, BCA methods (Thermo) determine protein concentration, add protease inhibitor cocktail (Roche) biotinylated pMHC molecules subpackage is stored in into -80 DEG C.
Using BIAcore Evaluation computed in software kinetic parameters, obtain the TCR molecules of solubility of the present invention with The kinetic profile that GLSNLTHVL-HLA A0201 complex is combined is as shown in figure 11 respectively.Collection of illustrative plates shows, what the present invention was obtained Soluble TCR molecules can be combined with GLSNLTHVL-HLA A0201 complex.Meanwhile, also this is have detected using said method The TCR molecules of invention solubility and other several irrelevant antigen small peptides and the binding activity of HLA complex, as a result show the present invention TCR molecules are with other irrelevant antigens without combination.
Embodiment 7PRAME antigen small peptide specificity TCR slow viruss are packed and are transfected with primary T cells
Quick mediation transient transfection (the Express-In-mediated transient of (a) by 293T cells Transfection slow viruss are prepared)
Using third generation slow viruss packaging system slow viruss of the packaging containing the gene of TCR needed for coding.Using quick Jie Lead transient transfection (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)), with 4 kinds of plasmid (one kind containing pLenti-RHAMMTRA-2A-TRB-IRES-NGFR described in embodiment 2 Slow virus carrier, and containing building 3 kinds of plasmids of infectiousness but other components necessary to non-replicating lentiviral particle) turn Dye 293T cells.
To be transfected, the 0th day kind cell, in 15 cm dishes, plants upper 1.7 × 107Individual 293T cells, make cell equal Even to be distributed on culture dish, degree of converging is slightly above 50%.1st day transfected plasmids, pack pLenti-RHAMMTRA-2A-TRB- IRES-NGFR and pLenti-eGFP pseudoviruss, by above expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV and PMD.2G is mixed, and the consumption of a 15 cm diameter plates is as follows：22.5 micrograms：15 micrograms:15 micrograms：7.5 microgram.Transfection examination The ratio of agent PEI-MAX and plasmid is 2:The usage amount of 1, each plate PEI-MAX is 120 micrograms.Concrete operations are：Expression Plasmid and packaging plasmid add 1800 microlitres of OPTI-MEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) 31985-070) culture medium Middle mix homogeneously, being stored at room temperature 5 minutes becomes DNA mixed liquors；Take respective amount PEI to mix with 1800 microlitres of OPTI-MEM culture medium Uniformly, being stored at room temperature 5 minutes becomes PEI mixed liquors.DNA mixed liquors and PEI mixed liquors are mixed and be stored at room temperature 30 minutes, then add 3150 microlitres of OPTI-MEM culture medium, it is added to after mix homogeneously and has been converted to 11.25 milliliters of OPTI- In the 293T cells of MEM, culture dish is gently rocked, make culture medium mix homogeneously, 37 DEG C/5%CO2Lower culture.5-7 is little for transfection When, remove transfection media, change into the DMEM containing 10% hyclone ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt)) complete medium, 37 DEG C/5%CO2Lower culture.Collect the training containing wrapped slow viruss within 3rd and the 4th day Foster base supernatant.To harvest the slow viruss of packaging, collected culture supernatant 3000g is centrifuged 15 minutes and removes cell debriss, 0.22 micron filters of Jing (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB) are filtered again, are finally used The concentration tube (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) UFC905096) of 50KD interceptions is concentrated, and is removed Most of supernatant, is finally concentrated to 1 milliliter, after decile subpackage -80 DEG C it is frozen.Taking pseudoviruss sample carries out virus titer survey Fixed, step is with reference to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit specification.Use as control, while also bag turns The pseudoviruss of pLenti-eGFP.
The lentiviruses transduction primary T cells of (b) with the φt cell receptor gene containing DAGE small peptide specificity
It is separated to CD8+T cells from the blood of healthy volunteer, then with the lentiviruses transduction packed.Count these thin Born of the same parents, in 48 orifice plates, containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10%FBS (Ji Bu can company (Gibco), Catalog number (Cat.No.) C10010500BT) 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture medium in 1 × 106 Individual cells/ml (0.5 milliliter/hole) and the AntiCD3 McAb for pre-washing/CD28 antibody-coating globule (T cell amplified matter, life Technologies, catalog number (Cat.No.) 11452D) overnight incubation stimulation altogether, cell：Pearl=3：1.
After stimulating overnight, according to the virus titer measured by p24ELISA test kits, add in the ratio of MOI=10 dense The slow viruss of the DAGE small peptide specific t-cell receptor gene of contracting, 32 DEG C, 900g centrifugations infection 1 hour.Infection is finished Slow virus infection liquid is removed afterwards, with the 1640 culture medium weights containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 Outstanding cell, cultivates 3 days under 37 DEG C/5%CO2.Next day carries out the second wheel infection in the same way.Second transduction was counted after 3 days Cell, diluting cells to 0.5 × 106Individual cells/ml.A cell was counted per two days, is replaced or is added and contain 50IU/ml The fresh culture of IL-2 and 10ng/ml IL-7, maintains cell 0.5 × 106-1×106Individual cells/ml.Opened from the 3rd day Begin by flow cytometry cell, started from the 5th day for function test (for example, the ELISPOT of IFN-γ release and non- Radioactivity cytotoxicity is detected).Started from the 10th day or when cell slows down division and size diminishes, stored frozen decile is thin Born of the same parents, at least 4 × 106Individual cell/pipe (1 × 107Individual cells/ml, 90%FBS/10%DMSO).
Embodiment 8 transduce TCR of the present invention T cell activation experiment
ELISPOT schemes
Carry out tests below to prove activating reaction of the T cell of TCR- transductions to target cell specificity.Using ELISPOT Readout of the IFN-γ yield of testing inspection as t cell activation.
Reagent
Test medium：10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), (Ji Bu can for RPMI1640 Company (Gibco), catalog number (Cat.No.) C11875500bt)
Lavation buffer solution (PBST)：0.01M PBS/0.05% polysorbas20s
PBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT)
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) (is caught and is detected and be anti-equipped with required every other reagent Body, Streptavidin-alkali phosphatase and BCIP/NBT solution)
Method
It is prepared by target cell
In this experiment, target cell used is T2 cells.Target cell is prepared in assay medium：Target cell concentration is adjusted to 2.0×105Individual/milliliter, takes 100 microlitres so as to obtain 2.0 × 10 per hole4Individual cells/well.
It is prepared by effector lymphocyte
Effector lymphocyte's (T cell) of this experiment is Jing flow cytometry expression DAGE of the present invention in embodiment 7 The CD8+T cells of small peptide specificity TCR, and with the CD8+T of same volunteer's untransfected TCR of the present invention as a control group.With anti- CD3/CD28 coatings pearl (T cell amplified matter, life technologies) stimulates T cell, special with DAGE small peptide is carried The lentiviruses transduction (according to embodiment 7) of different in nature tcr gene, containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10% FBS 1640 culture medium amplification until transduction after 9-12 days, then these cells are placed in test medium, 300g room temperature from The heart is washed for 10 minutes.Then cell is resuspended in test medium with 2 × required final concentration.It is same to process negative control Effector lymphocyte.
It is prepared by small peptide solution
Correspondence small peptide is added in corresponding target cell (T2) experimental group, final concentration of 1 μ of the small peptide in ELISPOT orifice plates is made g/ml。
ELISPOT
According to the description that manufacturer provides, preparation orifice plate as described below：1 is pressed with 10 milliliters of aseptic PBS of every block of plate：200 Anti-human IFN-γ capture antibodies are diluted, 100 microlitres dilution capture antibodies etc. point are added into each hole then.Orifice plate is incubated at 4 DEG C Overnight.After incubation, wash orifice plate to remove unnecessary capture antibodies.The RPMI 1640 for adding 100 microlitres/hole to contain 10%FBS Culture medium, and orifice plate is incubated at room temperature 2 hours to close orifice plate.Then culture medium is washed away from orifice plate, by light on paper Bullet and pat ELISPOT orifice plates with remove it is any remnants lavation buffer solution.
Then all components tested are added by ELISPOT orifice plates using following order：
100 microlitres of target cell 2*105Individual cells/ml (obtains about 2*10 altogether4Individual target cell/hole).
100 microlitres of effector lymphocyte (1*104Individual control effector lymphocyte/hole and PRAME TCR positive T cells/hole).
All holes prepare addition in duplicate.
Then incubate overnight (37 DEG C/5%CO of orifice plate2) second day, culture medium is abandoned, orifice plate 2 times is washed with distilled water, then is used Lavation buffer solution is washed 3 times, pats to remove the lavation buffer solution of remnants on napkin.Then pressed with the PBS containing 10%FBS 1：200 dilution detection antibodies, add each hole by 100 microlitres/hole.Orifice plate is incubated 2 hours under room temperature, then washed with lavation buffer solution 3 times, orifice plate is patted on napkin to remove excessive lavation buffer solution.
1 is pressed with the PBS containing 10%FBS：100 dilution Streptavidin-alkali phosphatases, by the chain of 100 microlitres of dilutions Mould Avidin-alkali phosphatase adds each hole and incubates orifice plate 1 hour at room temperature.Then 4 PBS are washed with lavation buffer solution Washing 2 times, pats orifice plate to remove excessive lavation buffer solution and PBS on napkin.Washing adds test kit to provide after finishing 100 microlitres/hole of BCIP/NBT solution developed.Orifice plate lucifuge is covered with masking foil during developing, 5-15 minutes are stood. The speckle of conventional sense development orifice plate, determines the Best Times of terminating reaction during this period.Remove BCIP/NBT solution and with pair Steam water to rinse orifice plate to stop developing reaction, dry, then orifice plate bottom is removed, orifice plate is dried at room temperature for until each hole It is completely dried, recycles immunodotting plate count meter (CTL, Celltech Ltd. (Cellular Technology Limited the)) speckle that counterdie is formed in counting orifice.
As a result
The T cell of inspection (as described above) TCR transductions of the present invention is tested to loading DAGE small peptide 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.
As shown in figure 12, the T cell of the TCR of the present invention that transduces has very experimental result to the target cell for loading its special small peptide Good activating reaction, and the substantially no activating reaction of T cell of the TCR of the present invention that do not transduce.
The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned teachings for having read the present invention, those skilled in the art can To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model limited by the application appended claims Enclose.
Sequence table
<110>Guangzhou Xiangxue Pharmaceutical Co
<120>The TCR of identification DAGE
<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 (10)

1. a kind of φt cell receptor (TCR), it is characterised in that the TCR can be tied with GLSNLTHVL-HLA A0201 complex Close；Preferably, described TCR includes TCR α chains variable domains and TCR β chain variable domains, it is characterised in that the TCR α chain variable domains CDR3 aminoacid sequence be AEIPSTGANNLF (SEQ ID NO:12)；And/or the CDR3 of the TCR β chain variable domains Aminoacid sequence is ASSQGGTQY (SEQ ID NO:15)；
It is highly preferred that 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)。
2. TCR as claimed in claim 1, it is characterised in which includes TCR α chains variable domains and TCR β chain variable domains, described TCR α chain variable domains are and SEQ ID NO:1 aminoacid sequence with least 90% sequence thereto；And/or the TCR β chains Variable domain is and SEQ ID NO：5 aminoacid sequences with least 90% sequence thereto.
3. TCR as claimed in claim 1, it is characterised in that C- the or N- ends of the α chains and/or β chains of the TCR are combined with Conjugate；Preferably, the conjugate for being combined with the φt cell receptor is detectable, therapeutic agent, PK modifications part or is appointed The combination of what these material；Preferably, the therapeutic agent is anti-CD 3 antibodies.
4. a kind of multivalent TCR complex, it is characterised in that comprising at least two TCR molecules, and at least one TCR therein Molecule is the TCR any one of the claims.
5. a kind of nucleic acid molecules, it is characterised in that the nucleic acid molecules include TCR point described in any of the above-described claim of coding The nucleotide sequence or its complementary series of son；
Preferably, nucleotide sequence SEQ ID NO of the described nucleic acid molecules comprising coding TCR α chain variable domains：2 or SEQ ID NO:33；And/or
Nucleotide sequence SEQ ID NO of the described nucleic acid molecules comprising coding TCR β chain variable domains：6 or SEQ ID NO:35.
6. a kind of carrier, it is characterised in that described carrier contains the nucleic acid molecules described in claim 5；Preferably, it is described Carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
7. a kind of detached host cell, it is characterised in that containing the load described in claim 26 in described host cell The nucleic acid molecules being integrated with body or chromosome described in the claim 5 of external source.
8. a kind of cell, it is characterised in that institute in the nucleic acid molecules or claim 6 described in the cell transduction claim 5 State carrier；Preferably, the cell is T cell or stem cell.
9. a kind of pharmaceutical composition, it is characterised in that the compositionss contain pharmaceutically acceptable carrier and claim The nucleic acid molecules described in the TCR complex described in TCR, claim 4, claim 5 or power any one of 1-3 Profit requires the cell described in 8.
10. the TCR complex or right described in the φt cell receptor or claim 4 any one of claim 1-3 Require the purposes of the cell described in 8, it is characterised in that for preparing the medicine for the treatment of tumor or autoimmune disease.
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CN108929378A (en) * 2017-05-22 2018-12-04 广东香雪精准医疗技术有限公司 A kind of T cell receptor identifying PRAME antigen and the nucleic acid for encoding this receptor
CN109251243A (en) * 2017-07-13 2019-01-22 中国科学院广州生物医药与健康研究院 A kind of T cell receptor identifying SAGE1 antigen and the nucleic acid for encoding this receptor
CN109400696A (en) * 2017-08-17 2019-03-01 广东香雪精准医疗技术有限公司 A kind of TCR identifying PRAME antigen small peptide
WO2019109821A1 (en) * 2017-12-06 2019-06-13 广东香雪精准医疗技术有限公司 High-affinity t cell receptor against prame
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WO2020057619A1 (en) * 2018-09-21 2020-03-26 广东香雪精准医疗技术有限公司 High affinity t cell receptor for recognizing afp antigen
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Publication number Priority date Publication date Assignee Title
US11111286B2 (en) 2017-03-23 2021-09-07 Immatics Biotechnologies Gmbh T cell receptors and immune therapy using the same against PRAME positive cancers
US11236145B2 (en) 2017-03-23 2022-02-01 Immatics Biotechnologies Gmbh T cell receptors and immune therapy using the same against PRAME positive cancers
CN110494160A (en) * 2017-03-23 2019-11-22 伊玛提克斯生物技术有限公司 T cell receptor and its immunization therapy for PRAME positive cancer
CN108690130A (en) * 2017-04-12 2018-10-23 广东香雪精准医疗技术有限公司 A kind of TCR of the identification from LMP1 antigen small peptides
CN108929378A (en) * 2017-05-22 2018-12-04 广东香雪精准医疗技术有限公司 A kind of T cell receptor identifying PRAME antigen and the nucleic acid for encoding this receptor
CN109251243A (en) * 2017-07-13 2019-01-22 中国科学院广州生物医药与健康研究院 A kind of T cell receptor identifying SAGE1 antigen and the nucleic acid for encoding this receptor
CN109251243B (en) * 2017-07-13 2021-10-19 中国科学院广州生物医药与健康研究院 T cell receptor for recognizing SAGE1 antigen and nucleic acid for encoding receptor
CN109400696A (en) * 2017-08-17 2019-03-01 广东香雪精准医疗技术有限公司 A kind of TCR identifying PRAME antigen small peptide
CN109879957B (en) * 2017-12-06 2022-03-18 香雪生命科学技术（广东）有限公司 High affinity T cell receptors for PRAME
CN109879957A (en) * 2017-12-06 2019-06-14 广东香雪精准医疗技术有限公司 For the high-affinity T cell receptor of PRAME
WO2019109821A1 (en) * 2017-12-06 2019-06-13 广东香雪精准医疗技术有限公司 High-affinity t cell receptor against prame
WO2020057619A1 (en) * 2018-09-21 2020-03-26 广东香雪精准医疗技术有限公司 High affinity t cell receptor for recognizing afp antigen
CN110950949A (en) * 2018-09-26 2020-04-03 广东香雪精准医疗技术有限公司 T cell receptor for recognizing SSX2 antigen
CN115073584A (en) * 2022-05-07 2022-09-20 深圳普瑞金生物药业股份有限公司 TCR for specifically recognizing PRAME antigen peptide and application thereof
CN115073584B (en) * 2022-05-07 2023-10-20 溧阳瑅赛生物医药有限公司 TCR capable of specifically recognizing PRAME antigen peptide and application thereof
WO2023216440A1 (en) * 2022-05-07 2023-11-16 溧阳瑅赛生物医药有限公司 Tcr specifically recognizing prame antigen peptide and use thereof

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