CN106632658A - TCR (T Cell Receptor) for identifying NY-ESO (New York-Esophageal Squamous Cell Carcinomas)-1 antigen short peptide - Google Patents
TCR (T Cell Receptor) for identifying NY-ESO (New York-Esophageal Squamous Cell Carcinomas)-1 antigen short peptide Download PDFInfo
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Abstract
The invention provides a TCR (T Cell Receptor) which is capable of specifically combining a short peptide SLLMWITQC derived from an NY-ESO (New York-Esophageal Squamous Cell Carcinomas)-1 antigen. The antigen short peptide SLLMWITQC and HLA A0201 can form a compound and can be delivered to the cell surface together. The invention also provides nucleic acid molecules for encoding the TCR and a carrier containing the nucleic acid molecules. In addition, the invention also provides cells for transducing the TCR.
Description
Technical field
The present invention relates to the TCR from NY-ESO-1 antigen small peptides is capable of identify that, the invention further relates on transduceing
The T cell of the NY-ESO-1 specificitys for stating TCR to obtain, and they are in prevention and treatment NY-ESO-1 phases
Purposes in related disorders.
Background technology
NY-ESO-1 belongs to tumor-testis antigen (Cancer-Testis Antigen, CTA) family, energy
Express in the tumor tissues of testis, ovary tissue and number of different types, and in other normal structures
Do not express, be the stronger tumor antigen of a species specificity.NY-ESO-1 is a kind of endogenous antigen, in cell
Micromolecule polypeptide is degraded to after interior generation, and shape is combined with MHC (main histocompatibility complex) molecule
Into complex, cell surface is presented to.SLLMWITQC is the small peptide derived from NY-ESO-1 antigens, is
A kind of target of NY-ESO-1 treating correlative diseases.Research shows that NY-ESO-1 is equal in kinds of tumors tissue
Have expression, neuroblastoma (Rodolfo M, et al., Cancer Res,
2003,63(20):6948-6955), sarcoma (Jungbhth A A et al., Int J Cancer, 200l,
94(2):252-256), malignant melanoma (Barrow C, et al., Clin Cancer Res, 2006,
12(3Pt 1):Have very high expression in 764-771), at the same carcinoma of prostate, bladder cancer, breast carcinoma,
Multiple myeloma, hepatocarcinoma, oral squamous cell carcinomas (Ries J, et al., Anticancer RES, 2009,
29(12):5125-5130) and the esophageal carcinoma (Fujita S, Clin Cancer Res, 2004,
10(19):Also there is higher expression in 6551-6558).For the treatment of above-mentioned disease, chemotherapy can be adopted
With the method such as radiation treatment, but all the normal cell of itself can be caused damage.
T cell adoptive immunotherapy is that the reaction-ive T cell to target cell antigen with specificity is proceeded to into disease
In human body so as to play a role for target cell.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.It is short by antigen in immune system
The combination of the TCR of peptide specific and small peptide-main histocompatibility complex (pMHC complex) causes T thin
The direct physical contact of born of the same parents and antigen-presenting cell (APC), then other cells of both T cell and 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 specificities plays immunological effect to its target cell.Therefore, ability
Field technique personnel are devoted to isolating the TCR for having specificity to NY-ESO-1 antigen small peptides, and should
TCR transduces T cell to obtain the T cell for having specificity to NY-ESO-1 antigen small peptides, so that they
Play a role in cellular immunotherapy.
The content of the invention
It is an object of the invention to provide a kind of φt cell receptor of identification NY-ESO-1 antigen small peptides.
A kind of a first aspect of the present invention, there is provided φt cell receptor (TCR), the TCR can be with
SLLMWITQC-HLA A0201 complex is combined.
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, described
The aminoacid sequence of the CDR3 of TCR α chain variable domains is AGFMDSNYQLI (SEQ ID NO:12)；With/
Or the aminoacid sequence of the CDR3 of the TCR β chain variable domains is ASSLGGSVLH (SEQ ID NO:15).
In another preference, 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
αCDR1-SIFNT(SEQ ID NO:10)
αCDR2-LYKAGEL(SEQ ID NO:11)
αCDR3-AGFMDSNYQLI(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
βCDR1-SGHVS(SEQ ID NO:13)
βCDR2-FQNEAQ(SEQ ID NO:14)
βCDR3-ASSLGGSVLH(SEQ ID NO:15)。
In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, described
TCR α chain variable domains are and SEQ ID NO:1 aminoacid sequence with least 90% sequence thereto；With/
Or the TCR β chain variable domains are and SEQ ID NO：The 5 aminoacid sequences with least 90% sequence thereto
Row.
In another preference, the TCR includes α chain variable domain amino acid sequence SEQ ID NO:1.
In another preference, the TCR includes β chain variable domain amino acid sequence SEQ ID NO:5.
In another preference, the TCR is α β heterodimers, and it includes TCR α chains constant region
TRAC*01 and TCR β chains constant region TRBC1*01 or TRBC2*01.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO：3 and/or described
The β chain amino acid sequences of TCR are SEQ ID NO:7.
In another preference, the TCR is solvable.
In another preference, the TCR is single-stranded.
In another preference, the TCR is to pass through peptide catenation sequence by α chains variable domain and β chains variable domain
It is formed by connecting.
In another preference, the TCR α chains variable region amino acid the 11st, 13,19,21,53,
76th, 89,91 or the 94th, and/or α chain J gene small peptides aminoacid inverse the 3rd, inverse the 5th
With one or more mutation in position or inverse the 7th；And/or the TCR is in β chain variable region amino acids
11st, 13,19,21,53,76,89,91 or the 94th, and/or β chain J gene small peptide amino
With one or more mutation, wherein aminoacid in sour 4th or reciprocal 6th reciprocal 2nd, reciprocal
Position Number is by the Position Number listed in IMGT (international immunogeneticses information system).
In another preference, the α chains variable domain amino acid sequence of the TCR includes SEQ ID NO:32
And/or the β chains variable domain amino acid sequence of the TCR includes SEQ ID NO:34.
In another preference, the aminoacid sequence of the TCR is SEQ ID NO:30.
In another preference, the TCR includes all or part of TCR α of (a) in addition to membrane spaning domain
Chain；And all or part of TCR β chains of (b) in addition to membrane spaning domain；
And (a) with (b) each self-contained functional variable domain, or comprising functional variable domain and institute
State at least a portion of TCR chain constant domains.
In another preference, cysteine residues form people between α the and β chain constant domains of the TCR
Work disulfide bond.
In another preference, the cysteine residues that artificial disulfide bond is formed in the TCR instead of choosing
From following one or more groups of sites：
The Ser57 of the Thr48 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser77 of the Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser17 of the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Asp59 of the Thr45 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Glu15 of the Ser15 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ser54 of the Arg53 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
The Ala19 of the Pro89 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s；
With
The Glu20 of the Tyr10 and TRBC1*01 or TRBC2*01 exons 1 of TRAC*01 exons 1s.
In another preference, the α chain amino acid sequences of the TCR are SEQ ID NO:26 and/or described
The β chain amino acid sequences of TCR are SEQ ID NO:28.
In another preference, artificial interchain is contained between the α chains variable region and β chains constant region of the TCR
Disulfide bond.
In another preference, it is characterised in that half Guang of artificial interchain disulfide bond is formed in the TCR
Histidine residue instead of selected from following one or more groups of sites：
46th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；
47th amino acids and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；
46th amino acids and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV；
Or
47th amino acids and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s of TRAV.
In another preference, the TCR is comprising α chains variable domain and β chains variable domain and except transmembrane structure
All or part of β chains constant domain beyond domain, but it does not contain α chain constant domains, the α chains of the TCR can
Variable domain and β chain formation heterodimers.
In another preference, the α chains of the TCR and/or C- the or N- ends of β chains are combined with conjugate.
In another preference, the conjugate combined with the φt cell receptor is detectable, treatment
The combination of agent, PK modifications part or any these materials.Preferably, the therapeutic agent is anti-CD 3 antibodies.
A kind of a second aspect of the present invention, there is provided multivalent TCR complex, it includes at least two TCR points
Son, and at least one TCR molecules therein are the TCR described in first aspect present invention.
A kind of a third aspect of the present invention, there is provided nucleic acid molecules, the nucleic acid molecules include the coding present invention
The nucleotide sequence or its complementary series of the TCR molecules described in first aspect.
In another preference, nucleotide sequence SEQ of the nucleic acid molecules comprising coding TCR α chain variable domains
ID NO：2 or SEQ ID NO:33.
In another preference, described nucleotide sequence of the nucleic acid molecules comprising coding TCR β chain variable domains
SEQ ID NO：6 or SEQ ID NO:35.
In another preference, nucleotide sequence SEQ ID NO of the nucleic acid molecules comprising coding TCR α chains：
4 and/or the nucleotide sequence SEQ ID NO comprising coding TCR β chains：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 or genome described in fourth aspect present invention described in the third aspect present invention of external source
Nucleic acid molecules.
A sixth aspect of the present invention, there is provided a kind of cell, described in the cell transduction third aspect present invention
Nucleic acid molecules or fourth aspect present invention described in carrier；Preferably, the 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, the compositionss contain pharmaceutically can be connect
The TCR complex described in TCR, second aspect present invention described in the carrier received and first aspect present invention,
The carrier described in nucleic acid molecules, fourth aspect present invention or the present invention the 6th described in third aspect present invention
Cell described in aspect.
A eighth aspect of the present invention, there is provided the φt cell receptor or the present invention described in first aspect present invention
Nucleic acid molecules described in TCR complex, third aspect present invention described in second aspect, the present invention the is cubic
The purposes of the cell described in carrier or sixth aspect present invention described in face, for preparation treatment tumor or certainly
The medicine of body immunological diseases.
A ninth aspect of the present invention, there is provided a kind of method for treating disease, including to object in need for the treatment of
Apply the φt cell receptor or the TCR described in second aspect present invention described in appropriate first aspect present invention
The carrier described in nucleic acid molecules, fourth aspect present invention or sheet described in complex, third aspect present invention
Invent the cell or the pharmaceutical composition described in seventh aspect present invention described in the 6th aspect；
Preferably, described disease be tumor, preferably described tumor include neuroblastoma, sarcoma,
Melanoma, carcinoma of prostate, bladder cancer, breast carcinoma, multiple myeloma, hepatocarcinoma, oral squamous cell carcinomas,
The esophageal carcinoma and gastric cancer, pulmonary carcinoma, squamous cell carcinoma of the head and neck, 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 between each technical characteristic for specifically describing, so as to constitute new or preferred skill
Art scheme.As space is limited, here is no longer tired out one by one and is stated.
Description of the drawings
Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e and Fig. 1 f are respectively TCR α chain variable domain amino acids
Sequence, TCR α chain variable domain nucleotide sequences, TCR α chain amino acid sequences, TCR α chain nucleotide sequences,
TCR α chain amino acid sequences with targeting sequencing and the TCR α chain nucleotide sequences with targeting sequencing.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f are respectively TCR β chain variable domain amino acids
Sequence, TCR β chain variable domain nucleotide sequences, TCR β chain amino acid sequences, TCR β chain nucleotide sequences,
TCR β chain amino acid sequences with targeting sequencing and the TCR β chain nucleotide sequences with targeting sequencing.
Fig. 3 is the CD8 of monoclonal cell+And the double positive staining results of the tetramer-PE.
Fig. 4 a and Fig. 4 b are respectively the aminoacid sequence and nucleotide sequence of sTCR α chains.
Fig. 5 a and Fig. 5 b are respectively the aminoacid sequence and nucleotide sequence of sTCR β chains.
Fig. 6 is the glue figure of the sTCR for 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 are respectively the aminoacid sequence and nucleotide sequence of single-stranded TCR.
Fig. 8 a and Fig. 8 b are respectively the aminoacid sequence and nucleotide sequence of single-stranded TCR α chains.
Fig. 9 a and Fig. 9 b are respectively the aminoacid sequence and nucleotide sequence of single-stranded TCR β chains.
Figure 10 a and Figure 10 b are respectively the aminoacid sequence and nucleoside of single-stranded TCR catenation sequences (linker)
Acid sequence.
Figure 11 is the glue figure of the soluble single-chain T CR for obtaining after purification.Left side swimming lane is molecular weight marker
(marker), right lanes are non-reduced glue.
Figure 12 is the BIAcore that sTCR of the present invention is combined with SLLMWITQC-HLA A0201 complex
Kinetic profile.
Figure 13 is that soluble single-chain T CR of the present invention is combined with SLLMWITQC--HLA A0201 complex
BIAcore kinetic profiles.
Specific embodiment
The present inventor have found and NY-ESO-1 antigen small peptide SLLMWITQC through extensively in-depth study
(SEQ ID NO:9) TCR that can be specifically bound, the antigen small peptide SLLMWITQC can be with HLA A0201
Form complex and be presented to cell surface together.Present invention also offers encoding the nucleic acid point of the TCR
Son and the carrier comprising the nucleic acid molecules.In addition, present invention also offers transduction TCR's of the present invention is thin
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
Different small peptides is to respective APC cell surfaces in Bai Kangyuan.The MHC of the mankind be commonly referred to HLA genes or
HLA complexs.
φt cell receptor (TCR), is the specific antigen peptide presented in main histocompatibility complex (MHC)
Unique receptor.In immune system, drawn by the combination of the TCR and pMHC complex of antigenic specificity
Send out T cell and antigen-presenting cell (APC) directly physical contact, then both T cell and APC its
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 specificities plays immunological effect to its target cell.
TCR is the surface of cell membrane existed in heterodimer form by α chains/β chains or γ chains/δ chains
Glycoprotein.TCR heterodimers are made up of α and β chains in 95% T cell, and 5% T cell has
By the TCR being made up of γ and δ chains.Heterogeneous dimerization TCR of natural α β has α chains and β chains, α chains and β
Chain constitutes the subunit of α β heterodimeric TCR.In a broad sense, each chains of α and β include variable region, connection
Area and constant region, β chains generally contain short variable region, but the variable region also between variable region and bonding pad
Often it is regarded as a part for bonding pad.Each variable region includes and is entrenched in frame structure (framework regions)
In 3 CDR (complementary determining region), CDR1, CDR2 and CDR3.CDR region determines TCR and pMHC
The combination of complex, wherein CDR3 are formed by variable region and bonding pad restructuring, are referred to as hypervariable region.TCR's
α and β chains are typically regarded as respectively two " domains " i.e. variable domain and constant domain, and variable domain can by what is connected
Become area and bonding pad to constitute.The sequence of TCR constant domains can be in international immunogeneticses information system (IMGT)
Public database in find, the such as constant domain sequence of TCR molecule alpha chains is " TRAC*01 ", TCR molecules
The constant domain sequence of β chains is " TRBC1*01 " or " TRBC2*01 ".Additionally, α the and β chains of TCR are also
Comprising transmembrane region and cytoplasmic region, cytoplasmic region is very short.
In the present invention, term " polypeptide of the present invention ", the 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 with C β interchains in the membrane-proximal region C α of natural TCR, be referred to as in the present invention " my god
Right 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 for 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, such as TRBC1*01 by the order from N-terminal to C-terminal successively
Or be P (proline) by the 60th aminoacid of the order from N-terminal to C-terminal successively in TRBC2*01,
The Pro60 of TRBC1*01 or TRBC2*01 exons 1s 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 1s, and for example TRBC1*01 or
It is Q (glutamine) by the 61st aminoacid of the order from N-terminal to C-terminal successively in TRBC2*01,
The Gln61 of TRBC1*01 or TRBC2*01 exons 1s then can be described it as in the present invention, also can be by it
The 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s are expressed as, other are by that analogy.This
In invention, the Position Number of the aminoacid sequence of variable region TRAV and TRBV, according to what is listed in IMGT
Position Number.Such as certain aminoacid in TRAV, the Position Number listed in IMGT is 46, then the present invention
In describe it as the amino acids of TRAV the 46th, other are by that analogy.In the present invention, other aminoacid
Sequence position numbers have specified otherwise, then by specified otherwise.
Detailed description of the invention
TCR molecules
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 it is a kind of can be with reference to the TCR molecules of SLLMWITQC-HLA A0201 complex.It is excellent
Selection of land, the TCR molecules are detached or purification.Respectively there are α the and β chains of the TCR 3 complementations to determine
Determine area (CDR).
One in the present invention is preferably carried out in mode, and the α chains of the TCR are comprising with following aminoacid
The CDR of sequence：
αCDR1-SIFNT(SEQ ID NO:10)
αCDR2-LYKAGEL(SEQ ID NO:11)
αCDR3-AGFMDSNYQLI(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
βCDR1-SGHVS(SEQ ID NO:13)
βCDR2-FQNEAQ(SEQ ID NO:14)
βCDR3-ASSLGGSVLH(SEQ ID NO:15)。
The CDR region aminoacid sequence of the invention described above can be embedded in any suitable frame structure and be made
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
The TCR molecules with corresponding function can be just designed or synthesized according to CDR region disclosed by the invention.Therefore,
TCR molecules of the present invention are referred to comprising above-mentioned α and/or β chains CDR region sequence and any suitable frame structure
TCR molecules.TCR α chain variable domains of the present invention are and SEQ ID NO:1 has at least 90%, preferably
95%, the more preferably aminoacid sequence of 98% sequence thereto；And/or TCR β chain variable domains of the present invention are
With SEQ ID NO：5 have at least 90%, preferably 95%, the more preferably amino of 98% sequence thereto
Acid sequence.
In a preference of the present invention, the TCR molecules of the present invention are be made up of α and β chains heterogeneous two
Aggressiveness.Specifically, on the one hand the α chains of the heterogeneous dimerization TCR molecule include variable domain and constant domain, institute
State CDR1 (SEQ ID NO of the α chains variable domain amino acid sequence comprising above-mentioned α chains：10)、CDR2(SEQ
ID NO：11) with CDR3 (SEQ ID NO:12).Preferably, the TCR molecules are variable comprising α chains
Domain amino acid sequence SEQ ID NO:1.It is highly preferred that the α chain variable domain amino acid sequences of the TCR molecules
It is classified as SEQ ID NO:1.On the other hand, the β chains of the heterogeneous dimerization TCR molecule include variable domain and perseverance
Localization, CDR1 (SEQ ID NO of the β chains variable domain amino acid sequence comprising above-mentioned β chains:13)、
CDR2(SEQ ID NO：14) with CDR3 (SEQ ID NO:15).Preferably, the TCR molecules bag
The variable domain amino acid sequence SEQ ID NO of chain containing β:5.It is highly preferred that the β chains of the TCR molecules are variable
Domain amino acid sequence is SEQ ID NO:5.
The present invention a preference in, the present invention TCR molecules be by the part or all of of α chains and/
Or the single chain TCR molecules of the part or all of composition of β chains.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 single-stranded comprising CDRs areas of the present invention
TCR molecules.Specifically, the single chain TCR molecules include V α, V β and C β, preferably according to from N
Hold being linked in sequence for C-terminal.
CDR1 (SEQ ID of the α chains variable domain amino acid sequence of the single chain TCR molecules comprising above-mentioned α chains
NO：10)、CDR2(SEQ ID NO：11) with CDR3 (SEQ ID NO:12).Preferably, it is described
Single chain TCR molecules include α chain variable domain amino acid sequence SEQ ID NO:1.It is highly preferred that described single-stranded
The α chains variable domain amino acid sequence of TCR molecules is SEQ ID NO:1.The β chains of the single chain TCR molecules
CDR1 (SEQ ID NO of the variable domain amino acid sequence comprising above-mentioned β chains：13)、CDR2(SEQ ID NO：
14) with CDR3 (SEQ ID NO：15).Preferably, the single chain TCR molecules include β chain variable domains
Aminoacid sequence SEQ ID NO:5.It is highly preferred that the β chain variable domain amino acids of the single chain TCR molecules
Sequence is SEQ ID NO:5.
In a preference of the present invention, the constant domain of the TCR molecules of the present invention is the constant domain of people.This
Art personnel know or can be by consulting pertinent texts or IMGT (international immunogeneticses information systems
System) public database obtaining the constant domain amino acid sequence of people.For example, TCR molecule alphas chain of the present invention
Constant domain sequence can be " TRAC*01 ", the constant domain sequence of TCR molecule β chains can be " TRBC1*01 "
Or " TRBC2*01 ".The 53rd of the aminoacid sequence be given in the TRAC*01 of IMGT is Arg,
This is expressed as：The Arg53 of TRAC*01 exons 1s, 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 chains is 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
Obtain the TCR molecules of solubility.The TCR molecules of solubility do 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 (such as cytotoxin compounds or immunostimulating compound) is transported to and presents the thin of specific antigen
Born of the same parents, in addition, sTCR can also 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 to NY-ESO-1 antigens
Small peptide has the sTCR of specificity.
To obtain sTCR, on the one hand, TCR of the present invention can be in the residual of 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 domains of the TCR
Form artificial interchain disulfide bond.Cysteine residues can be substituted in other ammonia of appropriate site in natural TCR
Base acid residue is forming artificial interchain disulfide bond.For example, replace the Thr48 of TRAC*01 exons 1s and take
For TRBC1*01 or TRBC2*01 exons 1s Ser57 cysteine residues forming disulfide bond.Draw
Other sites for entering cysteine residues to form disulfide bond can also be：The Thr45 of TRAC*01 exons 1s
With the Ser77 of TRBC1*01 or TRBC2*01 exons 1s；The Tyr10 of TRAC*01 exons 1s and
The Ser17 of TRBC1*01 or TRBC2*01 exons 1s；The Thr45 and TRBC1*01 of TRAC*01 exons 1s
Or the Asp59 of TRBC2*01 exons 1s；The Ser15 and TRBC1*01 of TRAC*01 exons 1s or
The Glu15 of TRBC2*01 exons 1s；The Arg53 and TRBC1*01 of TRAC*01 exons 1s or TRBC2*01
The Ser54 of exons 1；Show outside the Pro89 and TRBC1*01 of TRAC*01 exons 1s or TRBC2*01
The Ala19 of son 1；Or the Tyr10 and TRBC1*01 or TRBC2*01 exon of TRAC*01 exons 1s
1 Glu20.I.e. cysteine residues instead of above-mentioned α with arbitrary group of site in β chain constant domains.Can be
One or more C-terminal truncates of TCR constant domains of the present invention are most 50 or most 30 or most
The aminoacid of 15 or most 10 or most 8 or less, so that it does not include that cysteine is residual
Base lacks the purpose of natural disulphide bonds to reach, also can be by the cysteine residues by natural disulphide bonds are formed
Sport another aminoacid to reach above-mentioned purpose.
As described above, the TCR of the present invention may be embodied in the people introduced between the residue of itself α and β chain constant domain
Work disulfide bond.It should be noted that with or without the artificial disulfide bond of introducing mentioned above, the present invention between constant domain
TCR can be containing TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC of TCR
Constant domain sequence and TRBC1 or TRBC2 constant domains sequence can by being present in TCR in natural disulphide bonds connect
Connect.
To obtain sTCR, on the other hand, TCR of the present invention is additionally included in its hydrophobic core region to be occurred to dash forward
The TCR of change, the mutation in these hydrophobic core regions is preferably capable putting forward the stability of sTCR of the present invention
High mutation, as described in the patent documentation in Publication No. WO2014/206304.Such TCR can be
Undergo mutation the hydrophobic core position of its following variable domain：(α and/or β chains) variable region amino acid the 11st, 13,
19,21,53,76,89,91,94, and/or α chain J genes (TRAJ) small peptide amino acid positions
It is reciprocal 3rd, 5,7, and/or β chain J genes (TRBJ) small peptides amino acid position inverse the 2nd, 4,6,
Wherein the Position Number of aminoacid sequence is by the position listed in international immunogeneticses information system (IMGT)
Numbering.Those skilled in the art know above-mentioned international immunogeneticses information system, and can be according to the data base
Obtain Position Number of the amino acid residue of different TCR in IMGT.
The TCR that hydrophobic core region is undergone mutation in the present invention can be by a flexible peptide chain connection TCR α with
The variable domain of β chains and the solvable single-stranded TCR of stability that constitutes.It should be noted that flexible peptide chain can be with the present invention
It is the peptide chain of any suitable connection TCR α and β chain variable domains.The list built such as in the embodiment of the present invention 4
Chain sTCR, its α chains variable domain amino acid sequence is SEQ ID NO:32, the nucleotide sequence of coding
For SEQ ID NO:33；β chains 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
Artificial interchain disulfide bond is introduced between chain variable region and β chains constant region can be such that the stability of TCR significantly carries
It is high.Therefore, can also contain between the α chains variable region and β chains constant region of high-affinity TCR of the invention
Artificial interchain disulfide bond.Specifically, people is formed between the α chains variable region of the TCR and β chains 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 1s；47th amino acids of TRAV and TRBC1*01 or
61 amino acids of TRBC2*01 exons 1s；46th amino acids of TRAV and TRBC1*01 or
61st amino acids of TRBC2*01 exons 1s；Or TRAV the 47th amino acids and TRBC1*01 or
60th amino acids of TRBC2*01 exons 1s.Preferably, such TCR can be removed comprising (I)
All or part of TCR α chains beyond its membrane spaning domain, and (II) complete in addition to its membrane spaning domain
Portion or part TCR β chains, wherein (I) and (II) the variable domain comprising TCR chains and at least a portion
Constant domain, α chains and β chain formation heterodimers.It is highly preferred that such TCR can comprising α chains
Variable domain and β chains variable domain and all or part of β chains constant domain in addition to membrane spaning domain, but it does not wrap
The constant domain of chain containing α, α chains variable domain and the β chain formation heterodimers of the TCR.
The present invention TCR can also multivalence complex form provide.The multivalent TCR complex bag of the present invention
Containing the polymer that two, three, four or more TCR of the present invention combine and formed, p53 can be such as used
Four dimerization domains producing the tetramer, or multiple TCR of the present invention and another molecule combined and formed it is 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
With covalent manner combination.The conjugate includes that detectable (is diagnostic purpose, wherein the TCR
For detection present SLLMWITQC-HLA A0201 complex cell presence), therapeutic agent, PK (eggs
White kinases) modify the combination combination of part or any the above material or be coupled.
Detectable for diagnostic purposes is included but is not limited to：Fluorescence or luminous marker, radioactivity
Label, MRI (nuclear magnetic resonance) or CT (CT technology) contrast agent,
Or can produce the enzyme of detectable product.
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, cancerometastasis comment (Cancer metastasi s reviews) 24,539)；2. give birth to
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 Society) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene
Therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,
11631)；9. magnetic nanosphere；10. pro-drug activation enzymes (for example, DT- diaphorases (DTD) or 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 more than a kind of heterozygosis TCR of species sequence.
For example, research shows that Muridae TCR can be expressed more effectively in human T-cell than people TCR.Therefore,
TCR of the present invention can include the constant domain of people's variable domain and Mus.The defect of this method is possible to cause immunity to answer
Answer.Therefore, there should be regulation scheme 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 herein amino acid name 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
A second aspect of the present invention provides the nucleic acid point of coding first aspect present invention TCR molecule or part thereof
Son, the part can be one or more CDR, the variable domain of α and/or β chains, and α chains and/or
β chains.
The nucleotide sequence of coding first aspect present invention TCR molecule alpha chain CDR region is as follows：
αCDR1-agcatatttaacacc(SEQ ID NO:16)
αCDR2-ttatataaggctggtgaattg(SEQ ID NO:17)
αCDR3-gctggcttcatggatagcaactatcagttaatc(SEQ ID NO:18)
The nucleotide sequence of coding first aspect present invention TCR molecule β chain CDR regions is as follows：
βCDR1-tcgggtcatgtatcc(SEQ ID NO:19)
βCDR2-ttccagaatgaagctcaa(SEQ ID NO:20)
βCDR3-gccagcagcttagggggctctgtgctccac(SEQ ID NO:21)
Therefore, encoding the nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chains of the present invention includes SEQ ID NO:
16、SEQ ID NO:17 and SEQ ID NO:18, and/or encode the core of the present invention of TCR β chains 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 the nucleic acid molecules can be
RNA or DNA, and can include or not comprising intron.Preferably, the nucleoside of nucleic acid molecules of the present invention
Acid sequence is not comprising intron but can encode polypeptide of the present invention, for example, encode TCR α chain variable domains of the present invention
Nucleic acid molecules of the present invention nucleotide sequence include SEQ ID NO:2 and/or encode TCR β chains 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, the coding present invention
The nucleotide sequence of the nucleic acid molecules of the present invention of TCR α chain variable domains 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 domains 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 includes SEQ ID NO:4 and/or SEQ ID NO:
8.Or, the nucleotides sequence of nucleic acid molecules of the present invention is classified as SEQ ID NO:31.
It should be understood that due to the degeneracy of genetic code, different nucleotide sequences can encode identical polypeptide.
Therefore, encode TCR of the present invention nucleotide sequence can it is identical with the nucleotide sequence shown in accompanying drawing of the present invention or
It is the variant of degeneracy.Illustrating, " variant of degeneracy " is referred to one of example in the present invention
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 the optimization of end count numeral.Different cells is in the utilization of concrete codon
Different, can change the codon in sequence to increase expression according to the type of cell.Mammal
Cell and various other biological codon usage tables be well known to a person skilled in the art.
The present invention nucleic acid molecules full length sequence or its fragment generally can with but be not limited to PCR TRAP, weight
The method of group method or synthetic is obtained.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 the DNA sequence can be introduced ability
In domain in known various existing DNA moleculars (or such as carrier) and cell.DNA can be coding strand or non-
Coding strand.
Carrier
The invention further relates to the carrier of the nucleic acid molecules of the present invention is included, including expression vector, i.e., can be in body
Interior or vivoexpression construct.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, carrier can be transferred to the nucleotide of the present invention in cell, such as in T cell so that
The cell expresses the TCR of NY-ESO-1 antigenic specificities.Ideally, the carrier should be in T
Continual high levels ground expression in cell.
Cell
The invention further relates to the host cell produced with the carrier or coded sequence Jing genetic engineerings of the present invention.Institute
State the nucleic acid molecules that the present invention is integrated with carrier or chromosome containing the present invention in host cell.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.Such as, the cell can be isolated from periphery
Blood monocyte (PBMC), can be CD4+Helper T cell or CD8+Cytotoxic T cell.The cell
Can be in CD4+Helper T cell/CD8+In the mixing group of cytotoxic T cell.Usually, the cell can be used
Antibody (e.g., the antibody of anti-CD3 or anti-CD28) is activated, to allow them to easily receive transfection,
For example transfected with the carrier of the nucleotide sequence comprising coding TCR molecules of the present invention.
Alternatively, cell of the 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 expression of CD3 molecules will start the TCR molecules in the introducing of the surface expression of thymocyte cell.
There are many methods to be suitable for carrying out T cell transfection (e.g., with the DNA or RNA that encode TCR of the present invention
Robbins etc., (2008) J.Immunol.180:6116-6131).The T of expression 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 many conjunctions for carrying out adoptive treatment
Suitable method (e.g., Rosenberg etc., (2008) Nat Rev Cancer8 (4)：299-308).
NY-ESO-1 antigen-related diseases
The invention further relates to the method with NY-ESO-1 relevant diseases is treated and/or prevented in experimenter, its
The step of including adoptive transfer NY-ESO-1 specific T-cells to the experimenter.The NY-ESO-1 is special
Property the recognizable SLLMWITQC-HLA A0201 complex of T cell.
The T cell of the NY-ESO-1 specificitys of the present invention can be used to treating any presentation NY-ESO-1 antigens short
The NY-ESO-1 relevant diseases of peptide SLLMWITQC-HLA A0201 complex.Including but not limited to tumor,
Preferably described tumor includes neuroblastoma, sarcoma, melanoma, carcinoma of prostate, bladder cancer, breast
Adenocarcinoma, multiple myeloma, hepatocarcinoma, oral squamous cell carcinomas, the esophageal carcinoma and gastric cancer, pulmonary carcinoma, incidence
Squamous cell carcinoma, colon cancer, ovarian cancer etc..
Therapeutic Method
Can pass through to separate with the patient with NY-ESO-1 antigen-related diseases or the T cell of volunteer,
And the TCR of the present invention is imported in above-mentioned T cell, subsequently the cell of these genetic engineering modifications is fed back to
In patient body being treated.Therefore, the invention provides a kind of side for treating NY-ESO-1 relevant diseases
Method, including by the T cell of detached expression TCR of the present invention, it is preferable that the T cell is from patient's sheet
Body, is input in patient body.Usually, the T cell of patient is separated including (1), (2) use the present invention
Nucleic acid molecules can encode the nucleic acid molecules ex vivo transduction T cell of TCR molecules of the present invention, and (3) are by base
Because the T cell of engineering modification is input in patient body.Separate, the quantity of transfection and the cell for feeding back can be by
Doctor determines.
Main advantages of the present invention are：
(1) TCR of the invention can be with NY-ESO-1 antigen small peptide complex SLLMWITQC-HLA A0201
With reference to while the cell of the TCR of the present invention that transduceed can be by specific activation and to target cell with 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 houses)
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 can obtain from commercially available channel if no special instructions.
Embodiment 1 clones NY-ESO-1 antigen small peptide specific T-cells
Using synthesis small peptide SLLMWITQC (SEQ ID NO.:9；Beijing SBS Genetech gene technology company limited)
Stimulation comes from the peripheral blood lymphocyte (PBL) of the healthy volunteer that genotype is HLA-A0201.Will
SLLMWITQC small peptides and the HLA-A0201 renaturation with biotin labeling, prepare pHLA monoploid.These
Monoploid be combined into the tetramer of PE labellings with the Streptavidin (BD companies) of PE labellings, sort this four
Aggressiveness and anti-CD8-APC double positive cells.The cell of amplification sorting, and secondary sorting is carried out as stated above,
Subsequently carry out monoclonal with limiting dilution assay.Monoclonal cell tetramer staining, double positive gram for screening
It is grand as shown in Figure 3.
Embodiment 2 obtains the tcr gene and carrier of NY-ESO-1 antigen small peptide specific T-cell clones
Build
Use Quick-RNATMWhat is screened in MiniPrep (ZYMO research) extracting embodiments 1 is anti-
The total serum IgE of former small peptide SLLMWITQC specificitys, HLA-A0201 restricted T cell clone.CDNA's
Synthesis adopts the SMART RACE cDNA amplification kits of clontech, and the primer of employing is designed in people
The C-terminal conserved region of class tcr gene.Sequence is cloned in carrier T (TAKARA) and is sequenced.Should note
Meaning, the sequence is complementary seriess, not comprising intron.Jing is sequenced, the TCR's of this pair of positive colony expression
α chains and β chain-orderings 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
TCR α chain variable domain amino acid sequences, TCR α chain variable domain nucleotide sequences, TCR are respectively with Fig. 1 f
α chain amino acid sequences, TCR α chain nucleotide sequences, the TCR α chain amino acid sequences with targeting sequencing with
And the TCR α chain nucleotide sequences with targeting sequencing；Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and
Fig. 2 f are respectively TCR β chain variable domain amino acid sequences, TCR β chain variable domain nucleotide sequences, TCR β chains
Aminoacid sequence, TCR β chain nucleotide sequences, the TCR β chain amino acid sequences with targeting sequencing and tool
There are the TCR β chain nucleotide sequences of targeting sequencing.
Identified, α chains include the CDR with following aminoacid sequence：
αCDR1-SIFNT(SEQ ID NO:10)
αCDR2-LYKAGEL(SEQ ID NO:11)
αCDR3-AGFMDSNYQLI(SEQ ID NO:12)
β chains include the CDR with following aminoacid sequence：
βCDR1-SGHVS(SEQ ID NO:13)
βCDR2-FQNEAQ(SEQ ID NO:14)
βCDR3-ASSLGGSVLH(SEQ ID NO:15)
Respectively the full-length gene of TCR α chains and β chains is cloned into into slow viruss by overlapping (overlap) PCR
Expression vector pLenti (addgene).Specially：With overlap PCR by TCR α chains and TCR β chains
Full-length gene is attached and obtains TCR α -2A-TCR β fragments.By Lentiviral and TCR α
The connection of -2A-TCR β enzyme action obtains pLenti-TRA-2A-TRB-IRES-NGFR plasmids.Use as control, together
When also construction expression eGFP slow virus carrier pLenti-eGFP.With 293T/17 pack pseudoviruss again afterwards.
The expression of the solvable TCR of embodiment 3NY-ESO-1 antigen small peptide specificity, refolding and purification
To obtain solvable TCR molecules, α the and β chains of the TCR molecules of the present invention can only include respectively it
Introduce a cysteine residues in variable domain and portion constant domain, and the constant domain of α and β chains respectively
To form artificial interchain disulfide bond, the position for introducing cysteine residues is respectively TRAC*01 exons 1s
The Ser57 of Thr48 and TRBC2*01 exons 1s；The aminoacid sequence of its α chain is distinguished with nucleotide sequence
As shown in figures 4 a and 4b, the aminoacid sequence of its β chain and nucleotide sequence are respectively such as Fig. 5 a and Fig. 5 b
It is shown, introducing cysteine residues are 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 standard method described in) is by the objective gene sequence ECDC of above-mentioned TCR α and β chains into rear difference
Expression vector pET28a+ (Novagene) is inserted into, the cloning site of upstream and downstream is respectively NcoI and NotI.
Insert Fragment confirms errorless through sequencing.
The expression vector of TCR α and β chains is entered into 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 formed after α the and β chains expression of TCR is extracted by BugBuster Mix (Novagene),
And the repeated multiple times washing of Jing BugBuster solution, inclusion body is finally dissolved in 6M guanidine hydrochlorides, 10mM
Dithiothreitol, DTT (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), in 20mM Tris (pH 8.1).
TCR α and β chains after dissolving are with 1：1 mass ratio is quickly mixed in 5M carbamide, and 0.4M is smart
Propylhomoserin, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM β-mercapoethylamine
In (4 DEG C), final concentration of 60mg/mL.Solution is placed in the deionized water of 10 times of volumes thoroughly after mixing
Analysis (4 DEG C), changes deionized water into buffer (20mM Tris, pH 8.0) and continues at after 12 hours
4 DEG C are dialysed 12 hours.Solution after the completion of dialysis Jing 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
α and the dimeric TCR of 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 is determined through SDS-PAGE and is more than 90%, and concentration is determined by BCA methods.What the present invention was obtained
The SDS-PAGE glue figures of sTCR are as shown in Figure 6.
The generation of the soluble single-chain T CR of embodiment 4NY-ESO-1 antigen small peptide specificity
According to patent documentation WO2014/206304, using the method for rite-directed mutagenesises by embodiment 2
TCR α and the variable domain of β chains be built into one with flexible small peptide (linker) be connected it is stable solvable
Property single chain TCR molecules.The aminoacid sequence and nucleotide sequence of the single chain TCR molecules respectively such as Fig. 7 a and
Shown in Fig. 7 b.The aminoacid sequence and nucleotide sequence of its α chain variable domain is respectively such as Fig. 8 a and Fig. 8 b institutes
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 sequences and nucleotide sequence are respectively as as-shown-in figures 10 a and 10b.
By genes of interest Jing Nco I and the double digestions of Not I, and through Nco I and the double digestions of Not I
PET28a carriers connect.Connection product is converted to E.coli DH5 α, and coating contains the LB flat boards of kanamycin,
37 DEG C of inversion overnight incubations, picking positive colony enters performing PCR screening, positive recombinant is sequenced, really
Recombinant plasmid transformed is extracted after sequencing row are correct to E.coli BL21 (DE3), for expressing.
The expression of the soluble single-chain T CR of embodiment 5NY-ESO-1 antigen small peptide specificity, renaturation and pure
Change
The BL21 containing recombiant plasmid pET28a- template strands (DE 3) the bacterium colony whole that will be prepared in embodiment 4
It is inoculated in the LB culture medium containing kanamycin, it is 0.6-0.8 that 37 DEG C are cultivated to OD600, adds IPTG
To final concentration of 0.5mM, 37 DEG C are continued to cultivate 4h.5000rpm centrifugation 15min harvesting precipitations
Thing, with Bugbuster Master Mix (Merck) cell lysis precipitate, 6000rpm centrifugation 15min
Inclusion body is reclaimed, then is washed to remove cell debriss and membrane component with Bugbuster (Merck), 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 remove insoluble matter, supernatant with subpackage is carried out after BCA standard measures, in -80
DEG C save backup.
To in the single-stranded TCR inclusion body proteins of 5mg dissolvings, addition 2.5mL buffer (6M Gua-HCl,
50mM Tris-HCl pH 8.1,100mM NaCl, 10mM EDTA), DTT is added to final concentration
For 10mM, 37 DEG C of process 30min.With syringe to 125mL renaturation buffers (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 for 4kDa cellulose membrane bag filter, bag filter
In being placed in the water of 1L pre-coolings, 4 DEG C are slowly stirred overnight.After 17 hours, dialysis solution is changed into 1L pre-coolings
Buffer (20mM Tris-HCl pH 8.0), 4 DEG C continue dialyse 8h, then dialysis solution is changed into
Identical fresh buffer continues dialysed overnight.After 17 hours, 0.45 μm of membrane filtration of sample Jing, vacuum
By anion-exchange column (HiTrap Q HP, GE Healthcare) after degassing, 20mM Tris-HCl are used
The 0-1M NaCl linear gradient elution liquid purifying proteins that pH 8.0 is prepared, the elution fraction of collection is carried out
SDS-PAGE is analyzed, and solvent resistant column (Superdex 75 is further used after the concentration of the component comprising single-stranded TCR
10/300, GE Healthcare) purification is carried out, target components are also carried out SDS-PAGE analyses.
Elution fraction for BIAcore analyses further tests its purity using gel filtration.Condition is：
Chromatographic column Agilent Bio SEC-3 (7.8 × 300mm of 300A, φ), mobile phase is 150mM phosphate
Buffer, flow velocity 0.5mL/min, 25 DEG C of column temperature, ultraviolet detection wavelength 214nm.
The SDS-PAGE glue figures of the soluble single-chain T CR that the present invention is obtained are as shown in figure 11.
Embodiment 6 is combined and characterized
BIAcore is analyzed
This example demonstrated the TCR molecules of the present invention of solubility can be multiple with SLLMWITQC-HLA A0201
Compound specifically binds.
The TCR obtained in embodiment 3 and embodiment 5 is detected using BIAcore T200 real-time analyzers
The binding activity of molecule and SLLMWITQC-HLA A0201 complex.By the antibody of anti-Streptavidin
(GenScript) coupling buffer (10mM sodium-acetate buffers, pH 4.77) is added, then by antibody
The CM5 chips for being activated with EDC and NHS in advance are flow through, makes antibody be fixed on chip surface, finally use second
The hydrochloric acid solution of hydramine closes unreacted activating surface, completes coupling process, and coupling level is about 15,000
RU。
The Streptavidin for making low concentration flows through the chip surface of coated antibody, then will
SLLMWITQC-HLA A0201 complex flows through sense channel, another passage as reference channel, then by 0.05
The biotin of mM flows through chip 2min, the remaining combination of closing Streptavidin with the flow velocity of 10 μ L/min
Site.
The preparation process of above-mentioned SLLMWITQC-HLA A0201 complex is as follows：
A. purification
The E.coli bacterium solutions of 100ml abduction deliverings heavy chain or light chain are collected, in 4 DEG C of 8000g centrifugations
After 10min with 10ml PBS washing thallines once, afterwards with 5ml BugBuster Master Mix
Extraction Reagents (Merck) acutely shake resuspended thalline, and in room temperature rotation incubation 20min,
After 4 DEG C, 6000g centrifugation 15min, supernatant discarded collects 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 bodys of BugBuster, mix, 4 DEG C of 6000g are centrifuged 15min,
It is repeated twice, plus the resuspended inclusion bodys of 30ml 20mM Tris-HCl pH 8.0, mix, 4 DEG C of 6000g
Centrifugation 15min, finally dissolves inclusion body, SDS-PAGE detection bags with 20mM Tris-HCl 8M carbamide
Contain body purity, BCA test kits survey concentration.
B. renaturation
The small peptide SLLMWITQC (Beijing SBS Genetech gene technology company limited) of synthesis is dissolved in into 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 adds 3M guanidine hydrochlorides, 10mM sodium acetates, the further degeneration of 10mM EDTA dissolving, before renaturation.
By SLLMWITQC peptides with 25mg/L (final concentration) add renaturation buffer (0.4M L-Arginine, 100
MM Tris pH 8.3,2mM EDTA, 0.5mM GSSGs, 5mM reduced form gluathiones
Peptide, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add the light chain and 90mg/L of 20mg/L
Heavy chain (final concentration, heavy chain is added in three times, 8h/ time), renaturation carries out at least 3 days at 4 DEG C to complete
Into can SDS-PAGE detections renaturation success.
C. purification after renaturation
Renaturation buffer is changed as dialysis with the 20mM Tris pH 8.0 of 10 volumes, at least changes slow
Rush the ionic strength that liquid carrys out fully to reduce twice solution.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 volumes).Using Akta purification instrument (GE General Electric Co. Limited), 20mM Tris pH 8.0
The 0-400mM NaCl linear gradient liquid wash-out proteins of preparation, pMHC eluting about at 250mM NaCl,
Collect all peak components, SDS-PAGE detection purity.
D. biotinylation
With Millipore super filter tubes by the pMHC molecular concentrations of purification, while being 20mM by buffer exchange
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 enzymes (GST-BirA), incubation at room temperature is mixed
Overnight, whether SDS-PAGE detection biotinylations are complete for compound.
E. the complex after purifying biological elementization
With Millipore super filter tubes by the pMHC molecular concentrations 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 posts (GE General Electric Co. Limited), loading 1
Ml concentrated biotinylation pMHC molecules, then with PBS with 1ml/min flow velocity eluting.Biotin
The pMHC molecules of change occur in about 55ml as unimodal eluting.Merge the component containing protein, use
Millipore super filter tubes are concentrated, and BCA methods (Thermo) determine protein concentration, add albumen enzyme level
Biotinylated pMHC molecules subpackage is stored in -80 DEG C by agent cocktail (Roche).
Using BIAcore Evaluation computed in software kinetic parameters, the TCR of solubility of the present invention is obtained
The soluble single-chain T CR molecules that molecule and the present invention build are tied with SLLMWITQC-HLA A0201 complex
The kinetic profile of conjunction is respectively as shown in Figure 12 and Figure 13.Collection of illustrative plates shows, the sTCR that the present invention is obtained
Molecule and soluble single-chain T CR molecules can be combined with SLLMWITQC-HLA A0201 complex.Together
When, the TCR molecules and other several irrelevant antigen small peptides of solubility of the present invention is also have detected using said method
With the binding activity of HLA complex, as a result show TCR molecules of the present invention with other irrelevant antigens without combination.
The all documents referred in the present invention are all incorporated as in this application reference, just as each document
It is individually recited as with reference to such.In addition, it is to be understood that after the above-mentioned teachings for 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), it is characterised in that the TCR can be with SLLMWITQC-HLA
A0201 complex is combined；Preferably, described TCR includes TCR α chains variable domains and TCR β chain variable domains,
Characterized in that, the aminoacid sequence of the CDR3 of the TCR α chain variable domains is AGFMDSNYQLI (SEQ ID
NO:12)；And/or the aminoacid sequence of the CDR3 of the TCR β chain variable domains is ASSLGGSVLH (SEQ
ID NO:15)；
It is highly preferred that 3 complementary determining regions (CDR) of the TCR α chain variable domains are：
αCDR1-SIFNT(SEQ ID NO:10)
αCDR2-LYKAGEL(SEQ ID NO:11)
αCDR3-AGFMDSNYQLI(SEQ ID NO:12)；And/or
3 complementary determining regions of the TCR β chain variable domains are：
βCDR1-SGHVS(SEQ ID NO:13)
βCDR2-FQNEAQ(SEQ ID NO:14)
βCDR3-ASSLGGSVLH(SEQ ID NO:15)。
2. TCR as claimed in claim 1, it is characterised in that it includes TCR α chains variable domains and TCR
β chain variable domains, the TCR α chain variable domains are and SEQ ID NO:1 has at least 90% sequence thereto
Aminoacid sequence；And/or the TCR β chain variable domains are and SEQ ID NO：5 have at least 90% sequence
The aminoacid sequence of row homogeny.
3. TCR as claimed in claim 1, it is characterised in that the α chains of the TCR and/or β chains
C- or N- ends are combined with conjugate；Preferably, the conjugate for being combined with the φt cell receptor is detectable
The combination of label, therapeutic agent, PK modifications part or any these materials；Preferably, the therapeutic agent is
Anti-CD 3 antibodies.
4. a kind of multivalent TCR complex, it is characterised in that comprising at least two TCR molecules, and its
At least one of TCR molecules be the claims any one of TCR.
5. a kind of nucleic acid molecules, it is characterised in that the nucleic acid molecules will comprising any of the above-described right of coding
Ask the nucleotide sequence or its complementary series of described TCR molecules；
Preferably, nucleotide sequence SEQ ID NO of the described nucleic acid molecules comprising coding TCR α chain variable domains：
2 or SEQ ID NO:33；And/or
Nucleotide sequence SEQ ID NO of the described nucleic acid molecules comprising coding TCR β chain variable domains：6 or
SEQ ID NO:35。
6. a kind of carrier, it is characterised in that described carrier contains arbitrary described in claim 25-28
Nucleic acid molecules；Preferably, described carrier is viral vector；It is highly preferred that described carrier is slow disease
Poisonous carrier.
7. a kind of detached host cell, it is characterised in that contain claim in described host cell
Arbitrary described nucleic acid point in claim 25-28 of external source is integrated with carrier or chromosome described in 26
Son.
8. a kind of cell, it is characterised in that arbitrary described core in cell transduction claim 25-28
Carrier described in acid molecule or claim 29；Preferably, the cell is T cell or stem cell.
9. a kind of pharmaceutical composition, it is characterised in that the compositionss contain pharmaceutically acceptable carrier
And the TCR complex described in the TCR any one of claim 1-23, claim 24,
Cell in claim 25-28 described in arbitrary described nucleic acid molecules or claim 28.
10. described in the φt cell receptor or claim 24 any one of claim 1-23
The purposes of the cell described in TCR complex or claim 31, it is characterised in that swollen for preparing treatment
The medicine of tumor or autoimmune disease.
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| CN109251244A (en) * | 2017-07-13 | 2019-01-22 | 中国科学院广州生物医药与健康研究院 | A kind of identification is derived from the TCR of EBV memebrane protein LMP1 antigen |
| CN112442118A (en) * | 2019-08-30 | 2021-03-05 | 深圳普瑞金生物药业有限公司 | TCR and application thereof |
| CN113272435A (en) * | 2018-09-05 | 2021-08-17 | 加利福尼亚大学董事会 | NY-ESO-1 specific T cell receptor compositions restricted on multiple major histocompatibility complex molecules |
| WO2022093333A1 (en) * | 2020-10-27 | 2022-05-05 | T-Cure Biosciences, Inc. | Recombinant t-cell receptors that bind the ny-eso-1 and/or lage-1a cancer antigens |
| WO2022170509A1 (en) | 2021-02-09 | 2022-08-18 | 深圳普瑞金生物药业有限公司 | Tcr and application thereof |
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| CN109251244A (en) * | 2017-07-13 | 2019-01-22 | 中国科学院广州生物医药与健康研究院 | A kind of identification is derived from the TCR of EBV memebrane protein LMP1 antigen |
| CN109251244B (en) * | 2017-07-13 | 2021-10-26 | 中国科学院广州生物医药与健康研究院 | TCR (T cell receptor) for recognizing LMP1 antigen derived from EBV (Epstein-Barr Virus) membrane protein |
| CN113272435A (en) * | 2018-09-05 | 2021-08-17 | 加利福尼亚大学董事会 | NY-ESO-1 specific T cell receptor compositions restricted on multiple major histocompatibility complex molecules |
| EP3847265A4 (en) * | 2018-09-05 | 2023-02-15 | The Regents of University of California | Composition of ny-eso-1-specific t cell receptors restricted on multiple major histocompatibility complex molecules |
| CN112442118A (en) * | 2019-08-30 | 2021-03-05 | 深圳普瑞金生物药业有限公司 | TCR and application thereof |
| CN112442118B (en) * | 2019-08-30 | 2023-02-14 | 深圳普瑞金生物药业股份有限公司 | TCR and application thereof |
| WO2022093333A1 (en) * | 2020-10-27 | 2022-05-05 | T-Cure Biosciences, Inc. | Recombinant t-cell receptors that bind the ny-eso-1 and/or lage-1a cancer antigens |
| WO2022170509A1 (en) | 2021-02-09 | 2022-08-18 | 深圳普瑞金生物药业有限公司 | Tcr and application thereof |
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|---|---|
| CN106632658B (en) | 2020-10-27 |
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