CN106459179B - Identify the T cell receptor of RHAMM antigen small peptides - Google Patents

Identify the T cell receptor of RHAMM antigen small peptides Download PDF

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CN106459179B
CN106459179B CN201680001317.4A CN201680001317A CN106459179B CN 106459179 B CN106459179 B CN 106459179B CN 201680001317 A CN201680001317 A CN 201680001317A CN 106459179 B CN106459179 B CN 106459179B
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CN106459179A (en
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李懿
林燕梅
吴万里
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Xiangxue Life Science Technology (Guangdong) Co.,Ltd.
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Abstract

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

Description

Identify the T cell receptor of RHAMM antigen small peptides
Technical field
The present invention relates to can identify from RHAMM (Receptor for Hyaluronan-Mediated Moti l Ity, RHAMM) antigen small peptide TCR, the invention further relates to transduceing above-mentioned TCR the T cell of RHAMM specificity that obtains and Their purposes in RHAMM relevant diseases are prevented and treated.
Background technology
RHAMM also known as CD168 is the receptor of the hyaluronic acid as one of extracellular matrix components.RHAMM is in one kind Property antigen in source is degraded to micromolecule polypeptide after generating in the cell, and is combined with MHC (main histocompatibility complex) molecule Compound is formed, is presented to cell surface.ILSLELMKL (165-173) be derived from RHAMM small peptide (Greiner J, et al.,Blood 2005,106(3):938-945).Studies have shown that RHAMM has expression in kinds of tumors tissue, with white Blood disease (Greiner J, et al., Experimental hematology 2002,30 (9):1029-1035), colon cancer (Yamada Y,et al.,Japanese journal of cancer research:Gann 1999,90(9):987- 992), breast cancer (Wang C, et al., Clinical cancer research:an official journal of the American Association for Cancer Research 1998,4(3):It is 567-576) more prominent, in others Cancer, such as stomach cancer (Li H, et al., International journal of oncology 2000,17 (5):927- 932), kidney (Greiner J, et al., Experimental hematology 2002,30 (9):1029-1035), oral cavity Squamous cell carcinoma (Yamano Y, et al., International journal of oncology 2008,32 (5):1001- 1009), head and neck squamous cell carcinoma (Schmitt A, et al., International journal of oncology 2009,34(3):629-639) etc. also there is expression in tumour cells.Treatment for above-mentioned disease may be employed chemotherapy and put The methods of penetrating property is treated, but the normal cell of itself can all be damaged.
T cell adoptive immunotherapy is to be transferred to the reaction-ive T cell for having specificity to target cell antigen in patient body, It is made to play a role for target cell.T cell receptor (TCR) is a kind of memebrane protein on T cell surface, can be identified corresponding The antigen small peptide of target cell surface.In immune system, pass through TCR and the small peptide-main histocompatbility of antigen small peptide specificity The combination of complex (pMHC compounds) triggers T cell to be directly physically contacted with antigen presenting cell (APC), then T cell And other cell membrane surface molecules of both APC just interact, and cause a series of subsequent cell signals transmission and its His physiological reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, this field skill Art personnel are directed to isolating the TCR for having specificity to RHAMM antigens small peptide and TCR's T cell are transduceed to obtain pair RHAMM antigens small peptide has the T cell of specificity, so that they play a role in cellular immunotherapy.
The content of the invention
It is an object of the invention to provide a kind of T cell receptors for identifying RHAMM antigen small peptides.
The first aspect of the present invention, provides a kind of T cell receptor (TCR), and the TCR can be with ILSLELMKL-HLA Compound combines.
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chain variable domains For with SEQ ID NO:1 has the amino acid sequence of at least 90% sequence thereto；And/or the TCR β chains variable domain be with SEQ ID NO：5 have the amino acid sequence of at least 90% sequence thereto.
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, which is characterized in that described The amino acid sequence of the CDR3 of TCR α chain variable domains is AATNSGYALN (SEQ ID NO:12)；And/or the TCR β chains are variable The amino acid sequence of the CDR3 in domain is AWSVDGAEQY (SEQ ID NO:15).
In another preference, 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
α CDR1-DRVSQS (SEQ ID NO:10)
α CDR2-IYSNGD (SEQ ID NO:11)
α CDR3-AATNSGYALN (SEQ ID NO:12) and/or
3 complementary determining regions of the TCR β chain variable domains are：
β CDR1-GTSNPN (SEQ ID NO:13)
β CDR2-SVGIG (SEQ ID NO:14)
β CDR3-AWSVDGAEQY (SEQ ID NO:15)。
In another preference, the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.
In another preference, the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5.
In another preference, the TCR is α β heterodimers, and it includes TCR α chain constant region TRAC*01 and TCR β Chain constant region TRBC1*01 or TRBC2*01.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO：The β chain ammonia of the 3 and/or TCR Base acid sequence is SEQ ID NO:7.
In another preference, the TCR is soluble.
In another preference, the TCR is single-stranded.
In another preference, the TCR is to be formed by connecting by α chains variable domain with β chains variable domain by peptide catenation sequence.
In another preference, the TCR is in α chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or 94 and/or α chain J gene small peptides amino acid are 3rd reciprocal, it is one or more prominent to have in 5th reciprocal or 7th reciprocal Become；And/or the TCR is in β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th and/or β chains J Gene small peptide amino acid is 2nd reciprocal, has one or more mutation, wherein amino acid position in 4th reciprocal or 6th reciprocal Number is put by the Position Number listed in IMGT (international immunogenetics information system).
In another preference, the α chains variable domain amino acid sequence of the TCR includes SEQ ID NO:32 and/or described The β chains variable domain amino acid sequence of TCR includes SEQ ID NO:34.
In another preference, the amino acid sequence of the TCR is SEQ ID NO:30.
In another preference, the TCR includes all or part of TCR α chains (a) in addition to transmembrane domain；And (b) all or part of TCR β chains in addition to transmembrane domain；
And and (b) respectively contains functional variable domain (a), or includes functional variable domain and the TCR At least a portion of chain constant domain.
In another preference, cysteine residues form artificial disulfide bond between α the and β chain constant domains of the TCR.
In another preference, the cysteine residues of artificial disulfide bond are formed in the TCR instead of selected from following One or more groups of sites：
The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；With
The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO:The β chains of the 26 and/or TCR Amino acid sequence is SEQ ID NO:28.
In another preference, the α chains of the TCR and/or C- the or N- ends of β chains are combined with conjugate.
In another preference, the conjugate that is combined with the T cell receptor is detectable, therapeutic agent, PK are repaiied Decorations part or the combination of these any substances.Preferably, the therapeutic agent is anti-CD 3 antibodies.
The second aspect of the present invention provides a kind of multivalent TCR complex, it includes at least two TCR molecules, and its In at least one TCR molecules be the TCR described in first aspect present invention.
The third aspect of the present invention provides a kind of nucleic acid molecules, and the nucleic acid molecules, which include, encodes first party of the present invention The nucleotide sequence or its complementary series of TCR molecules described in face.
In another preference, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chain variable domains： 2 or SEQ ID NO:33.
In another preference, the nucleic acid molecules include the nucleotide sequence SEQ ID of coding TCR β chain variable domains NO：6 or SEQ ID NO:35.
In another preference, the nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR α chains：4 and/or Include the nucleotide sequence SEQ ID NO of coding TCR β chains：8.
The fourth aspect of the present invention, provides a kind of carrier, and the carrier contains the core described in third aspect present invention Acid molecule；Preferably, the carrier is viral vectors；It is highly preferred that the carrier is slow virus carrier.
The fifth aspect of the present invention provides a kind of separated host cell, contains the present invention in the host cell The nucleic acid molecules described in the third aspect present invention of external source are integrated in carrier or genome described in fourth aspect.
The sixth aspect of the present invention provides a kind of cell, the nucleic acid described in the cell transduction third aspect present invention Carrier described in molecule or fourth aspect present invention；Preferably, the cell is T cell or stem cell.
The seventh aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains pharmaceutically acceptable load The TCR compounds described in TCR, second aspect of the present invention, third aspect present invention institute described in body and first aspect present invention Carrier described in the nucleic acid molecules stated, fourth aspect present invention or the cell described in sixth aspect present invention.
The eighth aspect of the present invention provides T cell receptor or second aspect of the present invention described in first aspect present invention Nucleic acid molecules described in the TCR compounds, third aspect present invention, carrier or this hair described in fourth aspect present invention The purposes of cell described in bright 6th aspect is used to prepare the drug for the treatment of tumour or autoimmune disease.
The ninth aspect of the present invention provides a kind of method for treating disease, including giving object application in need for the treatment of suitable The TCR compounds described in T cell receptor or second aspect of the present invention, the present invention the 3rd described in the first aspect present invention of amount The carrier described in nucleic acid molecules, fourth aspect present invention described in aspect or cell or this hair described in sixth aspect present invention Pharmaceutical composition described in bright 7th aspect；
Preferably, the disease is acute myeloid system leukaemia, chronic myelocytic system leukaemia, acute lymphoblastic Leukaemia, chronic lymphocytic leukemia, Huppert's disease, melanoma, colon cancer, breast cancer, kidney, stomach cancer, bladder Transitional cell carcinoma, prostate cancer, oral squamous cell carcinoma and head and neck squamous cell carcinoma.
It is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively that TCR α chains variable domain amino acid sequence, TCR α chains are variable Domain nucleotide sequence, TCR α chain amino acid sequences, TCR α chains nucleotide sequence, the TCR α chain amino acid sequences with targeting sequencing And the TCR α chain nucleotide sequences with targeting sequencing.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively that TCR β chains variable domain amino acid sequence, TCR β chains are variable Domain nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, the TCR β chain amino acid sequences with targeting sequencing And the TCR β chain nucleotide sequences with targeting sequencing.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b are respectively the amino acid sequence and nucleotide sequence of sTCR α chains.
Fig. 5 a and Fig. 5 b are respectively the amino acid sequence and nucleotide sequence of sTCR β chains.
Fig. 6 is the glue figure of the sTCR obtained after purification.
Fig. 7 a and Fig. 7 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR.
Fig. 8 a and Fig. 8 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR α chains.
Fig. 9 a and Fig. 9 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR β chains.
Figure 10 a and Figure 10 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR catenation sequences (l inker).
Figure 11 is the glue figure of the soluble single-chain T CR obtained after purification.
Figure 12 is the BIAcore dynamics figures that sTCR of the present invention is combined with ILSLELMKL-HLA A0201 compounds Spectrum.
Figure 13 is the BIAcore power that soluble single-chain T CR of the present invention is combined with ILSLELMKL-HLA A0201 compounds Learn collection of illustrative plates.
Activation of the T cell that Figure 14 is transduction TCR of the present invention to the T2 cells of load or unsupported specific antigen small peptide Experimental result picture.
Killing of the T cell that Figure 15 is transduction TCR of the present invention to the T2 cells of load or unsupported specific antigen small peptide Experimental result picture.
Figure 16 is the T cell of transduction TCR of the present invention to special and non-specific cellular system killing experiments result figure.
Specific embodiment
The present inventor has found and RHAMM antigen small peptide ILSLELMKL (165-173) by in-depth study extensively (SEQ ID NO:9) TCR that can be specifically bound, the antigen small peptide ILSLELMKL can form compound with HLA A0201 And cell surface is presented to together.The present invention also provides the nucleic acid molecules for encoding the TCR and include the nucleic acid point The carrier of son.In addition, the cell the present invention also provides the TCR of the present invention that transduces.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, for The presentation of antigen has specificity, and different individuals has different MHC, can present small peptide different in a kind of proteantigen to respectively From APC cell surfaces.The MHC of the mankind is commonly referred to as HLA genes or HLA complexs.
T cell receptor (TCR) is the unique of specific antigen peptide of the presentation in main histocompatibility complex (MHC) Receptor.In immune system, trigger T cell and antigen presentation thin by the combination of the TCR and pMHC compounds of antigentic specificity Born of the same parents (APC) are directly physically contacted, and then other cell membrane surface molecules of both T cell and APC just interact, this A series of subsequent cell signals transmission and other physiological reactions are just caused, so that the T cell of different antigentic specificities Immunological effect is played to its target cell.
TCR is the glycoprotein of the cell membrane surface as existing for α chains/β chains or γ chains/δ chains in the form of heterodimer. TCR heterodimers are made of α and β chains in 95% T cell, and 5% T cell has the TCR being made of γ and δ chains.My god The heterogeneous dimerization TCR of right α β have α chains and β chains, and α chains and β chains form the subunit of α β heterodimerics TCR.In a broad sense, α and β are each Chain includes variable region, bonding pad and constant region, and β chains contain short variable region usually also between variable region and bonding pad, but should Variable region is often regarded as a part for bonding pad.Each variable region includes 3 be entrenched in frame structure (framework regions) A CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compounds, wherein CDR3 by Variable region and bonding pad recombinate, and are referred to as hypervariable region.α the and β chains of TCR generally regard that each there are two " structural domain " is i.e. variable as Domain and constant domain, variable domain are made of the variable region connected and bonding pad.The sequence of TCR constant domains can be in international immune genetic It learns and is found in the public database of information system (IMGT), if the constant domain sequence of TCR molecule alpha chains is " TRAC*01 ", TCR divides The constant domain sequence of sub- β chains is " TRBC1*01 " or " TRBC2*01 ".In addition, α the and β chains of TCR also include transmembrane region and kytoplasm Area, cytoplasmic region are very short.
In the present invention, term " polypeptide of the present invention ", " TCR of the invention ", " T cell receptor of the invention " is interchangeable makes With.
Detailed description of the invention
TCR molecules
In antigen processing pathways, antigen is degraded in the cell, is then carried by MHC molecule to cell surface.T is thin Born of the same parents' receptor can identify the peptide-MHC compounds of Antigen Presenting Cell surface.Therefore, the first aspect of the present invention provides one kind The TCR molecules of ILSLELMKL-HLA A0201 compounds can be specifically bound.Preferably, the TCR molecules be it is separated or Purifying.α the and β chains of the TCR respectively have 3 complementary determining regions (CDR).
One in the present invention is preferably carried out in mode, and the α chains of the TCR are included with following amino acid sequence CDR：
α CDR1-DRVSQS (SEQ ID NO:10)
α CDR2-IYSNGD (SEQ ID NO:11)
α CDR3-AATNSGYALN (SEQ ID NO:12)。
One in the present invention is preferably carried out in mode, and the β chains of the TCR are included with following amino acid sequence CDR：
β CDR1-GTSNPN (SEQ ID NO:13)
β CDR2-SVGIG (SEQ ID NO:14)
β CDR3-AWSVDGAEQY (SEQ ID NO:15)。
The CDR region amino acid sequence of the invention described above can be embedded into chimeric to prepare in any suitable frame structure TCR.As long as frame structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art's disclosed CDR region according to the present invention It can just design or synthesize the TCR molecules with corresponding function.Therefore, TCR molecules of the present invention refer to comprising above-mentioned α and/or β The TCR molecules of chain CDR region sequence and any suitable frame structure.TCR α chains variable domain of the present invention be and SEQ ID NO:1 tool There is the amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence thereto；And/or TCR β chains of the present invention can Variable domain be and SEQ ID NO：5 have at least 90%, preferably 95%, the amino acid sequence of more preferably 98% sequence thereto Row.
In the preference of the present invention, TCR molecules of the invention are the heterodimers being made of α and β chains.Specifically Ground, the α chains of the one side heterogeneous dimerization TCR molecules include variable domain and constant domain, the α chains variable domain amino acid sequence bag CDR1 (SEQ ID NO containing above-mentioned α chains：10)、CDR2(SEQ ID NO：And CDR3 (SEQ ID NO 11):12).Preferably, The TCR molecules include α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domains of the TCR molecules Amino acid sequence is SEQ ID NO:1.On the other hand, the β chains of the heterogeneous dimerization TCR molecules include variable domain and constant domain, The β chains variable domain amino acid sequence includes CDR1 (the SEQ ID NO of above-mentioned β chains:13)、CDR2(SEQ ID NO：14) and CDR3(SEQ ID NO:15).Preferably, the TCR molecules include β chain variable domain amino acid sequence SEQ ID NO:5.It is more excellent Selection of land, the β chains variable domain amino acid sequence of the TCR molecules is SEQ ID NO:5.
In the preference of the present invention, TCR molecules of the invention are part or all of and/or β chains the portions by α chains The single chain TCR molecules for dividing or all forming.Description in relation to single chain TCR molecules may be referred to document Chung et al (1994) Proc.Natl.Acad.Sc i.USA 91,12654-12658.According to document, those skilled in the art can be easy Ground builds the single chain TCR molecules for including CDRs areas of the present invention.Specifically, the single chain TCR molecules include V α, V β and C β, preferably Ground is according to being linked in sequence from N-terminal to C-terminal.
The α chains variable domain amino acid sequence of the single chain TCR molecules includes CDR1 (the SEQ ID NO of above-mentioned α chains：10)、 CDR2(SEQ ID NO：And CDR3 (SEQ ID NO 11):12).Preferably, the single chain TCR molecules include α chain variable domain ammonia Base acid sequence SEQ ID NO:1.It is highly preferred that the α chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO: 1.The β chains variable domain amino acid sequence of the single chain TCR molecules includes CDR1 (the SEQ ID NO of above-mentioned β chains：13)、CDR2 (SEQ ID NO：And CDR3 (SEQ ID NO 14)：15).Preferably, the single chain TCR molecules include β chain variable domain amino acids Sequence SEQ ID NO:5.It is highly preferred that the β chains variable domain amino acid sequence of the single chain TCR molecules is SEQ ID NO:5.
In the preference of the present invention, the constant domain of TCR molecules of the invention is the constant domain of people.Art technology Personnel know or can be obtained by consulting the public database of pertinent texts or IMGT (international immunogenetics information system) Obtain the constant domain amino acid sequence of people.For example, the constant domain sequence of TCR molecule alphas chain of the present invention can be " TRAC*01 ", TCR points The constant domain sequence of sub- β chains can be " TRBC1*01 " or " TRBC2*01 ".The amino acid sequence provided in the TRAC*01 of IMGT The 53rd for Arg, be expressed as herein：The Arg53 of TRAC*01 exons 1s, other and so on.Preferably, TCR of the present invention The amino acid sequence of molecule alpha chain is SEQ ID NO:The amino acid sequence of 3 and/or β chains is SEQ ID NO:7.
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.As immunoglobulin (antibody) is made The same for antigen recognizing molecule, at this moment TCR can also need to obtain soluble TCR points by development and application in diagnose and treat Son.Soluble TCR molecules do not include its transmembrane region.STCR has very extensive purposes, it cannot be only used for research TCR With the interaction of pMHC, it is also possible to make the diagnostic tool of detection infection or the marker as autoimmunity disease.Similarly, may be used Dissolubility TCR can be used to therapeutic agent (such as cytotoxin compounds or immunostimulating compound) being transported to presentation specificity The cell of antigen, in addition, sTCR can also with other molecules (e.g., anti-CD 3 antibodies) with reference to redirecting T cell, from And make the cell of its targeting presentation specific antigen.The present invention also obtains the solubility for having specificity to RHAMM antigens small peptide TCR。
To obtain sTCR, on the one hand, TCR of the present invention can be introduced between the residue of itself α and β chain constant domain The TCR of artificial disulfide bond.Cysteine residues form artificial interchain disulfide bond between α the and β chain constant domains of the TCR.Half Guang Histidine residue can be substituted in other amino acid residues of appropriate site in natural TCR to form artificial interchain disulfide bond.For example, Substitute the cysteine residues of the Thr48 of TRAC*01 exons 1s and the Ser57 of substitution TRBC1*01 or TRBC2*01 exons 1s To form disulfide bond.Cysteine residues are introduced to can also be to form other sites of disulfide bond：TRAC*01 exons 1s The Ser77 of Thr45 and TRBC1*01 or TRBC2*01 exons 1s；The Tyr10 and TRBC1*01 of TRAC*01 exons 1s or The Ser17 of TRBC2*01 exons 1s；Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s Asp59；The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 exons 1s Arg53 and TRBC1*01 or TRBC2*01 exons 1s Ser54；The Pro89 and TRBC1*01 of TRAC*01 exons 1s or The Ala19 of TRBC2*01 exons 1s；Or Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s Glu20.I.e. cysteine residues are instead of any group of site in above-mentioned α and β chain constant domains.It can be in TCR constant domains of the present invention One or more C-terminals truncate most 50 or most 30 or most 15 or most 10 or most 8 or less Amino acid so that its not including cysteine residues come achieve the purpose that lack natural disulphide bonds, also can be by the way that day will be formed The cysteine residues of right disulfide bond sport another amino acid to reach above-mentioned purpose.
As described above, the TCR of the present invention may be embodied in the artificial disulfide bond introduced between the residue of itself α and β chain constant domain. It should be noted that the artificial disulfide bond with or without introducing described above between constant domain, it is constant that TCR of the invention can contain TRAC Domain sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences of TCR can It is connected by the natural disulphide bonds being present in TCR.
To obtain sTCR, on the other hand, TCR of the present invention is additionally included in the TCR that its hydrophobic core region is undergone mutation, The mutation of these hydrophobic core regions is preferably capable the stability-enhanced mutation for making sTCR of the present invention, such as in publication number Described in patent document for WO2014/206304.Such TCR can undergo mutation in its following hydrophobic core position of variable domain： (α and/or β chains) variable region amino acid the 11st, 13,19,21,53,76,89,91,94 and/or α chain J genes (TRAJ) small peptide Amino acid position the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptides amino acid position reciprocal is 2nd, 4,6 reciprocal, wherein ammonia The Position Number of base acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).People in the art Member knows above-mentioned international immunogenetics information system, and the amino acid residue that different TCR can be obtained according to the database exists Position Number in IMGT.
The TCR that hydrophobic core region is undergone mutation in the present invention can be by α and the β chain of a flexible peptide chain link TCR can Variable domain and the solvable single-stranded TCR of stability formed.It should be noted that in the present invention flexible peptide chain can be any suitable connection TCR α with The peptide chain of β chain variable domains.Single chain soluble TCR, α the chain variable domain amino acid sequence such as built in the embodiment of the present invention 4 For SEQ ID NO:32, the nucleotides sequence of coding is classified as SEQ ID NO:33；β chains variable domain amino acid sequence is SEQ ID NO: 34, the nucleotides sequence of coding is classified as SEQ ID NO:35.
The present invention TCR can also multivalence complex form provide.The present invention multivalent TCR complex include two, Three, the four or more TCR of the present invention polymers that are combined and are formed, can such as be generated with four dimerization domains of p53 The tetramer or multiple TCR of the present invention and another molecule with reference to and the compound that is formed.The TCR compounds of the present invention can be used for body Outer or tracking in vivo or targeting present the cell of specific antigen, it can also be used to generate other multivalence TCR with such application and answer Close the intermediate of object.
The TCR of the present invention can be used alone, and can also be combined with conjugate with covalent or other modes, preferably with covalently side Formula combines.The conjugate includes detectable and (for diagnostic purpose, is presented wherein the TCR is used to detect The presence of the cell of ILSLELMKL-HLA A0201 compounds), therapeutic agent, PK (protein kinase) modified parts or it is any more than The combination of these substances combines or coupling.
Detectable for diagnostic purposes includes but not limited to：Fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.
The therapeutic agent that can be combined or be coupled with TCR of the present invention includes but not limited to：1. radionuclide (Koppe etc., 2005, metastasis of cancer comment (Cancer metastasi s reviews) 24,539)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gi ll ies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Hal in etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc Segment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；5. antibody ScFv segments (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold medal Nano particle/nanometer rods (Lapotko etc., 2005, cancer communication (Cancer letters) 239,36；Huang etc., 2006, it is beautiful Chemical Society of state magazine (Journal of the American Chemical Society) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,11631)；9. magnetic nanosphere；10. pro-drug activation enzymes are (for example, DT- diaphorases (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
In addition, the TCR of the present invention can also be comprising the heterozygosis TCR being derived from more than a kind of species sequence.For example, it grinds Study carefully display Muridae TCR can more effectively to express than people TCR in human T-cell.Therefore, TCR of the present invention can include people's variable domain With the constant domain of mouse.The defects of this method is possible to trigger immune response.Therefore, when it is used for adoptive T cell treatment There should be regulation scheme to carry out immunosupress, to allow to express the implantation of the T cell of Muridae.
It is to be understood that amino acid name is represented using international single English alphabet or three English alphabets herein, amino The correspondence of the single English alphabet and three English alphabets of sour title is as follows：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys (C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser (S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
Nucleic acid molecules
The second aspect of the present invention provides the nucleic acid molecules of coding first aspect present invention TCR molecules or part thereof, institute It can be partly one or more CDR to state, the variable domain and α chains and/or β chains of α and/or β chains.
The nucleotide sequence for encoding first aspect present invention TCR molecule alpha chain CDR regions is as follows：
α CDR1-gaccgagtttcccagtcc (SEQ ID NO:16)
α CDR2-atatactccaatggtgac (SEQ ID NO:17)
α CDR3-gccgctacaaattccgggtatgcactcaac (SEQ ID NO:18)
The nucleotide sequence for encoding first aspect present invention TCR molecule β chain CDR regions is as follows：
β CDR1-ggaacatcaaaccccaac (SEQ ID NO:19)
β CDR2-tccgttggtattggc (SEQ ID NO:20)
β CDR3-ctgaaagtggccgggtttaatctgctcatg(SEQ ID NO:21)
Therefore, encoding the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chains of the present invention includes SEQ ID NO:16、SEQ ID NO:17 and SEQ ID NO:18 and/or the nucleotide sequences of nucleic acid molecules of the present invention of coding TCR β chains of the present invention include SEQ ID NO:19、SEQ ID NO:20 and SEQ ID NO:21.
The nucleotide sequence of nucleic acid molecules of the present invention can be it is single-stranded or double-stranded, the nucleic acid molecules can be RNA or DNA, and can include or not comprising introne.Preferably, the nucleotide sequence of nucleic acid molecules of the present invention does not include introne But polypeptide of the present invention can be encoded, such as the nucleotide sequence bag of the nucleic acid molecules of the present invention of coding TCR α chain variable domains of the present invention Include SEQ ID NO:2 and/or the nucleotide sequences of nucleic acid molecules of the present invention of coding TCR β chain variable domains of the present invention include SEQ ID NO:6.Alternatively, the nucleotide sequence of the nucleic acid molecules of the present invention of coding TCR α chain variable domains of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequences of nucleic acid molecules of the present invention of coding TCR β chain variable domains of the present invention include SEQ ID NO:35.More Preferably, the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:4 and/or SEQ ID NO:8.It is alternatively, of the invention The nucleotides sequence of nucleic acid molecules is classified as SEQ ID NO:31.
It is to be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.Therefore, compile The nucleotide sequence of code book invention TCR can variant identical with the attached nucleotide sequence shown in figure of the present invention or degeneracy.With One of example in the present invention illustrates that " variant of degeneracy " refer to that coding has SEQ ID NO:1 protein sequence, But with SEQ ID NO:The 2 differentiated nucleotide sequence of sequence.
Nucleotide sequence can be through codon optimization.Different cells is above different in the utilization of specific codon , can the codon in sequence be changed to increase expression quantity according to the type of cell.Mammalian cell and various other The codon usage table of biology is well known to those skilled in the art.
The present invention nucleic acid molecules full length sequence or its segment usually can with but be not limited to PCR amplification method, recombination method or Artificial synthesized method obtains.At present, it is already possible to completely by chemical synthesis come obtain encoding TCR of the present invention (or its segment, Or derivatives thereof) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or Such as carrier) and cell in.DNA can be coding strand or noncoding strand.
Carrier
It, can in vivo or body including expression vector the invention further relates to the carrier for the nucleic acid molecules for including the present invention The construct of outer expression.Common carrier includes bacterial plasmid, bacteriophage and animals and plants virus.
Viral delivery systems include but not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, Retroviral vector, slow virus carrier, baculovirus vector.
Preferably, the nucleotide of the present invention can be transferred in cell by carrier, such as in T cell so that the cell table Up to the TCR of RHAMM antigentic specificities.Ideally, which can should express continual high levels in T cell.
Cell
The invention further relates to the host cells generated with the carrier or coded sequence of the present invention through genetic engineering.The host The nucleic acid molecules of the present invention are integrated in carrier or chromosome containing the present invention in cell.Host cell is selected from：Prokaryotic cell And eukaryocyte, such as Escherichia coli, yeast cells, Chinese hamster ovary celI etc..
In addition, the separated cell of the TCR present invention additionally comprises the expression present invention, particularly T cell.The T cell can spread out It is born from from the separated T cell of subject or can be the separated mixed cellularity group from subject, such as periphery hemolymph is thin The part of born of the same parents (PBL) group.Such as, which can be isolated from peripheral blood mononuclear cells (PBMC), can be CD4+Helper cell Or CD8+Cytotoxic T cell.The cell can be in CD4+Helper cell/CD8+In the mixing group of cytotoxic T cell.Generally Ground, the cell can use antibody (e.g., the antibody of anti-CD3 or anti-CD28) to activate, to allow them to easily receive to turn Dye, such as transfected with the carrier comprising the nucleotide sequence for encoding TCR molecules of the present invention.
Alternatively, cell of the invention can also be or derived from stem cell, such as candidate stem cell (HSC).Gene is turned Moving to HSC will not cause in cell surface expression TCR, because stem cell surface does not express CD3 molecules.However, when stem cell point It turns to when migrating to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecules will start in thymocyte The surface expression introducing TCR molecules.
There are many method be suitable for the DNA or RNA of coding TCR of the present invention carry out T cell transfection (e.g., the such as Robbins, (2008)J.Immunol.180:6116-6131).The T cell of expression TCR of the present invention can be used for adoptive immunotherapy.Ability Field technique personnel understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat Rev for carrying out adoptive treatment Cancer8(4)：299-308).
RHAMM antigen-related diseases
The invention further relates to being treated in subject and/or preventing the method with RHAMM relevant diseases, including adoptive Shift the step of RHAMM specific T-cells are to the subject.The RHAMM specific T-cells can recognize that ILSLELMKL-HLA A0201 compounds.
The T cell of the RHAMM specificity of the present invention can be used for treating any presentation RHAMM antigen small peptides ILSLELMKL- The RHAMM relevant diseases of HLA A0201 compounds.Including but not limited to acute myeloid system leukaemia, chronic myelocytic system are white Blood disease, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Huppert's disease, melanoma, colon cancer, mammary gland Cancer, kidney, stomach cancer, transitional cell carcinoma of the bladder, prostate cancer, oral squamous cell carcinoma and head and neck squamous cell carcinoma.
Therapy
Can by separation suffer from the patient of RHAMM antigen-related diseases or the T cell of volunteer, and by the present invention TCR is imported in above-mentioned T cell, then feeds back in patient body to treat by the cell that these genetic engineerings are modified.Therefore, The present invention provides a kind of method for treating RHAMM relevant diseases, including by the T cell of separated expression TCR of the present invention, preferably Ground, the T cell in itself, are input in patient body from patient.Usually, the T cell of (1) separation patient is included, (2) are with originally Invention nucleic acid molecules or the nucleic acid molecules ex vivo transduction T cell that can encode TCR molecules of the present invention, (3) modify genetic engineering T cell be input in patient body.The quantity of separation, transfection and the cell fed back can be determined by doctor.
Main advantages of the present invention are：
(1) TCR of the invention can be specifically bound with RHAMM antigen small peptide composite I LSLELMKL-HLA A0201, While the cell for the TCR of the present invention that transduceed can have very strong lethal effect by specific activation and to target cell.
Following specific embodiment, the present invention is further explained.It is to be understood that these embodiments be merely to illustrate the present invention and It is not used in and limits the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to normal condition, Such as (Sambrook and Russell et al., molecular cloning：Laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing houses) described in condition or according to the condition proposed by manufacturer.Unless In addition illustrate, otherwise percentage and number are calculated by weight.Unless otherwise stated, otherwise percentage and number are calculated by weight. Experiment material and reagent used can obtain unless otherwise instructed from commercially available channel in following embodiment.
Embodiment 1 clones RHAMM antigen small peptide specific T-cells
Come from genotype using small peptide ILSLELMKL (Beijing SBS Genetech gene technology Co., Ltd) stimulations are synthesized and be The peripheral blood lymphocytes (PBL) of the healthy volunteer of HLA-A0201.By ILSLELMKL small peptides with carrying biotin labeling HLA-A*0201 renaturation prepares pHLA monoploid.These monoploid are combined into the Streptavidin (BD companies) marked with PE The tetramer of PE marks, sorts the tetramer and anti-CD8-APC double positive cells.The cell of sorting is expanded, and as stated above Secondary sorting is carried out, then carries out monoclonal with limiting dilution assay.Monoclonal cell tetramer staining, what is screened is double positive Clone is as shown in Figure 3.
Embodiment 2 obtains the tcr gene of RHAMM antigen small peptide specific T-cell clones and the structure of carrier
Use Quick-RNATMThe antigen small peptide screened in MiniPrep (ZYMO research) extracting embodiments 1 The total serum IgE of ILSLELMKL specificity, HLA-A0201 restricted T cell clone.The synthesis of cDNA is using clontech's SMART RACE cDNA amplification kits, the primer of use are designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned It is sequenced on to carrier T (TAKARA).It should be noted that the sequence is complementary series, not comprising introne.Through sequencing, this pair of sun Property clonal expression TCR α chains and β chain-orderings structure respectively as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e With Fig. 1 f be respectively TCR α chains variable domain amino acid sequence, TCR α chain variable domains nucleotide sequence, TCR α chain amino acid sequences, TCR α chains nucleotide sequence, the TCR α chain amino acid sequences with targeting sequencing and the TCR α chain nucleotide with targeting sequencing Sequence；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chains variable domain amino acid sequence, TCR β chain variable domains Nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, have targeting sequencing TCR β chain amino acid sequences with And the TCR β chain nucleotide sequences with targeting sequencing.
Identified, α chains include the CDR with following amino acid sequence：
α CDR1-DRVSQS (SEQ ID NO:10)
α CDR2-IYSNGD (SEQ ID NO:11)
α CDR3-AATNSGYALN (SEQ ID NO:12)
β chains include the CDR with following amino acid sequence：
β CDR1-GTSNPN (SEQ ID NO:13)
β CDR2-SVGIG (SEQ ID NO:14)
β CDR3-AWSVDGAEQY (SEQ ID NO:15)
The full-length gene of TCR α chains and β chains is cloned into Lentiviral respectively by being overlapped (overlap) PCR pLenti(addgene).Specially：The full-length gene of TCR α chains and TCR β chains is attached to obtain TCR with overlap PCR α -2A-TCR β segments.Lentiviral and TCR α -2A-TCR β digestions are connected to obtain pLenti-RHAMMTRA-2A- TRB-IRES-NGFR plasmids.It is used as control, while the also slow virus carrier pLenti-eGFP of structure expression eGFP.Afterwards again Pseudovirus is packed with 293T.Detailed process is as described in Example 7.
Expression, refolding and the purifying of the solvable TCR of embodiment 3RHAMM antigens small peptide specificity
To obtain soluble TCR molecules, α the and β chains of TCR molecules of the invention can only include its variable domain and portion respectively Divide constant domain, and introduce a cysteine residues in the constant domain of α and β chains respectively to form artificial interchain disulfide bond, The position for introducing cysteine residues is respectively the Ser57 of the Thr48 and TRBC2*01 exons 1s of TRAC*01 exons 1s；Its α The amino acid sequence of chain is distinguished as shown in figures 4 a and 4b with nucleotide sequence, the amino acid sequence and nucleotide sequence of β chains Respectively as shown in figure 5 a and 5b, the cysteine residues of introducing are represented with overstriking letter.Pass through《Molecular Cloning: A Laboratory room hand Volume》Described in (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) The objective gene sequence of above-mentioned TCR α and β chains are inserted respectively into expression vector pET28a+ by standard method after synthesis (Novagene), the cloning site of upstream and downstream is NcoI and NotI respectively.Insert Fragment confirms errorless by sequencing.
The expression vector of TCR α and β chains is converted by chemical transformation into expression bacterium BL21 (DE3), bacterium respectively It is grown with LB culture solutions, in OD600It is induced when=0.6 with final concentration 0.5mM IPTG, the bag formed after α the and β chains expression of TCR Contain body to extract by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, forgive Body is finally dissolved in 6M guanidine hydrochlorides, 10mM dithiothreitol (DTT)s (DTT), 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris (pH 8.1) in.
Dissolved TCR α and β chains are with 1：1 mass ratio is quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Solution is placed in the deionized water of 10 times of volumes dialysis (4 DEG C) afterwards, 12 it is small when after change deionized water into buffer solution (20mM Tris, pH 8.0) continue at 4 DEG C dialysis 12 it is small when.Solution after the completion of dialysis after 0.45 μM of membrane filtration, by the moon from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains the successful α and β dimers of renaturation TCR is confirmed by SDS-PAGE glue.TCR then by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) it is further purified.TCR purity after purification is measured by SDS-PAGE is more than 90%, and concentration is by BCA methods It determines.The SDS-PAGE glue figures for the sTCR that the present invention obtains are as shown in Figure 6.
The generation of the soluble single-chain T CR of embodiment 4RHAMM antigens small peptide specificity
According to patent document WO2014/206304, using the method for rite-directed mutagenesis by TCR α and β in embodiment 2 The variable domain of chain has been built into the soluble single-chain T CR molecules of a stabilization with flexible small peptide (linker) connection.This is single-stranded Amino acid sequence and the nucleotide sequence difference of TCR molecules are as shown in figs. 7 a and 7b.The amino acid sequence of its α chain variable domain and Nucleotide sequence difference is as figures 8 a and 8 b show；The amino acid sequence and nucleotide sequence of its β chain variable domain are respectively such as Fig. 9 a Shown in Fig. 9 b；Amino acid sequence and the nucleotide sequence difference of its linker sequence are as as-shown-in figures 10 a and 10b.
By target gene through I double digestion of Nco I and Not, it is connected with by the pET28a carriers of I double digestion of Nco I and Not. Connection product converts and to E.coli DH5 α, is coated with the LB tablets containing kanamycins, 37 DEG C of inversion overnight incubations, positive gram of picking Grand progress PCR screenings, are sequenced positive recombinant, determine that sequence correctly extracts recombinant plasmid transformed to E.coli afterwards BL21 (DE3), for expressing.
Expression, renaturation and the purifying of the soluble single-chain T CR of embodiment 5RHAMM antigens small peptide specificity
BL21 (DE 3) bacterium colony containing recombinant plasmid pET28a- template strands prepared in embodiment 4 is all inoculated in In LB culture mediums containing kanamycins, 37 DEG C culture to OD600 be 0.6-0.8, add in IPTG to final concentration of 0.5mM, 37 DEG C continue to cultivate 4h.5000rpm centrifugation 15min harvest cell pellets, are cracked with Bugbuster Master Mix (Merck) Cell pellet, 6000rpm centrifugation 15min recycling inclusion bodys, then washed to remove cell with Bugbuster (Merck) Fragment and membrane component, 6000rpm centrifugation 15min, collect inclusion body.By solubilization of inclusion bodies in buffer solution (20mM Tris-HCl PH 8.0,8M urea) in, high speed centrifugation removal insoluble matter, supernatant is standby in -80 DEG C of preservations with being dispensed after BCA standard measures With.
In the single-stranded TCR inclusion body proteins dissolved to 5mg, 2.5mL buffer solutions (6M Gua-HCl, 50mM Tris- is added in HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration of 10mM, 37 DEG C of processing 30min.With injection Device is to 125mL renaturation buffers (100mM Tris-HCl pH 8.1,0.4M L-arginines, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) in treated single-stranded TCR, 4 DEG C of stirrings are added dropwise Then renaturation solution is packed into the cellulose membrane bag filter that interception is 4kDa by 10min, bag filter is placed in the water of 1L precoolings, 4 DEG C It is slowly stirred overnight.17 it is small when after, dialyzate changes into the buffer solutions (20mM Tris-HCl pH 8.0) of 1L precoolings, 4 DEG C after Then continuous dialysis 8h changes dialyzate into identical fresh buffer and continues dialysed overnight.17 it is small when after, sample through 0.45 μm filter Membrane filtration, by anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgas, with 20mM Tris-HCl The 0-1M NaCl linear gradient elution liquid purifying proteins that pH 8.0 is prepared, the elution fraction of collection carry out SDS-PAGE analyses, bag It is further carried out after component concentration containing single-stranded TCR with solvent resistant column (Superdex 7510/300, GE Healthcare) Purifying, target components also carry out SDS-PAGE analyses.
Elution fraction for BIAcore analyses further tests its purity using gel filtration.Condition is：Chromatographic column Agilent Bio SEC-3 (7.8 × 300mm of 300A, φ), mobile phase be 150mM phosphate buffers, flow velocity 0.5mL/min, 25 DEG C of column temperature, ultraviolet detection wavelength 214nm.
The SDS-PAGE glue figures for the soluble single-chain T CR that the present invention obtains are as shown in figure 11.
Embodiment 6 combines characterization
BIAcore is analyzed
It can be special with ILSLELMKL-HLA A0201 compounds this example demonstrated soluble TCR molecules of the present invention The opposite sex combines.
Detected using BIAcore T200 real-time analyzers the TCR molecules that are obtained in embodiment 3 and embodiment 5 with The combination activity of ILSLELMKL-HLA A0201 compounds.It is slow that the antibody (GenScript) of anti-Streptavidin is added in into coupling Then antibody is flowed through the CM5 chips activated in advance with EDC and NHS, made by fliud flushing (10mM sodium-acetate buffers, pH 4.77) Antibody is fixed on chip surface, finally closes unreacted activating surface with the hydrochloric acid solution of ethanolamine, completes coupling process, even Connection horizontal about 15,000RU.
The Streptavidin of low concentration is made to flow through the chip surface of coated antibody, then by ILSLELMKL-HLA A0201 compounds flow through sense channel, and another passage is as reference channel, then by the biotin of 0.05mM with the stream of 10 μ L/min Speed flows through chip 2min, closes the remaining binding site of Streptavidin.
The preparation process of above-mentioned ILSLELMKL-HLA A0201 compounds is as follows：
A. purify
The E.coli bacterium solutions of 100ml induced expressions heavy chain or light chain are collected, 10ml is used after 4 DEG C of 8000g centrifuge 10min PBS washing thallines are once, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) afterwards Thalline is resuspended in concussion, and is rotated in room temperature and be incubated 20min, after 4 DEG C, 6000g centrifugation 15min, supernatant discarding, collection forgives Body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Add 30ml The BugBuster of 10 times of dilution, mixing, 4 DEG C of 6000g centrifuge 15min；Supernatant discarding, the BugBuster that 30ml is added to dilute 10 times Inclusion body, mixing is resuspended, 4 DEG C of 6000g centrifuge 15min, are repeated twice, and add 30ml 20mM Tris-HCl pH 8.0 that bag is resuspended Contain body, mixing, 4 DEG C of 6000g centrifuge 15min, finally dissolve inclusion body, SDS-PAGE detections with 20mM Tris-HCl 8M urea Inclusion body purity, BCA kits survey concentration.
B. renaturation
The small peptide ILSLELMKL (Beijing SBS Genetech gene technology Co., Ltd) of synthesis is dissolved in DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain is dissolved with 8M urea, 20mM Tris pH 8.0,10mM DTT, is added in before renaturation 3M guanidine hydrochlorides, 10mM sodium acetates, 10mM EDTA are further denatured.ILSLELMKL peptides are added in into renaturation with 25mg/L (final concentration) Buffer solution (0.4M L-arginines, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced forms Glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, Heavy chain adds in three times, 8h/ times), renaturation carries out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detections renaturation success.
C. purified after renaturation
Make dialysis with the 20mM Tris pH 8.0 of 10 volumes to replace renaturation buffer, at least replace buffer solution and come twice Fully reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Instrument (the general electricity of GE is purified using Akta Gas company), the 0-400mM NaCl linear gradients liquid elution albumen that 20mM Tris pH 8.0 are prepared, pMHC is about in 250mM It is eluted at NaCl, collects all peak components, SDS-PAGE detection purity.
D. biotinylation
With Millipore super filter tubes by the pMHC molecular concentrations of purifying, while it is 20mM Tris pH by buffer exchange 8.0, then add in biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D- Biotin, 100 μ g/ml BirA enzymes (GST-BirA), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detections biotinylation Completely.
E. the compound after purifying biological elementization
PMHC molecular concentrations after biotinylation is marked with Millipore super filter tubes are to 1ml, using gel permeation chromatography The pMHC of purifying biological elementization, using Akta purifying instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibrations HiPrepTM 16/60S200HR columns (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated loading 1ml, then with PBS with 1ml/ Min flow velocitys elute.Biotinylated pMHC molecules occur in about 55ml as unimodal elution.Merge the group containing protein Point, it is concentrated with Millipore super filter tubes, BCA methods (Thermo) measure protein concentration, add in protease inhibitors cocktail (Roche) packing of biotinylated pMHC molecules is stored in -80 DEG C.
Using BIAcore Evaluation software computational dynamics parameters, obtain the TCR molecules of solubility of the invention with And the kinetic profile point that the soluble single-chain T CR molecules of the invention built are combined with ILSLELMKL-HLA A0201 compounds Not as shown in Figure 12 and Figure 13.Meanwhile also using the above method have detected solubility of the invention TCR molecules and other antigens it is short Peptide includes the combination activity of GILGFVFTL-HLA A0201 compounds and SLLMWITQC-HLA A0201 compounds, as a result shows Show TCR molecules of the present invention with other irrelevant antigens without combination.
Embodiment 7RHAMM antigen small peptide specificity TCRs slow virus is packed to be transfected with primary T cells
(a) (Express-In-mediated transient are transiently transfected by the quick mediation of 293T cells Transfection slow virus) is prepared
Utilize slow virus of the third generation slow virus packaging system packaging containing the gene of TCR needed for coding.It is situated between using quick Lead transient transfection (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)), with 4 kinds of plasmid (one kind containing pLenti-RHAMMTRA-2A-TRB-IRES-NGFR described in embodiment 2 Slow virus carrier and 3 kinds of plasmids containing other components necessary to structure infectiousness but non-replicating lentiviral particle) turn Contaminate 293T cells.
To be transfected, the 0th day kind cell, in 15 cm dishes, kind upper 1.7 × 107A 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 pseudovirus, by more than expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV and PMD.2G mixings, the dosage of a 15 cm diameter plates are 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 are 120 micrograms.Concrete operations are：Expression Plasmid and packaging plasmid add in 1800 microlitres of OPTI-MEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) 31985-070) culture mediums In be uniformly mixed, be stored at room temperature 5 minutes become DNA mixed liquors；Respective amount PEI is taken to be mixed with 1800 microlitres of OPTI-MEM culture mediums 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 3150 microlitres of OPTI-MEM culture mediums are added, it is added to has been converted to 11.25 milliliters of OPTI- after mixing In the 293T cells of MEM, culture dish is gently rocked, is uniformly mixed culture medium, cultivated under 37 DEG C/5%CO2.It is small to transfect 5-7 When, remove transfection media, change into containing 10% hyclone DMEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt)) complete medium is cultivated under 37 DEG C/5%CO2.Collect the training containing wrapped slow virus within 3rd and the 4th day Support base supernatant.For the slow virus of harvest packaging, collected culture supernatant 3000g is centrifuged 15 minutes removal cell fragments, It filters through 0.22 micron filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB), finally uses again 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, is frozen for -80 DEG C after decile packing.Pseudovirus sample is taken to carry out virus titer survey Fixed, step is with reference to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit specification.It is used as control, while also bag turns The pseudovirus of pLenti-eGFP.
(b) with the lentiviruses transduction primary T cells of the T cell receptor gene containing RHAMM antigens small peptide specificity
It is separated to CD8+T cells from the blood of healthy volunteer, then with the lentiviruses transduction packed.It is thin to count these Born of the same parents, in 48 orifice plates, containing 50IU/ml IL-2 and 10ng/ml IL-7 containing 10%FBS (Ji Bu can company (Gibco), Catalog number (Cat.No.) C10010500BT) 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture mediums in 1 × 106 A cells/ml (0.5 milliliter/hole) and AntiCD3 McAb/CD28 antibody-coating globule (T cell amplified matter, the life pre-washed Technologies, catalog number (Cat.No.) 11452D) be incubated overnight stimulates altogether, cell：Pearl=3：1.
After stimulating overnight, according to the virus titer that p24ELISA kits are measured, added in the ratio of MOI=10 dense The slow virus of the RHAMM antigen small peptide specific t-cell receptor genes of contracting, 32 DEG C, when 900g centrifugations infection 1 is small.Infection finishes Slow-virus infection liquid is removed afterwards, with the culture of 1640 containing the 10%FBS base weight containing 50IU/ml IL-2 and 10ng/ml IL-7 Cell is hanged, is cultivated 3 days under 37 DEG C/5%CO2.Next day carries out the second wheel infection in the same way.Second of transduction counts after 3 days Cell, diluting cells to 0.5 × 106A cells/ml.It counts a cell within every two days, replaces or add in and contain 50IU/ml The fresh culture of IL-2 and 10ng/ml IL-7 maintains cell 0.5 × 106-1×106A cells/ml.It was opened from the 3rd day Begin through flow cytometry cell, for function test (for example, the ELISPOT of IFN-γ release and non-since the 5th day Radioactivity cytotoxicity detects).Since the 10th day or when cell slows down division and size becomes smaller, stored frozen decile is thin Born of the same parents, at least 4 × 106A cell/pipe (1 × 107A cells/ml, 90%FBS/10%DMSO).
Embodiment 8 transduce TCR of the present invention T cell specific activation experiment
ELISPOT schemes
Tests below is carried out to prove activating reaction of the T cell to target cell specificity of TCR- transductions.Utilize ELISPOT Readout of the IFN-γ yield of testing inspection as t cell activation.
Reagent
Test medium：10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), (Ji Bu can by RPMI1640 Company (Gibco), catalog number (Cat.No.) C11875500bt)
Lavation buffer solution (PBST)：0.01M PBS/0.05% polysorbas20s
PBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT)
96 orifice plates of PVDF ELISPOT (Merck Mi Libo (Merck Mill ipore), catalog number (Cat.No.) MSIPS4510)
People's IFN-γ ELISPOT PVDF- enzyme reagent kits (BD) (capture equipped with required every other reagent and detection are anti- Body, Streptavidin-alkaline phosphatase and BCIP/NBT solution)
Method
It is prepared by target cell
Target cell used is T2 cells in this experiment.Target cell is prepared in assay medium：Target cell concentration is adjusted to 2.0×105A/milliliter takes 100 microlitres per hole so as to obtain 2.0 × 104A cells/well.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is to express RHAMM antigens of the present invention through flow cytometry in embodiment 7 The CD8+T cells of small peptide specificity TCR, and using same volunteer and the CD8+T of GFP is expressed as negative control effector cell. T cell is stimulated with AntiCD3 McAb/CD28 coating pearls (T cell amplified matter, life technologies), RHAMM antigens are short with carrying The lentiviruses transduction (according to embodiment 7) of peptide specific tcr gene, in containing containing 50IU/ml IL-2 and 10ng/ml IL-7 Then these cells are placed in test medium, 300g is normal by the 1640 culture mediums amplification of 10%FBS until 9-12 days after transduction Temperature centrifugation is washed for 10 minutes.Then by cell with 2 × required final concentration is resuspended in test medium.Similary processing is negative Compare effector cell.
It is prepared by small peptide solution
Corresponding small peptide is added in corresponding target cell (T2) experimental group, wherein specific small peptide is RHAMM (sequences： ILSLELMKL, number：P30A), non-specific small peptide is two small peptides for coming from antigen NY-ESO-1, and number is respectively P1A And the small peptide of P47 and 1 MAGE-A1, number P18.Make final concentration of 1 μ g/ml of the small peptide in ELISPOT orifice plates.
ELISPOT
According to the specification that manufacturer provides, prepare orifice plate as described below：1 is pressed with 10 milliliters of sterile PBS of every block of plate：200 It dilutes anti-human IFN-γ and captures antibody, 100 microlitres of dilution then is captured antibody etc. point adds in each hole.Orifice plate is incubated at 4 DEG C Overnight.After incubation, orifice plate is washed to remove extra capture antibody.Add in the RPMI 1640 that 10%FBS is contained in 100 microlitres/hole Culture medium, and incubate at room temperature orifice plate 2 it is small when to close orifice plate.Then culture medium is washed away from orifice plate, by light on paper Bullet and ELISPOT orifice plates are patted to remove the lavation buffer solution of any remnants.
RHAMM CD8+T cells (T cell of the special TCR transductions of RHAMM antigen small peptides, effector cell), CD8+T cells (negative control effector cell) and T2 cells (target cell).
Then all components of experiment are added in by ELISPOT orifice plates using following order：
100 microlitres of target cell 2*105A cells/ml (obtaining about 2*104 target cell/hole in total).
100 microlitres of effector cell (1*104A negative control effector cell/hole and RHAMM TCR positive T cells/hole).
All holes prepare addition in duplicate.
Then (37 DEG C/5%CO overnight of orifice plate is incubated2) second day, culture medium is abandoned, orifice plate is washed 2 times with distilled water, then uses Lavation buffer solution washs 3 times, pats to remove remaining lavation buffer solution on paper handkerchief.Then pressed with the PBS containing 10%FBS 1：200 dilution detection antibody, each hole is added in by 100 microlitres/hole.When incubation orifice plate 2 is small at room temperature, then washed with lavation buffer solution 3 times, orifice plate is patted on paper handkerchief to remove excessive lavation buffer solution.
1 is pressed with the PBS containing 10%FBS：100 dilution Streptavidin-alkaline phosphatases, by 100 microlitres of diluted chains Mould Avidin-alkaline phosphatase adds in each hole and when incubation orifice plate 1 is small at room temperature.Then 4 PBS are washed with lavation buffer solution Washing 2 times, pats orifice plate to remove excessive lavation buffer solution and PBS on paper handkerchief.Kit is added in after washing to provide 100 microlitres/hole of BCIP/NBT solution develop.It is protected from light during development with masking foil covering orifice plate, stands 5-15 minutes. The spot of conventional detection development orifice plate during this period determines to terminate the Best Times of reaction.Remove BCIP/NBT solution and with pair It steams water and rinses orifice plate to stop developing reaction, dry, then remove orifice plate bottom, be dried at room temperature for orifice plate until each hole It is completely dried, recycles immunodotting plate count meter (CTL, Celltech Ltd. (Cellular Technology Limited the)) spot that counterdie is formed in counting orifice.
As a result
The T cell (as described above) for examining TCR transductions of the present invention is tested by ELISPOT to resist load specificity RHAMM The IFN-γ release that the target cell of former small peptide ILSLELMKL and non-specific target cell react.Utilize graphpad prism6 Draw the ELSPOT amount of speckle observed in each hole.
Experimental result is as shown in figure 14, and the T cell for the TCR of the present invention that transduces only has the target cell for loading its special small peptide Activating reaction, and other are not loaded with the target cell of small peptide and the unrelated small peptide of load substantially without activating reaction.It is also possible to Find out that the activating reaction of the T cell for the TCR of the present invention that do not transduce is very poor.
Embodiment 9 transduce TCR of the present invention T cell non-radioactive cell toxicity test
The experiment is51Cr discharges the colorimetric alternate test of cell toxicity test, quantitative determines the breast discharged after cell cracking Acidohydrogenase (LDH).The LDH of release in the medium is detected using the enzyme reaction of coupling in 30 minutes, LDH can in enzyme reaction A kind of tetrazolium salts (INT) is made to be converted into red formazans (formazan).The amount of the red product of generation and the cell number of cracking It is directly proportional.Collection 490nm visible ray extinction Value Datas can be collected with 96 hole read plates of standard.
Material
CytoToxNon-radioactive cell toxicity detection (Pu Luomaige companies, G1780) contains substrate mixture, examination Test buffer solution, cracked solution and stop buffer.
Test medium：10%FBS (heat-inactivated, Ji Bu can company (Gibco)), without phenol red 90%RPMI1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt).
96 hole tissue culturing plate of micropore round bottom (Nucor Corporation (Nunc), catalog number (Cat.No.) 163320)
96 hole immuno plate Maxisorb (Nucor Corporation (Nunc), catalog number (Cat.No.) 442404)
Method
It is prepared by target cell
Target cell used is T2 cells in this experiment, and target cell is prepared in assay medium：Target cell concentration is adjusted to 1 ×106A/milliliter takes 100 microlitres per hole so as to obtain 1 × 105A cells/well.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is special through flow cytometry expression RHAMM antigens small peptide in embodiment 7 The CD8+T cells of different in nature TCR.Effector cell uses 1 with target cell ratio：1(1.0×106A/milliliter takes 100 microlitres per hole So as to obtain 1.0 × 105A cells/well) and 3：1(3.0×106A/milliliter takes 100 microlitres per hole so as to obtain 3.0 × 105It is a thin Born of the same parents/hole).
It is prepared by small peptide solution
Corresponding small peptide is added in corresponding target cell (T2) experimental group, wherein specific small peptide is RHAMM (sequences： ILSLELMKL, number：P30A), non-specific small peptide is the small peptide (number for coming from MAGE-A1：P18), small peptide is made to exist Final concentration of 1 μ g/ml in ELISPOT orifice plates.
(a) small peptide that various concentration is loaded by target cell (T2) detects effector cell's killing ability
Experiment prepares
All components of experiment are added in by 96 hole tissue culturing plate of micropore round bottom using following order：
- 100ul target cells (preparing as described above) add in each hole
- 100ul effector cells (preparing as described above) add in each hole
Control group is prepared as described below：
- do not load small peptide experimental group (not plus T2 cells of peptide)：Contain 100ul effector cell and 100ul target cells.
The spontaneous release of-effector cell：Only 100ul effector cell.
The spontaneous release of-target cell：Only 100ul target cells.
The maximum release of-target cell：Only 100ul target cells.
- volume correction compares：Only 200ul culture mediums.
- reagent culture medium compares：Only 200ul culture mediums.
All holes prepare in duplicate, and final volume is 200ul (inadequate is supplied with culture medium).
37 DEG C are incubated overnight.Before collecting all hole supernatants, in the maximum release control wells of target cell and volume correction control group After 20 μ l cell pyrolysis liquid mixings of middle addition, 37 DEG C incubate 45 minutes.
Tablet is centrifuged in 250g 4 minutes.The 50ul supernatants in each hole of test panel are transferred to 96 hole immuno plates The corresponding aperture of Maxisorb plates.Using buffer solution (12ml) reconstituted substrate mixture is tested, then add 50ul to each hole of tablet.It is flat Plate is protected from light incubation at room temperature 30 minutes after closeing the lid.50ul stop baths are added in into each hole of tablet to terminate reaction.It adds in and terminates After solution 1 it is small when inside counting record 490nm absorbance.
Result of calculation
From experimental group, the spontaneous release group of target cell and effector cell culture medium is deducted from all absorbances of release group The absorbance of background.
It brings the corrected value of above-mentioned middle acquisition into formula below, calculates each effect target than generated cytotoxicity Percentage.
% cytotoxicity=100 × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximum-volume correction Control-target cell is spontaneous)
As a result
By the non-radioactive cell toxicity detection T cell (as described above) for examining TCR transductions of the present invention to loading RHAMM The LDH releases that the target cell of antigen small peptide ILSLELMKL and non-specific target cell react.Utilize graphpad prism6 Draw 490nm visible rays light absorption value in each hole.
Experimental data statistical result is as shown in figure 15, and the T cell for the TCR of the present invention that transduces is only to having loaded small peptide The specific killing action of the T2 target cells of ILSLELMKL (RHAMM).And the target to not loading small peptide or the unrelated small peptide of load is thin Born of the same parents are without apparent lethal effect.And the T cell killing effect for the TCR of the present invention that do not transduce is very poor.
(b) load small peptide by different target cells and detect effector cell's specific killing ability
It is prepared by target cell
This experiment target cell used is IM9 (multiple myeloma cell line) and K562 (myeloma cell line), with new Test medium cell is resuspended to 1.0 × 106A/milliliter takes 100 microlitres per hole so as to obtain 1.0 × 105A cells/well.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is to express RHAMM specificity TCRs through flow cytometry in embodiment 3 CD8+T cells.Effector cell uses 1 with target cell ratio：1(1.0×106A/milliliter takes 100 microlitres per hole so as to obtain 1.0×105A cells/well) and 3：1(3.0×106A/milliliter takes 100 microlitres per hole so as to obtain 3.0 × 105A cells/well).
Experiment prepares
All components of experiment are added in by 96 hole tissue culturing plate of micropore round bottom using following order：
- 100ul target cells (preparing as described above) add in each hole
- 100ul effector cells (preparing as described above) add in each hole
Control group is prepared as described below：
- do not load small peptide experimental group：Contain 100ul effector cell and 100ul target cells.
The spontaneous release of-effector cell：Only 100ul effector cell.
The spontaneous release of-target cell：Only 100ul target cells.
The maximum release of-target cell：Only 100ul target cells.
- volume correction compares：Only 200ul culture mediums.
- reagent culture medium compares：Only 200ul culture mediums.
All holes prepare in duplicate, and final volume is 200ul (inadequate is supplied with culture medium).
Cell culture, sample treatment and detection and result of calculation method are identical with the present embodiment (a).
As a result
Figure 16 shows that the CD8+T cells for expressing TCR of the present invention have specific killing action to specificity target cell IM9. And to non-specific target cell K562 without lethal effect.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited It encloses.
Sequence table
<110>Guangzhou Xiangxue Pharmaceutical Co
<120>Identify the T cell receptor of RHAMM antigen small peptides
<130> P2016-1667
<150> 201510228111.2
<151> 2015-05-06
<160> 37
<170> PatentIn version 3.5
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Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
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Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
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Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
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Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
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Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Asn Ser Gly Tyr
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Ala Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro His
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cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
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gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gctacaaatt ccgggtatgc actcaacttc 300
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Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
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Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
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Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
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Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Asn Ser Gly Tyr
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Ala Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro His Ile
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Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
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Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
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Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu
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Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
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Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys
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Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
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cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
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gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gctacaaatt ccgggtatgc actcaacttc 300
ggcaaaggca cctcgctgtt ggtcacaccc catatccaga accctgaccc tgccgtgtac 360
cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 420
caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 480
gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 540
tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 600
ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 660
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aatctgctca tgacgctgcg gctgtggtcc agctag 756
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Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val Gly
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Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys Lys
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Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Val Asp
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ggcaggggcc tccagctgct cttctactcc gttggtattg gccagatcag ctctgaggtg 180
ccccagaatc tctcagcctc cagaccccag gaccggcagt tcatcctgag ttctaagaag 240
ctccttctca gtgactctgg cttctatctc tgtgcctgga gtgtagatgg ggccgagcag 300
tacttcgggc cgggcaccag gctcacggtc aca 333
<210> 7
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<212> PRT
<213>Artificial sequence
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Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val Gly
1 5 10 15
Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro Asn
20 25 30
Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu Phe
35 40 45
Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn Leu
50 55 60
Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys Lys
65 70 75 80
Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Val Asp
85 90 95
Gly Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 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
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tctcagacta ttcatcaatg gccagcgacc ctggtgcagc ctgtgggcag cccgctctct 60
ctggagtgca ctgtggaggg aacatcaaac cccaacctat actggtaccg acaggctgca 120
ggcaggggcc tccagctgct cttctactcc gttggtattg gccagatcag ctctgaggtg 180
ccccagaatc tctcagcctc cagaccccag gaccggcagt tcatcctgag ttctaagaag 240
ctccttctca gtgactctgg cttctatctc tgtgcctgga gtgtagatgg ggccgagcag 300
tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 360
gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 420
gtatgcctgg 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 cgtcacccag atcgtcagcg ccgaggcctg gggtagagca 720
gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 780
gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 840
gccatggtca agagaaagga ttccagaggc tag 873
<210> 9
<211> 9
<212> PRT
<213> RHAMM
<400> 9
Ile Leu Ser Leu Glu Leu Met Lys Leu
1 5
<210> 10
<211> 6
<212> PRT
<213>Artificial sequence
<400> 10
Asp Arg Val Ser Gln Ser
1 5
<210> 11
<211> 6
<212> PRT
<213>Artificial sequence
<400> 11
Ile Tyr Ser Asn Gly Asp
1 5
<210> 12
<211> 10
<212> PRT
<213>Artificial sequence
<400> 12
Ala Ala Thr Asn Ser Gly Tyr Ala Leu Asn
1 5 10
<210> 13
<211> 6
<212> PRT
<213>Artificial sequence
<400> 13
Gly Thr Ser Asn Pro Asn
1 5
<210> 14
<211> 5
<212> PRT
<213>Artificial sequence
<400> 14
Ser Val Gly Ile Gly
1 5
<210> 15
<211> 10
<212> PRT
<213>Artificial sequence
<400> 15
Ala Trp Ser Val Asp Gly Ala Glu Gln Tyr
1 5 10
<210> 16
<211> 18
<212> DNA
<213>Artificial sequence
<400> 16
gaccgagttt cccagtcc 18
<210> 17
<211> 18
<212> DNA
<213>Artificial sequence
<400> 17
atatactcca atggtgac 18
<210> 18
<211> 30
<212> DNA
<213>Artificial sequence
<400> 18
gccgctacaa attccgggta tgcactcaac 30
<210> 19
<211> 18
<212> DNA
<213>Artificial sequence
<400> 19
ggaacatcaa accccaac 18
<210> 20
<211> 15
<212> DNA
<213>Artificial sequence
<400> 20
tccgttggta ttggc 15
<210> 21
<211> 30
<212> DNA
<213>Artificial sequence
<400> 21
ctgaaagtgg ccgggtttaa tctgctcatg 30
<210> 22
<211> 272
<212> PRT
<213>Artificial sequence
<400> 22
Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser
1 5 10 15
Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu
20 25 30
Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp
35 40 45
Arg Val Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser
50 55 60
Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly
65 70 75 80
Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu
85 90 95
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala
100 105 110
Thr Asn Ser Gly Tyr Ala Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu
115 120 125
Val Thr Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg
130 135 140
Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp
145 150 155 160
Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr
165 170 175
Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser
180 185 190
Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe
195 200 205
Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser
210 215 220
Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn
225 230 235 240
Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu
245 250 255
Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
260 265 270
<210> 23
<211> 819
<212> DNA
<213>Artificial sequence
<400> 23
atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60
caacagaagg aggtggagca gaattctgga cccctcagtg ttccagaggg agccattgcc 120
tctctcaact gcacttacag tgaccgagtt tcccagtcct tcttctggta cagacaatat 180
tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 240
aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 300
cagcccagtg attcagccac ctacctctgt gccgctacaa attccgggta tgcactcaac 360
ttcggcaaag gcacctcgct gttggtcaca ccccatatcc agaaccctga ccctgccgtg 420
taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480
tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 540
ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 600
gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 660
agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 720
ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780
tttaatctgc tcatgacgct gcggctgtgg tccagctag 819
<210> 24
<211> 307
<212> PRT
<213>Artificial sequence
<400> 24
Met Leu Cys Ser Leu Leu Ala Leu Leu Leu Gly Thr Phe Phe Gly Val
1 5 10 15
Arg Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val
20 25 30
Gly Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro
35 40 45
Asn Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu
50 55 60
Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn
65 70 75 80
Leu Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys
85 90 95
Lys Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Val
100 105 110
Asp Gly Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr
115 120 125
Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro
130 135 140
Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu
145 150 155 160
Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn
165 170 175
Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys
180 185 190
Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu
195 200 205
Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
210 215 220
Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
225 230 235 240
Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg
245 250 255
Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser
260 265 270
Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala
275 280 285
Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp
290 295 300
Ser Arg Gly
305
<210> 25
<211> 924
<212> DNA
<213>Artificial sequence
<400> 25
atgctctgct ctctccttgc ccttctcctg ggcactttct ttggggtcag atctcagact 60
attcatcaat ggccagcgac cctggtgcag cctgtgggca gcccgctctc tctggagtgc 120
actgtggagg gaacatcaaa ccccaaccta tactggtacc gacaggctgc aggcaggggc 180
ctccagctgc tcttctactc cgttggtatt ggccagatca gctctgaggt gccccagaat 240
ctctcagcct ccagacccca ggaccggcag ttcatcctga gttctaagaa gctccttctc 300
agtgactctg gcttctatct ctgtgcctgg agtgtagatg gggccgagca gtacttcggg 360
ccgggcacca ggctcacggt cacagaggac ctgaaaaacg tgttcccacc cgaggtcgct 420
gtgtttgagc catcagaagc agagatctcc cacacccaaa aggccacact ggtatgcctg 480
gccacaggct tctaccccga ccacgtggag ctgagctggt gggtgaatgg gaaggaggtg 540
cacagtgggg tcagcacaga cccgcagccc ctcaaggagc agcccgccct caatgactcc 600
agatactgcc tgagcagccg cctgagggtc tcggccacct tctggcagaa cccccgcaac 660
cacttccgct gtcaagtcca gttctacggg ctctcggaga atgacgagtg gacccaggat 720
agggccaaac ccgtcaccca gatcgtcagc gccgaggcct ggggtagagc agactgtggc 780
ttcacctccg agtcttacca gcaaggggtc ctgtctgcca ccatcctcta tgagatcttg 840
ctagggaagg ccaccttgta tgccgtgctg gtcagtgccc tcgtgctgat ggccatggtc 900
aagagaaagg attccagagg ctag 924
<210> 26
<211> 204
<212> PRT
<213>Artificial sequence
<400> 26
Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Thr Asn Ser Gly Tyr
85 90 95
Ala Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro His Ile
100 105 110
Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
115 120 125
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val
130 135 140
Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu
145 150 155 160
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser
165 170 175
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile
180 185 190
Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200
<210> 27
<211> 612
<212> DNA
<213>Artificial sequence
<400> 27
cagaaagaag tggaacagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
ctcaactgca cttacagtga ccgagtttcc cagtccttct tctggtacag acaatattct 120
gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gctacaaatt ccgggtatgc actcaacttc 300
ggcaaaggca cctcgctgtt ggtcacaccc catatccaga accctgaccc tgccgtgtac 360
cagctgagag actctaagtc gagtgacaag tctgtctgcc tattcaccga ttttgattct 420
caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa atgtgtgcta 480
gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 540
tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 600
ccagaaagtt cc 612
<210> 28
<211> 241
<212> PRT
<213>Artificial sequence
<400> 28
Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val Gly
1 5 10 15
Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro Asn
20 25 30
Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu Phe
35 40 45
Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn Leu
50 55 60
Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys Lys
65 70 75 80
Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Val Asp
85 90 95
Gly Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 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
<400> 29
agccagacca ttcatcaatg gccagcgacc ctggtgcagc ctgtgggcag cccgctctct 60
ctggagtgca ctgtggaggg aacatcaaac cccaacctat actggtaccg acaggctgca 120
ggcaggggcc tccagctgct cttctactcc gttggtattg gccagatcag ctctgaggtg 180
ccccagaatc tctcagcctc cagaccccag gaccggcagt tcatcctgag ttctaagaag 240
ctccttctca gtgactctgg cttctatctc tgtgcctgga gtgtagatgg ggccgagcag 300
tacttcgggc cgggcaccag gctcacggtc acggaggacc 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> 246
<212> PRT
<213>Artificial sequence
<400> 30
Ala Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu
1 5 10 15
Gly Glu Asn Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln
20 25 30
Ser Phe Phe Trp Tyr Arg Gln Tyr Pro Gly Lys Ser Pro Glu Leu Ile
35 40 45
Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala
50 55 60
Gln Leu Asp Lys Ser Ser Gln Tyr Val Ser Leu Glu Ile Arg Asp Ile
65 70 75 80
Gln Pro Asn Asp Ser Ala Thr Tyr Phe Cys Ala Ala Thr Asn Ser Gly
85 90 95
Tyr Ala Leu Asn Phe Gly Lys Gly Thr Lys Leu Ser Val His Asn Gly
100 105 110
Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly
115 120 125
Gly Ser Glu Gly Gly Thr Gly Ser Gln Thr Ile His Gln Thr Pro Arg
130 135 140
Thr Leu Ser Val Pro Thr Gly Ser Pro Val Thr Leu Glu Cys Thr Val
145 150 155 160
Glu Gly Thr Ser Asn Pro Asn Leu Tyr Trp Tyr Arg Gln Asp Pro Gly
165 170 175
Arg Gly Leu Arg Leu Leu Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser
180 185 190
Ser Glu Val Pro Gln Arg Tyr Ser Ala Ser Arg Pro Gln Asp Arg Gln
195 200 205
Phe Glu Leu Ser Ile Lys Lys Val Thr Pro Ser Asp Ser Ala Phe Tyr
210 215 220
Leu Cys Ala Trp Ser Val Asp Gly Ala Glu Gln Tyr Phe Gly Pro Gly
225 230 235 240
Thr Arg Leu Glu Val Asp
245
<210> 31
<211> 738
<212> DNA
<213>Artificial sequence
<400> 31
gcacaaaaag aagttgaaca aaactcaggc ccgctgtcgg tcccggaagg cgaaaatgtc 60
tcaatcaact gtacctactc agatcgtgtg tcacagtcgt ttttctggta tcgtcaatat 120
ccgggcaaaa gtccggaact gattatgtca atctattcga acggcgataa agaagacggt 180
cgtttcacgg cacagctgga taaaagctct caatatgtta gcctggaaat tcgcgatatt 240
cagccgaacg acagcgcgac gtatttttgc gcggccacca actctggcta cgccctgaat 300
ttcggcaaag gcaccaaact gtcagtccat aatggcggtg gcagcgaagg tggcggttct 360
gaaggcggtg gcagtgaagg tggcggttcc gaaggcggca ccggttctca gacgattcac 420
caaaccccgc gtacgctgtc agtgccgacc ggctcgccgg ttaccctgga atgtacggtc 480
gaaggcacct caaacccgaa tctgtattgg tatcgtcaag atccgggccg tggtctgcgc 540
ctgctgtttt attcggtggg cattggtcag atcagttccg aagttccgca acgctacagc 600
gcatctcgtc cgcaggaccg ccaatttgaa ctgagtatca aaaaagtcac gccgagtgat 660
tccgcattct atctgtgcgc ttggtccgtg gacggtgctg aacagtactt tggtccgggc 720
acccgtctgg aagttgat 738
<210> 32
<211> 111
<212> PRT
<213>Artificial sequence
<400> 32
Ala Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu
1 5 10 15
Gly Glu Asn Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln
20 25 30
Ser Phe Phe Trp Tyr Arg Gln Tyr Pro Gly Lys Ser Pro Glu Leu Ile
35 40 45
Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala
50 55 60
Gln Leu Asp Lys Ser Ser Gln Tyr Val Ser Leu Glu Ile Arg Asp Ile
65 70 75 80
Gln Pro Asn Asp Ser Ala Thr Tyr Phe Cys Ala Ala Thr Asn Ser Gly
85 90 95
Tyr Ala Leu Asn Phe Gly Lys Gly Thr Lys Leu Ser Val His Asn
100 105 110
<210> 33
<211> 333
<212> DNA
<213>Artificial sequence
<400> 33
gcacaaaaag aagttgaaca aaactcaggc ccgctgtcgg tcccggaagg cgaaaatgtc 60
tcaatcaact gtacctactc agatcgtgtg tcacagtcgt ttttctggta tcgtcaatat 120
ccgggcaaaa gtccggaact gattatgtca atctattcga acggcgataa agaagacggt 180
cgtttcacgg cacagctgga taaaagctct caatatgtta gcctggaaat tcgcgatatt 240
cagccgaacg acagcgcgac gtatttttgc gcggccacca actctggcta cgccctgaat 300
ttcggcaaag gcaccaaact gtcagtccat aat 333
<210> 34
<211> 111
<212> PRT
<213>Artificial sequence
<400> 34
Ser Gln Thr Ile His Gln Thr Pro Arg Thr Leu Ser Val Pro Thr Gly
1 5 10 15
Ser Pro Val Thr Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro Asn
20 25 30
Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Arg Gly Leu Arg Leu Leu Phe
35 40 45
Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Arg Tyr
50 55 60
Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Glu Leu Ser Ile Lys Lys
65 70 75 80
Val Thr Pro Ser Asp Ser Ala Phe Tyr Leu Cys Ala Trp Ser Val Asp
85 90 95
Gly Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Glu Val Asp
100 105 110
<210> 35
<211> 333
<212> DNA
<213>Artificial sequence
<400> 35
tctcagacga ttcaccaaac cccgcgtacg ctgtcagtgc cgaccggctc gccggttacc 60
ctggaatgta cggtcgaagg cacctcaaac ccgaatctgt attggtatcg tcaagatccg 120
ggccgtggtc tgcgcctgct gttttattcg gtgggcattg gtcagatcag ttccgaagtt 180
ccgcaacgct acagcgcatc tcgtccgcag gaccgccaat ttgaactgag tatcaaaaaa 240
gtcacgccga gtgattccgc attctatctg tgcgcttggt ccgtggacgg tgctgaacag 300
tactttggtc cgggcacccg tctggaagtt gat 333
<210> 36
<211> 24
<212> PRT
<213>Artificial sequence
<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
<400> 37
ggcggtggca gcgaaggtgg cggttctgaa ggcggtggca gtgaaggtgg cggttccgaa 60
ggcggcaccg gt 72

Claims (32)

1. a kind of T cell receptor (TCR), which is characterized in that the TCR can be combined with ILSLELMKL-HLA compounds；
Also, 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
αCDR1-DRVSQS(SEQ ID NO:10)
αCDR2-IYSNGD(SEQ ID NO:11)
αCDR3-AATNSGYALN(SEQ ID NO:12) and
3 complementary determining regions of the TCR β chain variable domains are：
βCDR1-GTSNPN(SEQ ID NO:13)
βCDR2-SVGIG(SEQ ID NO:14)
βCDR3-AWSVDGAEQY(SEQ ID NO:15)。
2. TCR as described in claim 1, which is characterized in that the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1。
3. TCR as described in claim 1, which is characterized in that the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5。
4. TCR as described in claim 1, which is characterized in that the TCR is α β heterodimers, and it includes TCR α chains are constant Area TRAC*01 and TCR β chain constant regions TRBC1*01 or TRBC2*01.
5. TCR as described in claim 4, which is characterized in that the α chain amino acid sequences of the TCR are SEQ ID NO：3 And/or the β chain amino acid sequences of the TCR are SEQ ID NO:7.
6. the TCR as described in any in claim 1-3, which is characterized in that the TCR is soluble.
7. TCR as claimed in claim 6, which is characterized in that the TCR is single-stranded.
8. TCR as claimed in claim 7, which is characterized in that the TCR is to be connected by α chains variable domain with β chain variable domains by peptide Sequence is connect to be formed by connecting.
9. TCR as claimed in claim 8, which is characterized in that the TCR α chains variable region amino acid the 11st, 13,19,21, 53rd, in 76,89,91 or the 94th and/or α chain J gene small peptides amino acid inverses the 3rd, 5th reciprocal or inverse the 7th It is mutated with one or more；And/or the TCR in β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or 94th and/or β chain J gene small peptides amino acid is 2nd reciprocal, has one or more in 4th reciprocal or 6th reciprocal Mutation, wherein amino acid position number is by the Position Number listed in IMGT (international immunogenetics information system).
10. TCR as claimed in claim 9, which is characterized in that the α chains variable domain amino acid sequence of the TCR includes SEQ ID NO:The β chains variable domain amino acid sequence of the 32 and/or TCR includes SEQ ID NO:34.
11. TCR as claimed in claim 10, which is characterized in that the amino acid sequence of the TCR is SEQ ID NO:30.
12. TCR as claimed in claim 6, which is characterized in that the TCR include (a) whole in addition to transmembrane domain or Part TCR α chains；And all or part of TCR β chains of (b) in addition to transmembrane domain；
And and (b) respectively contains functional variable domain (a).
13. TCR as claimed in claim 12, which is characterized in that (a) and (b) each also includes the TCR chains constant domain At least a portion.
14. TCR as described in claim 12 or 13, which is characterized in that α and β chain of the cysteine residues in the TCR is constant Artificial disulfide bond is formed between domain.
15. TCR as claimed in claim 14, which is characterized in that the cysteine that artificial disulfide bond is formed in the TCR is residual Base is instead of selected from following one or more groups of sites：
The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；With
The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.
16. TCR as claimed in claim 15, which is characterized in that the α chain amino acid sequences of the TCR are SEQ ID NO:26 And/or the β chain amino acid sequences of the TCR are SEQ ID NO:28.
17. TCR as described in claim 1, which is characterized in that the α chains of the TCR and/or C- the or N- ends of β chains are combined with Conjugate.
18. TCR as claimed in claim 17, which is characterized in that the conjugate combined with the TCR for detectable, Therapeutic agent, PK modified parts or combination thereof.
19. TCR as claimed in claim 18, which is characterized in that the therapeutic agent is anti-CD 3 antibodies.
20. a kind of multivalent TCR complex, which is characterized in that comprising at least two TCR molecules, and at least one TCR therein TCR of the molecule any one of the claims.
21. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules are included any one of coding claim 1-19 The nucleotide sequence of TCR molecules or its complementary series.
22. nucleic acid molecules as claimed in claim 21, which is characterized in that it includes the nucleotides sequences of coding TCR α chain variable domains Arrange SEQ ID NO：2 or SEQ ID NO:33.
23. the nucleic acid molecules as described in claim 21 or 22, which is characterized in that it includes the nucleosides of coding TCR β chain variable domains Acid sequence SEQ ID NO：6 or SEQ ID NO:35.
24. nucleic acid molecules as claimed in claim 21, which is characterized in that it includes the nucleotide sequence SEQ of coding TCR α chains ID NO：4 and/or include coding TCR β chains nucleotide sequence SEQ ID NO：8.
25. a kind of carrier, which is characterized in that the carrier contains any nucleic acid molecules in claim 21-24.
26. carrier as claimed in claim 25, which is characterized in that the carrier is viral vectors.
27. carrier as claimed in claim 26, which is characterized in that the carrier is slow virus carrier.
28. a kind of separated host cell, which is characterized in that contain the load described in claim 25 in the host cell Any nucleic acid molecules in the claim 21-24 of external source are integrated in body or chromosome.
29. a kind of cell, which is characterized in that the cell transduction have the right in requirement 21-24 any nucleic acid molecules or Carrier described in claim 25.
30. cell as claimed in claim 29, which is characterized in that the cell is T cell or stem cell.
31. a kind of pharmaceutical composition, which is characterized in that the composition contains pharmaceutically acceptable carrier and claim The TCR compounds described in TCR, claim 20 any one of 1-19, any core in claim 21-24 Cell described in acid molecule or claim 29.
32. the TCR compounds or claim described in TCR or claim 20 any one of claim 1-19 The purposes of cell described in 29, which is characterized in that be used to prepare the drug for the treatment of tumour or autoimmune disease.
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CN109400697B (en) * 2017-08-17 2021-04-23 香雪生命科学技术（广东）有限公司 TCR (T cell receptor) for identifying PRAME (platelet-activating antigen) short peptide and related composition thereof
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