CN106478807A - The T cell receptor of identification MAGE-A3 - Google Patents
The T cell receptor of identification MAGE-A3 Download PDFInfo
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Abstract
The invention provides a kind of φt cell receptor (TCR) that can specifically bind the small peptide SYVKVLHHM derived from MAGE A3 antigen, described antigen small peptide SYVKVLHHM can with HLA A2402 formed complex and together be presented to cell surface.Present invention also offers encoding the nucleic acid molecules of described TCR and the carrier comprising described nucleic acid molecules.In addition, present invention also offers the cell of the TCR of the present invention that transduces.
Description
Technical field
The present invention relates to being capable of identify that the TCR from MAGE-A3 antigen small peptide, the invention still further relates in transduction
State the specific T cell of MAGE-A3 of TCR acquisition, and they are in prevention and treatment MAGE-A3 correlation disease
Purposes in disease.
Background technology
MAGE-A3, as a kind of autochthonous tumor antigen, is degraded to micromolecule polypeptide after generating in the cell,
And combine to form complex with MHC (main histocompatibility complex) molecule, it is presented to cell surface.
Research display, SYVKVLHHM is the small peptide derived from MAGE-A3.MAGE-A3 albumen is in kinds of tumors type
In all have expression, including melanoma, and other solid tumor such as gastric cancer, pulmonary carcinoma, esophageal carcinoma, bladder
Cancer, squamous cell carcinoma of the head and neck etc..For the treatment of above-mentioned disease, chemotherapy and radiation treatment can be adopted
Etc. method, but all the normal cell of itself can be caused damage.
T cell adoptive immunotherapy is to proceed to disease by having specific reaction-ive T cell to target cell antigen
So as to play a role for target cell in human body.φt cell receptor (TCR) is a kind of film on T cell surface
Albumen, it is capable of identify that the antigen small peptide of corresponding target cells.In immune system, short by antigen
The TCR of peptide specific causes T thin with the combination of small peptide-main histocompatibility complex (pMHC complex)
Born of the same parents and antigen-presenting cell (APC) directly physical contact, then T cell and other cells both APC
Membrane surface molecule just interacts, and causes a series of follow-up cell signal transmission and other physiology anti-
Should, so that the T cell of different antigenic specificity plays immunological effect to its target cell.Therefore, ability
Field technique personnel are devoted to isolating and have specific TCR to MAGE-A3 antigen small peptide, and by this TCR
Transduction T cell has specific T cell to obtain to MAGE-A3 antigen small peptide, so that they are thin
Play a role in born of the same parents' immunization therapy.
Content of the invention
It is an object of the invention to provide a kind of φt cell receptor of identification MAGE-A3 antigen small peptide.
A kind of a first aspect of the present invention, there is provided φt cell receptor (TCR), described TCR can be with
SYVKVLHHM-HLA A2402 complex combines.
In another preference, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, described
The aminoacid sequence of the CDR3 of TCR α chain variable domain is AGVMNSGNTPLV (SEQ ID NO:12)；With/
Or the aminoacid sequence of the CDR3 of described TCR β chain variable domain is ASSADIWETQY (SEQ ID NO:15).
In another preference, 3 complementary determining regions (CDR) of described TCR α chain variable domain are：
α CDR1-SVFSS (SEQ ID NO:10)
α CDR2-VVTGGEV (SEQ ID NO:11)
α CDR3-AGVMNSGNTPLV (SEQ ID NO:12)；And/or
3 complementary determining regions of described TCR β chain variable domain are：
β CDR1-SGDLS (SEQ ID NO:13)
β CDR2-YYNGEE (SEQ ID NO:14)
β CDR3-ASSADIWETQY (SEQ ID NO:15).
In another preference, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, described
TCR α chain variable domain is and SEQ ID NO:1 aminoacid sequence with least 90% sequence thereto；With/
Or described TCR β chain variable domain is and SEQ ID NO：The 5 aminoacid sequences with least 90% sequence thereto
Row.
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 single-stranded.
In another preference, described TCR is to pass 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 amino acid the 11st, 13,19,21,53,
76th, 89,91 or the 94th, and/or 3rd reciprocal of α chain J gene small peptide aminoacid, inverse the 5th
In position or inverse the 7th, there are one or more mutation；And/or described TCR is in β chain variable region amino acid
11st, 13,19,21,53,76,89,91 or the 94th, and/or β chain J gene small peptide amino
In 2nd reciprocal of acid, 4th reciprocal or 6th reciprocal, there are one or more mutation, wherein aminoacid
Position Number is by the Position Number listed in IMGT (international immunogeneticses 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 (b) all or part of TCR β chain 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 a portion of TCR chain constant domain.
In another preference, cysteine residues form people between α the and 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
From following one or more groups of sites：
Thr48 and TRBC1*01 of the TRAC*01 exons 1 or Ser57 of TRBC2*01 exons 1；
Thr45 and TRBC1*01 of the TRAC*01 exons 1 or Ser77 of TRBC2*01 exons 1；
Tyr10 and TRBC1*01 of the TRAC*01 exons 1 or Ser17 of TRBC2*01 exons 1；
Thr45 and TRBC1*01 of the TRAC*01 exons 1 or Asp59 of TRBC2*01 exons 1；
Ser15 and TRBC1*01 of the TRAC*01 exons 1 or Glu15 of TRBC2*01 exons 1；
Arg53 and TRBC1*01 of the TRAC*01 exons 1 or Ser54 of TRBC2*01 exons 1；
Pro89 and TRBC1*01 of the TRAC*01 exons 1 or Ala19 of TRBC2*01 exons 1；
With
Tyr10 and TRBC1*01 of the TRAC*01 exons 1 or Glu20 of TRBC2*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, between the α chain variable region of described TCR and β chain constant region, contain artificial interchain
Disulfide bond.
Form half Guang of artificial interchain disulfide bond in another preference it is characterised in that in described TCR
Histidine residue instead of selected from following one or more groups of sites：
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 removes transmembrane structure
All or part of β chain constant domain beyond domain, but it does not contain α chain constant domain, the α chain of described TCR can
Variable domain and β chain formation heterodimer.
In another preference, the α chain of described TCR and/or C- the or N- end of β chain are combined with conjugate.
In another preference, the conjugate being combined with described φt cell receptor is detectable, treatment
Part or the combination of these materials any are modified in agent, PK.Preferably, described therapeutic agent is anti-CD 3 antibodies.
A kind of a second aspect of the present invention, there is provided 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 kind of a third aspect of the present invention, there is provided nucleic acid molecules, described nucleic acid molecules comprise to encode the present invention
The nucleotide sequence of TCR molecule described in first aspect or its complementary series.
In another preference, described nucleic acid molecules comprise to encode the nucleotide sequence SEQ of TCR α chain variable domain
ID NO：2 or SEQ ID NO:33.
In another preference, described nucleic acid molecules comprise to encode the nucleotide sequence of TCR β chain variable domain
SEQ ID NO：6 or SEQ ID NO:35.
In another preference, described nucleic acid molecules comprise to encode the nucleotide sequence SEQ ID NO of TCR α chain：
4 and/or comprise encode TCR β chain nucleotide sequence SEQ ID NO：8.
A kind of a fourth aspect of the present invention, there is provided 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 kind of a fifth aspect of the present invention, there is provided detached host cell, contains in described host cell
It is integrated with carrier described in fourth aspect present invention or genome described in the third aspect present invention of external source
Nucleic acid molecules.
A kind of a sixth aspect of the present invention, there is provided 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 kind of a seventh aspect of the present invention, there is provided pharmaceutical composition, described compositionss contain and pharmaceutically can connect
TCR complex described in TCR described in the carrier being subject to and first aspect present invention, second aspect present invention,
Carrier described in nucleic acid molecules described in third aspect present invention, fourth aspect present invention or the present invention the 6th
Cell described in aspect.
A eighth aspect of the present invention, there is provided the φt cell receptor described in first aspect present invention or the present invention
Nucleic acid molecules described in TCR complex described in second aspect, third aspect present invention, present invention four directions
Carrier described in face or the purposes of the cell described in sixth aspect present invention, for preparation treatment tumor or certainly
The medicine of body immunological diseases.
A kind of a ninth aspect of the present invention, there is provided method treating disease, including to object in need for the treatment of
Apply the appropriate φt cell receptor described in first aspect present invention or the TCR described in second aspect present invention
Carrier described in nucleic acid molecules described in complex, third aspect present invention, 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 tumor, and preferably described tumor includes melanoma, and other are real
Body tumor for example gastric cancer, pulmonary carcinoma, esophageal carcinoma, bladder cancer, squamous cell carcinoma of the head and neck, carcinoma of prostate, breast carcinoma,
Colon cancer, ovarian cancer etc..
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (such as enforcement
Example) in can be combined with each other, thus constituting new or preferred skill between each technical characteristic of specifically describing
Art scheme.As space is limited, here is no longer tired out one by one and is stated.
Brief description
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 obtaining after purification.Leftmost side swimming lane is to go back virgin rubber, middle swimming lane
For molecular weight marker (marker), rightmost side swimming lane is non-reduced glue.
Fig. 7 a and Fig. 7 b is respectively aminoacid sequence and the nucleotide sequence of single-stranded TCR.
Fig. 8 a and Fig. 8 b is respectively aminoacid sequence and the nucleotide sequence of single-stranded TCR α chain.
Fig. 9 a and Fig. 9 b is respectively aminoacid sequence and the nucleotide sequence of single-stranded TCR β chain.
Figure 10 a and Figure 10 b is respectively aminoacid sequence and the nucleoside of single-stranded TCR catenation sequence (linker)
Acid sequence.
Figure 11 is the glue figure of the soluble single-chain T CR obtaining after purification.Left side swimming lane is molecular weight marker
(marker), right lanes are non-reduced glue.
The BIAcore that Figure 12 is combined with SYVKVLHHM-HLA A2402 complex for sTCR of the present invention
Kinetic profile.
Figure 13 is combined with SYVKVLHHM--HLA A2402 complex for soluble single-chain T CR of the present invention
BIAcore kinetic profile.
Specific embodiment
The present inventor, through extensively in-depth study, have found and MAGE-A3 antigen small peptide SYVKVLHHM
(SEQ ID NO:9) TCR that can specifically bind, described antigen small peptide SYVKVLHHM can be with HLA A2402
Form complex and be presented to cell surface together.Present invention also offers the nucleic acid encoding described TCR divides
Son and the carrier comprising described nucleic acid molecules.In addition, present invention also offers the thin of TCR of the present invention of transduceing
Born of the same parents.
Term
MHC molecule is the protein of immunoglobulin superfamily, can be I class or class Ⅱ[MHC.Cause
This, it has specificity for the presentation of antigen, and different individualities has different MHC, can present a hatching egg
In Bai Kangyuan, different small peptides 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 the specific antigen peptide presenting in main histocompatibility complex (MHC)
Unique receptor.In immune system, drawn by the combination of the TCR of antigenic specificity and pMHC complex
Send out T cell and antigen-presenting cell (APC) directly physical contact, then T cell and its both APC
He just interacts at cell membrane surface molecules, this just cause a series of follow-up cell signals 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 being existed with heterodimer form by α chain/β chain or γ chain/δ chain
Glycoprotein.In 95% T cell, TCR heterodimer is made up of α and β chain, and 5% T cell tool
By the TCR being made up of γ and δ chain.Heterogeneous dimerization TCR of natural 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
Area and constant region, β chain generally contains short variable region also between variable region and bonding pad, but this variable region
Often it is regarded as a part for bonding pad.Each variable region comprises to be 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 are formed by variable region and bonding pad restructuring, are referred to as hypervariable region.TCR's
α and β chain is typically regarded as respectively two " domains " i.e. variable domain and constant domain, and variable domain can by connect
Become area and bonding pad to constitute.The sequence of TCR constant domain can be in international immunogeneticses information system (IMGT)
Public database in find, such as 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 the and β chain of TCR is also
Comprise transmembrane region and cytoplasmic region, cytoplasmic region is very short.
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 " is used interchangeably.
Native interchain disulfide bond and artificial interchain disulfide bond
There is one group of disulfide bond in the membrane-proximal region C α of natural TCR with C β interchain, be referred to as in the present invention " my god
So interchain disulfide bond ".In the present invention, will be manually-injected, the position of position and native interchain disulfide bond
Different interchain covalent disulfide bonds are referred to as " artificial interchain disulfide bond ".
For convenience of the position describing disulfide bond, TRAC*01 and TRBC1*01 or TRBC2*01 ammonia in the present invention
The Position Number of base acid sequence carries out Position Number by order successively from N-terminal to C-terminal, such as TRBC1*01
Or in TRBC2*01, be P (proline) by the 60th aminoacid of order successively from N-terminal to C-terminal,
The Pro60 of TRBC1*01 or TRBC2*01 exons 1 then can be described it as in the present invention, also can be by it
Be expressed as the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1, and for example TRBC1*01 or
In TRBC2*01, it is Q (L-Glutamine) by the 61st aminoacid of order successively from N-terminal to C-terminal,
The Gln61 of TRBC1*01 or TRBC2*01 exons 1 then can be described it as in the present invention, also can be by it
It is expressed as the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1, other are by that analogy.This
In invention, the Position Number of the aminoacid sequence of variable region TRAV and TRBV, according to list in IMGT
Position Number.As certain aminoacid in TRAV, the Position Number listed in IMGT is 46, then the present invention
In describe it as TRAV the 46th amino acids, other are by that analogy.In the present invention, other aminoacid
Sequence position numbers have specified otherwise, then press specified otherwise.
Detailed Description Of The Invention
TCR molecule
In antigen processing pathways, antigen is degraded in the cell, 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 a kind of can be in conjunction with the TCR molecule of SYVKVLHHM-HLA A2402 complex.Excellent
Selection of land, described TCR molecule is detached or purification.α the and β chain of this TCR respectively has 3 complementations and determines
Determine area (CDR).
One in the present invention is preferably carried out in mode, and the α chain of described TCR comprises there is following aminoacid
The CDR of sequence：
α CDR1-SVFSS (SEQ ID NO:10)
α CDR2-VVTGGEV (SEQ ID NO:11)
α CDR3-AGVMNSGNTPLV (SEQ ID NO:12)；And/or
3 complementary determining regions of described TCR β chain variable domain are：
β CDR1-SGDLS (SEQ ID NO:13)
β CDR2-YYNGEE (SEQ ID NO:14)
β CDR3-ASSADIWETQY (SEQ ID NO:15).
The CDR region aminoacid sequence of the invention described above can be embedded into make in any suitable frame structure
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 suitable frame structure
TCR molecule.TCR α chain variable domain of the present invention is and SEQ ID NO:1 has at least 90%, preferably
95%, the aminoacid sequence of more preferably 98% sequence thereto；And/or TCR β chain variable domain of the present invention is
With SEQ ID NO：5 have at least 90%, preferably 95%, the amino of more preferably 98% sequence thereto
Acid sequence.
In a preference of the present invention, the TCR molecule of the present invention is be made up of α and β chain heterogeneous two
Aggressiveness.Specifically, on the one hand the α chain of described heterogeneous dimerization TCR molecule comprises variable domain and constant domain, institute
State CDR1 (the SEQ ID NO that α chain variable domain amino acid sequence comprises above-mentioned α chain：10)、CDR2(SEQ
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 comprises CDR1 (the SEQ ID NO of above-mentioned β chain:13)、
CDR2(SEQ ID NO：14) and CDR3 (SEQ ID NO:15).Preferably, described TCR molecule bag
The variable domain amino acid sequence SEQ ID NO of chain containing β: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 part or all of composition of β chain.Description about single chain TCR molecules may be referred to
Document Chung et al (1994) Proc.Natl.Acad.Sci.USA 91,12654-12658.
According to document, those skilled in the art can easily build and comprise the single-stranded of CDRs area of the present invention
TCR molecule.Specifically, described single chain TCR molecules comprise V α, V β and C β, preferably according to from N
Hold being linked in sequence of C-terminal.
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 comprise α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that it is described single-stranded
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 comprises CDR1 (the SEQ ID NO of above-mentioned β chain：13)、CDR2(SEQ ID NO：
14) and CDR3 (SEQ ID NO：15).Preferably, described single chain TCR molecules comprise 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 or can be by consulting pertinent texts or IMGT (international immunogeneticses information system
System) the constant domain amino acid sequence to obtain people for the public database.For example, 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 being given in the TRAC*01 of IMGT is Arg,
This is expressed as：The Arg53 of TRAC*01 exons 1, 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 be SEQ ID NO:
7.
Naturally occurring TCR is a kind of memebrane protein, is stabilized by its transmembrane region.Such as immunoglobulin
(antibody) is the same as antigen recognizing molecule, and TCR can also be developed and be applied to diagnose and treat, and at this moment need
The TCR molecule of solubility to be obtained.The TCR molecule of solubility does not include its transmembrane region.STCR has
Very extensive purposes, it cannot be only used for studying the interaction of TCR and pMHC it is also possible to make detection sense
The diagnostic tool of dye or the mark as autoimmune disease.Similarly, sTCR can be used to by
Therapeutic agent (as cytotoxin compounds or immunostimulating compound) is transported to and presents the thin of specific antigen
Born of the same parents, in addition, sTCR also can combine to redirect T with other molecules (e.g., anti-CD 3 antibodies)
Cell, so that its targeting presents the cell of specific antigen.The present invention also obtain short to MAGE-A3 antigen
Peptide has specific sTCR.
For obtaining sTCR, on the one hand, TCR of the present invention can be residual in itself α and β chain constant domain
The TCR of artificial disulfide bond is introduced between base.Cysteine residues are between α the and 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.For example, replace the Thr48 of TRAC*01 exons 1 and take
Cysteine residues for the Ser57 of TRBC1*01 or TRBC2*01 exons 1 to form disulfide bond.Draw
Other sites entering cysteine residues to form disulfide bond can also be：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 of TRAC*01 exons 1 or TRBC2*01
The Ser54 of exons 1；Aobvious outside Pro89 and TRBC1*01 of TRAC*01 exons 1 or TRBC2*01
The Ala19 of son 1；Or Tyr10 the and TRBC1*01 or TRBC2*01 exon of TRAC*01 exons 1
1 Glu20.I.e. cysteine residues instead of above-mentioned α and arbitrary group of site in 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 its not include cysteine residual
Base, to reach the purpose of disappearance natural disulphide bonds, also can pass through the cysteine residues will form natural disulphide bonds
Sport another aminoacid to reach above-mentioned purpose.
As described above, the TCR of the present invention may be embodied in the people introducing between the residue of itself α and β chain constant domain
Work disulfide bond.It should be noted that with or without the artificial disulfide bond of 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, TCR of the present invention is additionally included in its hydrophobic core region and occurs to dash forward
The TCR becoming, the mutation in these hydrophobic core regions is preferably capable making the stability of sTCR of the present invention carry
High mutation, as described in the patent documentation in Publication No. WO2014/206304.Such TCR can be
Undergo mutation in the hydrophobic core position of its following variable domain：(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,
The Position Number of wherein aminoacid sequence presses the position listed in international immunogeneticses information system (IMGT)
Numbering.Those skilled in the art know above-mentioned world immunogeneticses 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 solvable single-stranded TCR of stability that constitutes.It should be noted that flexible peptide chain is permissible in the present invention
It is the peptide chain of any suitable connection TCR α and β chain variable domain.As the list building 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.
In addition, for stability, patent documentation 201510260322.4 also discloses the α in TCR
Introducing artificial interchain disulfide bond between chain variable region and β chain constant region can make the stability of TCR significantly carry
High.Therefore, can also contain between the α chain variable region of the high-affinity TCR of the present invention and β chain constant region
Artificial interchain disulfide bond.Specifically, form people between the α chain variable region of described TCR and β chain constant region
The cysteine residues of work interchain disulfide bond instead of：46th amino acids of TRAV and TRBC1*01 or
60th amino acids of TRBC2*01 exons 1；47th amino acids of TRAV and TRBC1*01 or
61 amino acids of TRBC2*01 exons 1；46th amino acids of TRAV and TRBC1*01 or
61st amino acids of TRBC2*01 exons 1；Or the 47th amino acids of TRAV and TRBC1*01 or
60th amino acids of TRBC2*01 exons 1.Preferably, such TCR can comprise () and removes
All or part of TCR α chain beyond its membrane spaning domain, and () is complete in addition to its membrane spaning domain
Portion or part TCR β chain, wherein () and () all comprise variable domain and at least a portion of TCR chain
Constant domain, α chain and β chain formation heterodimer.It is highly preferred that such TCR can comprise α chain can
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 wrap
The constant domain of chain containing α, 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 bag of the present invention
Containing two, three, the four or more TCR of the present invention polymer that combines and formed, such as p53 can be used
Four dimerization domain producing the tetramer, or multiple TCR of the present invention and another molecule be combined 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 such application.
The TCR of the present invention can be used alone, and also can be combined with covalent or other modes with conjugate, preferably
Combined with covalent manner.Described conjugate includes detectable (for diagnostic purpose, wherein said TCR
For detection present SYVKVLHHM-HLA A2402 complex cell presence), therapeutic agent, PK (egg
White kinases) modify the combination combination of part or any the above material or be coupled.
Detectable for diagnostic purposes includes but is not limited to：Fluorescence or luminous marker, radioactivity
Label, MRI (nuclear magnetic resonance) or CT (CT technology) contrast agent,
Or the enzyme of detectable product can be produced.
The therapeutic agent that can be combined with TCR of the present invention or be coupled is included but is not limited to：1. radionuclide
(Koppe etc., 2005, (Cancer metastasis reviews) 24,539 is commented in cancerometastasis)；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
The factor such as IL-2 etc. (Gillies etc., 1992, NAS's proceeding (PNAS) 89,1428；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 piece
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 Soc iety) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene
Therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,
11631)；9. magnetic nanosphere；10. pro-drug activation enzymes (for example, DT- diaphorase (DTD) or xenyl water
Solution enzyme-sample protein (BPHL))；11. chemotherapeutics (for example, cisplatin) or any type of nano-particle etc..
In addition, the TCR of the present invention can also be comprising derived from exceeding a kind of heterozygosis TCR of species sequence.
For example, 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, regulation scheme should be had to carry out immunosuppressant when it is used for adoptive T cell treatment, with
Allow the implantation of the T cell of expression Muridae.
It should be understood that amino acid name herein adopts international single English alphabet or three the English alphabets
Show, single English alphabet of amino acid name is as follows with the corresponding relation of three English alphabets：Ala(A)、Arg(R)、
Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、
Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、
Tyr(Y)、Val(V).
Nucleic acid molecules
The nucleic acid that a second aspect of the present invention provides coding first aspect present invention TCR molecule or part thereof divides
Son, described can be partly one or more CDR, the variable domain of 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-agtgttttttccagc(SEQ ID NO:16)
α CDR2-gtagttacgggtggagaagtg(SEQ ID NO:17)
α CDR3-gcaggggtgatgaattcaggaaacacacctcttgtc(SEQ ID NO:18)
The nucleotide sequence of coding first aspect present invention TCR molecule β chain CDR region is as follows：
β CDR1-tctggagacctctct(SEQ ID NO:19)
β CDR2-tattataatggagaagag(SEQ ID NO:20)
β CDR3-gccagcagcgccgacatatgggagacccagtac(SEQ ID NO:21)
Therefore, the nucleotide sequence encoding the nucleic acid molecules of the present invention of TCR α chain of the present invention includes SEQ ID NO:
16、SEQ ID NO:17 and SEQ ID NO:18, and/or encode the core of the present invention of TCR β chain of the present invention
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 single-stranded or double-stranded, 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 comprise intron but can encode polypeptide of the present invention, for example, encode TCR α chain variable domain of the present invention
The nucleotide sequence of nucleic acid molecules of the present invention include SEQ ID NO:2 and/or encode TCR β chain of the present invention
The nucleotide sequence of the nucleic acid molecules of the present invention of variable domain includes SEQ ID NO:6.Or, encode the present invention
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.
It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.
Therefore, encode TCR of the present invention nucleotide sequence can identical with the nucleotide sequence shown in accompanying drawing of the present invention or
It is the variant of degeneracy.To be illustrated with the one of example in the present invention, " variant of degeneracy " refers to
Coding has SEQ ID NO:1 protein sequence, but with SEQ ID NO:The 2 differentiated nucleic acid of sequence
Sequence.
Nucleotide sequence can be through codon optimization.Different cells concrete codon using on be
Different, codon in sequence can be changed to increase expression according to the type of cell.Mammal
The codon usage table of cell and multiple other biological be 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 known various existing DNA moleculars (or as carrier) and cell in domain.DNA can be coding strand or non-
Coding strand.
Carrier
The invention still further relates to comprising the carrier of the nucleic acid molecules of the present invention, including expression vector, that is, can be in body
The construct of interior or vivoexpression.Conventional carrier includes bacterial plasmid, phage and animals and plants virus.
Viral delivery systems include but is not limited to adenovirus vector, adeno-associated viruses (AAV) carrier, herpess
Viral vector, retroviral vector, slow virus carrier, baculovirus vector.
Preferably, the nucleotide of the present invention can be transferred in cell for carrier, such as in T cell so that
This cell expresses the TCR of MAGE-A3 antigenic specificity.Ideally, this carrier should be in T
Continual high levels ground expression in cell.
Cell
The invention still further relates to the host cell being produced through genetic engineering with carrier or the coded sequence of the present invention.Institute
State the nucleic acid molecules being integrated with the present invention in the carrier containing the present invention in host cell or chromosome.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..
In addition, present invention additionally comprises the detached cell of the TCR of the expression present invention, particularly T cell.Should
T cell can derived from from the detached T cell of experimenter, or can be that detached mixing is thin from experimenter
The part of born of the same parents group, such as 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 in CD4+Helper T cell/CD8+In the mixing group of cytotoxic T cell.Usually, this cell can be used
Antibody (e.g., antibody of anti-CD3 or anti-CD28) activates, to allow them to easily accept transfection,
For example transfected with the carrier comprising the nucleotide sequence encoding TCR molecule of the present invention.
Alternatively, the cell of the present invention can also be or derived from stem cell, such as hematopoietic stem cell (HSC).
Gene transfer to HSC is not result in cell surface expression TCR, because stem cell surface does not express CD3
Molecule.However, migrating to the lymphoid precursor of thymus (lymphoid precursor) when stem cell is divided into
When, the TCR molecule of expression this introducing of surface expression in thymocyte cell by startup of CD3 molecule.
Many methods are had to be suitable for carrying out T cell transfection (e.g., with DNA or RNA encoding TCR of the present invention
Robbins etc., (2008) J.Immunol.180:6116-6131).The T expressing TCR of the present invention is thin
Born of the same parents can 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).
MAGE-A3 antigen-related disease
The invention still further relates to treating in experimenter and/or preventing the method with MAGE-A3 relevant disease, its
Step including adoptive transfer MAGE-A3 specific T-cells to this experimenter.This MAGE-A3 specificity
T cell can recognize that SYVKVLHHM-HLA A2402 complex.
The specific T cell of MAGE-A3 of the present invention can be used for treating any presentation MAGE-A3 antigen small peptide
The MAGE-A3 relevant disease of SYVKVLHHM-HLA A2402 complex.Including but not limited to tumor is such as black
Melanoma, and other entity tumors for example gastric cancer, pulmonary carcinoma, esophageal carcinoma, bladder cancer, squamous cell carcinoma of the head and neck,
Carcinoma of prostate, breast carcinoma, colon cancer, ovarian cancer etc..
Therapeutic Method
Can be suffered from and the patient of MAGE-A3 antigen-related disease or the T cell of volunteer by separating, and
The TCR of the present invention is imported in above-mentioned T cell, the cell subsequently modified these genetic engineerings feeds back to disease
Treated in human body.Therefore, the invention provides a kind of method treating MAGE-A3 relevant disease,
Including by the T cell of detached expression TCR of the present invention it is preferable that this T cell derive from patient itself,
It is input in patient body.Usually, separate the T cell of patient including (1), (2) use nucleic acid of the present invention
Molecule or the nucleic acid molecules ex vivo transduction T cell that can encode TCR molecule of the present invention, (3) are by gene work
The T cell that journey is modified is input in patient body.The quantity of the cell separate, transfecting and feeding back can be by doctor
Determine.
Main advantages of the present invention are：
(1) TCR of the present invention can be with MAGE-A3 antigen small peptide complex SYVKVLHHM-HLA A2402
In conjunction with, the TCR of the present invention that simultaneously transduceed cell can have by specific activation and to target cell very strong
Lethal effect.
Following specific embodiment, is expanded on further the present invention.It 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 examples
In experiment material used and reagent all can obtain from commercially available channel if no special instructions.
Embodiment 1 clones MAGE-A3 antigen small peptide specific T-cells
Using synthesis small peptide SYVKVLHHM (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.Will
SYVKVLHHM small peptide and the HLA-A2402 renaturation with biotin labeling, prepare pHLA monoploid.These
Monoploid and the tetramer being combined into PE labelling with the Streptavidin (BD company) of PE labelling, sort this four
Aggressiveness and anti-CD8-APC double positive cells.The cell of amplification sorting, and carry out secondary sorting as stated above,
Subsequently carry out monoclonal with limiting dilution assay.Monoclonal cell tetramer staining, double positive gram screening
Grand as shown in Figure 3.
Embodiment 2 obtains the tcr gene of MAGE-A3 antigen small peptide specific T-cell clones and the structure of carrier
Build
Use Quick-RNATMScreen in MiniPrep (ZYMO research) extracting embodiment 1 is anti-
The total serum IgE of the restricted T cell clone of former small peptide SYVKVLHHM specificity, HLA-A2402.CDNA's
Using the SMART RACE cDNA amplification kit of clontech, the primer of employing is design in people for synthesis
The C-terminal conserved region of class tcr gene.Sequence is cloned in carrier T (TAKARA) and is sequenced.Should note
Meaning, this sequence is complementary seriess, does not comprise intron.Through sequencing, the TCR's of this pair of positive colony expression
α chain and β chain-ordering structure are distinguished 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-SVFSS (SEQ ID NO:10)
α CDR2-VVTGGEV (SEQ ID NO:11)
α CDR3-AGVMNSGNTPLV (SEQ ID NO:12)
β chain comprises the CDR with following aminoacid sequence：
β CDR1-SGDLS (SEQ ID NO:13)
β CDR2-YYNGEE (SEQ ID NO:14)
β CDR3-ASSADIWETQY (SEQ ID NO:15)
The full-length gene of TCR α chain and β chain is cloned into respectively by slow viruss by overlapping (overlap) PCR
Expression vector pLenti (addgene).It is specially：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
- 2A-TCR β enzyme action connects and obtains pLenti-TRA-2A-TRB-IRES-NGFR plasmid.Use as comparison, with
When also construction expression eGFP slow virus carrier pLenti-eGFP.Pack pseudoviruss with 293T/17 more afterwards.
The expression of the solvable TCR of embodiment 3 MAGE-A3 antigen small peptide specificity, refolding and purification
For obtaining solvable TCR molecule, α the and β chain of the TCR molecule of the present invention can only comprise it respectively
Cysteine residues are introduced respectively in variable domain and portion constant domain, and the constant domain of α and β chain
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 as Fig. 5 a and Fig. 5 b
Shown, the cysteine residues of introducing with overstriking and underline letter representation.Pass through<<Molecular Cloning: A Laboratory
Room handbook>>(Molecular Cloning a Laboratory Manual) (third edition, Sambrook and
Russell the difference after synthesis by the objective gene sequence of above-mentioned TCR α and β chain of the standard method described in)
It is inserted into expression vector pET28a+ (Novagene), the cloning site of upstream and downstream is NcoI and NotI respectively.
Insert Fragment confirms errorless through sequencing.
The expression vector of TCR α and β chain is entered expression antibacterial BL21 by chemical transformation conversion respectively
(DE3), antibacterial is grown with LB culture fluid, in OD600Induced with final concentration 0.5mM IPTG when=0.6,
The inclusion body being formed after α the and β chain expression of TCR 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 after dissolving and β chain are with 1：1 mass ratio is quickly mixed in 5M carbamide, 0.4M essence
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 into buffer (20mM Tris, pH 8.0) after 12 hours and continues at
Dialyse 12 hours for 4 DEG C.Solution after the completion of dialysis after 0.45 μM of membrane filtration, by the moon from
Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purification.Eluting peak contains renaturation success
The dimeric TCR of α and β confirmed by SDS-PAGE glue.TCR subsequently passes through gel permeation chromatography
(HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) is further purified.Purification
TCR purity afterwards measures through SDS-PAGE and is more than 90%, and 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 the specific soluble single-chain T CR of embodiment 4 MAGE-A3 antigen small peptide
According to patent documentation WO2014/206304, using rite-directed mutagenesises method by embodiment 2
TCR α and the variable domain of 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 as Fig. 7 a and
Shown in Fig. 7 b.The aminoacid sequence of its α chain variable domain and nucleotide sequence are respectively as Fig. 8 a and Fig. 8 b institute
Show；The aminoacid sequence of its β chain variable domain and nucleotide sequence are respectively as shown in figures 9 a and 9b；Its
The aminoacid sequence of linker sequence and nucleotide sequence are respectively 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.Connection product converts to E.coli DH5 α, and coating contains the LB flat board of kanamycin,
37 DEG C of inversion overnight incubation, picking positive colony enters performing PCR screening, positive recombinant is sequenced, really
Sequencing row correctly extract recombinant plasmid transformed afterwards to E.coli BL21 (DE3), for expressing.
The expression of the specific soluble single-chain T CR of embodiment 5 MAGE-A3 antigen small peptide, renaturation and purification
Will be whole for the BL21 containing recombiant plasmid pET28a- template strand (DE 3) bacterium colony of preparation in embodiment 4
It is inoculated in the LB culture medium containing kanamycin, 37 DEG C are cultivated to OD600 is 0.6-0.8, add IPTG
To final concentration of 0.5mM, 37 DEG C are continued culture 4h.5000rpm centrifugation 15min harvesting precipitation
Thing, with Bugbuster Master Mix (Merck) cell lysis precipitate, 6000rpm centrifugation 15min
Reclaim inclusion body, then washed with Bugbuster (Merck) to remove cell debriss and membrane component, 6000
Rpm is centrifuged 15min, collects inclusion body.By solubilization of inclusion bodies in buffer (20mM Tris-HCl pH 8.0,8
M carbamide) in, high speed centrifugation removes insoluble matter, and supernatant carries out subpackage with after BCA standard measure, in -80
DEG C save backup.
To in the single-stranded TCR inclusion body protein of 5mg dissolving, 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 of 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 single-stranded TCR after the above-mentioned process of Deca, 4
DEG C stirring 10min, then by renaturation solution load interception be 4kDa cellulose membrane bag filter, bag filter
It is placed in the water of 1L pre-cooling, 4 DEG C are slowly stirred overnight.After 17 hours, dialysis solution is changed into 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 of membrane filtration, vacuum
Pass through anion-exchange column (HiTrap Q HP, GE Healthcare) after degassing, use 20mM Tris-HCl
The 0-1M NaCl linear gradient elution liquid purifying protein that pH 8.0 prepares, the elution fraction of collection is carried out
SDS-PAGE analyzes, and the component comprising single-stranded TCR uses solvent resistant column (Superdex 75 after concentrating further
10/300, GE Healthcare) carry out purification, target components are also carried out SDS-PAGE analysis.
Elution fraction for BIAcore analysis tests its purity using gel filtration further.Condition is：
Chromatographic column Agilent Bio SEC-3 (300A, φ 7.8 × 300mm), mobile phase is 150mM phosphate
Buffer, flow velocity 0.5mL/min, 25 DEG C of column temperature, 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
This example demonstrated the TCR molecule of the present invention of solubility can be multiple with SYVKVLHHM-HLA A2402
Compound specifically binds.
Detect the TCR obtaining in embodiment 3 and embodiment 5 using BIAcore T200 real-time analyzer
Molecule and the binding activity of SYVKVLHHM-HLA A2402 complex.Antibody by anti-Streptavidin
(GenScript) add coupling buffer (10mM sodium-acetate buffer, pH 4.77), then by antibody
Flow through the CM5 chip being 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, coupling level is about 15,000
RU.
Make the Streptavidin of low concentration flow through the chip surface of coated antibody, then will
SYVKVLHHM-HLA A2402 complex flows through sense channel, another passage 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 SYVKVLHHM-HLA A2402 complex is as follows：
A. purification
Collect the E.coli bacterium solution of 100ml abduction delivering heavy chain or light chain, be centrifuged in 4 DEG C of 8000g
Use 10ml PBS washing thalline once after 10min, use 5ml BugBuster Master Mix afterwards
Extraction Reagents (Merck) acutely shakes resuspended thalline, and rotates incubation 20min in room temperature,
After 4 DEG C, 6000g be centrifuged 15min, supernatant discarded, collect inclusion body.
Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation incubation 5min；
Plus 30ml dilutes 10 times of BugBuster, mix, 4 DEG C of 6000g are centrifuged 15min；Supernatant discarded,
Plus 30ml dilutes 10 times of the resuspended inclusion body of BugBuster, mix, 4 DEG C of 6000g are centrifuged 15min,
It is repeated twice, plus the resuspended inclusion body of 30ml 20mM Tris-HCl pH 8.0, mix, 4 DEG C of 6000g
Centrifugation 15min, finally uses 20mM Tris-HCl 8M carbamide to dissolve inclusion body, SDS-PAGE detection bag
Contain body purity, BCA test kit surveys concentration.
B. renaturation
The small peptide SYVKVLHHM (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, to dissolve, adds 3M guanidine hydrochloride, 10mM Sodium Acetate Trihydrate, the further degeneration of 10mM EDTA before renaturation.
By SYVKVLHHM 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, are cooled to 4 DEG C), then sequentially add 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 complete at 4 DEG C
Become, can SDS-PAGE detection renaturation success.
C. purification after renaturation
Change renaturation buffer as dialysis with the 20mM Tris pH 8.0 of 10 volumes, at least change slow
Rush the ionic strength that liquid fully reduces solution twice.With 0.45 μm of 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).Using Akta purification instrument (GE General Electric Co. Limited), 20mM Tris pH 8.0
The 0-400mM NaCl linear gradient liquid wash-out protein prepared, pMHC eluting about at 250mM NaCl,
Collect all peaks component, SDS-PAGE detects purity.
D. biotinylation
With Millipore super filter tube by the pMHC molecular concentration of purification, buffer exchange is 20mM simultaneously
Tris pH 8.0, be subsequently adding biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10
MM MgOAc, 50 μM of D-Biotin, 100 μ g/ml BirA enzyme (GST-BirA), incubated at room is mixed
Overnight, whether SDS-PAGE detection biotinylation is complete for compound.
E. the complex after purifying biological elementization
With Millipore super filter tube by the pMHC molecular concentration after biotinylation labelling to 1ml, using solidifying
The biotinylated pMHC of glue filtration chromatography, using Akta purification instrument (GE General Electric Co. Limited), uses
Filtered PBS pre-equilibration HiPrepTM16/60 S200HR post (GE General Electric Co. Limited), loads 1
The concentrated biotinylation pMHC molecule of ml, then uses PBS with 1ml/min flow velocity eluting.Biotin
The pMHC molecule changed occurs as unimodal eluting in about 55ml.Merge the component containing protein, use
Millipore super filter tube concentrates, and BCA method (Thermo) measures protein concentration, adds albumen enzyme level
Biotinylated pMHC molecule subpackage is saved in -80 DEG C by agent cocktail (Roche).
Using BIAcore Evaluation computed in software kinetic parameter, obtain the TCR of solubility of the present invention
The soluble single-chain T CR molecule of molecule and present invention structure and SYVKVLHHM-HLA A2402 complex are tied
The kinetic profile closing is respectively as shown in Figure 12 and Figure 13.Collection of illustrative plates shows, the sTCR that the present invention obtains
Molecule and soluble single-chain T CR molecule can be combined with SYVKVLHHM-HLA A2402 complex.With
When, also have detected TCR molecule and other several irrelevant antigen small peptides of solubility of the present invention using said method
With the binding activity of HLA complex, result show TCR molecule of the present invention be all no combined with other irrelevant antigen.
The all documents referring in the present invention are all incorporated as reference 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,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (10)
1. a kind of φt cell receptor (TCR) is it is characterised in that described TCR can be with SYVKVLHHM-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 AGVMNSGNTPLV (SEQ
ID NO:12)；And/or the aminoacid sequence of the CDR3 of described TCR β chain variable domain is ASSADIWETQY
(SEQ ID NO:15)；
It is highly preferred that 3 complementary determining regions (CDR) of described TCR α chain variable domain are：
α CDR1-SVFSS(SEQ ID NO:10)
α CDR2-VVTGGEV(SEQ ID NO:11)
α CDR3-AGVMNSGNTPLV(SEQ ID NO:12)；And/or
3 complementary determining regions of described TCR β chain variable domain are：
β CDR1-SGDLS(SEQ ID NO:13)
β CDR2-YYNGEE(SEQ ID NO:14)
β CDR3-ASSADIWETQY(SEQ ID NO:15).
2. TCR as claimed in claim 1 is 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 and SEQ ID NO:1 has at least 90% sequence thereto
Aminoacid sequence；And/or described TCR β chain variable domain is and SEQ ID NO：5 have at least 90% sequence
The aminoacid sequence of row homogeny.
3. TCR as claimed in claim 1 is 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 to detect
Label, therapeutic agent, PK modify part or the combination of these materials any；Preferably, described therapeutic agent is
Anti-CD 3 antibodies.
4. a kind of multivalent TCR complex is it is characterised in that comprise at least two TCR molecules, and its
At least one of TCR molecule be the claims any one of TCR.
5. a kind of nucleic acid molecules it is characterised in that described nucleic acid molecules comprise to encode any of the above-described right will
Ask the described nucleotide sequence of TCR molecule or its complementary series；
Preferably, described nucleic acid molecules comprise to encode the nucleotide sequence SEQ ID NO of TCR α chain variable domain：
2 or SEQ ID NO:33；And/or
Described nucleic acid molecules comprise to encode the nucleotide sequence SEQ ID NO of TCR β chain variable domain：6 or
SEQ ID NO:35.
6. a kind of carrier is it is characterised in that described carrier contains the nucleic acid molecules described in claim 5；
Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow virus carrier.
7. a kind of detached host cell is it is characterised in that contain claim in described host cell
It is integrated with the nucleic acid molecules described in the claim 5 of external source in carrier described in 26 or chromosome.
8. a kind of cell is it is characterised in that the nucleic acid molecules described in described cell transduction claim 5 or power
Profit requires carrier described in 6；Preferably, described cell is T cell or stem cell.
9. a kind of pharmaceutical composition is it is characterised in that described compositionss contain pharmaceutically acceptable carrier
And the TCR complex described in the TCR any one of claim 1-3, claim 4, power
Profit requires the cell described in nucleic acid molecules or claim 8 described in 5.
10. the φt cell receptor any one of claim 1-3 or the TCR described in claim 4
The purposes of the cell described in complex or claim 8 it is characterised in that for preparation treatment tumor or
The medicine of autoimmune disease.
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| CN201510734101.6A CN106478807B (en) | 2015-10-30 | 2015-10-30 | Identify the T cell receptor of MAGE-A3 |
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| CN108929378A (en) * | 2017-05-22 | 2018-12-04 | 广东香雪精准医疗技术有限公司 | A kind of T cell receptor identifying PRAME antigen and the nucleic acid for encoding this receptor |
| CN109563147A (en) * | 2016-03-23 | 2019-04-02 | 英美偌科有限公司 | T cell receptor |
| CN109836490A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | It is a kind of to target the T cell receptor of MAGE-A3, T cell receptor gene modification T cell and its preparation method and application |
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| CN108929378A (en) * | 2017-05-22 | 2018-12-04 | 广东香雪精准医疗技术有限公司 | A kind of T cell receptor identifying PRAME antigen and the nucleic acid for encoding this receptor |
| CN109836490A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | It is a kind of to target the T cell receptor of MAGE-A3, T cell receptor gene modification T cell and its preparation method and application |
| CN109837248A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | A kind of T cell receptor gene modification T cell and its preparation method and application for the targeting MAGE-A3 that TCR is knocked out |
| CN110016074A (en) * | 2018-01-08 | 2019-07-16 | 中国科学院广州生物医药与健康研究院 | MAGE-A3 humanization T cell receptor |
| CN110016074B (en) * | 2018-01-08 | 2021-03-30 | 中国科学院广州生物医药与健康研究院 | MAGE-A3 humanized T cell receptor |
| 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 |
| JP2023518276A (en) * | 2020-03-20 | 2023-04-28 | エックスライフエスシー リミテッド | A high-affinity TCR that recognizes the AFP antigen |
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| CN117946246A (en) * | 2023-02-23 | 2024-04-30 | 暨南大学 | T cell receptor for recognizing MAGE-A3 antigen short peptide and application thereof |
| CN117946247A (en) * | 2023-02-23 | 2024-04-30 | 暨南大学 | T cell receptor for recognizing MAGE-A3 antigen short peptide and application thereof |
| CN117946246B (en) * | 2023-02-23 | 2024-08-06 | 暨南大学 | T cell receptor for recognizing MAGE-A3 antigen short peptide and application thereof |
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