CN106459179A - T cell receptor for recognizing rhamm antigen short-chain polypeptide - Google Patents
T cell receptor for recognizing rhamm antigen short-chain polypeptide Download PDFInfo
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- CN106459179A CN106459179A CN201680001317.4A CN201680001317A CN106459179A CN 106459179 A CN106459179 A CN 106459179A CN 201680001317 A CN201680001317 A CN 201680001317A CN 106459179 A CN106459179 A CN 106459179A
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Abstract
Provided in the present invention is a T cell receptor (TCR) capable of binding specifically antigen short-chain polypeptide ILSLELMKL derived from RHAMM. The antigen short-chain polypeptide ILSLELMKL is capable of forming a complex with HLA A0201 and being presented together on the surface of a cell. Also provided in the present invention are a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule. In addition, also provided in the present invention is a cell for transducing the TCR of the present invention.
Description
The present invention relates to can recognize from RHAMM (Receptor for Hyaluronan-Mediated Motility, RHAMM) the TCR of antigen small peptide, the invention further relates to transduce above-mentioned TCR come the specific T cells of RHAMM obtained, and they prevention and treatment RHAMM relevant diseases in purposes.
RHAMM also known as CD168, is the acceptor of the hyaluronic acid as one of extracellular matrix components.RHAMM is a kind of endogenous antigen, and micromolecule polypeptide is degraded to after generating in the cell, and combines to form compound with MHC (main histocompatibility complex) molecule, is presented to cell surface.ILSLELMKL (165-173) is small peptide (Greiner J, et al., Blood 2005,106 (3) derived from RHAMM:938-945).Studies have shown that RHAMM has expression in kinds of tumors tissue, with leukaemia (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):567-576) more protrude, in other cancers, 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), OSCC (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 cell., can be using methods such as chemotherapy and radiation treatments for the treatment of above-mentioned disease, but the normal cell of itself can all be caused damage.
T cell adoptive immunotherapy is that will there is specific reaction-ive T cell to be transferred in patient body target cell antigen, it is played a role for target cell.φt cell receptor (TCR) is a kind of memebrane protein on T cell surface, and it can recognize the antigen small peptide of corresponding target cells.In immune system, T cell is triggered directly to be physically contacted with antigen presenting cell (APC) by the combination of the specific TCR of antigen small peptide and small peptide-main histocompatibility complex (pMHC compounds), then other cell membrane surface molecules of both T cell and APC just interact, cause a series of follow-up cell signal transmission and other physiological reactions, so that the T cell of different antigentic specificities plays immunological effect to its target cell.Therefore, those skilled in the art, which are directed to isolating, has specific TCR to RHAMM antigen small peptides, and to obtain TCR T cells of transduceing are had into specific T cell to RHAMM antigen small peptides, so that they play a role in cellular immunotherapy.
The content of the invention
It is an object of the invention to provide a kind of φt cell receptor of identification RHAMM antigen small peptides.
The first aspect of the present invention can be combined there is provided a kind of φt cell receptor (TCR), the TCR with ILSLELMKL-HLA compounds.
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, and the TCR α chains variable domain is and SEQ ID NO:1 has the amino acid sequence of at least 90% sequence thereto；And/or the TCR β chains variable domain is and 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, it is characterised in that the CDR3 of TCR α chain variable domains amino acid sequence is AATNSGYALN (SEQ ID NO:
12)；And/or the CDR3 of TCR β chain variable domains amino acid sequence 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 regions TRBC1*01 or TRBC2*01.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO：3 and/or TCR β chain amino acid sequences are SEQ ID NO:7.
In another preference, the TCR is solvable.
In another preference, the TCR is single-stranded.
In another preference, the TCR is to be formed by connecting by α chains variable domain with β chains variable domain by peptide catenation sequence.
In another preference, the TCR has one or more mutation in α chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th, and/or α chain J gene small peptides amino acid inverse the 3rd, 5th reciprocal or inverse the 7th；And/or the TCR β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th, and/or there are one or more mutation in β chain J gene small peptides amino acid inverse the 2nd, 4th reciprocal or inverse the 6th, wherein amino acid position number is 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 TCR β chains variable domain amino acid sequence 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 membrane spaning domain；And all or part of TCR β chains of (b) in addition to membrane spaning domain；
And with (b) each self-contained functional variable domain (a), or at least a portion comprising functional variable domain and the TCR chains constant domain.
In another preference, cysteine residues form artificial disulfide bond between α the and β chain constant domains of the TCR.
In another preference, the cysteine residues that artificial disulfide bond is formed in the TCR instead of selected from following one or more groups of sites：
The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；With
The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO:26 and/or described
TCR β chain amino acid sequences are 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 combined with the φt cell receptor is the combination of detectable, therapeutic agent, PK modified parts or these any materials.Preferably, the therapeutic agent is anti-CD 3 antibodies.
The second aspect of the present invention is there is provided a kind of multivalent TCR complex, and it includes at least two TCR molecules, and at least one TCR molecule therein is the TCR described in first aspect present invention.
The third aspect of the present invention includes the nucleotide sequence or its complementary series of the TCR molecules described in coding first aspect present invention there is provided a kind of nucleic acid molecules, the nucleic acid molecules.
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, described nucleic acid molecules include the nucleotide sequence SEQ ID NO of coding TCR β chain variable domains：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 coding TCR β chains nucleotide sequence SEQ ID NO：8.
The fourth aspect of the present invention contains the nucleic acid molecules described in third aspect present invention there is provided a kind of carrier, described carrier；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
The fifth aspect of the present invention contains the nucleic acid molecules being integrated with the carrier or genome described in fourth aspect present invention described in the third aspect present invention of external source there is provided a kind of host cell of separation in described host cell.
The sixth aspect of the present invention is there is provided a kind of cell, the nucleic acid molecules described in the cell transduction third aspect present invention or the carrier described in fourth aspect present invention；Preferably, the cell is T cell or stem cell.
The seventh aspect of the present invention, there is provided a kind of pharmaceutical composition, the composition contains pharmaceutically acceptable carrier and TCR, the TCR compounds described in second aspect of the present invention, the nucleic acid molecules described in third aspect present invention, the carrier described in fourth aspect present invention or the cell described in sixth aspect present invention described in first aspect present invention.
The eighth aspect of the present invention, there is provided the purposes of the TCR compounds described in the φt cell receptor or second aspect of the present invention described in first aspect present invention, the nucleic acid molecules described in third aspect present invention, the carrier described in fourth aspect present invention or the cell described in sixth aspect present invention, the medicine for preparing treatment tumour or autoimmune disease.
The ninth aspect of the present invention, there is provided a kind of method for treating disease, including to the object for needing to treat using the φt cell receptor or the TCR compounds described in second aspect of the present invention, the nucleic acid molecules described in third aspect present invention, the carrier described in fourth aspect present invention or the cell described in sixth aspect present invention or the pharmaceutical composition described in seventh aspect present invention described in appropriate first aspect present invention；
Preferably, described disease is acute myeloid system leukaemia, chronic myelocytic system leukaemia, ALL, chronic lymphocytic leukemia, Huppert's disease, melanoma, colon cancer, breast cancer, kidney, stomach cancer, TCCB, prostate cancer, OSCC and head and neck squamous cell carcinoma.
It should be understood that within the scope of the present invention, can be combined with each other between above-mentioned each technical characteristic of the invention and each technical characteristic specifically described in below (eg embodiment), so as to constitute new or preferred technical scheme.As space is limited, no longer tire out one by one herein and state.
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively TCR α chains variable domain amino acid sequence, TCR α 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 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 TCR β chains variable domain amino acid sequence, TCR β chain variable domains nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, the TCR β chain amino acid sequences with targeting sequencing 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 single-stranded TCR amino acid sequence and nucleotide sequence.
Fig. 8 a and Fig. 8 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR α chains.
Fig. 9 a and Fig. 9 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR β chains.
Figure 10 a and Figure 10 b are respectively the amino acid sequence and nucleotide sequence of single-stranded TCR catenation sequences (linker).
Figure 11 is the soluble single-chain T CR obtained after purification glue figure.
Figure 12 is the BIAcore kinetic profiles that sTCR of the present invention is combined with ILSLELMKL-HLA A0201 compounds.
Figure 13 is the BIAcore kinetic profiles that soluble single-chain T CR of the present invention is combined with ILSLELMKL-HLA A0201 compounds.
The T cell that Figure 14 is transduction TCR of the present invention is to load or the activation experiment result figure of the T2 cells of unsupported specific antigen small peptide.
The T cell that Figure 15 is transduction TCR of the present invention is to load or the killing experiments result figure of the T2 cells of unsupported specific antigen small peptide.
Figure 16 is transduction TCR of the present invention T cell to special and non-specific cellular system killing experiments result figure.
The present inventor have found and RHAMM antigen small peptide ILSLELMKL (165-173) (SEQ ID NO by in-depth study extensively:9) TCR that can be specifically bound, the antigen small peptide ILSLELMKL can with HLA A0201 formation compound and together be presented to cell surface.Present invention also offers encode the nucleic acid molecules of the TCR and include the carrier of the nucleic acid molecules.In addition, present invention also offers the cell for 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, it has specificity for the presentation of antigen, and different individuals has different MHC, can present small peptides different in a kind of proteantigen to respective APC cell surfaces.The MHC of the mankind is commonly referred to as HLA genes or HLA complexs.
φt cell receptor (TCR), is the unique acceptor for presenting the specific antigen peptide in main histocompatibility complex (MHC).In immune system, trigger T cell to be directly physically contacted with antigen presenting cell (APC) by the combination of the TCR and pMHC compounds of antigentic specificity, then both T cell and APC its
He just interacts at cell membrane surface molecules, and this just causes a series of follow-up cell signal transmission and other physiological reactions, so that the T cell of different antigentic specificities plays immunological effect to its target cell.
TCR is the glycoprotein of the cell membrane surface existed by α chains/β chains or γ chains/δ chains in heterodimer form.TCR heterodimers are made up of α and β chains in 95% T cell, and 5% T cell has the TCR being made up of γ and δ chains.The natural heterogeneous dimerization TCR of α β have α chains and β chains, and α chains and β chains constitute α β heterodimerics TCR subunit.In a broad sense, each chains of α and β include variable region, bonding pad and constant region, and β chains generally contain short variable region also between variable region and bonding pad, but the variable region is often regarded as a part for bonding pad.Each variable region includes 3 CDR (complementary determining region) being entrenched in frame structure (framework regions), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR and pMHC compounds, and wherein CDR3 is formed by variable region and bonding pad restructuring, is referred to as hypervariable region.TCR α and β chains, which are typically regarded as, respectively two " domains " i.e. variable domain and constant domain, and variable domain is made up of the variable region and bonding pad connected.The sequence of TCR constant domains can be found in the public database of international immunogenetics information system (IMGT), constant domain sequence such as TCR molecule alpha chains is " TRAC*01 ", and the constant domain sequence of TCR molecule β chains is " TRBC1*01 " or " TRBC2*01 ".In addition, TCR α and β chains also include transmembrane region and cytoplasmic region, cytoplasmic region is very short.
In the present invention, term " polypeptide of the present invention ", " TCR " of the invention, " φt cell receptor of the invention " are used interchangeably.
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.φt cell receptor can recognize the peptide-MHC compounds of Antigen Presenting Cell surface.Therefore, the first aspect of the present invention can specifically bind the TCR molecules of ILSLELMKL-HLA A0201 compounds there is provided a kind of.Preferably, the TCR molecules are separation 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 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)。
One in the present invention is preferably carried out in mode, and the β chains of the TCR 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 CDR region amino acid sequence of the invention described above can be embedded into any suitable frame structure to prepare chimeric TCR.As long as frame structure is compatible with the TCR of present invention CDR region, those skilled in the art can just design or synthesize the TCR molecules with corresponding function according to CDR region disclosed by the invention.Therefore, TCR molecules of the present invention refer to the TCR molecules comprising above-mentioned α and/or β chains CDR region sequence and any suitable frame structure.TCR α chains variable domain of the present invention be and SEQ ID NO:1 has at least 90%, preferably 95%, the more preferably amino acid sequence of 98% sequence thereto；And/or TCR β chains variable domain of the present invention is and SEQ ID NO：5 have at least 90%, preferably 95%, the more preferably amino acid sequence of 98% sequence thereto.
In the preference of the present invention, TCR molecules of the invention are the heterodimers being made up of α and β chains.Specifically, on the one hand the α chains of the heterogeneous dimerization TCR molecules include variable domain and constant domain, and the α chains variable domain amino acid sequence includes CDR1 (the SEQ ID NO of above-mentioned α chains：10)、CDR2(SEQ ID NO：11) with CDR3 (SEQ ID NO:12).Preferably, the TCR molecules are variable comprising α chains
Domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chains variable domain amino acid sequence of the TCR molecules is SEQ ID NO:1.On the other hand, the β chains of the heterogeneous dimerization TCR molecules include variable domain and constant domain, and 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) with CDR3 (SEQ ID NO:15).Preferably, the TCR molecules include β chain variable domain amino acid sequence SEQ ID NO:5.It is highly preferred that the β chains variable domain amino acid sequence of the TCR molecules is SEQ ID NO:5.
In the preference of the present invention, TCR molecules of the invention by α chains part or all of and/or β chains the single chain TCR molecules partly or entirely constituted.Description about single chain TCR molecules may be referred to document Chung et al (1994) Proc.Natl.Acad.Sci.USA 91,12654-12658.According to document, those skilled in the art can easily build 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 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：11) with CDR3 (SEQ ID NO:12).Preferably, the single chain TCR molecules include α chain variable domain amino 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：14) with CDR3 (SEQ ID NO：15).Preferably, the single chain TCR molecules include β chain variable domain amino acid sequence SEQ ID NO:5.It is highly preferred that the β chains variable domain amino acid sequence of the 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.Those skilled in the art know or can obtain the constant domain amino acid sequence of people by consulting the public database of pertinent texts or IMGT (international immunogenetics information system).For example, the constant domain sequence of TCR molecule alphas chain of the present invention can be " TRAC*01 ", the constant domain sequence of TCR molecule β chains can be " TRBC1*01 " or " TRBC2*01 ".The 53rd of the amino acid sequence provided in IMGT TRAC*01 is Arg, is expressed as herein：The Arg53 of TRAC*01 exons 1s, other are by that analogy.Preferably, the amino acid sequence of TCR molecule alphas chain of the present invention is SEQ ID NO:3, and/or the amino acid sequence of β chains is SEQ ID NO:7.
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.As immunoglobulin (antibody) as antigen recognizing molecule, TCR can also be developed applied to diagnosis and treat, and at this moment need to obtain soluble TCR molecules.Soluble TCR molecules do not include its transmembrane region.STCR has very extensive purposes, and it cannot be only used for studying TCR and pMHC interaction, it is also possible to make the diagnostic tool or the mark as autoimmunity disease of detection infection.Similarly, sTCR can be used to being transported to therapeutic agent (such as cytotoxin compounds or immunostimulating compound) into the cell for presenting specific antigen, in addition, sTCR can also be with other molecules (such as, anti-CD 3 antibodies) combine to redirect T cell, so that its targeting presents the cell of specific antigen.The present invention also obtain has specific sTCR to RHAMM antigen small peptides.
To obtain sTCR, on the one hand, TCR of the present invention can be the TCR that artificial disulfide bond is introduced between the residue of itself α and β chain constant domain.Cysteine residues form artificial interchain disulfide bond between α the and β chain constant domains of the TCR.Cysteine residues can be substituted in other amino acid residues of appropriate site in natural TCR to form artificial interchain disulfide bond.For example, the Thr48 of substitution TRAC*01 exons 1s forms disulfide bond with the Ser57 of substitution TRBC1*01 or TRBC2*01 exons 1s cysteine residues.Cysteine residues are introduced to can also be with other sites for forming disulfide bond：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 Arg53 and TRBC1*01 or TRBC2*01 of TRAC*01 exons 1s
The Ser54 of exons 1；The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；Or the Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.I.e. cysteine residues instead of above-mentioned α and any group of site in β chain constant domains.The amino acid of most 50 or most 30 or most 15 or most 10 or most 8 or less can be truncated in one or more C-terminals of TCR constant domains of the present invention, so that it does not include cysteine residues to reach the purpose of missing natural disulphide bonds, also above-mentioned purpose can be reached by the way that the cysteine residues for forming natural disulphide bonds are sported into another amino acid.
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, TCR of the invention can contain TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences.TCR TRAC constant domains sequence and TRBC1 or TRBC2 constant domains sequence can be connected by the natural disulphide bonds being present in TCR.
To obtain sTCR, on the other hand, TCR of the present invention is additionally included in the TCR that its hydrophobic core region is undergone mutation, and the mutation in these hydrophobic core regions is preferably capable making the stability-enhanced mutation of sTCR of the present invention, as described in the patent document in Publication No. WO2014/206304.Such TCR can undergo mutation in its following hydrophobic core position of variable domain：(α and/or β chains) variable region amino acid the 11st, 13,19,21,53,76,89,91,94, and/or α chain J gene (TRAJ) small peptides amino acid position is reciprocal 3rd, 5,7, and/or β chain J gene (TRBJ) small peptides amino acid position inverse the 2nd, 4,6, the wherein Position Number of amino acid sequence presses the Position Number listed in international immunogenetics information system (IMGT).Those skilled in the art know above-mentioned international immunogenetics information system, and Position Number of the different TCR amino acid residue in IMGT can be obtained according to the database.
The TCR that hydrophobic core region is undergone mutation in the present invention can be the solvable single-stranded TCR of stability being made up of the variable domain of α and the β chain of a flexible peptide chain connection TCR.It should be noted that flexible peptide chain can be any suitable connection TCR α and β chain variable domains peptide chain in the present invention.The single chain soluble TCR such as built in the embodiment of the present invention 4, its α chains variable domain amino acid sequence is SEQ ID NO:32, the nucleotides sequence of coding is classified as SEQ ID NO:33；β chains variable domain amino acid sequence is SEQ ID NO:34, the nucleotides sequence of coding is classified as SEQ ID NO:35.
The present invention TCR can also multivalence complex form provide.The multivalent TCR complex of the present invention comprising two, three, four or more TCR of the present invention be combined formed by polymer, can such as produce the tetramer with p53 four dimerization domains, or multiple TCR of the present invention with another molecule the compound with reference to formed by.The TCR compounds of the present invention are available for external or tracking in vivo or the cell of targeting presentation specific antigen, it can also be used to produce the intermediate of other multivalent TCR complex with such application.
The TCR of the present invention can be used alone, and can also be combined, preferably be combined with covalent manner with covalent or other modes with conjugate.The combination that the conjugate includes detectable (being diagnostic purpose, wherein the TCR is used for the presence for detecting the cell for presenting ILSLELMKL-HLA A0201 compounds), therapeutic agent, PK (protein kinase) modified parts or any the above material is combined or is coupled.
Detectable for diagnostic purposes includes but is not limited to：Fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (CT technology) contrast agent can produce the enzyme of detectable product.
The therapeutic agent that can be combined or be coupled with TCR of the present invention includes but is not limited to：1. radionuclide (Koppe etc., 2005, metastasis of cancer comment (Cancer metastasis reviews) 24,539)；2. biological poison (Chaudhary etc., 1989, natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. (Gillies etc., 1992, NAS's proceeding (PNAS) 89,1428 such as cell factor such as IL-2；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc fragment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；
5. antibody scFv fragment (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold nano grain/nanometer rods (Lapotko etc., 2005, cancer communication (Cancer letters) 239,36；Huang etc., 2006, U.S. chemical institute magazine (Journal of the American Chemical Society) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,11631)；9. magnetic nanosphere；10. pro-drug activation enzymes (for example, DT- diaphorases (DTD) or biphenyl base hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
In addition, the TCR of the present invention can also be comprising the heterozygosis TCR being derived from more than a kind of species sequence.For example, there is research to show that Muridae TCR can be expressed more effectively in human T-cell than people TCR.Therefore, the constant domain that TCR of the present invention can be comprising people's variable domain and mouse.The defect of this method is possible to trigger immune response.Therefore, there should be regulation scheme to carry out immunosupress when it is used for adoptive T cell treatment, with the implantation for the T cell for allowing to express Muridae.
It should be understood that amino acid name represents that the corresponding relation of the single English alphabet and three English alphabets of amino acid name is as follows using international single English alphabet or three English alphabets herein：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, and the part can be one or more CDR, the variable domain of α and/or β chains, and α chains 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 sequence of the nucleic acid molecules of the present invention of coding TCR β chains of the present invention includes 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 single-stranded or double-stranded, and 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 can encode polypeptide of the present invention, for example, encode the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chain variable domains of the present invention and 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.Or, encoding the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chain variable domains of the present invention includes SEQ ID NO:33 and/or the nucleotide sequences of nucleic acid molecules of the present invention of coding TCR β chain variable domains of the present invention include SEQ ID NO:35.It is highly preferred that the nucleotide sequence of nucleic acid molecules of the present invention includes SEQ ID NO:4 and/or SEQ ID NO:8.Or, the nucleotides sequence of nucleic acid molecules of the present invention is classified as SEQ ID NO:31.
It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.Therefore, the nucleotide sequence for encoding TCR of the present invention can variant identical or degeneracy with the nucleotide sequence shown in accompanying drawing of the present invention.Illustrated with one of example in the present invention, " variant of degeneracy " refers 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 be changed the codon in sequence to increase expression quantity according to the type of cell.Mammalian cell and various other biological codon usage tables be well known to a person skilled in the art.
The nucleic acid molecules full length sequence or its fragment of the present invention generally can with but be not limited to PCR TRAPs, recombination method or artificial synthesized method and obtain.At present, it is already possible to obtain encoding TCR of the present invention (or its fragment, or derivatives thereof) DNA sequence dna by chemical synthesis completely.Then the DNA sequence dna can be introduced into various existing DNA moleculars (or such as carrier) as known in the art and cell.DNA can be coding strand or noncoding strand.
Carrier
The invention further relates to the carrier of the nucleic acid molecules comprising the present invention, including expression vector, i.e., the construct that can be expressed in vivo or in vitro.Conventional carrier includes bacterial plasmid, bacteriophage and animals and plants virus.
Viral delivery systems include but is not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpesvirus vector, retroviral vector, slow virus carrier, baculovirus vector.
Preferably, the nucleotides of the present invention can be transferred in cell by carrier, such as in T cell so that the cell expresses the TCR of RHAMM antigentic specificities.Ideally, the carrier be able to should be expressed continual high levels in T cell.
Cell
The invention further relates to the host cell produced with the carrier or coded sequence of the present invention through genetic engineering.The nucleic acid molecules of the present invention are integrated with carrier or chromosome containing the present invention in the host cell.Host cell is selected from：Prokaryotic and eukaryotic, such as Escherichia coli, yeast cells, Chinese hamster ovary celI.
In addition, present invention additionally comprises the cell of the TCR of expression present invention separation, particularly T cell.The T cell can be derived from the T cell separated from subject, or can be the mixed cellularity group separated from subject, such as part of PBLC (PBL) group.Such as, the cell can be isolated from PMBC (PBMC), can be CD4+Helper cell or CD8+Cytotoxic T cell.The cell can be in CD4+Helper cell/CD8+In the mixing group of cytotoxic T cell.Usually, the cell can be activated with antibody (e.g., anti-CD3 or anti-CD28 antibody), to allow them to easily receive transfection, for example, be 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 transfer to HSC will not be caused in cell surface expression TCR, because stem cell surface does not express CD3 molecules.However, when stem cell is divided into and migrated to the lymphoid precursor of thymus gland (lymphoid precursor), the expression of CD3 molecules will start TCR molecules in the introducing of the surface expression of thymocyte.
There are many methods to be suitable for carrying out T cell transfection (e.g., the such as Robbins, (2008) J.Immunol.180 with the DNA or RNA for encoding TCR of the present invention:6116-6131).Expression TCR of the present invention T cell can be used for adoptive immunotherapy.Those skilled in the art understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat Rev Cancer8 (4) for carrying out adoptive treatment：299-308).
RHAMM antigen-related diseases
The invention further relates to be treated in subject and/or prevent the method with RHAMM relevant diseases, it includes the step of adoptive transfer RHAMM specific T-cells are to the subject.The RHAMM specific T-cells can recognize that ILSLELMKL-HLA A0201 compounds.
The specific T cells of RHAMM of the present invention can be used for the RHAMM relevant diseases for treating any presentation RHAMM antigens small peptide ILSLELMKL-HLA A0201 compounds.Including but not limited to acute myeloid system leukaemia, chronic myelocytic system leukaemia, ALL, chronic lymphocytic leukemia, Huppert's disease, melanoma, colon cancer, breast cancer, kidney, stomach cancer, TCCB, prostate cancer, OSCC and head and neck squamous cell carcinoma.
Treatment method
It can be imported by separating with the patient with RHAMM antigen-related diseases or the T cell of volunteer, and by the TCR of the present invention in above-mentioned T cell, the cell for then modifying these genetic engineerings feeds back in patient body to be treated.Therefore, the invention provides a kind of method for treating RHAMM relevant diseases, including by expression TCR of the present invention of separation T cell, it is preferable that the T cell derives from patient in itself, is input in patient body.Usually, the T cell of patient is separated including (1), (2) with nucleic acid molecules of the present invention or the nucleic acid molecules ex vivo transduction T cell of TCR molecules of the present invention can be encoded, the T cell that genetic engineering is modified is input in patient body by (3).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, is expanded on further the present invention.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as (Sambrook and Russell et al., molecular cloning：Laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing houses) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are calculated by weight.Unless otherwise indicated, otherwise percentage and number are calculated by weight.Experiment material and reagent used can be obtained from commercially available channel unless otherwise instructed in following examples.
Embodiment 1 clones RHAMM antigen small peptide specific T-cells
Come from PBLC (PBL) of the genotype for HLA-A0201 healthy volunteer using small peptide ILSLELMKL (Beijing SBS Genetech gene technology Co., Ltd) stimulations are synthesized.By ILSLELMKL small peptides and the HLA-A*0201 renaturation with biotin labeling, pHLA monoploid is prepared.These monoploid are combined into the tetramer of PE marks with the Streptavidin (BD companies) marked with PE, sort the tetramer and anti-CD8-APC double positive cells.The cell of sorting is expanded, and carries out secondary sorting as stated above, then monoclonal is carried out with limiting dilution assay.Monoclonal cell tetramer staining, the double positive colonies screened are as shown in Figure 3.
Embodiment 2 obtains the tcr gene of RHAMM antigen small peptide specific T-cell clones and the structure of carrier
Use Quick-RNATMThe total serum IgE of the T cell that antigen small peptide ILSLELMKL is specific, HLA-A0201 the is restricted clone screened in MiniPrep (ZYMO research) extracting embodiments 1.CDNA synthesis is using clontech SMART RACE cDNA amplification kits, and the primer of use is designed in the C-terminal conserved region of mankind's tcr gene.Sequence is cloned into carrier T (TAKARA) and is sequenced.It should be noted that the sequence is complementary series, not comprising introne.Through sequencing, respectively as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively TCR α chains variable domain amino acid sequence, TCR α 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 sequences with targeting sequencing to the TCR of this pair of positive colony expression α chains and β chain-orderings structure；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chains variable domain amino acid sequence, TCR β chain variable domains nucleotide sequence, TCR β chain amino acid sequences, TCR β chains nucleotide sequence, the TCR β chain amino acid sequences with targeting sequencing 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 by Lentiviral pLenti (addgene) by overlapping (overlap) PCR respectively.Specially：The full-length gene of TCR α chains and TCR β chains is attached with overlap PCR and obtains TCR α -2A-TCR β fragments.Lentiviral and TCR α -2A-TCR β digestions connection are obtained into pLenti-RHAMMTRA-2A-TRB-IRES-NGFR plasmids.Used as control, while also construction expression eGFP slow virus carrier pLenti-eGFP.Pack pseudovirus with 293T again afterwards.Detailed process is as described in Example 7.
The solvable TCR of the RHAMM antigens small peptide of embodiment 3 specificity expression, refolding and purifying
To obtain solvable TCR molecules, α the and β chains of the TCR molecules of the present invention can only include its variable domain and portion constant domain respectively, and a cysteine residues are introduced 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；With nucleotide sequence difference as shown in figures 4 a and 4b, with nucleotide sequence difference as shown in figure 5 a and 5b, the cysteine residues of introducing are represented the amino acid sequence of its β chain the amino acid sequence of its α chain with overstriking letter.Pass through《Molecular Cloning: A Laboratory room handbook》(Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell) described in standard method above-mentioned TCR α and β chains objective gene sequence are inserted respectively into expression vector pET28a+ (Novagene) after synthesis, 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 entered into expression bacterium BL21 (DE3) by chemical transformation conversion respectively, bacterium is grown with LB nutrient solutions, in OD600Induced when=0.6 with final concentration 0.5mM IPTG, the inclusion body formed after TCR α and β chains expression is extracted by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, inclusion body is finally dissolved in 6M guanidine hydrochlorides, 10mM dithiothreitol (DTT)s (DTT), in 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris (pH 8.1).
TCR α and β chains after dissolving are with 1：1 mass ratio is quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Solution is placed in the deionized water of 10 times of volumes dialysis (4 DEG C) after mixing, deionized water is changed into buffer solution (20mM Tris, pH 8.0) after 12 hours and continues at 4 DEG C and dialyse 12 hours.Solution after the completion of dialysis is purified after 0.45 μM of membrane filtration by anion-exchange column (HiTrap Q HP, 5ml, GE Healthcare).The TCR that eluting peak contains the successful α and β dimers of renaturation is confirmed by SDS-PAGE glue.TCR is then further purified by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare).TCR purity after purification is determined by SDS-PAGE is more than 90%, and concentration is determined by BCA methods.The SDS-PAGE glue figures for the sTCR that the present invention is obtained are as shown in Figure 6.
The specific soluble single-chain T CR of RHAMM antigen small peptides of embodiment 4 generation
According to patent document WO2014/206304, TCR α in embodiment 2 have been built into the soluble single-chain T CR molecules of a stabilization being connected with flexible small peptide (linker) with the variable domain of β chains using the method for rite-directed mutagenesis.Amino acid sequence and the nucleotide sequence difference of the single chain TCR molecules are as shown in figs. 7 a and 7b.Amino acid sequence and the nucleotide sequence difference of its α chain variable domain are as figures 8 a and 8 b show；Amino acid sequence and the nucleotide sequence difference of its β chain variable domain are as shown in figures 9 a and 9b；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 Nco I and the double digestions of Not I, it is connected with the pET28a carriers by Nco I and the double digestions of Not I.Connection product is converted to E.coli DH5 α, is coated with the LB flat boards containing kanamycins, and 37 DEG C of inversion overnight incubations, picking positive colony enters performing PCR screening, positive recombinant is sequenced, really
Extracting recombinant plasmid transformed is to E.coli BL21 (DE3) after sequencing row are correct, for expressing.
The specific soluble single-chain T CR of RHAMM antigen small peptides of embodiment 5 expression, renaturation and purifying
BL21 (DE 3) bacterium colony containing recombinant plasmid pET28a- template strands prepared in embodiment 4 is all inoculated in the LB culture mediums containing kanamycins, it is 0.6-0.8 that 37 DEG C, which are cultivated to OD600, IPTG to final concentration of 0.5mM is added, 37 DEG C are continued to cultivate 4h.5000rpm centrifuges 15min harvesting sediments, with Bugbuster Master Mix (Merck) cell lysis sediment, 6000rpm centrifugations 15min reclaims inclusion body, washed again with Bugbuster (Merck) to remove cell fragment and membrane component, 6000rpm centrifuges 15min, collects inclusion body.By solubilization of inclusion bodies in buffer solution (20mM Tris-HCl pH 8.0,8M urea), high speed centrifugation removes insoluble matter, and supernatant is saved backup with being dispensed after BCA standard measures in -80 DEG C.
In the single-stranded TCR inclusion body proteins dissolved to 5mg, 2.5mL buffer solutions (6M Gua-HCl, 50mM Tris-HCl pH 8.1,100mM NaCl, 10mM EDTA) are added, DTT to final concentration of 10mM, 37 DEG C of processing 30min is added.With syringe to 125mL renaturation buffers (100mM Tris-HCl pH 8.1,0.4M L-arginines, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) the middle single-stranded TCR being added dropwise after above-mentioned processing, 4 DEG C of stirring 10min, then renaturation solution is loaded into the cellulose membrane bag filter that interception is 4kDa, bag filter is placed in the water of 1L precoolings, and 4 DEG C are slowly stirred overnight.After 17 hours into, dialyzate is changed to the buffer solution (20mM Tris-HCl pH 8.0) of 1L precoolings, 4 DEG C are continued the 8h that dialyses, then changing dialyzate into identical fresh buffer continues dialysed overnight.After 17 hours, sample is through 0.45 μm of membrane filtration, pass through anion-exchange column (HiTrap Q HP after vacuum outgas, GE Healthcare), the 0-1M NaCl linear gradient elution liquid purifying proteins prepared with 20mM Tris-HCl pH 8.0, the elution fraction of collection carries out SDS-PAGE analyses, solvent resistant column (Superdex 7510/300 is further used after component concentration comprising single-stranded TCR, GE Healthcare) purified, target components also carry out SDS-PAGE analyses.
Its purity is further tested using gel filtration for the elution fraction that BIAcore is analyzed.Condition is：Chromatographic column Agilent Bio SEC-3 (7.8 × 300mm of 300A, φ), mobile phase is 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 is obtained are as shown in figure 11.
Embodiment 6, which is combined, to be characterized
BIAcore is analyzed
It can be specifically bound this example demonstrated soluble TCR molecules of the present invention with ILSLELMKL-HLA A0201 compounds.
The binding activity of the TCR molecules and ILSLELMKL-HLA A0201 compounds obtained in embodiment 3 and embodiment 5 is detected using BIAcore T200 real-time analyzers.The antibody (GenScript) of anti-Streptavidin is added into coupling buffer (10mM sodium-acetate buffers, pH 4.77), then antibody flowed through to the CM5 chips activated in advance with EDC and NHS, antibody is set to be fixed on chip surface, finally unreacted activating surface is closed with the hydrochloric acid solution of monoethanolamine, coupling process is completed, coupling level is about 15,000RU.
The Streptavidin of low concentration is set to flow through the chip surface of coated antibody, then ILSLELMKL-HLA A0201 compounds are flowed through into sense channel, another passage is used as reference channel, 0.05mM biotin is flowed through into chip 2min with 10 μ L/min flow velocity again, the remaining binding site of Streptavidin is closed.
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, in using 10ml PBS washing thallines once after 4 DEG C of 8000g centrifugations 10min, afterwards with 5ml BugBuster Master Mix
Acutely thalline is resuspended in concussion to Extraction Reagents (Merck), and is incubated 20min in room temperature rotation, after 4 DEG C, 6000g centrifugation 15min, supernatant discarding collects inclusion body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Plus 30ml dilutes 10 times of BugBuster, mix, 4 DEG C of 6000g centrifuge 15min；Supernatant discarding, plus 30ml dilutes 10 times of BugBuster resuspension inclusion bodys, mix, 4 DEG C of 6000g centrifuge 15min, are repeated twice, plus inclusion body is resuspended in 30ml 20mM Tris-HCl pH 8.0, mix, 4 DEG C of 6000g centrifuge 15min, finally with 20mM Tris-HCl 8M urea dissolving inclusion body, SDS-PAGE detects inclusion body purity, and 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, and 3M guanidine hydrochlorides, 10mM sodium acetates, 10mM EDTA are added before renaturation and is further denatured.ILSLELMKL peptides are added into renaturation buffer (0.4M L-arginines, 100mM Tris pH 8.3,2mM EDTA, 0.5mM GSSG, 5mM reduced glutathiones, 0.2mM PMSF with 25mg/L (final concentration), it is cooled to 4 DEG C), then 20mg/L light chain and 90mg/L heavy chain (final concentration are sequentially added, heavy chain is added 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 change renaturation buffer, at least change the ionic strength that buffer solution carrys out fully to reduce twice solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded on HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Using Akta purifying instrument (GE General Electric Co. Limited), the 0-400mM NaCl linear gradients liquid elution albumen that 20mM Tris pH 8.0 are prepared, pMHC is eluted about at 250mM NaCl, collects all peak components, SDS-PAGE detection purity.
D. biotinylation
With Millipore super filter tubes by the pMHC molecular concentrations of purifying, it is simultaneously 20mM Tris pH 8.0 by buffer exchange, then 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) are added, incubation at room temperature mixture is stayed overnight, and whether SDS-PAGE detection biotinylations are complete.
E. the compound after purifying biological elementization
PMHC molecular concentrations after biotinylation is marked with Millipore super filter tubes, using the biotinylated pMHC of gel filtration chromatography, purify instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibrations HiPrep to 1ml using AktaTMThen 16/60S200HR posts (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated loading 1ml are eluted with PBS with 1ml/min flow velocitys.Biotinylated pMHC molecules occur in about 55ml as unimodal elution.Merge the component containing protein, concentrated with Millipore super filter tubes, BCA methods (Thermo) determine protein concentration, add protease inhibitors cocktail (Roche) and the packing of biotinylated pMHC molecules is stored in -80 DEG C.
Using BIAcore Evaluation software computational dynamics parameters, obtain the kinetic profile that the soluble single-chain T CR molecules that the TCR molecules and the present invention of solubility of the invention build are combined with ILSLELMKL-HLA A0201 compounds and distinguish as shown in Figure 12 and Figure 13.Simultaneously, the TCR molecules that also have detected solubility of the invention using the above method include the binding activity of GILGFVFTL-HLA A0201 compounds and SLLMWITQC-HLA A0201 compounds with other antigen small peptides, as a result show TCR molecules of the present invention with other irrelevant antigens without combination.
The RHAMM antigen small peptide specificity TCRs slow virus of embodiment 7 is packed to be transfected with primary T cells
(a) (Express-In-mediated transient transfection) is transiently transfected by the quick mediation of 293T cells and prepares slow virus
The slow virus containing TCR gene needed for coding is packed using third generation slow virus packaging system.Transiently transfected (Express-In-mediated transient transfection) (open Biosys Corp. (Open Biosystems)) using quick mediation, with 4 kinds of plasmids (a kind of slow virus carrier containing pLenti-RHAMMTRA-2A-TRB-IRES-NGFR described in embodiment 2, and 3 kinds of plasmids containing other components necessary to building infectiousness but non-replicating lentiviral particle) transfection 293T cells.
To be transfected, the 0th day kind cell, in 15 cm dishes, plants upper 1.7 × 107Individual 293T cells, make cell be evenly distributed on culture dish, degree of converging is slightly above 50%.1st day transfected plasmids, pack pLenti-RHAMMTRA-2A-TRB-IRES-NGFR and pLenti-eGFP pseudovirus, above expression plasmid and packaging plasmid pMDLg/pRRE, pRSV-REV and pMD.2G are mixed, the consumption of a 15 cm diameter plates is as follows：22.5 micrograms：15 micrograms:15 micrograms：7.5 microgram.The ratio of transfection reagent PEI-MAX and plasmid is 2:1, each plate PEI-MAX usage amount are 120 micrograms.Concrete operations are：Expression plasmid and packaging plasmid are added and are well mixed in 1800 microlitres of OPTI-MEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) 31985-070) culture medium, being stored at room temperature 5 minutes turns into DNA mixed liquors；Respective amount PEI is taken to be well mixed with 1800 microlitres of OPTI-MEM culture mediums, being stored at room temperature 5 minutes turns into PEI mixed liquors.DNA mixed liquors and PEI mixed liquors are mixed and be stored at room temperature 30 minutes, 3150 microlitres of OPTI-MEM culture mediums are added again, it is added to after well mixed in the 293T cells for having been converted to 11.25 milliliters of OPTI-MEM, gently rock culture dish, culture medium is well mixed, is cultivated under 37 DEG C/5%CO2.Transfection 5-7 hour, removes transfection media, changes the DMEM ((Ji Bu can company (Gibco), catalog number (Cat.No.) C11995500bt) containing 10% hyclone into) complete medium, culture under 37 DEG C/5%CO2.Collect the culture medium supernatant containing wrapped slow virus within 3rd and the 4th day.For the slow virus of harvest packaging, collected culture supernatant 3000g is centrifuged 15 minutes and removes cell fragment, again through 0.22 micron filter (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) SLGP033RB) filtering, finally use concentration tube (the Merck Mi Libo (Merck Millipore) of 50KD interceptions, catalog number (Cat.No.) UFC905096) concentrated, remove most of supernatant, 1 milliliter is finally concentrated to, -80 DEG C freeze after decile packing.Pseudovirus sample is taken to carry out virus titer measure, step is with reference to p24ELISA (Clontech, catalog number (Cat.No.) 632200) kit specification.Used as control, while also bag turns pLenti-eGFP pseudovirus.
(b) with the lentiviruses transduction primary T cells containing the specific φt cell receptor gene of RHAMM antigen small peptides
It is separated to CD8+T cells from the blood of healthy volunteer, then with the lentiviruses transduction packed.Count these cells, in 48 orifice plates, containing 10%FBS, (Ji Bu can company (Gibco) containing 50IU/ml IL-2 and 10ng/ml IL-7, catalog number (Cat.No.) C10010500BT) 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt) culture mediums in 1 × 106Individual cells/ml (0.5 milliliter/hole) and the AntiCD3 McAb/CD28 antibody-coating globule (T cell amplified matter, life technologies, catalog number (Cat.No.) 11452D) pre-washed are incubated overnight stimulate altogether, cell：Pearl=3：1.
After stimulating overnight, the virus titer measured according to p24 ELISA kits adds the slow virus of the RHAMM antigen small peptide specific t-cell receptor genes concentrated, 32 DEG C, 900g centrifugations infection 1 hour in MOI=10 ratio.Infection removes slow-virus infection liquid after finishing, and is resuspended under cell, 37 DEG C/5%CO2 and cultivated 3 days with 1640 culture mediums containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7.Next day carries out the second wheel infection in the same way.Second of transduction counts cell, diluting cells to 0.5 × 10 after 3 days6Individual cells/ml.Count a cell within every two days, replace or add the fresh culture containing 50IU/ml IL-2 and 10ng/ml IL-7, maintain cell 0.5 × 106-1×106Individual cells/ml.Flow cytometry cell was begun through from the 3rd day, function test (for example, ELISPOT and non-radioactive cell toxicity detection of IFN-γ release) was used for since the 5th day.Since the 10th day or when cell slows down division and size diminishes, stored frozen etc. point cell, at least 4 × 106Individual cell/pipe (1 × 107Individual 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.By the use of ELISPOT testing inspections IFN-γ yield as t cell activation readout.
Reagent
Test medium：10%FBS (Ji Bu can company (Gibco), catalog number (Cat.No.) 16000-044), RPMI1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt)
Lavation buffer solution (PBST)：0.01M PBS/0.05% polysorbas20s
PBS (Ji Bu can company (Gibco), catalog number (Cat.No.) C10010500BT)
The orifice plates of PVDF ELISPOT 96 (Merck Mi Libo (Merck Millipore), catalog number (Cat.No.) MSIPS4510)
People's IFN-γ ELISPOT PVDF- enzyme reagent kits (BD) are equipped with required every other reagent (catching and detection antibody, Streptavidin-alkaline phosphatase and BCIP/NBT solution)
Method
It is prepared by target cell
Target cell used is T2 cells in this experiment.Target cell is prepared in assay medium：Target cell concentration is adjusted to 2.0 × 105Individual/milliliter, 100 microlitres are taken per hole to obtain 2.0 × 104Individual cells/well.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is the CD8+T cells for expressing RHAMM antigens small peptide specificity TCR of the present invention in embodiment 7 through flow cytometry, and using same volunteer and expresses GFP CD8+T and be used as negative control effector cell.With AntiCD3 McAb/CD28 coating pearl (T cell amplified matters, life technologies) stimulate T cell, with the lentiviruses transduction for carrying RHAMM antigen small peptide specificity TCR genes (according to embodiment 7), in the amplification of 1640 culture mediums containing 10%FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 until 9-12 days after transduction, then these cells are placed in test medium, the centrifugation of 300g normal temperature is washed for 10 minutes.Then by cell with 2 × required final concentration is resuspended in test medium.Same processing negative control effector cell.
It is prepared by small peptide solution
Correspondence small peptide is added in corresponding target cell (T2) experimental group, wherein specific small peptide is RHAMM (sequences：ILSLELMKL, numbering：P30A), non-specific small peptide is two small peptides for coming from antigen NY-ESO-1, numbering respectively P1A and P47, and 1 MAGE-A1 small peptide, and numbering is P18.Make final concentration of 1 μ g/ml of the small peptide in ELISPOT orifice plates.
ELISPOT
The specification provided according to manufacturer, prepares orifice plate as described below：1 is pressed with 10 milliliters of sterile PBS of every block of plate：The 200 anti-human IFN-γs of dilution catch antibody, and 100 microlitres of dilution then is caught into antibody etc. point adds each hole.Orifice plate is incubated at 4 DEG C to stay overnight.After incubation, wash orifice plate to remove unnecessary seizure antibody.The culture mediums of RPMI 1640 that 10%FBS is contained in 100 microlitres/hole are added, and incubate orifice plate at room temperature 2 hours to close orifice plate.Then culture medium is washed away from orifice plate, by the way that ELISPOT orifice plates are flicked and patted on paper to remove any remaining lavation buffer solution.
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 by ELISPOT orifice plates using following order：
100 microlitres of target cell 2*105Individual cells/ml (obtaining about 2*104 target cell/hole altogether).
100 microlitres of effector cell (1*104Individual negative control effector cell/hole and RHAMM TCR positive T cells/hole).
All holes prepare addition in duplicate.
Then incubate orifice plate and stay overnight (37 DEG C/5%CO2) second day, culture medium is abandoned, orifice plate is washed with distilled water 2 times, then washed 3 times with lavation buffer solution, pat to remove remnants lavation buffer solution on paper handkerchief.Then 1 is pressed with the PBS containing 10%FBS：200 dilution detection antibody, each hole is added by 100 microlitres/hole.Orifice plate being incubated at room temperature 2 hours, then being washed 3 times with lavation buffer solution, 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, add each hole by the Streptavidin of 100 microlitres of dilutions-alkaline phosphatase and incubate orifice plate at room temperature 1 hour.Then 4 PBS are washed with lavation buffer solution to wash 2 times, and orifice plate is patted on paper handkerchief to remove excessive lavation buffer solution and PBS.Developed in washing adds kit offer 100 microlitres/hole of BCIP/NBT solution after finishing.Orifice plate lucifuge is covered with masking foil during developing, 5-15 minutes are stood.The spot of conventional detection development orifice plate, determines the Best Times of terminating reaction during this period.Remove BCIP/NBT solution and rinse orifice plate to stop developing reaction with distilled water, dry, then orifice plate bottom is removed, orifice plate is dried at room temperature for until each hole is completely dried, recycle the spot of counterdie formation in immunodotting plate count meter (CTL, Celltech Ltd. (Cellular Technology Limited)) counting orifice.
As a result
The IFN-γ (as described above) for examining the T cell that TCR of the present invention transduces to react the target cell and non-specific target cell that load specificity RHAMM antigen small peptides ILSLELMKL is tested by ELISPOT to discharge.The ELSPOT amount of speckle observed in each hole is drawn using graphpad prism6.
As shown in figure 14, the T cell for the TCR of the present invention that transduces only has activating reaction to experimental result to the target cell for loading its special small peptide, and the target cell that other are not loaded with small peptide and the unrelated small peptide of load does not have activating reaction substantially.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, the lactic dehydrogenase (LDH) discharged after quantitative determination cell cracking.The LDH of release in the medium is detected using the enzyme reaction of coupling in 30 minutes, LDH can make a kind of tetrazolium salts (INT) be converted into red formazans (formazan) in enzyme reaction.The amount of the red product of generation is directly proportional to the cell number cracked.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, experiment buffer solution, cracked solution and stop buffer.
Test medium：10%FBS (heat-inactivated, Ji Bu can company (Gibco)), without phenol red 90%RPMI 1640 (Ji Bu can company (Gibco), catalog number (Cat.No.) C11875500bt).
The hole tissue culturing plate of micropore round bottom 96 (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 × 106Individual/milliliter, 100 microlitres are taken per hole to obtain 1 × 105Individual cells/well.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is the CD8+T cells through flow cytometry expression RHAMM antigen small peptide specificity TCRs in embodiment 7.Effector cell uses 1 with target cell ratio：1(1.0×106Individual/milliliter, 100 microlitres are taken per hole to obtain 1.0 × 105Individual cells/well) and 3：1(3.0×106Individual/milliliter, 100 microlitres are taken per hole to obtain 3.0 × 105Individual cells/well).
It is prepared by small peptide solution
Correspondence small peptide is added in corresponding target cell (T2) experimental group, wherein specific small peptide is RHAMM (sequences：ILSLELMKL, numbering：P30A), non-specific small peptide is the small peptide (numbering for coming from MAGE-A1：P18), final concentration of 1 μ g/ml of the small peptide in ELISPOT orifice plates is made.
(a) small peptide for loading various concentrations by target cell (T2) detects that effector cell kills ability
Experiment prepares
All components of experiment are added by the hole tissue culturing plate of micropore round bottom 96 using following order：
- 100ul target cells (preparing as described above) add each hole
- 100ul effector cells (preparing as described above) add each hole
Control group is prepared as described below：
- the small peptide experimental group T2 cells of peptide (not plus) is not loaded：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 is compareed：Only 200ul culture mediums.
- reagent culture medium is compareed：Only 200ul culture mediums.
All holes are prepared in duplicate, and final volume is 200ul (inadequate is supplied with culture medium).
37 DEG C are incubated overnight.Collect before all hole supernatants, added in target cell maximum release control wells and volume correction control group after the mixing of 20 μ l cell pyrolysis liquids, 37 DEG C incubate 45 minutes.
Flat board is centrifuged in 250g 4 minutes.The 50ul supernatants in each hole of test panel are transferred to the respective aperture of 96 hole immuno plate Maxisorb plates.Using buffer solution (12ml) reconstituted substrate mixture is tested, then add 50ul to each hole of flat board.Lucifuge incubation at room temperature 30 minutes after flat board closes the lid.50ul stop baths are added into each hole of flat board with terminating reaction.Add the absorbance that inside counting in 1 hour after stop bath records 490nm.
Result of calculation
From experimental group, the spontaneous release group of target cell and effector cell from all absorbances of release group in deduct culture medium background absorbance.
Bring the corrected value of above-mentioned middle acquisition into formula below, calculate each effect target than produced percentage of cytotoxicity.
% cytotoxicity=100 × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximum-volume correction control-target cell is spontaneous)
As a result
Discharged by the non-radioactive cell toxicity detection LDH (as described above) for examining the T cell that TCR of the present invention transduces to react the target cell and non-specific target cell that load RHAMM antigen small peptides ILSLELMKL.490nm visible ray light absorption values in each hole are drawn using graphpad prism6.
As shown in figure 15, the T cell for the TCR of the present invention that transduces is only to the specific killing action for the T2 target cells for having loaded small peptide ILSLELMKL (RHAMM) for experimental data statistical result.And to not loading the target cell of small peptide or the unrelated small peptide of load without obvious 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), and cell is resuspended to 1.0 × 10 with new test medium6Individual/milliliter, 100 microlitres are taken per hole to obtain 1.0 × 105Individual cells/well.
It is prepared by effector cell
Effector cell's (T cell) of this experiment is the CD8+T cells through flow cytometry expression RHAMM specificity TCRs in embodiment 3.Effector cell uses 1 with target cell ratio：1(1.0×106Individual/milliliter, 100 microlitres are taken per hole to obtain 1.0 × 105Individual cells/well) and 3：1(3.0×106Individual/milliliter, 100 microlitres are taken per hole to obtain 3.0 × 105Individual cells/well).
Experiment prepares
All components of experiment are added by the hole tissue culturing plate of micropore round bottom 96 using following order：
- 100ul target cells (preparing as described above) add each hole
- 100ul effector cells (preparing as described above) add each hole
Control group is prepared as described below：
- small peptide experimental group is not loaded：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 is compareed：Only 200ul culture mediums.
- reagent culture medium is compareed：Only 200ul culture mediums.
All holes are prepared 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 documents referred in the present invention are all incorporated as reference in this application, are individually recited just as each document as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims limited range.
Claims (31)
- A kind of φt cell receptor (TCR), it is characterised in that the TCR can be combined with ILSLELMKL-HLA compounds.
- TCR as claimed in claim 1, it is characterised in that it includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chains variable domain is and SEQ ID NO:1 has the amino acid sequence of at least 90% sequence thereto；And/or the TCR β chains variable domain is and SEQ ID NO：5 have the amino acid sequence of at least 90% sequence thereto.
- TCR as claimed in claim 1 or 2, it includes TCR α chains variable domains and TCR β chain variable domains, it is characterised in that the CDR3 of TCR α chain variable domains amino acid sequence is AATNSGYALN (SEQ ID NO:12)；And/or the CDR3 of TCR β chain variable domains amino acid sequence is AWSVDGAEQY (SEQ ID NO:15).
- As claim 3 TCR, it is characterised in that 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:And/or 3 complementary determining regions of the TCR β chain variable domains are 12)：β CDR1-GTSNPN (SEQ ID NO:13)β CDR2-SVGIG (SEQ ID NO:14)β CDR3-AWSVDGAEQY (SEQ ID NO:15)。
- TCR as described in any of the above claim, it is characterised in that the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.
- TCR as described in any of the above claim, it is characterised in that the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5.
- TCR as described in any of the above claim, it is characterised in that the TCR is α β heterodimers, it includes TCR α chain constant region TRAC*01 and TCR β chain constant regions TRBC1*01 or TRBC2*01.
- TCR as described in claim 7, it is characterised in that the α chain amino acid sequences of the TCR are SEQ ID NO：3 and/or TCR β chain amino acid sequences are SEQ ID NO:7.
- TCR as described in any in claim 1-6, it is characterised in that the TCR is solvable.
- TCR as claimed in claim 9, it is characterised in that the TCR is single-stranded.
- TCR as claimed in claim 10, it is characterised in that the TCR is to be formed by connecting by α chains variable domain with β chains variable domain by peptide catenation sequence.
- TCR as claimed in claim 11, it is characterized in that, the TCR has one or more mutation in α chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th, and/or α chain J gene small peptides amino acid inverse the 3rd, 5th reciprocal or inverse the 7th；And/or the TCR β chains variable region amino acid the 11st, 13,19,21,53,76,89,91 or the 94th, and/or there are one or more mutation in β chain J gene small peptides amino acid inverse the 2nd, 4th reciprocal or inverse the 6th, wherein amino acid position number is by the Position Number listed in IMGT (international immunogenetics information system).
- TCR as claimed in claim 12, it is characterised in that the α chains variable domain amino acid sequence of the TCR includes SEQ ID NO:32 and/or TCR β chains variable domain amino acid sequence includes SEQ ID NO:34.
- TCR as claimed in claim 13, it is characterised in that the amino acid sequence of the TCR is SEQ ID NO:30.
- TCR as claimed in claim 9, it is characterised in that the TCR includes all or part of TCR α chains (a) in addition to membrane spaning domain；And all or part of TCR β chains of (b) in addition to membrane spaning domain；And with (b) each self-contained functional variable domain (a), or at least a portion comprising functional variable domain and the TCR chains constant domain.
- TCR as claimed in claim 15, it is characterised in that cysteine residues form artificial disulfide bond between α the and β chain constant domains of the TCR.
- TCR as claimed in claim 16, it is characterised in that the cysteine residues that artificial disulfide bond is formed in the TCR instead of selected from following one or more groups of sites：The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；WithThe Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.
- TCR as claimed in claim 17, it is characterised in that the α chain amino acid sequences of the TCR are SEQ ID NO:26 and/or TCR β chain amino acid sequences are SEQ ID NO:28.
- TCR as described in any of the above claim, it is characterised in that the α chains of the TCR and/or C- the or N- ends of β chains are combined with conjugate.
- φt cell receptor as claimed in claim 19, it is characterised in that the conjugate combined with the φt cell receptor is the combination of detectable, therapeutic agent, PK modified parts or these any materials.Preferably, the therapeutic agent is anti-CD 3 antibodies.
- A kind of multivalent TCR complex, it is characterised in that comprising at least two TCR molecules, and at least one TCR molecule therein is the TCR any one of the claims.
- A kind of nucleic acid molecules, it is characterised in that the nucleic acid molecules include the nucleotide sequence or its complementary series for encoding the TCR molecules described in any of the above-described claim.
- Nucleic acid molecules as claimed in claim 22, it is characterised in that it includes the nucleotide sequence SEQ ID NO of coding TCR α chain variable domains：2 or SEQ ID NO:33.
- Nucleic acid molecules as described in claim 22 or 23, it is characterised in that it includes the nucleotide sequence SEQ ID NO of coding TCR β chain variable domains：6 or SEQ ID NO:35.
- Nucleic acid molecules as claimed in claim 22, it is characterised in that it includes the nucleotide sequence SEQ ID NO of coding TCR α chains：4 and/or include coding TCR β chains nucleotide sequence SEQ ID NO：8.
- A kind of carrier, it is characterised in that described carrier contains any described nucleic acid molecules in claim 22-25；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
- A kind of host cell of separation, it is characterised in that containing being integrated with any described nucleic acid molecules in the claim 22-25 of external source in the carrier or chromosome described in claim 26 in described host cell.
- A kind of cell, it is characterised in that carrier described in the nucleic acid molecules or claim 26 described in any in the cell transduction claim 22-25；Preferably, the cell is T cell or stem cell.
- A kind of pharmaceutical composition, characterized in that, the composition contains pharmaceutically acceptable carrier and the TCR any one of claim 1-20, the TCR compounds described in claim 21, the cell in claim 22-25 described in any described nucleic acid molecules or claim 28.
- The purposes of the TCR compounds described in φt cell receptor or claim 21 any one of claim 1-20 or the cell described in claim 28, it is characterised in that swollen for preparing treatment The medicine of knurl or autoimmune disease.
- A kind of method for treating disease, it is characterised in that it includes the object to needs treatment applies any described TCR in appropriate claim 1-20, TCR compounds described in claim 21, the cell described in claim 28 or the pharmaceutical composition described in claim 29；Preferably, described disease is acute myeloid system leukaemia, chronic myelocytic system leukaemia, ALL, chronic lymphocytic leukemia, Huppert's disease, melanoma, colon cancer, breast cancer, kidney, stomach cancer, TCCB, prostate cancer, OSCC and head and neck squamous cell carcinoma.
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| CN2015102281112 | 2015-05-06 | ||
| CN201510228111.2A CN106188274A (en) | 2015-05-06 | 2015-05-06 | Identify the φt cell receptor of RHAMM antigen small peptide |
| PCT/CN2016/077679 WO2016177195A1 (en) | 2015-05-06 | 2016-03-29 | T cell receptor for recognizing rhamm antigen short-chain polypeptide |
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| WO2023241391A1 (en) * | 2022-06-17 | 2023-12-21 | 香雪生命科学技术（广东）有限公司 | Tcr molecule binding to ssx2 antigen and application thereof |
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| EA201992131A1 (en) * | 2017-03-15 | 2020-02-06 | Фред Хатчинсон Кэнсер Рисерч Сентер | HIGH-AFFINE SPECIFIC TO MAGE-A1 TCR AND THEIR APPLICATION |
| CN108690130B (en) * | 2017-04-12 | 2021-04-23 | 香雪生命科学技术（广东）有限公司 | TCR for recognizing LMP1 antigen-derived short peptide |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103619882A (en) * | 2011-04-01 | 2014-03-05 | 纪念斯隆-凯特琳癌症中心 | T cell receptor-like antibodies specific for a WT1 peptide presented by HLA-A2 |
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|---|---|---|---|---|
| CN103619882A (en) * | 2011-04-01 | 2014-03-05 | 纪念斯隆-凯特琳癌症中心 | T cell receptor-like antibodies specific for a WT1 peptide presented by HLA-A2 |
Non-Patent Citations (1)
| Title |
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| GREINER，J.等: "Identification and characterization of epitopes of the receptor for hyaluronic acid-mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2-positive patients with acute myeloid leukemia.", 《BLOOD》 * |
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| WO2023241391A1 (en) * | 2022-06-17 | 2023-12-21 | 香雪生命科学技术（广东）有限公司 | Tcr molecule binding to ssx2 antigen and application thereof |
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| CN106188274A (en) | 2016-12-07 |
| WO2016177195A1 (en) | 2016-11-10 |
| CN106459179B (en) | 2018-06-05 |
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