CN106188274A - Identify the φt cell receptor of RHAMM antigen small peptide - Google Patents
Identify the φt cell receptor of RHAMM antigen small peptide Download PDFInfo
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- CN106188274A CN106188274A CN201510228111.2A CN201510228111A CN106188274A CN 106188274 A CN106188274 A CN 106188274A CN 201510228111 A CN201510228111 A CN 201510228111A CN 106188274 A CN106188274 A CN 106188274A
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention provides a kind of can the specific binding φt cell receptor (TCR) derived from RHAMM antigen small peptide ILSLELMKL, described antigen small peptide ILSLELMKL can with HLA A0201 formed complex and together be presented to cell surface.Present invention also offers the nucleic acid molecules encoding described TCR and the carrier comprising described nucleic acid molecules.It addition, present invention also offers the cell of the TCR of the present invention that transduces.
Description
Technical field
The present invention relates to be capable of identify that and be derived from RHAMM (Receptor for Hyaluronan-Mediated
Moti l ity, RHAMM) TCR of antigen small peptide, the RHAMM that the above-mentioned TCR that the invention still further relates to transduce obtains
Specific T cell, and they are in the purposes prevented and in treatment RHAMM relevant disease.
Background technology
RHAMM has another name called CD168, is the receptor of the hyaluronic acid as one of extracellular matrix components.RHAMM is
A kind of endogenous antigen, is degraded to micromolecule polypeptide after intracellular generation, and with MHC (main histocompatibility
Complex) molecule combination formation complex, it is presented to cell surface.ILSLELMKL (165-173) is to spread out
It is conigenous the small peptide (Greiner J, et al., Blood 2005,106 (3): 938-945) of RHAMM.Research
Display, RHAMM all has expression in kinds of tumors tissue, with leukemia (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 carcinoma
(Wang C,et al.,Clinical cancer research:an official journal of the
American Association for Cancer Research 1998,4 (3): 567-576) the most prominent,
In other cancer, as gastric cancer (Li H, et al., International journal of oncology 2000,
17 (5): 927-932), renal carcinoma (Greiner J, et al., Experimental hematology 2002,
30 (9): 1029-1035), oral squamous cell carcinoma (Yamano Y, et al., International journal
Of oncology 2008,32 (5): 1001-1009), squamous cell carcinoma of the head and neck (Schmitt A, et al.,
International journal of oncology 2009,34 (3): 629-639) etc. the most equal in tumor cell
There is expression.For the treatment of above-mentioned disease, the method such as chemotherapy and radiation treatment can be used, but all can be to self
Normal cell cause damage.
T cell adoptive immunotherapy is to proceed to patient by having specific reaction-ive T cell to target cell antigen
Internal so that it is to play a role for target cell.φt cell receptor (TCR) is a kind of memebrane protein on T cell surface,
It is capable of identify that the antigen small peptide of corresponding target cells.In immune system, specific by antigen small peptide
TCR and the combination of small peptide-main histocompatibility complex (pMHC complex) cause T cell thin with antigen presentation
Born of the same parents (APC) directly physical contact, then just there is phase in T cell and other cell membrane surface molecules both APC
Interaction, causes a series of follow-up cell signal transmission and other physiological reactions, so that different antigen-specific
Property T cell to its target cell play immunological effect.Therefore, those skilled in the art are devoted to isolate RHAMM
Antigen small peptide has specific TCR, and obtains this TCR transduction T cell to RHAMM antigen small peptide tool
There is specific T cell, so that they play a role in cellular immunotherapy.
Summary of the invention
It is an object of the invention to provide a kind of φt cell receptor identifying RHAMM antigen small peptide.
A first aspect of the present invention, it is provided that a kind of φt cell receptor (TCR), described TCR can be with
ILSLELMKL-HLA complex combines.
In another preference, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, described
TCR α chain variable domain is to have the aminoacid sequence of at least 90% sequence thereto with SEQ ID NO:1；With/
Or described TCR β chain variable domain is to have the aminoacid sequence of at least 90% sequence thereto with SEQ ID NO:5
Row.
In another preference, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, and it is special
Levy and be, the aminoacid sequence of the CDR3 of described TCR α chain variable domain be AATNSGYALN (SEQ ID NO:
12)；And/or the aminoacid sequence of the CDR3 of described TCR β chain variable domain be AWSVDGAEQY (SEQ ID NO:
15)。
In another preference, 3 complementary determining regions (CDR) of described TCR α chain variable domain 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 described TCR β chain variable domain 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, described TCR comprises α chain variable domain amino acid sequence SEQ ID NO:1.
In another preference, described TCR comprises β chain variable domain amino acid sequence SEQ ID NO:5.
In another preference, described TCR is α β heterodimer, and it comprises 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 sequence of described TCR is SEQ ID NO:3 and/or described
The β chain amino acid sequence of TCR is SEQ ID NO:7.
In another preference, described TCR is solvable.
In another preference, described TCR is strand.
In another preference, described TCR is to be passed through peptide catenation sequence by α chain variable domain with β chain variable domain
It is formed by connecting.
In another preference, described TCR α chain variable region aminoacid the 11st, 13,19,21,53,
76,89,91 or the 94th, and/or 3rd reciprocal of α chain J gene small peptide aminoacid, inverse the 5th
Position or inverse the 7th has one or more sudden change；And/or described TCR is at β chain variable region aminoacid
11st, 13,19,21,53,76,89,91 or the 94th, and/or β chain J gene small peptide amino
2nd reciprocal of acid, inverse the 4th or 6th reciprocal has one or more sudden change, wherein aminoacid
Position Number is by the Position Number listed in IMGT (international immunogenetics information system).
In another preference, the α chain variable domain amino acid sequence of described TCR comprises SEQ ID NO:32
And/or the β chain variable domain amino acid sequence of described TCR comprises SEQ ID NO:34.
In another preference, the aminoacid sequence of described TCR is SEQ ID NO:30.
In another preference, described TCR includes (a) all or part of TCR α in addition to membrane spaning domain
Chain；And all or part of TCR β chain that (b) is in addition to membrane spaning domain；
And (a) with (b) each self-contained functional variable domain, or comprise functional variable domain and institute
State at least some of of TCR chain constant domain.
In another preference, cysteine residues forms people between α and the β chain constant domain of described TCR
Work disulfide bond.
In another preference, the cysteine residues forming artificial disulfide bond in described TCR instead of choosing
One or more groups site from following:
The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
The Ala19 of Pro89 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1；
With
The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1.
In another preference, the α chain amino acid sequence of described TCR is SEQ ID NO:26 and/or described
The β chain amino acid sequence of TCR is SEQ ID NO:28.
In another preference, the α chain of described TCR and/or C-or the N-end of β chain are combined with conjugate.
In another preference, the conjugate being combined with described φt cell receptor is detectable, treatment
Agent, PK modify part or the combination of these materials any.Preferably, described therapeutic agent is anti-CD 3 antibodies.
A second aspect of the present invention, it is provided that a kind of multivalent TCR complex, it comprises at least two TCR and divides
Son, and at least one TCR molecule therein is the TCR described in first aspect present invention.
A third aspect of the present invention, it is provided that a kind of nucleic acid molecules, described nucleic acid molecules comprises code book invention
The nucleotide sequence of the TCR molecule described in first aspect or its complementary series.
In another preference, described nucleic acid molecules comprises the nucleotide sequence SEQ of coding TCR α chain variable domain
ID NO:2 or SEQ ID NO:33.
In another preference, described nucleic acid molecules comprises the nucleotide sequence of coding TCR β chain variable domain
SEQ ID NO:6 or SEQ ID NO:35.
In another preference, described nucleic acid molecules comprise coding TCR α chain nucleotide sequence SEQ ID NO:
4 and/or comprise coding TCR β chain nucleotide sequence SEQ ID NO:8.
A fourth aspect of the present invention, it is provided that a kind of carrier, described carrier contains third aspect present invention institute
The nucleic acid molecules stated；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow
Viral vector.
A fifth aspect of the present invention, it is provided that the host cell of a kind of separation, contains in described host cell
Carrier described in fourth aspect present invention or genome are integrated and has described in the third aspect present invention of external source
Nucleic acid molecules.
A sixth aspect of the present invention, it is provided that a kind of cell, described in described cell transduction third aspect present invention
Nucleic acid molecules or fourth aspect present invention described in carrier；Preferably, described cell is T cell or dry thin
Born of the same parents.
A seventh aspect of the present invention, it is provided that a kind of pharmaceutical composition, described compositions contains and pharmaceutically can connect
TCR described in the carrier being subject to and first aspect present invention, the TCR complex described in second aspect present invention,
Nucleic acid molecules described in third aspect present invention, the carrier described in fourth aspect present invention or the present invention the 6th
Cell described in aspect.
A eighth aspect of the present invention, it is provided that the φt cell receptor described in first aspect present invention or the present invention
TCR complex described in second aspect, the nucleic acid molecules described in third aspect present invention, present invention four directions
Carrier described in face or the purposes of the cell described in sixth aspect present invention, be used for preparing treatment tumor or from
The medicine of body immunological diseases.
A ninth aspect of the present invention, it is provided that a kind of method treating disease, including to the object needing treatment
Use the φt cell receptor described in appropriate first aspect present invention or the TCR described in second aspect present invention
Nucleic acid molecules described in complex, third aspect present invention, the carrier described in fourth aspect present invention or basis
Invent the cell described in the 6th aspect or the pharmaceutical composition described in seventh aspect present invention；
Preferably, described disease is acute myeloid system leukemia, chronic myelocytic system leukemia, acute
Lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, melanoma, colon cancer,
Breast carcinoma, renal carcinoma, gastric cancer, transitional cell carcinoma of bladder, carcinoma of prostate, oral squamous cell carcinoma and head and neck
Portion's squamous cell carcinoma.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (as implemented
Example) in can be combined with each other between each technical characteristic of specifically describing, thus constitute new or preferred skill
Art scheme.As space is limited, the most tired at this state.
Accompanying drawing explanation
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively TCR α chain variable domain amino acid
Sequence, TCR α chain variable domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence,
The TCR α chain amino acid sequence with targeting sequencing and the TCR α chain nucleotide sequence 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 chain variable domain amino acid
Sequence, TCR β chain variable domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence,
The TCR β chain amino acid sequence with targeting sequencing and the TCR β chain nucleotide sequence with targeting sequencing.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining result of the tetramer-PE.
Fig. 4 a and Fig. 4 b is respectively aminoacid sequence and the nucleotide sequence of sTCR α chain.
Fig. 5 a and Fig. 5 b is respectively aminoacid sequence and the nucleotide sequence of sTCR β chain.
Fig. 6 is the glue figure of the sTCR obtained after purification.
Fig. 7 a and Fig. 7 b is respectively aminoacid sequence and the nucleotide sequence of strand TCR.
Fig. 8 a and Fig. 8 b is respectively aminoacid sequence and the nucleotide sequence of strand TCR α chain.
Fig. 9 a and Fig. 9 b is respectively aminoacid sequence and the nucleotide sequence of strand TCR β chain.
Figure 10 a and Figure 10 b is respectively aminoacid sequence and the nucleoside of strand TCR catenation sequence (l inker)
Acid sequence.
Figure 11 is the glue figure of the soluble single-chain T CR obtained after purification.
Figure 12 is the BIAcore that sTCR of the present invention is combined with ILSLELMKL-HLA A0201 complex
Kinetic profile.
Figure 13 is that soluble single-chain T CR and ILSLELMKL-HLA A0201 complex of the present invention is combined
BIAcore kinetic profile.
Detailed description of the invention
The present inventor, through extensively in-depth study, have found and RHAMM antigen small peptide ILSLELMKL
(165-173) TCR that (SEQ ID NO:9) can be specific binding, described antigen small peptide ILSLELMKL
Can with HLA A0201 formed complex and together be presented to cell surface.Present invention also offers coding institute
State the nucleic acid molecules of TCR and comprise the carrier of described nucleic acid molecules.It addition, present invention also offers transduction
The cell of TCR of the present invention.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Cause
This, it is presented for antigen has specificity, and different individualities has different MHC, can present a hatching egg
Small peptides different in Bai Kangyuan is to respective APC cell surface.The MHC of the mankind be commonly referred to HLA gene or
HLA complex.
φt cell receptor (TCR), is to present the specific antigen peptide in main histocompatibility complex (MHC)
Unique receptor.In immune system, drawn by the combination of TCR with the pMHC complex of antigenic specificity
Send out T cell and antigen-presenting cell (APC) directly physical contact, then T cell and its both APC
He just interacts by cell membrane surface molecules, this just cause a series of follow-up cell signal transmission and
Other physiological reactions, so that the T cell of different antigenic specificity plays immunological effect to its target cell.
TCR is the surface of cell membrane existed with heterodimer form by α chain/β chain or γ chain/δ chain
Glycoprotein.In the T cell of 95%, TCR heterodimer is made up of α and β chain, and the T cell of 5% tool
By the TCR being made up of γ and δ chain.Natural heterogeneous dimerization TCR of α β has α chain and β chain, α chain and β
Chain constitutes the subunit of α β heterodimeric TCR.In a broad sense, each chain of α and β comprises variable region, connection
District and constant region, β chain generally the most also between variable region and bonding pad containing short variable region, but this variable region
Often regard as a part for bonding pad.Each variable region comprises and is entrenched in frame structure (framework regions)
In 3 CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines TCR and pMHC
The combination of complex, wherein CDR3 is recombinated by variable region and bonding pad and forms, and is referred to as hypervariable region.TCR's
α and β chain is typically regarded as and is respectively arranged with two " domain " i.e. variable domains and constant domain, and variable domain can by connect
Become district and bonding pad to constitute.The sequence of TCR constant domain can be international immunogenetics information system (IMGT)
Public data storehouse in find, if the constant domain sequence of TCR molecule alpha chain is " TRAC*01 ", TCR molecule
The constant domain sequence of β chain is " TRBC1*01 " or " TRBC2*01 ".Additionally, α and the β chain of TCR is also
Comprising cross-film district and cytoplasmic region, cytoplasmic region is the shortest.
In the present invention, term " polypeptide of the present invention ", " TCR of the present invention ", " T of the present invention is thin
Born of the same parents' receptor " it is used interchangeably.
Detailed Description Of The Invention
TCR molecule
In antigen processing pathways, antigen is degraded intracellular, is then carried to cell by MHC molecule
Surface.φt cell receptor is capable of identify that the peptide-MHC complex of Antigen Presenting Cell surface.Therefore, the present invention
First aspect provide and a kind of can divide the TCR of specific binding ILSLELMKL-HLA A0201 complex
Son.Preferably, described TCR molecule is to separate or purification.α and the β chain of this TCR respectively has 3
Complementary determining region (CDR).
Being preferably carried out in mode of the present invention, the α chain of described TCR comprises and has following aminoacid
The CDR of sequence:
α CDR1-DRVSQS (SEQ ID NO:10)
α CDR2-IYSNGD (SEQ ID NO:11)
α CDR3-AATNSGYALN (SEQ ID NO:12)。
Being preferably carried out in mode of the present invention, the β chain of described TCR comprises and has following aminoacid
The CDR of 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 aminoacid sequence of the invention described above can be embedded in any applicable frame structure and make
Standby chimeric 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 root
Just can design according to CDR region disclosed by the invention or synthesize the TCR molecule with corresponding function.Therefore,
TCR molecule of the present invention refers to comprise above-mentioned α and/or β chain CDR region sequence and any applicable frame structure
TCR molecule.TCR α chain variable domain of the present invention is for have at least 90% with SEQ ID NO:1, preferably
95%, the aminoacid sequence of more preferably 98% sequence thereto；And/or TCR β chain variable domain of the present invention is
At least 90% is had, the amino of preferably 95%, more preferably 98% sequence thereto with SEQ ID NO:5
Acid sequence.
In a preference of the present invention, the TCR molecule of the present invention is heterogeneous two be made up of α with β chain
Aggressiveness.Specifically, the α chain of the most described heterogeneous dimerization TCR molecule comprises variable domain and constant domain, institute
State CDR1 (SEQ ID NO:10), CDR2 (SEQ that α chain variable domain amino acid sequence comprises above-mentioned α chain
ID NO:11) and CDR3 (SEQ ID NO:12).Preferably, to comprise α chain variable for described TCR molecule
Domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domain amino acid sequence of described TCR molecule
It is classified as SEQ ID NO:1.On the other hand, the β chain of described heterogeneous dimerization TCR molecule comprises variable domain and perseverance
Localization, described β chain variable domain amino acid sequence comprise above-mentioned β chain CDR1 (SEQ ID NO:13),
CDR2 (SEQ ID NO:14) and CDR3 (SEQ ID NO:15).Preferably, described TCR molecule bag
Containing β chain variable domain amino acid sequence SEQ ID NO:5.It is highly preferred that the β chain of described TCR molecule is variable
Domain amino acid sequence is SEQ ID NO:5.
In a preference of the present invention, the TCR molecule of the present invention be by α chain partly or entirely and/
Or the single chain TCR molecules of the partly or entirely composition of β chain.Description about single chain TCR molecules is 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 strand comprising CDRs district of the present invention
TCR molecule.Specifically, described single chain TCR molecules comprises V α, V β and C β, preferably according to from N
End being linked in sequence to C end.
The α chain variable domain amino acid sequence of described single chain TCR molecules comprises CDR1 (the SEQ ID of above-mentioned α chain
NO:10), CDR2 (SEQ ID NO:11) and CDR3 (SEQ ID NO:12).Preferably, described
Single chain TCR molecules comprises α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that described strand
The α chain variable domain amino acid sequence of TCR molecule is SEQ ID NO:1.The β chain of described single chain TCR molecules
Variable domain amino acid sequence comprise above-mentioned β chain CDR1 (SEQ ID NO:13), CDR2 (SEQ ID NO:
14) and CDR3 (SEQ ID NO:15).Preferably, described single chain TCR molecules comprises β chain variable domain
Aminoacid sequence SEQ ID NO:5.It is highly preferred that the β chain variable domain amino acid of described single chain TCR molecules
Sequence is SEQ ID NO:5.
In a preference of the present invention, the constant domain of the TCR molecule of the present invention is the constant domain of people.This
Skilled person knows maybe can be by consulting pertinent texts or IMGT (world immunogenetics information system
System) public data storehouse obtain the constant domain aminoacid sequence of people.Such as, TCR molecule alpha chain of the present invention
Constant domain sequence can be " TRAC*01 ", the constant domain sequence of TCR molecule β chain can be " TRBC1*01 "
Or " TRBC2*01 ".The 53rd of the aminoacid sequence be given in the TRAC*01 of IMGT is Arg,
This is expressed as: the Arg53 of TRAC*01 exons 1, and other are by that analogy.Preferably, TCR of the present invention
The aminoacid sequence of molecule alpha chain is SEQ ID NO:3, and/or the aminoacid sequence of β chain is SEQ ID NO:
7。
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its cross-film district.Such as immunoglobulin
(antibody) is the same as antigen recognizing molecule, and TCR can also be developed and be applied to diagnosis and treatment, at this moment needs
The TCR molecule of solubility to be obtained.The TCR molecule of solubility does not include its cross-film district.STCR has
Purposes the most widely, it cannot be only used for studying the interaction of TCR Yu pMHC, it is possible to is used as detection sense
The diagnostic tool of dye or as the mark of autoimmune disease.Similarly, sTCR can be used to by
Therapeutic agent (such as cytotoxin compounds or immunostimulating compound) is transported to present the thin of specific antigen
Born of the same parents, it addition, sTCR also can combine with other molecules (e.g., anti-CD 3 antibodies) redirects T
Cell, so that its targeting presents the cell of specific antigen.The present invention also obtain RHAMM antigen small peptide
There is specific sTCR.
For obtaining sTCR, on the one hand, TCR of the present invention can be residual at itself α and β chain constant domain
The TCR of artificial disulfide bond is introduced between base.Cysteine residues is between α and the β chain constant domain of described TCR
Form artificial interchain disulfide bond.Cysteine residues can be substituted in other ammonia of appropriate site in natural TCR
Base acid residue is to form artificial interchain disulfide bond.Such as, replace the Thr48 of TRAC*01 exons 1 and take
Cysteine residues for the Ser57 of TRBC1*01 or TRBC2*01 exons 1 forms disulfide bond.Draw
Enter cysteine residues to form other sites of disulfide bond it may also is that the Thr45 of TRAC*01 exons 1
Ser77 with TRBC1*01 or TRBC2*01 exons 1；The Tyr10 of TRAC*01 exons 1 and
The Ser17 of TRBC1*01 or TRBC2*01 exons 1；Thr45 and TRBC1*01 of TRAC*01 exons 1
Or the Asp59 of TRBC2*01 exons 1；Ser15 and TRBC1*01 of TRAC*01 exons 1 or
The Glu15 of TRBC2*01 exons 1；Arg53 and TRBC1*01 or TRBC2*01 of TRAC*01 exons 1
The Ser54 of exons 1；Show outside Pro89 and TRBC1*01 or TRBC2*01 of TRAC*01 exons 1
The Ala19 of son 1；Or Tyr10 and TRBC1*01 or the TRBC2*01 exon of TRAC*01 exons 1
The Glu20 of 1.Arbitrary group of site during i.e. cysteine residues instead of above-mentioned α Yu β chain constant domain.Can be
Most 50 or most 30 or most of one or more C-terminal truncates of TCR constant domain of the present invention
15 or most 10 or most 8 or less aminoacid, so that it does not include that cysteine is residual
Base reaches to lack the purpose of natural disulphide bonds, it is possible to by the cysteine residues by forming natural disulphide bonds
Sport another aminoacid to reach above-mentioned purpose.
As it has been described above, the TCR of the present invention may be embodied in the people introduced between the residue of itself α and β chain constant domain
Work disulfide bond.It should be noted that the artificial disulfide bond with or without introducing mentioned above, the present invention between constant domain
TCR all can contain TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequence.The TRAC of TCR
Constant domain sequence and TRBC1 or TRBC2 constant domain sequence can be connected by the natural disulphide bonds being present in TCR
Connect.
For obtaining sTCR, on the other hand, it is prominent that TCR of the present invention is additionally included in the generation of its hydrophobic core region
The TCR become, the sudden change in these hydrophobic core regions is preferably capable making the stability of sTCR of the present invention carry
High sudden change, as described in the patent documentation of Publication No. WO2014/206304.Such TCR can be
Undergo mutation in its following variable domain hydrophobic core position: (α and/or β chain) variable region amino acid the 11st, 13,
19,21,53,76,89,91,94, and/or α chain J gene (TRAJ) small peptide amino acid position
3rd, 5,7 reciprocal, and/or 2nd, 4,6 reciprocal of β chain J gene (TRBJ) small peptide amino acid position,
Wherein the Position Number of aminoacid sequence is by the position listed in international immunogenetics information system (IMGT)
Numbering.Those skilled in the art know above-mentioned international immunogenetics information system, and can be according to this data base
Obtain the amino acid residue of the different TCR Position Number in IMGT.
The TCR that in the present invention, undergo mutation in hydrophobic core region can be by a flexible peptide chain connect the α of TCR with
The variable domain of β chain and the stability solvable strand TCR that constitutes.It should be noted that in the present invention, flexible peptide chain is permissible
It it is the peptide chain of any applicable connection TCR α and β chain variable domain.Such as the list built in the embodiment of the present invention 4
Chain sTCR, its α chain variable domain amino acid sequence is SEQ ID NO:32, the nucleotide sequence of coding
For SEQ ID NO:33；β chain variable domain amino acid sequence is SEQ ID NO:34, the nucleotides sequence of coding
It is classified as SEQ ID NO:35.
The TCR of the present invention can also multivalence complex form provide.The multivalent TCR complex bag of the present invention
Containing two, three, the four or more TCR of the present invention polymer that combines and formed, as p53 can be used
Four dimerization domain produce the tetramer, or multiple TCR of the present invention be combined with another molecule and formed compound
Thing.The TCR complex of the present invention can be used for external or internal tracking or targeting presents the cell of specific antigen,
Can also be used for producing the intermediate of other multivalent TCR complex with this type of application.
The TCR of the present invention can be used alone, it is possible to is combined, preferably with covalency or other modes with conjugate
Combine with covalent manner.Described conjugate includes that detectable is (for diagnostic purpose, wherein said TCR
Present the existence of cell of ILSLELMKL-HLA A0201 complex for detection), therapeutic agent, PK (egg
White kinases) modify part or the combination combination of any the above material or coupling.
Detectable for diagnostic purposes includes but not limited to: fluorescence or luminous marker, radioactivity
Label, MRI (nuclear magnetic resonance) or CT (CT technology) contrast agent,
Maybe can produce the enzyme that can detect product.
Can be combined with TCR of the present invention or the therapeutic agent of coupling includes but not limited to: 1. radionuclide
(Koppe etc., 2005, cancerometastasis comment (Cancer metastasis reviews) 24,539)；2. raw
Thing poison (Chaudhary etc., 1989, natural (Nature) 339,394；Epel etc., 2002, cancer is exempted from
Epidemiology and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell
(Gillies etc., 1992, institute of NAS periodical (PNAS) 89,1428 such as the factor such as IL-2；Card
Deng, 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,
345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc sheet
Section (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 (such as, DT-diaphorase (DTD) or xenyl water
Solve enzyme-sample protein (BPHL))；11. chemotherapeutics (such as, cisplatin) or any type of nano-particle etc..
It addition, the TCR of the present invention can also is that heterozygosis TCR comprised derived from exceeding a kind of species sequence.
Such as, research display Muridae TCR is had can more effectively to express than people TCR in human T-cell.Therefore,
TCR of the present invention can comprise the constant domain of people's variable domain and Mus.The defect of this method is possible to cause immunity to answer
Answer.Therefore, should there is regulation scheme to carry out immunosuppressant when it is treated for adoptive T cell, with
Allow to express the implantation of the T cell of Muridae.
Should be understood that amino acid name uses international single English alphabet or three the English alphabets herein
Showing, single English alphabet of amino acid name and the corresponding relation of three English alphabets are 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
A second aspect of the present invention provides the nucleic acid of coding first aspect present invention TCR molecule or its part and divides
Son, described part can be the variable domain of one or more CDR, α and/or β chain, and α chain and/or
β chain.
The nucleotide sequence of coding first aspect present invention TCR molecule alpha chain CDR region 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 of coding first aspect present invention TCR molecule β chain CDR region 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, the nucleotide sequence of nucleic acid molecules of the present invention of code book invention TCR α chain includes SEQ ID NO:
The core of the present invention of 16, SEQ ID NO:17 and SEQ ID NO:18, and/or code book invention TCR β chain
The nucleotide sequence of acid molecule 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 strand or double-strand, and this nucleic acid molecules can be
RNA or DNA, and can comprise or not comprise intron.Preferably, the nucleoside of nucleic acid molecules of the present invention
Acid sequence does not comprises intron but can code book invention polypeptide, such as code book invention TCR α chain variable domain
The nucleotide sequence of nucleic acid molecules of the present invention include SEQ ID NO:2 and/or code book invention TCR β chain
The nucleotide sequence of the nucleic acid molecules of the present invention of variable domain includes SEQ ID NO:6.Or, code book is invented
The nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chain variable domain includes SEQ ID NO:33 and/or coding
The nucleotide sequence of the nucleic acid molecules of the present invention of TCR β chain variable domain of the present invention includes SEQ ID NO:35.
It is highly preferred that the nucleotide sequence of nucleic acid molecules of the present invention comprises 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.
Should be understood that the degeneracy due to genetic code, different nucleotide sequences can encode identical polypeptide.
Therefore, code book invention TCR nucleotide sequence can identical with the nucleotide sequence shown in accompanying drawing of the present invention or
It it is the variant of degeneracy.Illustrating with one of them example in the present invention, " variant of degeneracy " refers to
Coding has a protein sequence of SEQ ID NO:1, but with the differentiated nucleic acid of sequence of SEQ ID NO:2
Sequence.
Nucleotide sequence can be through codon optimized.Different cells is in the utilization of concrete codon
Different, can be according to the type of cell, the codon changed in sequence increases expression.Mammal
The codon usage table of cell and multiple other biological is to well known to a person skilled in the art.
The nucleic acid molecules full length sequence of the present invention or its fragment generally can with but be not limited to PCR TRAP, weight
The method of group method or synthetic obtains.At present, it is already possible to obtain code book by chemosynthesis completely
The DNA sequence of invention TCR (or its fragment, or derivatives thereof).Then this DNA sequence can be introduced ability
In various existing DNA moleculars (or such as carrier) known in territory and cell.DNA can be coding strand or non-
Coding strand.
Carrier
The invention still further relates to the carrier of the nucleic acid molecules comprising the present invention, including expression vector, i.e. can be at body
In or the construct of vivoexpression.Conventional carrier includes bacterial plasmid, phage and animals and plants virus.
Viral delivery systems includes but not limited to adenovirus vector, adeno-associated virus (AAV) carrier, herpes
Viral 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
This cell expresses the TCR of RHAMM antigenic specificity.Ideally, this carrier should be thin at T
In born of the same parents, continual high levels ground is expressed.
Cell
The invention still further relates to the host cell using the carrier of the present invention or coded sequence to produce through genetic engineering.Institute
State in the carrier or chromosome containing the present invention in host cell and integrate the nucleic acid molecules having the present invention.Host is thin
Born of the same parents are selected from: prokaryotic cell and eukaryotic cell, such as escherichia coli, yeast cells, Chinese hamster ovary celI etc..
It addition, present invention additionally comprises the cell of the separation of the TCR expressing the present invention, particularly T cell.Should
T cell can derived from the T cell separated from experimenter, or can be that the mixing separated from experimenter is thin
Born of the same parents group, the such as part of peripheral blood lymphocyte (PBL) group.As, this cell can be isolatable from periphery
Blood monocyte (PBMC), can be CD4+Helper T cell or CD8+Cytotoxic T cell.This cell
Can be at CD4+Helper T cell/CD8+In the mixing group of cytotoxic T cell.Usually, this cell can be used
Antibody (e.g., the antibody of anti-CD3 or anti-CD28) activates, in order to allow them to be easier to accept transfection,
Such as transfect with the carrier of the nucleotide sequence comprising code book invention TCR molecule.
Alternatively, the cell of the present invention can also is that or derived from stem cell, such as hematopoietic stem cell (HSC).
Gene is transferred to HSC and is not result at cell surface expression TCR, because stem cell surface does not express CD3
Molecule.But, migrate to the lymphoid precursor (lymphoid precursor) of thymus when stem cell is divided into
Time, expressing the TCR molecule of this introducing of surface expression started at thymocyte cell of CD3 molecule.
There are many methods to be suitable for DNA or RNA of code book invention TCR and carry out T cell transfection (e.g.,
Robbins etc., (2008) J.Immunol.180:6116-6131).The T expressing TCR of the present invention is thin
Born of the same parents may be used for adoptive immunotherapy.Those skilled in the art understand that the many conjunctions carrying out adoptive treatment
Suitable method (e.g., Rosenberg etc., (2008) Nat Rev Cancer8 (4): 299-308).
RHAMM antigen-related disease
The invention still further relates to treat in experimenter and/or prevent and the method for RHAMM relevant disease, its bag
Include adoptive transfer RHAMM specific T-cells to the step of this experimenter.This RHAMM specific T-cells
Recognizable ILSLELMKL-HLA A0201 complex.
The specific T cell of RHAMM of the present invention can be used for treating any presents RHAMM antigen small peptide
The RHAMM relevant disease of ILSLELMKL-HLA A0201 complex.Include but not limited to acute myeloid system
Leukemia, chronic myelocytic system leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,
Multiple myeloma, melanoma, colon cancer, breast carcinoma, renal carcinoma, gastric cancer, transitional cell carcinoma of bladder,
Carcinoma of prostate, oral squamous cell carcinoma and squamous cell carcinoma of the head and neck.
Therapeutic Method
The T cell of the patient with RHAMM antigen-related disease or volunteer can be suffered from by separation, and will
The TCR of the present invention imports in above-mentioned T cell, subsequently the cell that these genetic engineerings are modified is fed back to patient
Internal treat.Therefore, the invention provides a kind of method treating RHAMM relevant disease, including
T cell by the expression TCR of the present invention of separation, it is preferable that this T cell derives from patient itself, input
In patient body.Usually, separating the T cell of patient including (1), (2) use nucleic acid molecules of the present invention
Or can code book invention TCR molecule nucleic acid molecules ex vivo transduction T cell, genetic engineering is repaiied by (3)
The T cell of decorations is input in patient body.The quantity of the cell separate, transfecting and feeding back can be determined by doctor.
Main advantages of the present invention are:
(1) TCR of the present invention can be special with RHAMM antigen small peptide composite I LSLELMKL-HLA A0201
Anisogamy, the cell of the TCR of the present invention that simultaneously transduceed can have by specific activation and to target cell
The strongest lethal effect.
Following specific embodiment, is expanded on further the present invention.Should be understood that these embodiments are merely to illustrate
The present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally according to normal condition, such as (Sambrook and Russell et al., molecular cloning: laboratory
Handbook (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing house)
Described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and
Number is calculated by weight.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.Following example
Experiment material and reagent used by the most all can obtain from commercially available channel.
Embodiment 1 clones RHAMM antigen small peptide specific T-cells
Utilize synthesis small peptide ILSLELMKL (Beijing SBS Genetech gene technology company limited) to stimulate and come from base
Peripheral blood lymphocyte (PBL) because of healthy volunteer that type is HLA-A0201.ILSLELMKL is short
Peptide, with biotin labeled HLA-A*0201 renaturation, prepares pHLA monoploid.These monoploid and use
The Streptavidin (BD company) of PE labelling is combined into the tetramer of PE labelling, sorts this tetramer and resists
The double positive cell of-CD8-APC.The cell of amplification sorting, and carry out secondary sorting as stated above, use subsequently
Limiting dilution assay carries out monoclonal.Monoclonal cell tetramer staining, the double positive colonies screened such as figure
Shown in 3.
Embodiment 2 obtains the tcr gene of RHAMM antigen small peptide specific T-cell clones and the structure of carrier
Use Quick-RNATMScreen in MiniPrep (ZYMO research) extracting embodiment 1 is anti-
The total serum IgE of former small peptide ILSLELMKL specificity, HLA-A0201 restrictive T cell clone.CDNA's
Synthesis uses the SMART RACE cDNA amplification kit of clontech, and the primer of employing is that design is people
The C end conserved region of class tcr gene.Sequence is cloned in carrier T (TAKARA) and checks order.Should note
Meaning, this sequence is complementary series, does not comprise intron.Through order-checking, the TCR's of this pair of positive colony expression
α chain and β chain-ordering structure are distinguished the most as depicted in figs. 1 and 2, Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e
It is respectively TCR α chain variable domain amino acid sequence, TCR α chain variable domain nucleotide sequence, TCR with Fig. 1 f
α chain amino acid sequence, TCR α chain nucleotide sequence, have the TCR α chain amino acid sequence of targeting sequencing with
And there is the TCR α chain nucleotide sequence of targeting sequencing；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and
Fig. 2 f is respectively TCR β chain variable domain amino acid sequence, TCR β chain variable domain nucleotide sequence, TCR β chain
Aminoacid sequence, TCR β chain nucleotide sequence, the TCR β chain amino acid sequence with targeting sequencing and tool
There is the TCR β chain nucleotide sequence of targeting sequencing.
Identified, α chain comprises the CDR with following aminoacid sequence:
α CDR1-DRVSQS (SEQ ID NO:10)
α CDR2-IYSNGD (SEQ ID NO:11)
α CDR3-AATNSGYALN (SEQ ID NO:12)
β chain comprises the CDR with following aminoacid sequence:
β CDR1-GTSNPN (SEQ ID NO:13)
β CDR2-SVGIG (SEQ ID NO:14)
β CDR3-AWSVDGAEQY (SEQ ID NO:15)
Respectively the full-length gene of TCR α chain and β chain is cloned into slow virus by overlapping (overlap) PCR
Expression vector pLenti (addgene).Particularly as follows: with overlap PCR by TCR α chain and TCR β chain
Full-length gene is attached obtaining TCR α-2A-TCR β fragment.By Lentiviral and TCR α
The connection of-2A-TCR β enzyme action obtains pLenti-RHAMMTRA-2A-TRB-IRES-NGFR plasmid.As comparison
With, the most also slow virus carrier pLenti-eGFP of construction expression eGFP.Pack with 293T/17 the most again
Pseudovirus.
Expression, refolding and the purification of the embodiment 3 RHAMM antigen solvable TCR of small peptide specificity
For obtaining solvable TCR molecule, α and the β chain of the TCR molecule of the present invention can the most only comprise it
Variable domain and portion constant territory, and the constant domain of α and β chain introduce a cysteine residues respectively
To form artificial interchain disulfide bond, the position introducing cysteine residues is respectively TRAC*01 exons 1
The Ser57 of Thr48 and TRBC2*01 exons 1；The aminoacid sequence of its α chain is with nucleotide sequence respectively
As shown in figures 4 a and 4b, the aminoacid sequence of its β chain and nucleotide sequence are respectively such as Fig. 5 a and Fig. 5 b
Shown in, the cysteine residues of introducing is with overstriking letter representation.By " Molecular Cloning: A Laboratory room handbook
" (Molecular Cloning a Laboratory Manual) (third edition, Sambrook and Russell)
Described in standard method by the genes of interest sequence of above-mentioned TCR α and β chain through synthesis after be inserted respectively into table
Reaching carrier pET28a+ (Novagene), the cloning site of upstream and downstream is NcoI and NotI respectively.Insert
Fragment confirms errorless through order-checking.
Convert to enter by chemical transformation respectively by the expression vector of TCR α and β chain and express antibacterial BL21
(DE3), antibacterial LB culture fluid grows, in OD600Induce with final concentration 0.5mM IPTG when=0.6,
The inclusion body that α and the β chain of TCR is formed after expressing is extracted by BugBuster Mix (Novagene),
And through the repeated multiple times washing of BugBuster solution, inclusion body is finally dissolved in 6M guanidine hydrochloride, 10mM
Dithiothreitol, DTT (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), in 20mM Tris (pH 8.1).
TCR α and β chain after dissolving are quickly mixed in 5M carbamide, 0.4M essence with the mass ratio of 1:1
Propylhomoserin, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM β-mercapoethylamine
In (4 DEG C), final concentration of 60mg/mL.After mixing, solution is placed in the deionized water of 10 times of volumes thoroughly
Analysis (4 DEG C), changes deionized water buffer (20mM Tris, pH 8.0) into and continues at after 12 hours
Dialyse 12 hours for 4 DEG C.Solution after having dialysed after the membrane filtration of 0.45 μM, by the moon from
Sub-exchange column (HiTrap Q HP, 5ml, GE Healthcare) purification.Eluting peak contains renaturation success
The dimeric TCR of α and β confirmed by SDS-PAGE glue.TCR passes through gel permeation chromatography subsequently
(HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) is further purified.Purification
After TCR purity measure more than 90% through SDS-PAGE, concentration is determined by BCA method.The present invention obtains
The SDS-PAGE glue figure of sTCR is as shown in Figure 6.
The generation of embodiment 4 RHAMM antigen small peptide specific soluble single-chain T CR
According to patent documentation WO2014/206304, utilize the method for rite-directed mutagenesis by embodiment 2
The variable domain of TCR α and β chain be built into one with flexible small peptide (linker) be connected stable solvable
Property single chain TCR molecules.The aminoacid sequence of this single chain TCR molecules and nucleotide sequence respectively such as Fig. 7 a and
Shown in Fig. 7 b.The aminoacid sequence of its α chain variable domain and nucleotide sequence are respectively such as Fig. 8 a and Fig. 8 b institute
Show；The aminoacid sequence of its β chain variable domain and nucleotide sequence are the most as shown in figures 9 a and 9b；Its
The aminoacid sequence of linker sequence and nucleotide sequence are the most as as-shown-in figures 10 a and 10b.
By genes of interest through Nco I and Not I double digestion, and through Nco I and Not I double digestion
PET28a carrier connects.Connecting product to convert to E.coli DH5 α, coating contains the LB flat board of kanamycin,
Being inverted overnight incubation for 37 DEG C, picking positive colony carries out PCR screening, checks order positive recombinant, really
Fixed sequence extracts recombinant plasmid transformed the most afterwards to E.coli BL21 (DE3), is used for expressing.
Expression, renaturation and the purification of embodiment 5 RHAMM antigen small peptide specific soluble single-chain T CR
By whole for BL21 (DE 3) bacterium colony containing recombiant plasmid pET28a-template strand of preparation in embodiment 4
Being inoculated in the LB culture medium containing kanamycin, 37 DEG C are cultivated to OD600 is 0.6-0.8, adds IPTG
To final concentration of 0.5mM, 37 DEG C are continued to cultivate 4h.5000rpm is centrifuged 15min harvesting precipitation
Thing, with Bugbuster Master Mix (Merck) cell lysis precipitate, 6000rpm is centrifuged 15min
Reclaim inclusion body, then carry out washing to remove cell debris and membrane component with Bugbuster (Merck), 6000
Rpm is centrifuged 15min, collects inclusion body.By solubilization of inclusion bodies at buffer (20mM Tris-HCl pH 8.0,8
M carbamide) in, high speed centrifugation removes insoluble matter, carries out subpackage, in-80 after supernatant BCA standard measure
DEG C save backup.
In the strand TCR inclusion body protein that 5mg dissolves, addition 2.5mL buffer (6M Gua-HCl,
50mM Tris-HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration
For 10mM, 37 DEG C process 30min.With syringe to 125mL renaturation buffer (100mM Tris-HCl
PH 8.1,0.4M L-arginine, 5M carbamide, 2mM EDTA, 6.5mM β
-mercapthoethylamine, 1.87mM Cystamine) in drip the strand TCR after above-mentioned process, 4
DEG C stirring 10min, then renaturation solution is loaded the cellulose membrane bag filter that interception is 4kDa, bag filter
Being placed in the water of 1L pre-cooling, 4 DEG C are slowly stirred overnight.After 17 hours into, dialysis solution is changed 1L pre-cooling
Buffer (20mM Tris-HCl pH 8.0), 4 DEG C continue dialysis 8h, then dialysis solution is changed into
Identical fresh buffer continues dialysed overnight.After 17 hours, sample is through 0.45 μm membrane filtration, vacuum
By anion-exchange column (HiTrap Q HP, GE Healthcare) after degassing, use 20mM Tris-HCl
The 0-1M NaCl linear gradient elution liquid purifying protein of pH 8.0 preparation, the elution fraction of collection is carried out
SDS-PAGE analyzes, and the component comprising strand TCR uses solvent resistant column (Superdex 75 after concentrating further
10/300, GE Healthcare) it is purified, target components is also carried out SDS-PAGE and analyzes.
The elution fraction analyzed for BIAcore uses gel filtration to test its purity further.Condition is:
Chromatographic column Agi lent Bio SEC-3 (300A, φ 7.8 × 300mm), flowing is 150mM phosphate mutually
Buffer, flow velocity 0.5mL/min, column temperature 25 DEG C, ultraviolet detection wavelength 214nm.
The SDS-PAGE glue figure of the soluble single-chain T CR that the present invention obtains is as shown in figure 11.
Embodiment 6 combines and characterizes
BIAcore analyzes
The TCR molecule of the present invention that this example demonstrated solubility can be multiple with ILSLELMKL-HLA A0201
Compound is specific binding.
Use the TCR obtained in BIAcore T200 real-time analyzer detection embodiment 3 and embodiment 5
Molecule is active with the combination of ILSLELMKL-HLA A0201 complex.Antibody by anti-Streptavidin
(GenScript) coupling buffer (10mM sodium-acetate buffer, pH 4.77) is added, then by antibody
Flow through the CM5 chip activated with EDC and NHS in advance, make antibody be fixed on chip surface, finally use second
The hydrochloric acid solution of hydramine closes unreacted activating surface, completes coupling process, and coupling level is about 15,000
RU。
The Streptavidin making low concentration flows through the chip surface of coated antibody, then will
ILSLELMKL-HLA A0201 complex flows through sense channel, and another passage is as reference channel, then by 0.05
The biotin of mM flows through chip 2min with the flow velocity of 10 μ L/min, closes the remaining combination of Streptavidin
Site.
The preparation process of above-mentioned ILSLELMKL-HLA A0201 complex is as follows:
A. purification
Collect 100ml abduction delivering heavy chain or the E.coli bacterium solution of light chain, be centrifuged in 4 DEG C of 8000g
After 10min with 10ml PBS washing thalline once, afterwards with 5ml BugBuster Master Mix
Extraction Reagents (Merck) acutely shakes resuspended thalline, and hatches 20min in room temperature rotation,
Afterwards in 4 DEG C, 6000g is centrifuged 15min, supernatant discarded, collects inclusion body.
Being resuspended in by above-mentioned inclusion body in 5ml BugBuster Master Mix, room temperature rotates hatches 5min；
Adding 30ml and dilute the BugBuster of 10 times, mixing, 4 DEG C of 6000g are centrifuged 15min；Supernatant discarded,
Adding 30ml and dilute the resuspended inclusion body of BugBuster of 10 times, mixing, 4 DEG C of 6000g are centrifuged 15min,
It is repeated twice, adds the 30ml resuspended inclusion body of 20mM Tris-HCl pH 8.0, mixing, 4 DEG C of 6000g
Centrifugal 15min, finally dissolves inclusion body with 20mM Tris-HCl 8M carbamide, and SDS-PAGE detects bag
Containing body purity, BCA test kit surveys concentration.
B. renaturation
The small peptide ILSLELMKL (Beijing SBS Genetech gene technology company limited) of synthesis is dissolved in DMSO extremely
The concentration of 20mg/ml.The inclusion body of light chain and heavy chain 8M carbamide, 20mM Tris pH 8.0,10mM
DTT dissolves, and adds 3M guanidine hydrochloride, 10mM sodium acetate, the further degeneration of 10mM EDTA before renaturation.
By ILSLELMKL peptide with 25mg/L (final concentration) add renaturation buffer (0.4M L-arginine, 100
MM Tris pH 8.3,2mM EDTA, 0.5mM GSSG, 5mM reduced form gluathione
Peptide, 0.2mM PMSF, be cooled to 4 DEG C), then it is sequentially added into light chain and the 90mg/L of 20mg/L
Heavy chain (final concentration, heavy chain adds in three times, 8h/ time), renaturation carries out at least 3 days to the completeest at 4 DEG C
Becoming, can SDS-PAGE detection renaturation success.
C. purification after renaturation
Change renaturation buffer with the 20mM Tris pH 8.0 of 10 volumes as dialysis, at least change slow
Rush liquid and fully reduce the ionic strength of solution for twice.By 0.45 μm cellulose acetate sheets mistake after dialysis
Filter protein solution, is then loaded on HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column
(5ml bed volume).Utilize Akta purification instrument (GE General Electric Co. Limited), 20mM Tris pH 8.0
The 0-400mM NaCl linear gradient liquid eluted protein of preparation, pMHC about eluting at 250mM NaCl,
Collecting all peaks component, SDS-PAGE detects purity.
D. biotinylation
With Millipore super filter tube by the pMHC molecular concentration of purification, it is 20mM by buffer exchange simultaneously
Tris pH 8.0, be subsequently adding biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10
MM MgOAc, 50 μMs of D-Biotin, 100 μ g/ml BirA enzyme (GST-BirA), incubated at room is mixed
Overnight, SDS-PAGE detection biotinylation is the most complete for compound.
E. the complex after purifying biological element
With Millipore super filter tube by the pMHC molecular concentration after biotinylation labelling to 1ml, use solidifying
The biotinylated pMHC of glue filtration chromatography, utilizes Akta purification instrument (GE General Electric Co. Limited), uses
Filtered PBS pre-equilibration HiPrepTM16/60S200HR post (GE General Electric Co. Limited), loads 1
Biotinylation pMHC molecule concentrated for ml, then with PBS with 1ml/min flow velocity eluting.Biotin
The pMHC molecule changed occurs as unimodal eluting when about 55ml.Merge the component containing protein, use
Millipore super filter tube concentrates, and BCA method (Thermo) measures protein concentration, adds protease suppression
Biotinylated pMHC molecule subpackage is saved in-80 DEG C by agent cocktail (Roche).
Utilize BIAcore Evaluation computed in software kinetic parameter, obtain the TCR of solubility of the present invention
The soluble single-chain T CR molecule that molecule and the present invention build is tied with ILSLELMKL-HLA A0201 complex
The kinetic profile closed is the most as shown in Figure 12 and Figure 13.Meanwhile, said method is also utilized to have detected this
The TCR molecule of bright solubility and other antigen small peptides include GILGFVFTL-HLA A0201 complex and
The combination activity of SLLMWITQC-HLA A0201 complex, result shows TCR molecule of the present invention and other nothings
Close antigen all without combining.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document
It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention,
The present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within this Shen equally
Please appended claims limited range.
Claims (10)
1. a φt cell receptor (TCR), it is characterised in that described TCR can be with ILSLELMKL-HLA
Complex combines；
Preferably, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, and described TCR α chain can
Variable domain is to have the aminoacid sequence of at least 90% sequence thereto with SEQ ID NO:1；And/or described TCR
β chain variable domain is to have the aminoacid sequence of at least 90% sequence thereto with SEQ ID NO:5；
It is highly preferred that described TCR comprises TCR α chain variable domain and TCR β chain variable domain, described TCR α chain
The aminoacid sequence of the CDR3 of variable domain is AATNSGYALN (SEQ ID NO:12)；And/or described TCR
The aminoacid sequence of the CDR3 of β chain variable domain is AWSVDGAEQY (SEQ ID NO:15).
2. TCR as claimed in claim 1, it is characterised in that 3 of described TCR α chain variable domain are mutual
Mend and determine that district (CDR) is:
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 described TCR β chain variable domain are:
β CDR1-GTSNPN (SEQ ID NO:13)
β CDR2-SVGIG (SEQ ID NO:14)
β CDR3-AWSVDGAEQY (SEQ ID NO:15)。
3. the TCR as described in any of the above claim, it is characterised in that the α chain of described TCR and/or
C-or the N-end of β chain is combined with conjugate；
Preferably, the conjugate being combined with described φt cell receptor is that detectable, therapeutic agent, PK repair
Decorations part or the combination of these materials any；It is further preferred that described therapeutic agent is anti-CD 3 antibodies.
4. a multivalent TCR complex, it is characterised in that comprise at least two TCR molecule, and its
In at least one TCR molecule be the TCR according to any one of the claims.
5. a nucleic acid molecules, it is characterised in that described nucleic acid molecules comprises any of the above-described right of coding to be wanted
Ask nucleotide sequence or its complementary series of described TCR molecule；
Preferably, described nucleic acid molecules comprise coding TCR α chain variable domain nucleotide sequence SEQ ID NO:
2 or SEQ ID NO:33；
It is highly preferred that described nucleic acid molecules comprise coding TCR β chain variable domain nucleotide sequence SEQ ID NO:
6 or SEQ ID NO:35.
6. a carrier, it is characterised in that described carrier contains the nucleic acid described in claim 5 and divides
Son；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
7. the host cell separated, it is characterised in that containing claim 6 in described host cell
Described in carrier or chromosome in integrate and have the nucleic acid molecules described in the claim 5 of external source.
8. a cell, it is characterised in that in described cell transduction claim 5, arbitrary described nucleic acid divides
Carrier described in son or claim 6；Preferably, described cell is T cell or stem cell.
9. a pharmaceutical composition, it is characterised in that described compositions contains pharmaceutically acceptable carrier
And TCR according to any one of claim 1-3, the TCR complex described in claim 4, power
Profit requires the nucleic acid molecules described in 5 or the cell described in claim 8.
10. the φt cell receptor according to any one of claim 1-3 or the TCR described in claim 4
Nucleic acid molecules described in complex or claim 5 or the carrier described in claim 6 or power
Profit requires the purposes of the cell described in 8, it is characterised in that be used for preparing treatment tumor or autoimmune disease
Sick medicine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510228111.2A CN106188274A (en) | 2015-05-06 | 2015-05-06 | Identify the φt cell receptor of RHAMM antigen small peptide |
| CN201680001317.4A CN106459179B (en) | 2015-05-06 | 2016-03-29 | Identify the T cell receptor of RHAMM antigen small peptides |
| 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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| CN201510228111.2A CN106188274A (en) | 2015-05-06 | 2015-05-06 | Identify the φt cell receptor of RHAMM antigen small peptide |
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| CN201680001317.4A Active CN106459179B (en) | 2015-05-06 | 2016-03-29 | Identify the T cell receptor of RHAMM antigen small peptides |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690130A (en) * | 2017-04-12 | 2018-10-23 | 广东香雪精准医疗技术有限公司 | A kind of TCR of the identification from LMP1 antigen small peptides |
| CN109400697A (en) * | 2017-08-17 | 2019-03-01 | 广东香雪精准医疗技术有限公司 | A kind of TCR of PRAME antigen small peptide and its compositions related of identifying |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110582299A (en) * | 2017-03-15 | 2019-12-17 | 弗雷德哈钦森癌症研究中心 | High affinity MAGE-A1 specific TCRs and uses thereof |
| CN117304301A (en) * | 2022-06-17 | 2023-12-29 | 香雪生命科学技术（广东）有限公司 | TCR molecule combined with SSX2 antigen and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6082997B2 (en) * | 2011-04-01 | 2017-02-22 | メモリアル スローン−ケタリング キャンサー センター | T cell receptor-like antibody to WT1 peptide presented by HLA-A2 |
-
2015
- 2015-05-06 CN CN201510228111.2A patent/CN106188274A/en active Pending
-
2016
- 2016-03-29 WO PCT/CN2016/077679 patent/WO2016177195A1/en active Application Filing
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690130A (en) * | 2017-04-12 | 2018-10-23 | 广东香雪精准医疗技术有限公司 | A kind of TCR of the identification from LMP1 antigen small peptides |
| CN109400697A (en) * | 2017-08-17 | 2019-03-01 | 广东香雪精准医疗技术有限公司 | A kind of TCR of PRAME antigen small peptide and its compositions related of identifying |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2016177195A1 (en) | 2016-11-10 |
| CN106459179A (en) | 2017-02-22 |
| CN106459179B (en) | 2018-06-05 |
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Effective date of registration: 20171101 Address after: 510663, No. 2, Jinfeng Road, Science Town, Guangzhou hi tech Industrial Development Zone, Guangdong province (first) Applicant after: Guangdong Xiangxue precision medical technology Co Ltd Address before: 510663 Guangdong city of Guangzhou province Luogang District Science City Jinfeng Road No. 2 Applicant before: Guangzhou Xiangxue Pharmaceutical Co., ltd. |
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Application publication date: 20161207 |