CN106084036A - Identify the φt cell receptor of DAGE small peptide - Google Patents
Identify the φt cell receptor of DAGE small peptide Download PDFInfo
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- CN106084036A CN106084036A CN201510741810.7A CN201510741810A CN106084036A CN 106084036 A CN106084036 A CN 106084036A CN 201510741810 A CN201510741810 A CN 201510741810A CN 106084036 A CN106084036 A CN 106084036A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention provides a kind of can the φt cell receptor (TCR) of the specific binding small peptide RYLFPPLFM derived from DAGE, described antigen small peptide RYLFPPLFM can be formed with HLA A2402 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 the TCR being derived from DAGE small peptide, the specific T cell of PRAME that the above-mentioned TCR that the invention still further relates to transduce obtains, and their purposes in prevention and treatment PRAME relevant disease.
Background technology
PRAME is melanoma specific antigen (preferentially expressed antigen of melanoma, PRAME), expression (Ikeda H is had in 88% onset and 95% transfer melanoma, et al.Immunity, 1997,6 (2): 199-208), normal skin tissue and benign melanocyte are not expressed.PRAME is degraded to micromolecule polypeptide after intracellular generation, and is combined formation complex with MHC (main histocompatibility complex) molecule, is presented to cell surface.RYLFPPLFM is derived from the small peptide of DAGE, is a kind of target of PRAME treating correlative diseases.Except melanoma, PRAME also expresses in kinds of tumors, (the van't Veer LJ such as including squamous cell lung carcinoma, breast carcinoma, renal cell carcinoma, tumor of head and neck, Huo Jiejin lymphomas, sarcoma and medulloblastoma, et al.Nature, 2002,415 (6871): 530-536；Boon K, et al.Oncogene, 2003,22 (48): 7687-7694) it addition, its PRAME in leukemia also has notable expression, acute lymphoblastic leukemia 17%～42%, acute myeloblastic leukemia 30%～64% (SteinbachD, et al.Cancer Genet Cytogene, 2002,138 (1): 89-91).For the treatment of above-mentioned disease, the method such as chemotherapy and radiation treatment can be used, but all the normal cell of self can be caused damage.
T cell adoptive immunotherapy is to proceed in patient body by having specific reaction-ive T cell to target cell antigen so that it is play a role for target cell.φt cell receptor (TCR) is a kind of memebrane protein on T cell surface, and it is capable of identify that the antigen small peptide of corresponding target cells.In immune system, T cell and antigen-presenting cell (APC) directly physical contact is caused by the combination of the specific TCR of antigen small peptide with small peptide-main histocompatibility complex (pMHC complex), then T cell and other cell membrane surface molecules both APC just interact, 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.Therefore, those skilled in the art are devoted to isolate NY-ESO-1 antigen small peptide are had specific TCR, and T cell of being transduceed by this TCR obtains and NY-ESO-1 antigen small peptide is had 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 DAGE small peptide.
A first aspect of the present invention, it is provided that a kind of φt cell receptor (TCR), described TCR can be combined with RYLFPPLFM-HLA A2402 complex.
In another preference, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, and the aminoacid sequence of the CDR3 of described TCR α chain variable domain is VVNHFIDSGYALN (SEQ ID NO:12)；And/or the aminoacid sequence of the CDR3 of described TCR β chain variable domain is ATSPDASNEKLF (SEQ ID NO:15).
In another preference, 3 complementary determining regions (CDR) of described TCR α chain variable domain are:
α CDR1-NSASQS (SEQ ID NO:10)
α CDR2-VYSSGN (SEQ ID NO:11)
α CDR3-VVNHFIDSGYALN (SEQ ID NO:12)；And/or
3 complementary determining regions of described TCR β chain variable domain are:
β CDR1-KGHDR (SEQ ID NO:13)
β CDR2-SFDVKD (SEQ ID NO:14)
βCDR3-ATSPDASNEKLF (SEQ ID NO:15)。
In another preference, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, and described TCR α chain 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.
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 be the β chain amino acid sequence of SEQ ID NO:3 and/or described TCR be 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 formed by connecting by peptide catenation sequence with β chain variable domain by α chain variable domain.
In another preference, described TCR is in the 11st, 13,19,21,53,76,89,91 or the 94th, α chain variable region aminoacid, and/or has one or more sudden change in 3rd reciprocal of α chain J gene small peptide aminoacid, inverse the 5th or 7th reciprocal；And/or described TCR is in the 11st, 13,19,21,53,76,89,91 or the 94th, β chain variable region aminoacid, and/or 2nd reciprocal of β chain J gene small peptide aminoacid, inverse the 4th or 6th reciprocal have one or more sudden change, wherein amino acid position number is by the Position Number listed in IMGT (international immunogenetics information system).
In another preference, the β chain variable domain amino acid sequence that the α chain variable domain amino acid sequence of described TCR comprises SEQ ID NO:32 and/or 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 chain in addition to membrane spaning domain；And all or part of TCR β chain that (b) is in addition to membrane spaning domain；
And (a) and (b) each self-contained functional variable domain, or comprise at least some of of functional variable domain and described TCR chain constant domain.
In another preference, cysteine residues forms artificial disulfide bond between α and the β chain constant domain of described TCR.
In another preference, the cysteine residues forming artificial disulfide bond in described TCR instead of selected from one or more groups following site:
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 be the β chain amino acid sequence of SEQ ID NO:26 and/or described TCR be SEQ ID NO:28.
In another preference, containing artificial interchain disulfide bond between α chain variable region and the β chain constant region of described TCR.
In another preference, it is characterised in that the cysteine residues forming artificial interchain disulfide bond in described TCR instead of selected from one or more groups following site:
46th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1；
47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1；
46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1；Or
47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1.
In another preference, described TCR comprises α chain variable domain and β chain variable domain and all or part of β chain constant domain in addition to membrane spaning domain, but it does not contain α chain constant domain, the α chain variable domain of described TCR and β chain formation heterodimer.
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, therapeutic agent, PK modification 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 molecule, 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 nucleotide sequence or its complementary series of coding TCR molecule described in first aspect present invention.
In another preference, described nucleic acid molecules comprises the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:33 of coding TCR α chain variable domain.
In another preference, described nucleic acid molecules comprises the nucleotide sequence SEQ ID NO:6 or SEQ ID NO:35 of coding TCR β chain variable domain.
In another preference, described nucleic acid molecules comprises the nucleotide sequence SEQ ID NO:4 encoding TCR α chain and/or the nucleotide sequence SEQ ID NO:8 comprising coding TCR β chain.
A fourth aspect of the present invention, it is provided that a kind of carrier, described carrier contains the nucleic acid molecules described in third aspect present invention；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
A fifth aspect of the present invention, it is provided that the host cell of a kind of separation, has the nucleic acid molecules described in third aspect present invention of external source containing integrating in the carrier described in fourth aspect present invention or genome in described host cell.
A sixth aspect of the present invention, it is provided that a kind of cell, the nucleic acid molecules described in described cell transduction third aspect present invention or the carrier described in fourth aspect present invention；Preferably, described cell is T cell or stem cell.
A seventh aspect of the present invention, providing a kind of pharmaceutical composition, described compositions contains the TCR described in pharmaceutically acceptable carrier and first aspect present invention, the TCR complex described in second 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.
A eighth aspect of the present invention, provide the φt cell receptor described in first aspect present invention or the purposes of nucleic acid molecules, the carrier described in fourth aspect present invention or the cell described in sixth aspect present invention described in the TCR complex described in second aspect present invention, third aspect present invention, for preparing treatment tumor or the medicine of autoimmune disease.
A ninth aspect of the present invention, provide a kind of method treating disease, use the φt cell receptor described in appropriate first aspect present invention or nucleic acid molecules, the carrier described in fourth aspect present invention or the cell described in sixth aspect present invention described in the TCR complex described in second aspect present invention, third aspect present invention or the pharmaceutical composition described in seventh aspect present invention including to the object needing treatment.
Preferably, described disease is tumor, the most described tumor includes that melanoma, squamous cell lung carcinoma, breast carcinoma, renal cell carcinoma, tumor of head and neck, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukemia (include but not limited to, acute lymphoblastic leukemia, acute myeloblastic leukemia), gastric cancer, pulmonary carcinoma, esophageal carcinoma, bladder cancer, squamous cell carcinoma of the head and neck, carcinoma of prostate, colon cancer, ovarian cancer etc..
In should be understood that within the scope of the present invention, can be combined with each other between above-mentioned each technical characteristic and each technical characteristic specifically described in below (eg embodiment) of the present invention, thus constitute new or preferred technical 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 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, there is the TCR α chain amino acid sequence of targeting sequencing 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 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, there is the TCR β chain amino acid sequence of targeting sequencing and there is the TCR β chain nucleotide sequence of 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.Leftmost side swimming lane is for going back virgin rubber, and middle swimming lane is molecular weight marker (marker), and rightmost side swimming lane is non-reduced glue.
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 nucleotide sequence of strand TCR catenation sequence (linker).
Figure 11 is the glue figure of the soluble single-chain T CR obtained after purification.Left side swimming lane is molecular weight marker (marker), and right lanes is non-reduced glue.
Figure 12 is the BIAcore kinetic profile that sTCR of the present invention is combined with RYLFPPLFM-HLA A2402 complex.
Detailed description of the invention
The present inventor is through extensively in-depth study, have found can be specific binding with DAGE small peptide RYLFPPLFM (SEQ ID NO:9) TCR, described antigen small peptide RYLFPPLFM can with HLA A2402 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.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Therefore, it is presented for antigen has specificity, and different individualities has different MHC, can present small peptides different in a kind of proteantigen to respective APC cell surface.The MHC of the mankind is commonly referred to HLA gene or HLA complex.
φt cell receptor (TCR), is the unique receptor presenting the specific antigen peptide in main histocompatibility complex (MHC).In immune system, T cell and antigen-presenting cell (APC) directly physical contact is caused by the combination of TCR Yu the pMHC complex of antigenic specificity, then T cell and other cell membrane surface molecules both APC just interact, this just causes 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 glycoprotein of the surface of cell membrane existed with heterodimer form by α chain/β chain or γ chain/δ chain.In the T cell of 95%, TCR heterodimer is made up of α and β chain, and the T cell of 5% has 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 and constitutes the subunit of α β heterodimeric TCR.In a broad sense, each chain of α and β comprises variable region, bonding pad and constant region, and β chain the most also contains short variable region between variable region and bonding pad, but this variable region often regards as a part for bonding pad.Each variable region comprises 3 CDR (complementary determining region) being entrenched in frame structure (framework regions), CDR1, CDR2 and CDR3.CDR region determines the combination of TCR Yu pMHC complex, and wherein CDR3 is recombinated by variable region and bonding pad and forms, and is referred to as hypervariable region.α and the β chain of TCR is typically regarded as and is respectively arranged with two " domain " i.e. variable domains and constant domain, and variable domain is made up of the variable region connected and bonding pad.The sequence of TCR constant domain can find in the public data storehouse of international immunogenetics information system (IMGT), if the constant domain sequence of TCR molecule alpha chain is " TRAC*01 ", the constant domain sequence of TCR molecule β chain is " TRBC1*01 " or " TRBC2*01 ".Additionally, α and the β chain of TCR also comprises 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 ", " φt cell receptor of the present invention " are used interchangeably.
Native interchain disulfide bond and artificial interchain disulfide bond
There is one group of disulfide bond at the membrane-proximal region C α of natural TCR with C β interchain, the present invention is referred to as " native interchain disulfide bond ".In the present invention, by manually-injected, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond is referred to as " artificial interchain disulfide bond ".
nullFor convenience of the position describing disulfide bond，In the present invention, the Position Number of TRAC*01 Yu TRBC1*01 or TRBC2*01 aminoacid sequence carries out Position Number by from N end to C end order successively，In TRBC1*01 or TRBC2*01，By being P (proline) from N end to C end the 60th aminoacid of order successively，The present invention then can describe it as the Pro60 of TRBC1*01 or TRBC2*01 exons 1，Also the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1 can be stated that，And for example in TRBC1*01 or TRBC2*01，By being Q (glutamine) from N end to C end the 61st aminoacid of order successively，The present invention then can describe it as the Gln61 of TRBC1*01 or TRBC2*01 exons 1，Also the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1 can be stated that，Other are by that analogy.In the present invention, the Position Number of the aminoacid sequence of variable region TRAV Yu TRBV, according to the Position Number listed in IMGT.Such as certain aminoacid in TRAV, the Position Number listed in IMGT is 46, then describe it as TRAV the 46th amino acids in the present invention, and other are by that analogy.In the present invention, other amino acid whose Sequence position numbers have specified otherwise, then by specified otherwise.
Detailed Description Of The Invention
TCR molecule
In antigen processing pathways, antigen is degraded intracellular, is then carried to cell surface by MHC molecule.φt cell receptor is capable of identify that the peptide-MHC complex of Antigen Presenting Cell surface.Therefore, a first aspect of the present invention provide a kind of can be in conjunction with the TCR molecule of RYLFPPLFM-HLA A2402 complex.Preferably, described TCR molecule is to separate or purification.α and the β chain of this TCR respectively has 3 complementary determining regions (CDR).
Being preferably carried out in mode of the present invention, the α chain of described TCR comprises the CDR with following aminoacid sequence:
α CDR1-NSASQS (SEQ ID NO:10)
α CDR2-VYSSGN (SEQ ID NO:11)
α CDR3-VVNHFIDSGYALN (SEQ ID NO:12)；And/or
3 complementary determining regions of described TCR β chain variable domain are:
β CDR1-KGHDR (SEQ ID NO:13)
β CDR2-SFDVKD (SEQ ID NO:14)
β CDR3-ATSPDASNEKLF (SEQ ID NO:15)。
The CDR region aminoacid sequence of the invention described above can be embedded in any applicable frame structure and prepare 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 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 the TCR molecule of any applicable frame structure.TCR α chain variable domain of the present invention for have at least 90% with SEQ ID NO:1, the aminoacid sequence of preferably 95%, more preferably 98% sequence thereto；And/or TCR β chain variable domain of the present invention is for have at least 90% with SEQ ID NO:5, the aminoacid sequence of preferably 95%, more preferably 98% sequence thereto.
In a preference of the present invention, the TCR molecule of the present invention is the heterodimer being made up of α with β chain.Specifically, the α chain of the most described heterogeneous dimerization TCR molecule comprises variable domain and constant domain, and described α chain variable domain amino acid sequence comprises the CDR1 (SEQ ID NO:10) of above-mentioned α chain, CDR2 (SEQ ID NO:11) and CDR3 (SEQ ID NO:12).Preferably, described TCR molecule comprises α chain variable 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 is SEQ ID NO:1.On the other hand, the β chain of described heterogeneous dimerization TCR molecule comprises variable domain and constant domain, and described β chain variable domain amino acid sequence comprises the CDR1 (SEQ ID NO:13) of above-mentioned β chain, CDR2 (SEQ ID NO:14) and CDR3 (SEQ ID NO:15).Preferably, described TCR molecule comprises β chain variable domain amino acid sequence SEQ ID NO:5.It is highly preferred that the β chain variable domain amino acid sequence of described TCR molecule is SEQ ID NO:5.
In a preference of the present invention, the TCR molecule of the present invention is by the part or all of of α chain and/or the single chain TCR molecules partly or entirely formed 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 single chain TCR molecules comprising CDRs district of the present invention.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 the CDR1 (SEQ ID NO:10) of above-mentioned α chain, 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 the α chain variable domain amino acid sequence of described single chain TCR molecules is SEQ ID NO:1.The β chain variable domain amino acid sequence of described single chain TCR molecules comprises the CDR1 (SEQ ID NO:13) of above-mentioned β chain, CDR2 (SEQ ID NO:14) and CDR3 (SEQ ID NO:15).Preferably, described single chain TCR molecules comprises β chain variable domain amino acid sequence SEQ ID NO:5.It is highly preferred that the β chain variable domain amino acid sequence of described single chain TCR molecules 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.Those skilled in the art know the constant domain aminoacid sequence that maybe can obtain people by consulting the public data storehouse of pertinent texts or IMGT (international immunogenetics information system).Such as, the constant domain sequence of TCR molecule alpha chain of the present invention can be " TRAC*01 ", and 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, is expressed as at this: the Arg53 of TRAC*01 exons 1, and other are by that analogy.Preferably, the aminoacid sequence of TCR molecule alpha chain of the present invention 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.As immunoglobulin (antibody) is as antigen recognizing molecule, TCR can also be developed and be applied to diagnosis and treatment, at this moment needs to obtain the TCR molecule of solubility.The TCR molecule of solubility does not include its cross-film district.STCR has purposes the most widely, and it cannot be only used for studying the interaction of TCR Yu pMHC, it is possible to be used as to detect the diagnostic tool infected or the mark as autoimmune disease.Similarly, sTCR can be used to be transported to present by therapeutic agent (such as cytotoxin compounds or immunostimulating compound) cell of specific antigen, additionally, sTCR also can with other molecules (as, anti-CD 3 antibodies) combine redirect T cell, so that its targeting presents the cell of specific antigen.The present invention also obtain has specific sTCR to DAGE small peptide.
For obtaining sTCR, on the one hand, TCR of the present invention can be the TCR introducing artificial disulfide bond between the residue of itself α and β chain constant domain.Cysteine residues forms artificial interchain disulfide bond between α and the β chain constant domain of described TCR.Cysteine residues can be substituted in natural TCR other amino acid residues of appropriate site to form artificial interchain disulfide bond.Such as, the cysteine residues of the Thr48 replacing TRAC*01 exons 1 and the Ser57 replacing TRBC1*01 or TRBC2*01 exons 1 forms disulfide bond.Introduce cysteine residues to form other sites of disulfide bond it may also is that the Ser77 of Thr45 and TRBC1*01 or 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；Or the Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1.Arbitrary group of site during i.e. cysteine residues instead of above-mentioned α Yu β chain constant domain.Can be at one or more C-terminal truncates of TCR constant domain of the present invention most 50 or most 30 or most 15 or most 10 or most 8 or less aminoacid, so that it does not include that cysteine residues reaches to lack the purpose of natural disulphide bonds, it is possible to reach above-mentioned purpose by the cysteine residues forming natural disulphide bonds is sported another aminoacid.
As it has been 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 mentioned above between constant domain, the TCR of the present invention all can contain TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequence.The TRAC constant domain sequence of TCR and TRBC1 or TRBC2 constant domain sequence can be connected by the natural disulphide bonds being present in TCR.
For obtaining sTCR, on the other hand, TCR of the present invention is additionally included in the TCR undergone mutation in its hydrophobic core region, and the sudden change in these hydrophobic core regions is preferably capable making the stability-enhanced sudden change of sTCR of the present invention, as described in the patent documentation of Publication No. WO2014/206304.Such TCR can 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 3rd, 5,7 reciprocal of α chain J gene (TRAJ) small peptide amino acid position, and/or β chain J gene (TRBJ) small peptide amino acid position the reciprocal 2nd, 4,6, wherein the Position Number of aminoacid sequence is by the Position Number listed in international immunogenetics information system (IMGT).Those skilled in the art know above-mentioned international immunogenetics information system, and can obtain the amino acid residue of different TCR Position Number in IMGT according to this data base.
The stability solvable strand TCR that the TCR that in the present invention, undergo mutation in hydrophobic core region can be the variable domain of α Yu the β chain being connected TCR by a flexible peptide chain and constitute.It should be noted that in the present invention, flexible peptide chain can be the peptide chain of any applicable connection TCR α and β chain variable domain.Such as the single chain soluble TCR built in the embodiment of the present invention 4, its α chain variable domain amino acid sequence is SEQ ID NO:32, and the nucleotides sequence of coding is classified as SEQ ID NO:33；β chain variable domain amino acid sequence is SEQ ID NO:34, and the nucleotides sequence of coding is classified as SEQ ID NO:35.
It addition, for stability, patent documentation 201510260322.4 also discloses and introduces artificial interchain disulfide bond between the α chain variable region of TCR and β chain constant region the stability of TCR can be made to significantly improve.Therefore, artificial interchain disulfide bond can also be contained between α chain variable region and the β chain constant region of the high-affinity TCR of the present invention.Specifically, the cysteine residues forming artificial interchain disulfide bond between the α chain variable region of described TCR and β chain constant region instead of: the 46th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1；47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1；46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1；Or the 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1.Preferably, such TCR can comprise () all or part of TCR α chain in addition to its membrane spaning domain, () all or part of TCR β chain in addition to its membrane spaning domain, wherein () and () all comprises the variable domain of TCR chain and at least some of constant domain, α chain and β chain formation heterodimer.It is highly preferred that such TCR can comprise α chain variable domain and β chain variable domain and all or part of β chain constant domain in addition to membrane spaning domain, but it does not contain α chain constant domain, the α chain variable domain of described TCR and β chain formation heterodimer.
The TCR of the present invention can also multivalence complex form provide.The multivalent TCR complex of the present invention comprises two, three, the four or more TCR of the present invention polymer that combines and formed, as produced the tetramer, or the complex that multiple TCR of the present invention is combined with another molecule and is formed with four dimerization domain of p53.The TCR complex of the present invention can be used for external or internal tracking or targeting presents the cell of specific antigen it can also be used to produce 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 with covalency or other modes with conjugate, preferably combines with covalent manner.Described conjugate includes that detectable (for diagnostic purpose, wherein said TCR presents the existence of the cell of RYLFPPLFM-HLA A2402 complex for detection), therapeutic agent, PK (protein kinase) 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, radioactive marker, MRI (nuclear magnetic resonance) or CT (CT technology) contrast agent, maybe can produce and can detect the enzyme of 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. 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, institute of NAS periodical (PNAS) 89,1428 such as cytokine 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 (such as, DT-diaphorase (DTD) or xenyl hydrolytic 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 immunne response.Therefore, should there is regulation scheme to carry out immunosuppressant when it is treated for adoptive T cell, to allow to express the implantation of the T cell of Muridae.
It should be understood that, amino acid name uses international single English alphabet or three English alphabets to represent herein, 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 coding first aspect present invention TCR molecule or the nucleic acid molecules of its part, and 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-aacagtgcttctcagtct (SEQ ID NO:16)
α CDR2-gtatactccagtggtaat (SEQ ID NO:17)
α CDR3-gtggtgaaccattttatagattccgggtatgcactcaac (SEQ ID NO:18)
The nucleotide sequence of coding first aspect present invention TCR molecule β chain CDR region is as follows:
β CDR1-aagggtcatgataga (SEQ ID NO:19)
β CDR2-tcctttgatgtcaaagat (SEQ ID NO:20)
β CDR3-gccaccagtcccgacgcttctaatgaaaaactgttt (SEQ ID NO:21)
Therefore, the nucleotide sequence of the nucleic acid molecules of the present invention of code book invention TCR α chain 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 code book invention TCR β chain 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 nucleotide sequence of nucleic acid molecules of the present invention does not comprises intron but code book invention polypeptide, the such as nucleotide sequence of nucleic acid molecules of the present invention of code book invention TCR α chain variable domain can include that the nucleotide sequence of nucleic acid molecules of the present invention of SEQ ID NO:2 and/or code book invention TCR β chain variable domain includes SEQ ID NO:6.Or, the nucleotide sequence of the nucleic acid molecules of the present invention of code book invention TCR α chain variable domain includes that the nucleotide sequence of the nucleic acid molecules of the present invention of SEQ ID NO:33 and/or code book invention TCR β chain variable domain 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, the nucleotide sequence of code book invention TCR can or the variant of degeneracy identical with the nucleotide sequence shown in accompanying drawing of the present invention.Illustrating with one of them example in the present invention, " variant of degeneracy " refers to encode the protein sequence with SEQ ID NO:1, but with the differentiated nucleotide sequence of sequence of SEQ ID NO:2.
Nucleotide sequence can be through codon optimized.Different cells is different in the utilization of concrete codon, can be according to the type of cell, and the codon changed in sequence increases expression.The codon usage table of mammalian 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, recombination method or synthetic method obtain.At present, it is already possible to obtained the DNA sequence of code book invention TCR (or its fragment, or derivatives thereof) completely by chemosynthesis.Then this DNA sequence can be introduced in 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 still further relates to the carrier of the nucleic acid molecules comprising the present invention, including expression vector, i.e. can in vivo 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, herpesvirus vector, retroviral vector, slow virus carrier, baculovirus vector.
Preferably, the nucleotide of the present invention can be transferred in cell by carrier, such as in T cell so that this cell expresses the specific TCR of DAGE.Ideally, this carrier should be expressed in T cell continual high levels.
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.The carrier containing the present invention in described host cell or chromosome are integrated and has the nucleic acid molecules of the present invention.Host cell is 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.This T cell can derived from the T cell separated from experimenter, or can be the mixed cellularity group separated from experimenter, the such as part of peripheral blood lymphocyte (PBL) group.As, this cell can be isolatable from peripheral blood lymphocytes (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 use antibody (e.g., the antibody of anti-CD3 or anti-CD28) to activate, in order to allows 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, when stem cell is divided into lymphoid precursor (the lymphoid precursor) migrating to thymus, expressing the TCR molecule of this introducing of surface expression started at thymocyte cell of CD3 molecule.
Have many methods be suitable for code book invention TCR DNA or RNA carry out T cell transfection (e.g., Robbins etc., (2008) J.Immunol.180:6116-6131).The T cell expressing TCR of the present invention may be used for adoptive immunotherapy.Those skilled in the art understand that carry out adoptive treatment many appropriate method (e.g., Rosenberg etc., (2008) Nat Rev Cancer8 (4): 299-308).
DAGE relevant disease
The invention still further relates to treat in experimenter and/or prevent and the method for PRAME relevant disease, it includes that adoptive transfer PRAME specific T-cells is to the step of this experimenter.This PRAME specific T-cells can recognize that RYLFPPLFM-HLA A2402 complex.
The specific T cell of PRAME of the present invention can be used for treating any PRAME relevant disease presenting DAGE small peptide RYLFPPLFM-HLA A2402 complex, such as tumor.Described tumor includes but not limited to that melanoma, squamous cell lung carcinoma, breast carcinoma, renal cell carcinoma, tumor of head and neck, Huo Jiejin lymphomas, sarcoma, medulloblastoma, leukemia (include but not limited to, acute lymphoblastic leukemia, acute myeloblastic leukemia), gastric cancer, pulmonary carcinoma, esophageal carcinoma, bladder cancer, squamous cell carcinoma of the head and neck, carcinoma of prostate, colon cancer, ovarian cancer etc..
Therapeutic Method
Can be suffered from the T cell of the patient with DAGE relevant disease or volunteer by separation, and the TCR of the present invention is imported in above-mentioned T cell, treat in subsequently the cell that these genetic engineerings are modified being fed back to patient body.Therefore, the invention provides a kind of method treating PRAME relevant disease, including the T cell of the expression TCR of the present invention that will separate, it is preferable that this T cell derives from patient 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 can code book invention TCR molecule nucleic acid molecules ex vivo transduction T cell, the T cell that genetic engineering is modified is input in patient body by (3).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 combined with DAGE small peptide complex RYLFPPLFM-HLA A2402, and the cell of the TCR of the present invention that simultaneously transduceed can be by specific activation and have the strongest lethal effect to target cell.
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 limit the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as (Sambrook and Russell et al., molecular cloning: laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing house) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.Experiment material used in following example and reagent the most all can obtain from commercially available channel.
Embodiment 1 clones DAGE small peptide specific T-cells
Utilize synthesis small peptide RYLFPPLFM (SEQ ID NO.:9；Beijing SBS Genetech gene technology company limited) stimulate the peripheral blood lymphocyte (PBL) coming from the healthy volunteer that genotype is HLA-A2402.By RYLFPPLFM small peptide with biotin labeled HLA-A2402 renaturation, prepare pHLA monoploid.These monoploid and the tetramer being combined into PE labelling with the Streptavidin (BD company) of PE labelling, sort this tetramer and the double positive cell of anti-CD8-APC.The cell of amplification sorting, and carry out secondary sorting as stated above, carry out monoclonal with limiting dilution assay subsequently.Monoclonal cell tetramer staining, the double positive colonies screened are as shown in Figure 3.
Embodiment 2 obtains the tcr gene of DAGE small peptide specific T-cell clones and the structure of carrier
Use Quick-RNATMMiniPrep (ZYMO research) extracts antigen small peptide RYLFPPLFM specificity, the total serum IgE of HLA-A2402 restrictive T cell clone screened in embodiment 1.The synthesis of cDNA uses the SMART RACE cDNA amplification kit of clontech, the primer of employing to be that design is in the C end conserved region of mankind's tcr gene.Sequence is cloned in carrier T (TAKARA) and checks order.It should be noted that this sequence is complementary series, do not comprise intron.Through order-checking, the α chain of the TCR that this pair of positive colony is expressed and β chain-ordering structure the most 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 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, have the TCR α chain amino acid sequence of targeting sequencing and have 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 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, there is the TCR β chain amino acid sequence of targeting sequencing and there is the TCR β chain nucleotide sequence of targeting sequencing.
Identified, α chain comprises the CDR with following aminoacid sequence:
α CDR1-NSASQS (SEQ ID NO:10)
α CDR2-VYSSGN (SEQ ID NO:11)
α CDR3-VVNHFIDSGYALN (SEQ ID NO:12)
β chain comprises the CDR with following aminoacid sequence:
β CDR1-KGHDR (SEQ ID NO:13)
β CDR2-SFDVKD (SEQ ID NO:14)
β CDR3-ATSPDASNEKLF (SEQ ID NO:15)
Respectively the full-length gene of TCR α chain and β chain is cloned into Lentiviral pLenti (addgene) by overlapping (overlap) PCR.Particularly as follows: be attached obtaining TCR α-2A-TCR β fragment by the full-length gene of TCR α chain and TCR β chain with overlap PCR.Lentiviral and TCR α-2A-TCR β enzyme action are connected and obtains pLenti-TRA-2A-TRB-IRES-NGFR plasmid.As comparison use, the most also slow virus carrier pLenti-eGFP of construction expression eGFP.Pseudovirus is packed the most again with 293T/17.
Expression, refolding and the purification of the embodiment 3 DAGE 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 its variable domain and portion constant territory, and the constant domain of α and β chain introduces a cysteine residues respectively to form artificial interchain disulfide bond, introduce the Ser57 that the position of cysteine residues is respectively Thr48 and the TRBC2*01 exons 1 of TRAC*01 exons 1；With nucleotide sequence the most as shown in figures 4 a and 4b, with nucleotide sequence the most as shown in figure 5 a and 5b, the cysteine residues of introducing is with overstriking and underlines letter representation for the aminoacid sequence of its β chain for the aminoacid sequence of its α chain.By " Molecular Cloning: A Laboratory room handbook " (Molecular Cloning a Laboratory the 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 expression vector pET28a+ (Novagene), the cloning site of upstream and downstream is NcoI and NotI respectively.Insert Fragment confirms errorless through order-checking.
Converting 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 with the mass ratio of 1:1,0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, in 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.After mixing, solution is placed in the deionized water of 10 times of volumes dialysis (4 DEG C), after 12 hours, deionized water is changed into buffer (20mM Tris, pH 8.0) and continues at 4 DEG C of dialysis 12 hours.Solution after having dialysed is after the membrane filtration of 0.45 μM, by anion-exchange column (HiTrap Q HP, 5ml, GE Healthcare) purification.Eluting peak is contained the renaturation dimeric TCR of successful α and β and is confirmed by SDS-PAGE glue.TCR is further purified by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) subsequently.TCR purity after purification measures more than 90% through SDS-PAGE, and concentration is determined by BCA method.The SDS-PAGE glue figure of the sTCR that the present invention obtains is as shown in Figure 6.
The generation of embodiment 4 DAGE small peptide specific soluble single-chain T CR
According to patent documentation WO2014/206304, utilize the method for rite-directed mutagenesis that the variable domain of TCR α and β chain in embodiment 2 has been built into a stable soluble single-chain T CR molecule being connected with flexible small peptide (linker).The aminoacid sequence of this single chain TCR molecules and nucleotide sequence are the most as shown in figs. 7 a and 7b.The aminoacid sequence of its α chain variable domain and nucleotide sequence are the most as figures 8 a and 8 b show；The aminoacid sequence of its β chain variable domain and nucleotide sequence are the most as shown in figures 9 a and 9b；The aminoacid sequence of its 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, it is connected with the pET28a carrier through Nco I and Not I double digestion.Connect product to convert to E.coli DH5 α, the coating LB flat board containing kanamycin, be inverted overnight incubation for 37 DEG C, picking positive colony carries out PCR screening, positive recombinant is checked order, determines that sequence extracts recombinant plasmid transformed the most afterwards to E.coli BL21 (DE3), be used for expressing.
Expression, renaturation and the purification of embodiment 5 DAGE small peptide specific soluble single-chain T CR
BL21 (DE 3) bacterium colony containing recombiant plasmid pET28a-template strand of preparation in embodiment 4 is all inoculated in the LB culture medium containing kanamycin, 37 DEG C of cultivations are 0.6-0.8 to OD600, adding IPTG to final concentration of 0.5mM, 37 DEG C are continued to cultivate 4h.5000rpm is centrifuged 15min harvesting precipitate, with Bugbuster Master Mix (Merck) cell lysis precipitate, 6000rpm is centrifuged 15min and reclaims inclusion body, carry out washing to remove cell debris and membrane component with Bugbuster (Merck) again, 6000rpm is centrifuged 15min, collects inclusion body.By solubilization of inclusion bodies in buffer (20mM Tris-HCl pH 8.0,8M carbamide), high speed centrifugation removes insoluble matter, carries out subpackage, save backup in-80 DEG C after supernatant BCA standard measure.
In the strand TCR inclusion body protein that 5mg dissolves, adding 2.5mL buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration of 10mM, 37 DEG C process 30min.With syringe to 125mL renaturation buffer (100mM Tris-HClpH 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 of stirring 10min, then renaturation solution loads the cellulose membrane bag filter that interception is 4kDa, and bag filter is placed in the water of 1L pre-cooling, and 4 DEG C are slowly stirred overnight.After 17 hours, dialysis solution changing the buffer (20mM Tris-HCl pH 8.0) of 1L pre-cooling into, 4 DEG C are continued dialysis 8h, then dialysis solution changes into identical fresh buffer and continues dialysed overnight.After 17 hours, sample is through 0.45 μm membrane filtration, by anion-exchange column (HiTrap Q HP after vacuum outgas, GE Healthcare), 0-1M NaCl linear gradient elution liquid purifying protein with 20mM Tris-HCl pH 8.0 preparation, the elution fraction collected carries out SDS-PAGE analysis, the component comprising strand TCR uses solvent resistant column (Superdex 75 10/300 after concentrating further, 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 Agilent Bio SEC-3 (300A, φ 7.8 × 300mm), and flowing is 150mM phosphate buffer mutually, 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 specific binding with RYLFPPLFM-HLA A2402 complex.
Use the combination activity of TCR molecule and the RYLFPPLFM-HLA A2402 complex obtained in BIAcore T200 real-time analyzer detection embodiment 3 and embodiment 5.The antibody (GenScript) of anti-Streptavidin is added coupling buffer (10mM sodium-acetate buffer, pH 4.77), then antibody is flow through the CM5 chip activated with EDC and NHS in advance, antibody is made to be fixed on chip surface, finally close unreacted activating surface with the hydrochloric acid solution of ethanolamine, completing coupling process, coupling level is about 15,000RU.
The Streptavidin making low concentration flows through the chip surface of coated antibody, then RYLFPPLFM-HLA A2402 complex is flow through sense channel, another passage is as reference channel, again the biotin of 0.05mM is flow through chip 2min with the flow velocity of 10 μ L/min, close the remaining binding site of Streptavidin.
The preparation process of above-mentioned RYLFPPLFM-HLA A2402 complex is as follows:
A. purification
Collect 100ml abduction delivering heavy chain or the E.coli bacterium solution of light chain, after 4 DEG C of 8000g are centrifuged 10min with 10ml PBS washing thalline once, resuspended thalline is acutely shaken afterwards with 5ml BugBuster Master Mix Extraction Reagents (Merck), and hatch 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, add 30ml and dilute the resuspended inclusion body of BugBuster of 10 times, mixing, 4 DEG C of 6000g are centrifuged 15min, are repeated twice, add the 30ml resuspended inclusion body of 20mM Tris-HCl pH 8.0, mixing, 4 DEG C of 6000g are centrifuged 15min, finally dissolve inclusion body with 20mM Tris-HCl 8M carbamide, SDS-PAGE detects inclusion body purity, and BCA test kit surveys concentration.
B. renaturation
The small peptide RYLFPPLFM (Beijing SBS Genetech gene technology company limited) of synthesis is dissolved in the concentration of DMSO to 20mg/ml.The inclusion body 8M carbamide of light chain and heavy chain, 20mM Tris pH 8.0,10mM DTT dissolve, and add 3M guanidine hydrochloride, 10mM sodium acetate, the further degeneration of 10mM EDTA before renaturation.RYLFPPLFM peptide is added renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3,2mM EDTA, 0.5mM GSSG, 5mM reduced glutathion, 0.2mM PMSF with 25mg/L (final concentration), it is cooled to 4 DEG C), then the light chain of 20mg/L and the heavy chain (final concentration of 90mg/L it are sequentially added into, heavy chain adds in three times, 8h/ time), renaturation 4 DEG C carry out at least 3 days to completing, SDS-PAGE detection can renaturation success.
C. purification after renaturation
Change renaturation buffer with the 20mM Tris pH 8.0 of 10 volumes as dialysis, at least change buffer and fully reduce the ionic strength of solution for twice.With 0.45 μm 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 volume).Utilizing Akta purification instrument (GE General Electric Co. Limited), the 0-400mM NaCl linear gradient liquid eluted protein of 20mM Tris pH 8.0 preparation, pMHC about eluting at 250mM NaCl, collect all peaks component, SDS-PAGE detects purity.
D. biotinylation
With Mi ll ipore super filter tube by the pMHC molecular concentration of purification, it is 20mM Tris pH 8.0 by buffer exchange simultaneously, it is subsequently adding biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μMs of D-Biotin, 100 μ g/ml BirA enzyme (GST-BirA), overnight, SDS-PAGE detection biotinylation is the most complete for incubated at room mixture.
E. the complex after purifying biological element
With Millipore super filter tube by the pMHC molecular concentration after biotinylation labelling to 1ml, use the biotinylated pMHC of gel filtration chromatography, utilize Akta purification instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibration HiPrepTM16/60S200HR post (GE General Electric Co. Limited), loads the concentrated biotinylation pMHC molecule of 1ml, then with PBS with 1ml/min flow velocity eluting.Biotinylated pMHC molecule occurs as unimodal eluting when about 55ml.Merging the component containing protein, concentrate with Millipore super filter tube, BCA method (Thermo) measures protein concentration, adds protease inhibitor cocktail (Roche) and biotinylated pMHC molecule subpackage is saved in-80 DEG C.
Utilizing BIAcore Evaluation computed in software kinetic parameter, the kinetic profile that the soluble single-chain T CR molecule of the TCR molecule and present invention structure that obtain solubility of the present invention is combined with RYLFPPLFM-HLA A2402 complex is as shown in figure 12.Collection of illustrative plates shows, soluble TCR molecules and soluble single-chain T CR molecule that the present invention obtains can be combined with RYLFPPLFM-HLA A2402 complex.Meanwhile, also utilizing said method to have detected the TCR molecule of solubility of the present invention and the combination activity of other several irrelevant antigen small peptides with HLA complex, result shows that TCR molecule of the present invention and other irrelevant antigen are all without combining.
The all documents mentioned in the present invention are incorporated as reference the most in this application, are individually recited as with reference to like that just as each document.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, these equivalent form of values fall within the application appended claims limited range equally.
Claims (10)
1. a φt cell receptor (TCR), it is characterised in that described TCR can be with RYLFPPLFM-HLA
A2402 complex combines；Preferably, described TCR comprises TCR α chain variable domain and TCR β chain variable domain,
It is characterized in that, the aminoacid sequence of the CDR3 of described TCR α chain variable domain is VVNHFIDSGYALN (SEQ
ID NO:12)；And/or the aminoacid sequence of the CDR3 of described TCR β chain variable domain is ATSPDASNEKLF
(SEQ ID NO:15)；
It is highly preferred that 3 complementary determining regions (CDR) of described TCR α chain variable domain are:
αCDR1-NSASQS (SEQ ID NO:10)
αCDR2-VYSSGN (SEQ ID NO:11)
αCDR3-VVNHFIDSGYALN (SEQ ID NO:12)；And/or
3 complementary determining regions of described TCR β chain variable domain are:
βCDR1-KGHDR (SEQ ID NO:13)
βCDR2-SFDVKD (SEQ ID NO:14)
βCDR3-ATSPDASNEKLF (SEQ ID NO:15)。
2. TCR as claimed in claim 1, it is characterised in that it comprises TCR α chain variable domain and TCR
β chain variable domain, described TCR α chain variable domain is for have at least 90% sequence thereto with SEQ ID NO:1
Aminoacid sequence；And/or described TCR β chain variable domain is for have at least 90% sequence with SEQ ID NO:5
The aminoacid sequence of row homogeny.
3. TCR as claimed in claim 1, it is characterised in that the α chain of described TCR and/or β chain
C-or N-end is combined with conjugate；Preferably, the conjugate being combined with described φt cell receptor is for detecting
Label, therapeutic agent, PK modify part or the combination of these materials any；Preferably, 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；And/or
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 in claim 25-28 arbitrary described
Nucleic acid molecules；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow sick
Poisonous carrier.
7. the host cell separated, it is characterised in that containing claim in described host cell
Carrier described in 26 or chromosome are integrated and has arbitrary described nucleic acid in claim 25-28 of external source to divide
Son.
8. a cell, it is characterised in that arbitrary described core in described cell transduction claim 25-28
Carrier described in acid molecule or claim 29；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 the TCR according to any one of claim 1-23, the TCR complex described in claim 24,
Cell described in arbitrary described nucleic acid molecules or claim 28 in claim 25-28.
10. described in φt cell receptor according to any one of claim 1-23 or claim 24
The purposes of the cell described in TCR complex or claim 31, it is characterised in that be used for preparing treatment swollen
Tumor or the medicine of autoimmune disease.
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| CN201510741810.7A CN106084036A (en) | 2015-11-02 | 2015-11-02 | Identify the φt cell receptor of DAGE small peptide |
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| CN109400696A (en) * | 2017-08-17 | 2019-03-01 | 广东香雪精准医疗技术有限公司 | A kind of TCR identifying PRAME antigen small peptide |
| CN109879957A (en) * | 2017-12-06 | 2019-06-14 | 广东香雪精准医疗技术有限公司 | For the high-affinity T cell receptor of PRAME |
| US11111286B2 (en) | 2017-03-23 | 2021-09-07 | Immatics Biotechnologies Gmbh | T cell receptors and immune therapy using the same against PRAME positive cancers |
| WO2021185368A1 (en) * | 2020-03-20 | 2021-09-23 | 香雪生命科学技术（广东）有限公司 | High-affinity tcr for recognizing afp antigen |
| WO2022012284A1 (en) * | 2020-07-11 | 2022-01-20 | 成都益安博生物技术有限公司 | Peripheral blood tcr marker for melanoma, and detection kit and use thereof |
| US11236145B2 (en) | 2017-03-23 | 2022-02-01 | Immatics Biotechnologies Gmbh | T cell receptors and immune therapy using the same against PRAME positive cancers |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11111286B2 (en) | 2017-03-23 | 2021-09-07 | Immatics Biotechnologies Gmbh | T cell receptors and immune therapy using the same against PRAME positive cancers |
| US11236145B2 (en) | 2017-03-23 | 2022-02-01 | Immatics Biotechnologies Gmbh | T cell receptors and immune therapy using the same against PRAME positive cancers |
| CN109400696A (en) * | 2017-08-17 | 2019-03-01 | 广东香雪精准医疗技术有限公司 | A kind of TCR identifying PRAME antigen small peptide |
| CN109879957A (en) * | 2017-12-06 | 2019-06-14 | 广东香雪精准医疗技术有限公司 | For the high-affinity T cell receptor of PRAME |
| CN109879957B (en) * | 2017-12-06 | 2022-03-18 | 香雪生命科学技术（广东）有限公司 | High affinity T cell receptors for PRAME |
| WO2021185368A1 (en) * | 2020-03-20 | 2021-09-23 | 香雪生命科学技术（广东）有限公司 | High-affinity tcr for recognizing afp antigen |
| WO2022012284A1 (en) * | 2020-07-11 | 2022-01-20 | 成都益安博生物技术有限公司 | Peripheral blood tcr marker for melanoma, and detection kit and use thereof |
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Effective date of registration: 20171102 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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