CN113667008A - High-affinity T cell receptor for recognizing AFP antigen - Google Patents

High-affinity T cell receptor for recognizing AFP antigen Download PDF

Info

Publication number
CN113667008A
CN113667008A CN202010414690.0A CN202010414690A CN113667008A CN 113667008 A CN113667008 A CN 113667008A CN 202010414690 A CN202010414690 A CN 202010414690A CN 113667008 A CN113667008 A CN 113667008A
Authority
CN
China
Prior art keywords
ser
tcr
leu
gly
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010414690.0A
Other languages
Chinese (zh)
Inventor
李懿
彭真
孙含丽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiangxue Life Science Technology Guangdong Co ltd
Original Assignee
Xiangxue Life Science Technology Guangdong Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xiangxue Life Science Technology Guangdong Co ltd filed Critical Xiangxue Life Science Technology Guangdong Co ltd
Priority to CN202010414690.0A priority Critical patent/CN113667008A/en
Priority to PCT/CN2021/093947 priority patent/WO2021228255A1/en
Priority to TW110117492A priority patent/TW202144404A/en
Publication of CN113667008A publication Critical patent/CN113667008A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001181—Alpha-feto protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464481—Alpha-feto protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma

Abstract

The present invention provides a T Cell Receptor (TCR) having the property of binding to the TSSELMAITR-HLA a1101 complex; and the binding affinity of the TCR to the TSSELMAITR-HLA A1101 complex is at least 2-fold greater than the binding affinity of a wild-type TCR to the TSSELMAITR-HLA A1101 complex. The invention also provides fusion molecules of such TCRs with therapeutic agents. Such TCRs can be used alone or in combination with therapeutic agents to target TSSELMAITR-HLA a1101 complex-presenting tumor cells.

Description

High-affinity T cell receptor for recognizing AFP antigen
Technical Field
The present invention relates to the field of biotechnology, and more specifically to T Cell Receptors (TCRs) capable of recognizing polypeptides derived from AFP proteins. The invention also relates to the preparation and use of said receptors.
Background
Only two types of molecules are able to recognize antigens in a specific manner. One of which is an immunoglobulin or antibody; the other is the T Cell Receptor (TCR), which is a cell membrane surface glycoprotein that exists as a heterodimer from the α chain/β chain or the γ chain/δ chain. The composition of the TCR repertoire of the immune system is produced by v (d) J recombination in the thymus, followed by positive and negative selection. In the peripheral environment, TCRs mediate the specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells, and are therefore critical for the cellular immune function of the immune system.
TCRs are the only receptors for specific antigenic peptides presented on the Major Histocompatibility Complex (MHC), and such exogenous or endogenous peptides may be the only signs of cellular abnormalities. In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.
The MHC class I and II molecular ligands corresponding to the TCR are also proteins of the immunoglobulin superfamily but are specific for presentation of antigens, with different individuals having different MHC, and thereby presenting different short peptides of a single protein antigen to the cell surface of the respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.
AFP (alpha Fetoprotein), also known as alpha Fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently inhibited. In liver cancer, AFP expression is activated (Butterfield et al.J. Immunol.,2001, Apr 15; 166(8): 5300-8). AFP is processed into antigenic peptides after intracellular production, and is bound to MHC (major histocompatibility complex) molecules to form complexes, which are presented on the cell surface. TSSELMAITR are short peptides derived from the AFP antigen, which are one of the targets for the treatment of AFP-related diseases.
Thus, the TSSELMAITR-HLA A1101 complex provides a marker for targeting of TCRs to tumor cells. The TCR capable of combining the TSSELMAITR-HLA A1101 complex has high application value in treating tumors. For example, TCRs capable of targeting the tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or to be transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for administration to patients in a therapeutic process known as adoptive immunotherapy. For the former purpose, the ideal TCR is of higher affinity, enabling the TCR to reside on the targeted cell for a long period of time. For the latter purpose, it is preferred to use a medium affinity TCR. Accordingly, those skilled in the art are working to develop TCRs that target tumor cell markers that can be used to meet different objectives.
Disclosure of Invention
It is an object of the present invention to provide a TCR with a higher affinity for the TSSELMAITR-HLA a1101 complex.
It is a further object of the present invention to provide a method for preparing a TCR of the above type and uses thereof.
In a first aspect of the invention, there is provided a T Cell Receptor (TCR) comprising an alpha chain variable domain and a beta chain variable domain, which has the activity of binding TSSELMAITR-HLA a1101 complex, and the amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID No. 1 and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID No. 2.
In a preferred embodiment, the amino acid sequence of the TCR a chain variable domain and the amino acid sequence of the TCR β chain variable domain are not the amino acid sequence of the wild-type TCR a chain variable domain and the amino acid sequence of the wild-type TCR β chain variable domain at the same time.
In a further preferred embodiment, the amino acid sequence of the variable domain of the TCR alpha chain is not the amino acid sequence shown in SEQ ID NO 1, and/or
The amino acid sequence of the variable domain of the TCR beta chain is not the amino acid sequence shown in SEQ ID NO. 2.
In another preferred embodiment, the α chain variable domain of the TCR comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the sequence set forth in SEQ ID No. 1.
In another preferred embodiment, the β chain variable domain of the TCR is an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the sequence set forth in SEQ ID No. 2.
In another preferred embodiment, the amino acid sequence of the variable domain of the TCR alpha chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO. 2.
In another preferred embodiment, the amino acid sequence of the variable domain of the TCR β chain has at least 95% sequence homology with the amino acid sequence set forth in SEQ ID NO. 2.
In another preferred embodiment, the reference sequence of the 3 CDR regions (complementarity determining regions) of the variable domain of the TCR alpha chain is as follows,
CDR1α：YGATPY
CDR2α：YFSGDTLV
CDR3 α: AVVATDSWGKLQ, and CDR3 α contains at least one of the following mutations:
residues before mutation Post-mutation residues
Position 3V of CDR3 alpha A or P
Position 4A of CDR3 alpha S or D or G
Position 5T of CDR3 α L or M or Y or I
Position 6D of CDR3 α K or Q or S or E or A or H or N or P or R
In another preferred embodiment, the amino acid mutation in CDR 3a comprises:
residues before mutation Post-mutation residues
Position 3V of CDR3 alpha A
In another preferred embodiment, the amino acid mutation in CDR 3a comprises:
residues before mutation Post-mutation residues
Position 4A of CDR3 alpha S
In another preferred embodiment, the amino acid mutation in CDR 3a comprises:
residues before mutation Post-mutation residues
Position 3V of CDR3 alpha A
Position 4A of CDR3 alpha S
In another preferred embodiment, the number of amino acid mutations in CDR 3a is 2-5, specifically 2 or 3 or 4 or 5, preferably 3 or 4.
In another preferred embodiment, the TCR has at least 2-fold greater affinity for the TSSELMAITR-HLA a1101 complex than a wild-type TCR.
In another preferred embodiment, the CDR3 α of the TCR α chain is selected from AVASLKSWGKLQ, AVASMDSWGKLQ, AVASMQSWGKLQ, AVASYQSWGKLQ and AVASLSSWGKLQ.
In another preferred embodiment, the 3 CDRs of the TCR β chain variable domain are:
CDR1β：SGHVS；
CDR2 β: FQNEAQ; and
CDR3β：ASSLVAGARTDTQY。
in another preferred embodiment, the amino acid sequence of the variable domain of TCR β chain is SEQ ID NO 2.
In another preferred embodiment, the reference sequence of 3 CDRs of the TCR beta chain variable domain is as follows,
CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3 β: ASSLVAGARTDTQY, and CDR3 β contains at least one of the following mutations:
residues before mutation Post-mutation residues
Position 3S of CDR3 beta T
L at position 4 of CDR3 beta M or W
V at position 5 of CDR3 beta L or I
Position 6A of CDR3 beta G
In another preferred example, the TCR α chain variable domain comprises CDR1 α, CDR2 α, and CDR3 α, wherein the amino acid sequence of CDR1 α is YGATPY, the amino acid sequence of CDR2 α is YFSGDTLV, and the amino acid sequence of CDR3 α is: AV [3 AlX 1] [3 AlX 2] [3 AlX 3] [3 AlX 4] SWGKLQ, wherein [3 AlX 1] is V or A or P, and/or [3 AlX 2] is A or S or D or G, and/or [3 AlX 3] is T or L or M or Y or I, and/or [3 AlX 4] is D or K or Q or S or E or A or H or N or P or R.
In another preferred embodiment, the TCR has a mutation in the alpha chain variable domain shown in SEQ ID NO. 1 selected from one or more of V94A/P, A95S/D/G, T96L/M/Y/I, D97K/Q/S/E/A/H/N/P/R, wherein the numbering of the amino acid residues is as shown in SEQ ID NO. 1.
In another preferred example, the TCR β chain variable domain comprises CDR1 β, CDR2 β, and CDR3 β, wherein the amino acid sequence of CDR1 β is SGHVS, the amino acid sequence of CDR2 β is FQNEAQ, and the amino acid sequence of CDR3 β is: AS [3 betaX 1] [3 betaX 2] [3 betaX 3] [3 betaX 4] GARTDTQY, wherein [3 betaX 1] is S or T, and/or [3 betaX 2] is L or M or W, and/or [3 betaX 3] is V or L or I, and/or [3 betaX 4] is A or G.
In another preferred embodiment, the TCR has a mutation in the beta chain variable domain shown in SEQ ID NO. 2 selected from one or more of S95T, L96M/W, V97L/I, A98G, wherein the numbering of the amino acid residues is as shown in SEQ ID NO. 2.
In another preferred embodiment, the TCR has CDRs selected from the group consisting of:
Figure BDA0002494521140000041
Figure BDA0002494521140000051
in another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is an α β heterodimeric TCR comprising an α chain TRAC constant region sequence and a β chain TRBC1 or TRBC2 constant region sequence.
In another preferred embodiment, the TCR comprises (i) all or part of a TCR α chain, except for its transmembrane domain, and (ii) all or part of a TCR β chain, except for its transmembrane domain, wherein both (i) and (ii) comprise the variable domain and at least part of the constant domain of the TCR chain.
In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain constant region and the β chain constant region.
In another preferred embodiment, the cysteine residues forming the artificial interchain disulfide bond between the constant regions of the TCR α and β chains are substituted at one or more groups of sites selected from:
thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;
ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;
arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;
pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01;
and Tyr10 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu20 of TRBC2 × 01 exon 1.
In another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR is one of SEQ ID NOs 1, 13-32; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 33-36.
In another preferred embodiment, the TCR is selected from the group consisting of:
Figure BDA0002494521140000061
in another preferred embodiment, the TCR is a single chain TCR.
In another preferred embodiment, the TCR is a single chain TCR consisting of an alpha chain variable domain and a beta chain variable domain linked by a flexible short peptide sequence (linker).
In another preferred embodiment, the C-or N-terminus of the α chain and/or β chain of the TCR is conjugated to a conjugate, preferably a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these.
In another preferred embodiment, the therapeutic agent that binds to the TCR is an anti-CD 3 antibody linked to the C-or N-terminus of the α or β chain of the TCR.
In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in any one of the preceding claims.
In a third aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention or a multivalent TCR complex according to the second aspect of the invention, or a complement thereof.
In a fourth aspect of the invention, there is provided a vector comprising the nucleic acid molecule of the third aspect of the invention.
In a fifth aspect of the invention, there is provided a host cell comprising a vector or chromosome of the fourth aspect of the invention and, integrated therein, an exogenous nucleic acid molecule of the third aspect of the invention.
In a sixth aspect of the invention, there is provided an isolated cell expressing a TCR according to the first aspect of the invention.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth aspect of the invention, there is provided a method of treating a disease, comprising administering to a subject in need thereof an amount of a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, preferably the disease is an AFP-positive tumour, more preferably the tumour is liver cancer, most preferably the tumour is hepatocellular carcinoma.
In a ninth aspect, the present invention provides the use of a TCR of the first aspect of the invention, or a TCR complex of the second aspect of the invention, or a cell of the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumour, preferably the tumour is an AFP-positive tumour, more preferably the tumour is liver cancer, most preferably the tumour is hepatocellular carcinoma.
In a tenth aspect of the invention, there is provided a method of preparing a T cell receptor according to the first aspect of the invention, comprising the steps of:
(i) culturing a host cell according to the fifth aspect of the invention, thereby expressing a T-cell receptor according to the first aspect of the invention;
(ii) isolating or purifying said T cell receptor.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Fig. 1a and 1b show the amino acid sequences of the wild-type TCR α and β chain variable domains, respectively, capable of specifically binding to the TSSELMAITR-HLA a1101 complex.
FIGS. 2a and 2b show the amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain, respectively, of a single chain template TCR constructed in accordance with the invention.
FIGS. 3a and 3b are the DNA sequences of the alpha chain variable domain and beta chain variable domain, respectively, of a single-chain template TCR constructed in accordance with the invention.
FIGS. 4a and 4b show the amino acid sequence and DNA sequence of the linker peptide (linker) of the single-chain template TCR constructed according to the present invention.
FIGS. 5a and 5b show the amino acid sequence and DNA sequence, respectively, of a single-stranded template TCR constructed in accordance with the invention.
FIGS. 6a and 6b are amino acid sequences of soluble reference TCR α and β chains, respectively, of the invention.
Fig. 7(1) - (20) show the α chain variable domain amino acid sequences of the heterodimeric TCR with high affinity for the TSSELMAITR-HLA a1101 complex, respectively, with mutated residues underlined.
Fig. 8(1) - (4) show the β chain variable domain amino acid sequences of the heterodimeric TCR with high affinity for the TSSELMAITR-HLA a1101 complex, respectively, with mutated residues underlined.
Fig. 9a and 9b show the extracellular amino acid sequences of the wild-type TCR α and β chains, respectively, capable of binding specifically to the TSSELMAITR-HLA a1101 complex.
Fig. 10a and 10b show the amino acid sequences of the wild-type TCR α and β chains, respectively, capable of specifically binding to the TSSELMAITR-HLA a1101 complex.
FIG. 11 is a graph of the binding of a soluble reference TCR, i.e., a wild-type TCR, to the TSSELMAITR-HLA A1101 complex.
Fig. 12a and 12b are experimental results of the activation function of effector cells transfected with the high affinity TCR of the present invention against short peptide-loaded T2 cells.
FIGS. 13a and 13b are experimental results of the activation function of effector cells transfected with the high affinity TCR of the invention against tumor cell lines.
Fig. 14 is an experimental result of the killing function of effector cells transfected with the high affinity TCR of the present invention against gradient short peptide-loaded T2 cells.
Fig. 15a, 15b and 15c are experimental results of the killing function of effector cells transfected with the high affinity TCRs of the invention against tumor cell lines.
Detailed Description
The present inventors, through extensive and intensive studies, have obtained a high affinity T Cell Receptor (TCR) that recognizes TSSELMAITR short peptides (derived from the AFP protein), the TSSELMAITR short peptide being presented as a peptide-HLA a1101 complex. The high affinity TCR has 3 CDR regions in its alpha chain variable domain:
CDR1α：YGATPY
CDR2α：YFSGDTLV
CDR3 α: AVVATDSWGKLQ; and/or in the 3 CDR regions of its beta chain variable domain:
CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3 β: ASSLVAGARTDTQY; and, the affinity and/or binding half-life of the inventive TCR, after mutation, to the TSSELMAITR-HLA a1101 complex described above is at least 2-fold that of a wild-type TCR.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
Term(s) for
T Cell Receptor (TCR)
The TCR may be described using the international immunogenetics information system (IMGT). Native α β heterodimeric TCRs have an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linker region and a constant region, and the beta chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered part of the linker region. The TCR connecting region is defined by the unique TRAJ and TRBJ of IMGT, and the TCR constant region is defined by the TRAC and TRBC of IMGT.
Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in a framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different types of V α and V β, respectively. In the IMGT system, the α chain constant domain has the following symbols: TRAC 01, wherein "TR" denotes a T cell receptor gene; "A" represents an alpha chain gene; c represents a constant region; ". 01" indicates allele 1. The beta-strand constant domain has the following symbols: TRBC1 x 01 or TRBC2 x 01, wherein "TR" denotes a T cell receptor gene; "B" represents a beta chain gene; c represents a constant region; ". 01" indicates allele 1. The constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes, "C1" and "C2". The constant region gene sequences of the TCR alpha and beta chains can be obtained by those skilled in the art from published IMGT databases.
The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of linked variable regions and linked regions. Thus, in the description and claims of this application, the "TCR α chain variable domain" refers to the linked TRAV and TRAJ regions, and likewise the "TCR β chain variable domain" refers to the linked TRBV and TRBD/TRBJ regions. The 3 CDRs of the TCR α chain variable domain are CDR1 α, CDR2 α and CDR3 α, respectively; the 3 CDRs of the TCR β chain variable domain are CDR1 β, CDR2 β and CDR3 β, respectively. The framework sequences of the TCR variable domains of the invention may be murine or human, preferably human. The constant domain of the TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion.
In the present invention, the amino acid sequences of the α and β chain variable domains of the wild-type TCR capable of binding the TSSELMAITR-HLA A1101 complex are SEQ ID NO 1 and SEQ ID NO 2, respectively, as shown in FIG. 1a and FIG. 1 b. The alpha chain amino acid sequence and the beta chain amino acid sequence of the soluble "reference TCR" are respectively SEQ ID NO. 11 and SEQ ID NO. 12, as shown in FIG. 6a and FIG. 6 b. The extracellular amino acid sequence of the alpha chain and the extracellular amino acid sequence of the beta chain of the wild-type TCR are SEQ ID NO 37 and SEQ ID NO 38 respectively, as shown in FIG. 9a and FIG. 9 b. The TCR sequences used in the present invention are of human origin. The alpha chain amino acid sequence and the beta chain amino acid sequence of the wild-type TCR are respectively SEQ ID NO 39 and SEQ ID NO 40, as shown in FIGS. 10a and 10 b. In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Natural interchain disulfide bond and artificial interchain disulfide bond
A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".
For convenience of description, the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are position-numbered in the order from the N-terminus to the C-terminus, such as TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid is P (proline) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Pro60 in the invention, or TRBC1 × 01 or TRBC2 × 01 exon 1, and as TRBC1 × 01 or TRBC2 × 01, and the 61 st amino acid is Q (glutamine) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2, and as glbc 8201 or TRBC 8536. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.
Tumor(s)
The term "tumor" is meant to include all types of cancer cell growth or carcinogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or the stage of infection. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignancies of different organ systems, such as sarcomas, squamous carcinomas of the lung and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx. Squamous carcinoma of the lung includes malignant tumors, such as, for example, most cancers of the colon, rectum, renal cell, liver, lung, small cell, small intestine and esophagus. Metastatic lesions of the above-mentioned cancers can likewise be treated and prevented using the methods and compositions of the present invention.
Detailed Description
It is well known that the α chain variable domain and β chain variable domain of a TCR each contain 3 CDRs, similar to the complementarity determining regions of an antibody. CDR3 interacts with antigen short peptides, CDR1 and CDR2 interact with HLA. Thus, the CDRs of the TCR molecule determine their interaction with the antigen short peptide-HLA complex. The amino acid sequences of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the wild-type TCR capable of binding the antigen short peptide TSSELMAITR to the HLA A1101 complex (i.e., TSSELMAITR-HLA A1101 complex) are SEQ ID NO 1 and SEQ ID NO 2, respectively, which were the first findings of the present inventors. It has the following CDR regions: alpha chain variable domain CDR1 a: YGATPY
CDR2α：YFSGDTLV
CDR3α：AVVATDSWGKLQ
And the beta chain variable domain CDR1 beta: SGHVS
CDR2 β: FQNEAQ and
CDR3β：ASSLVAGARTDTQY。
the invention obtains the high affinity TCR with the affinity of TSSELMAITR-HLA A1101 complex being at least 2 times that of the wild TCR and TSSELMAITR-HLA A1101 complex by carrying out mutation screening on the CDR region.
Further, the TCR of the invention is an α β heterodimeric TCR, the α chain variable domain of which comprises at least 85% of the amino acid sequence set forth in SEQ ID No. 1; preferably, at least 90%; more preferably, at least 92%; more preferably, at least 94% (e.g., can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of sequence homology; and/or the β chain variable domain of the TCR comprises at least 90%, preferably at least 92%, of the amino acid sequence set forth as SEQ ID No. 2; more preferably, at least 94% (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology) of sequence homology.
Further, the TCR of the invention is a single chain TCR, the α chain variable domain of which comprises at least 85%, preferably at least 90% of the amino acid sequence shown in SEQ ID No. 3; more preferably, at least 92%; most preferably, at least 94% (e.g., can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of sequence homology; and/or the β chain variable domain of the TCR comprises at least 85%, preferably at least 90% of the amino acid sequence set forth as SEQ ID No. 4; more preferably, at least 92%; most preferably, at least 94%; (e.g., can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of sequence homology.
The 3 CDRs of the variable domain of the wild-type TCR alpha chain of SEQ ID NO. 1, i.e., CDR1, CDR2 and CDR3, are located at positions 27-32, 50-57 and 92-103 of SEQ ID NO. 1, respectively. Accordingly, the amino acid residue numbering is as shown in SEQ ID NO. 1, 94V is the 3 rd position V of CDR3 alpha, 95A is the 4 th position A of CDR3 alpha, 96T is the 5 th position T of CDR3 alpha, 97D is the 6 th position D of CDR3 alpha.
The invention provides a TCR having the property of binding TSSELMAITR-HLA a1101 complex and comprising an alpha chain variable domain and a beta chain variable domain, wherein the TCR is mutated in the alpha chain variable domain as shown in SEQ ID No. 1, the mutated amino acid residue positions comprising one or more of 94V, 95A, 96T and 97D, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID No. 1.
Preferably, the TCR α chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of: 94A or 94P; 95S or 95D or 95G; 96L or 96M or 96Y or 96I; 97K or 97Q or 97S or 97E or 97A or 97H or 97N or 97P or 97R, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO. 1.
More specifically, the specific form of the mutation in the variable domain of the alpha chain includes one or more of V94A/P, A95S/D/G, T96L/M/Y/I, D97K/Q/S/E/A/H/N/P/R.
The 3 CDRs of the variable domain of the wild-type TCR beta chain of the invention SEQ ID NO. 2, i.e., CDR1, CDR2 and CDR3, are located at positions 27-31, 49-54 and 93-106 of SEQ ID NO. 2, respectively. Accordingly, the amino acid residue numbering is as shown in SEQ ID NO. 2, 95S is the 3 rd position S of CDR3 beta, 96L is the 4 th position L of CDR3 beta, 97V is the 5 th position V of CDR3 beta, 98A is the 6 th position A of CDR3 beta.
The invention provides a TCR having the property of binding TSSELMAITR-HLA a1101 complex and comprising a β chain variable domain and a β chain variable domain, wherein the TCR is mutated in the β chain variable domain as shown in SEQ ID No. 2, the mutated amino acid residue positions comprising one or more of 95S, 96L, 97V, 98A, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID No. 2.
Preferably, the TCR β chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of: 95T; 96M or 96W; 97L or 97I; 98G, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO. 2.
More specifically, the specific form of the mutation in the variable domain of the beta chain includes one or more of S95T, L96M/W, V97L/I, A98G.
It should be understood that the amino acid names herein are given by the international single english letter designation, and the three english letters abbreviation corresponding to the amino acid names are: 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) and Val (V);
in the present invention, Pro60 or 60P both represent proline at position 60. In addition, the expression of a specific form of the mutation described in the present invention, for example, "V94A/P" means that V at position 94 is substituted by A or by G, and so on.
The reference TCR was obtained by mutating Thr48 of exon 1 of the α chain constant region TRAC 01 of the wild-type TCR to cysteine and Ser57 of exon 1 of the β chain constant region TRBC1 x 01 or TRBC2 x 01 to cysteine according to site-directed mutagenesis methods well known to those skilled in the art, the amino acid sequences of which are SEQ ID NO:11 and SEQ ID NO:12, respectively, as shown in fig. 6a and 6b, and the mutated cysteine residues are shown in bold letters. The cysteine substitutions described above enable artificial interchain disulfide bonds to be formed between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, enabling more convenient assessment of binding affinity and/or binding half-life between the TCR and the TSSELMAITR-HLA a1101 complex. It will be appreciated that the CDR regions of the TCR variable region determine their affinity for the pMHC complex and therefore cysteine substitutions in the TCR constant region as described above do not have an effect on the binding affinity and/or binding half-life of the TCR. Thus, in the present invention, the measured binding affinity between the reference TCR and the TSSELMAITR-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the TSSELMAITR-HLA A1101 complex. Similarly, if the binding affinity between the inventive TCR and the TSSELMAITR-HLA a1101 complex is determined to be at least 10-fold greater than the binding affinity between the reference TCR and the TSSELMAITR-HLA a1101 complex, i.e., equivalent to the binding affinity between the inventive TCR and the TSSELMAITR-HLA a1101 complex being at least 10-fold greater than the binding affinity between the wild-type TCR and the TSSELMAITR-HLA a1101 complex.
Binding affinity (equilibrium constant K to dissociation) can be determined by any suitable methodDInversely proportional) and binding half-life (denoted T)1/2) For example, surface plasmon resonance. It will be appreciated that doubling the affinity of the TCR will result in KDAnd (4) halving. T is1/2Calculated as In2 divided by dissociation rate (K)off). Thus, T1/2Doubling can result in KoffAnd (4) halving. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, e.g. 3 times or more, using the same assay protocol, and the results are averaged. In a preferred embodiment, the surface plasmon resonance (BIAcore) method of the examples herein is used to detect the affinity of soluble TCRs, provided that: the temperature is 25 ℃, and the pH value is 7.1-7.5. The method detects the dissociation equilibrium constant K of the reference TCR to the TSSELMAITR-HLA A1101 complexD2.98E-05M, i.e., 29.8. mu.M, the dissociation equilibrium constant K of the wild-type TCR for the TSSELMAITR-HLA A1101 complex is considered to beDAlso 29.8. mu.M. Doubling of the affinity due to TCR will result in KDHalving, so if the dissociation equilibrium constant K of the high affinity TCR for the TSSELMAITR-HLA A1101 complex is detectedD2.98E-06M, i.e., 2.98. mu.M, indicates that the high affinity TCR has 10 times the affinity for the TSSELMAITR-HLA A1101 complex as compared to the wild type TCR for the TSSELMAITR-HLA A1101 complex. K is well known to those skilled in the artDConversion between units of value, i.e. 1M 106μ M, 1000nM for 1 μ M and 1000pM for 1 nM. In the present invention, the affinity of the TCR to the TSSELMAITR-HLA a1101 complex is at least 2-fold that of the wild-type TCR.
The mutation may be performed using any suitable method, including but not limited to those based on Polymerase Chain Reaction (PCR), cloning based on restriction enzymes, or Ligation Independent Cloning (LIC) methods. These methods are detailed in a number of standard molecular biology texts. For more details on Polymerase Chain Reaction (PCR) mutagenesis and Cloning by restriction enzymes, see Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (third edition) CSHL Press. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6).
The method of producing the TCRs of the invention may be, but is not limited to, screening a diverse library of phage particles displaying such TCRs for a TCR with high affinity for the TSSELMAITR-HLA-A1101 complex, as described in the literature (Li, et al (2005) Nature Biotech 23(3): 349-.
It will be appreciated that genes expressing the α and β chain variable domain amino acids of a wild type TCR, or genes expressing slightly modified α and β chain variable domain amino acids of a wild type TCR, may be used to make a template TCR. The alterations required to produce the high affinity TCRs of the invention are then introduced into the DNA encoding the variable domains of the template TCR.
The high affinity TCR of the invention comprises an alpha chain variable domain amino acid sequence of one of SEQ ID NO 1, 13-32; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 33-36. In the present invention, the amino acid sequences of the α chain variable domain and the β chain variable domain that form the heterodimeric TCR molecule are preferably selected from table 1 below:
TABLE 1
Figure BDA0002494521140000151
For the purposes of the present invention, the inventive TCRs are moieties having at least one TCR α and/or TCR β chain variable domain. They typically comprise both a TCR α chain variable domain and a TCR β chain variable domain. They may be α β heterodimers or single chain forms or any other form that is stable. In adoptive immunotherapy, the entire long chain (containing both cytoplasmic and transmembrane domains) of an α β heterodimeric TCR can be transfected. The TCRs of the invention are useful as targeting agents for delivering therapeutic agents to antigen presenting cells or in combination with other molecules to produce bifunctional polypeptides for targeting effector cells, where the TCRs are preferably in soluble form.
For stability, it is disclosed in the prior art that the introduction of an artificial interchain disulfide bond between the α and β chain constant domains of a TCR enables soluble and stable TCR molecules to be obtained, as described in patent document PCT/CN 2015/093806. Thus, the inventive TCR may be one in which an artificial interchain disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a substitution of Thr48 for exon 1 of TRAC × 01 and a substitution of Ser57 for exon 1 of TRBC1 × 01 or TRBC2 × 01 form disulfide bonds. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include cysteine residues for the purpose of deleting the native interchain disulphide bond, or by mutating the cysteine residues forming the native interchain disulphide bond to another amino acid.
As described above, the TCRs of the invention may comprise an artificial interchain disulfide bond introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native interchain disulfide bonds present in the TCR.
In addition, for stability, patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.
For stability, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that improve the stability of the inventive TCRs, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 of the reciprocal amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2,4,6 of the reciprocal amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.
More specifically, the TCR with the mutated hydrophobic core region of the present invention may be a high stability single chain TCR comprising a flexible peptide chain connecting the variable domains of the α chain and β chain of the TCR. The CDR regions of the variable region of the TCR determine the affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can stabilize the TCR without affecting the affinity with the short peptide-HLA complex. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The template chain for screening high affinity TCR constructed in example 1 of the present invention is the above-described high stability single chain TCR comprising the hydrophobic core mutation. Using a TCR with higher stability, the affinity between the TCR and the TSSELMAITR-HLA-A1101 complex can be more conveniently assessed.
The CDR regions of the alpha chain variable domain and the beta chain variable domain of the single-chain template TCR are completely identical to the CDR regions of the wild-type TCR. That is, the 3 CDRs of the α chain variable domain are CDR1 α: YGATPY; CDR2 α: YFSGDTLV; CDR3 α: AVVATDSWGKLQ and the 3 CDRs of the β chain variable domain are CDR1 β: SGHVS; CDR2 β: FQNEAQ; CDR3 β: ASSLVAGARTDTQY are provided. The amino acid sequence (SEQ ID NO:9) and the nucleotide sequence (SEQ ID NO:10) of the single-chain template TCR are shown in FIGS. 5a and 5b, respectively. Thus, a single-chain TCR composed of an alpha chain variable domain and a beta chain variable domain having high affinity for the TSSELMAITR-HLA A1101 complex was selected.
The α β heterodimer of the present invention having high affinity for the TSSELMAITR-HLA-A1101 complex is obtained by transferring the CDR regions of the α and β chain variable domains of the selected high affinity single-chain TCR to the corresponding positions of the α chain variable domain (SEQ ID NO:1) and β chain variable domain (SEQ ID NO:2) of the wild-type TCR.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the TSSELMAITR-HLA-a1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
Antibodies or fragments thereof that bind to the TCRs of the invention include anti-T cell or NK-cell determining antibodies, such as anti-CD 3 or anti-CD 28 or anti-CD 16 antibodies, whose binding to the TCR directs effector cells to better target cells. A preferred embodiment is the binding of a TCR of the invention to an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody. Specifically, the fusion molecule of the TCR and the anti-CD 3 single-chain antibody comprises a TCR alpha chain variable domain amino acid sequence which is one of SEQ ID NO 1 and SEQ ID NO 13-32; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 33-36.
The invention also relates to nucleic acid molecules encoding the inventive TCRs. The nucleic acid molecules of the invention may be in the form of DNA or in the form of RNA. The DNA may be the coding strand or the non-coding strand. For example, a nucleic acid sequence encoding a TCR of the present invention may be identical to or a degenerate variant of a nucleic acid sequence as set out in the figures of the present invention. By way of illustration of the meaning of "degenerate variant", as used herein, is meant a nucleic acid sequence which encodes a protein sequence having SEQ ID NO. 3, but differs from the sequence of SEQ ID NO. 5.
The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as genetically engineered host cells with the vectors or coding sequences of the invention.
The invention also includes isolated cells, particularly T cells, expressing a TCR of the invention. There are many methods suitable for T cell transfection using DNA or RNA encoding the high affinity TCRs of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the high affinity TCRs of the invention may be used for adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Rev Cancer 8(4): 299-308).
The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention.
The invention also provides a method of treating a disease comprising administering to a subject in need thereof an amount of a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention, or a pharmaceutical composition of the invention.
In the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Addition of one or several amino acids at the C-terminus and/or N-terminus will not generally alter the structure and function of the protein. Thus, the TCR of the invention also includes TCRs in which up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid (especially outside the CDR regions) of the TCR of the invention has been replaced by amino acids of similar or analogous nature, and still retain its functionality.
The invention also includes TCRs that are slightly modified from the TCRs of the invention. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the inventive TCR, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that result from glycosylation modifications made during synthesis and processing or during further processing steps of the inventive TCR. Such modification may be accomplished by exposing the TCR to an enzyme that effects glycosylation, such as mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to improve their resistance to proteolysis or to optimize solubility.
The TCR of the invention, the TCR complex or the TCR-transfected T cell of the invention may be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCRs, multivalent TCR complexes or cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.
In addition, the TCRs of the invention may be used alone, or in combination or coupling with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
Pharmaceutically acceptable carriers in therapeutic compositions can comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.
Once formulated, the compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated may be an animal; especially a human.
When the pharmaceutical composition of the present invention is used for practical treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, injections, oral agents and the like are exemplified.
These pharmaceutical compositions may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may be carried out in a conventional manner according to the dosage form.
The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the inventive TCR may be incorporated into a pellet or microcapsule carried by a slow release polymer, which pellet or microcapsule is then surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.
When the pharmaceutical composition of the present invention is used for practical treatment, the TCR or TCR complex of the present invention or the cells presenting the TCR of the present invention as an active ingredient can be determined reasonably according to the body weight, age, sex, degree of symptoms of each patient to be treated, and finally the reasonable amount is decided by a physician.
The main advantages of the invention are:
(1) the high affinity TCRs of the invention have at least 2-fold greater affinity and/or binding half-life for the TSSELMAITR-HLA-a1101 complex than wild-type TCRs.
(2) The high affinity TCRs of the invention are capable of specifically binding to the TSSELMAITR-HLA a1101, while cells transfected with the high affinity TCRs of the invention are capable of being specifically activated and proliferating.
(3) The effector cells transfected with the high affinity TCRs of the invention have a strong specific killing effect.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.
Materials and methods
The experimental materials used in the examples of the present invention are commercially available as such, unless otherwise specified, wherein e.coli DH5 α is available from Tiangen, e.coli BL21(DE3) is available from Tiangen, e.coli Tuner (DE3) is available from Novagen, and plasmid pET28a is available from Novagen.
Example 1 Generation of Stable Single chain TCR template chains with hydrophobic core mutations
The invention utilizes a site-directed mutagenesis method, and constructs a stable single-chain TCR molecule formed by connecting TCR alpha and beta chain variable domains by a flexible short peptide (linker) according to the patent document WO2014/206304, wherein the amino acid and DNA sequences of the stable single-chain TCR molecule are SEQ ID NO. 9 and SEQ ID NO. 10 respectively, as shown in FIG. 5a and FIG. 5 b. And the single-chain TCR molecule is taken as a template to screen the high-affinity TCR molecule. The amino acid sequences of the alpha chain variable domain (SEQ ID NO:3) and the beta chain variable domain (SEQ ID NO:4) of the template strand are shown in FIGS. 2a and 2 b; the corresponding DNA sequences are SEQ ID NO 5 and SEQ ID NO 6, respectively, as shown in FIGS. 3a and 3 b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are shown in SEQ ID NO 7 and 8, respectively, as shown in FIGS. 4a and 4 b.
The target gene carrying the template strand was digested simultaneously with Nco I and Not I, and ligated to pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli BL21(DE3) for expression.
Example 2 expression, renaturation and purification of the Stable Single-chain TCR constructed in example 1
The entire colony of BL21(DE3) containing the template strand of the recombinant plasmid pET28a prepared in example 1 was inoculated in LB medium containing kanamycin and cultured at 37 ℃ to OD6000.6-0.8, IPTG was added to a final concentration of 0.5mM and incubation was continued for 4h at 37 ℃. The cell pellet was harvested by centrifugation at 5000rpm for 15min, the cell pellet was lysed by Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and centrifuged at 6000rpm for 15min to collect the inclusion bodies. The inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantified by BCA method and split charged, and stored at-80 ℃ for further use.
To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30 min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM beta-mercaptoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4 ℃ for 10min, and then the renaturation solution was filled into a cellulose membrane dialysis bag with a cut-off of 4kDa, and the bag was placed in 1L of precooled water and stirred slowly at 4 ℃ overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20mM Tris-HCl pH 8.0), dialysis was continued at 4 ℃ for 8h, and then dialysis was continued overnight with the same fresh buffer. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare), the protein was purified using a 0-1M NaCl linear gradient eluent formulated in 20mM Tris-HCl pH 8.0, the collected fractions were subjected to SDS-PAGE analysis, the fractions containing single-stranded TCR were concentrated and then further purified using a gel filtration column (Superdex 7510/300, GE Healthcare), and the target fraction was also subjected to SDS-PAGE analysis.
The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions are as follows: the chromatographic column Agilent Bio SEC-3(300A, phi 7.8X 300mM) and the mobile phase are 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214 nm.
Example 3 binding characterisation
BIAcore analysis
The BIAcore T200 real-time assay system was used to detect the binding activity of the TCR molecules to the TSSELMAITR-HLA-A1101 complex. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10mM sodium acetate buffer, pH 4.77), and then the antibody was passed through CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process at a coupling level of about 15,000 RU. The conditions are as follows: the temperature is 25 ℃, and the PH value is 7.1-7.5.
The low concentration of streptavidin was flowed over the antibody coated chip surface, then TSSELMAITR-HLA-A1101 complex was flowed over the detection channel, the other channel served as a reference channel, and 0.05mM biotin was flowed over the chip at a flow rate of 10. mu.L/min for 2min to block the remaining binding sites of streptavidin. The affinity was determined by single cycle kinetic assay, in which the TCR was diluted with HEPES-EP buffer (10mM HEPES,150mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4) to several different concentrations, passed over the chip surface sequentially at a flow rate of 30. mu.L/min, with a binding time of 120s for each injection, and dissociated for 600s after the end of the last injection. At the end of each assay run, the chip was regenerated with 10mM Gly-HCl pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software.
The TSSELMAITR-HLA-A1101 complex is prepared as follows:
a. and (3) purification: collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.
Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5 min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15 min; discarding the supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation: the synthesized short peptide TSSELMAITR (Beijing Baisheng Gene technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 8M Urea, 20mM Tris pH 8.0, 10mM DTT and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. TSSELMAITR peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure of the renaturation.
c. And (3) purification after renaturation: the renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5ml bed volume). The protein was eluted using an Akta purifier (GE general electric) with a 0-400mM NaCl linear gradient prepared in 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, the peak fractions were collected, and the purity was checked by SDS-PAGE.
d. Biotinylation: the purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while displacing the buffer to 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. mu. M D-Biotin, 100. mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine the completion of biotinylation.
e. Purification of biotinylated complex: the biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using Akta purifier (GE general electric Co., Ltd.)TM16/60S200 HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein are combined together,concentration was performed using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by addition of the protease inhibitor cocktail (Roche).
Example 4 Generation of high affinity TCR
Phage display technology is one means of generating libraries of TCR high affinity variants to screen for high affinity variants. The TCR phage display and screening methods described by Li et al ((2005) Nature Biotech 23(3):349-354) were applied to the single-chain TCR templates in example 1. Libraries of high affinity TCRs were created by mutating the CDR regions of the template chains and panning was performed. The phage library after several rounds of panning has specific binding with corresponding antigen, and monoclonal is picked from the phage library and analyzed.
Mutations in the CDR regions of the selected high affinity single chain TCR were introduced into the corresponding sites in the variable domain of the α β heterodimeric TCR and their affinity for the TSSELMAITR-HLA-A1101 complex was determined by BIAcore. The introduction of the high affinity mutation points in the CDR regions described above is performed by site-directed mutagenesis methods well known to those skilled in the art. The amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in FIG. 1a (SEQ ID NO:1) and FIG. 1b (SEQ ID NO:2), respectively.
It should be noted that to obtain a more stable soluble TCR for more convenient assessment of binding affinity and/or binding half-life between the TCR and the TSSELMAITR-HLA A1101 complex, the α β heterodimeric TCR may be a TCR in which a cysteine residue is introduced in the constant region of the α and β chains, respectively, to form an artificial interchain disulfide bond, in this example the amino acid sequences of the TCR α and β chains after introduction of the cysteine residue are shown in FIGS. 6a (SEQ ID NO:11) and 6b (SEQ ID NO:12), respectively, and the introduced cysteine residue is shown in bold letters.
Extracellular sequence genes of TCR α and β chains to be expressed were synthesized and inserted into expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning a Laboratory Manual (third edition, Sambrook and Russell), with upstream and downstream Cloning sites being NcoI and NotI, respectively. Mutations in CDR regions are introduced by overlap pcr (overlap pcr), well known to those skilled in the art. The insert was confirmed by sequencing without error.
Example 5 expression, renaturation and purification of high affinity TCR
The expression vectors of TCR alpha and beta chains are transformed into expression bacteria BL21(DE3) by chemical transformation method, and the bacteria are grown in LB culture solution and OD600Inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times at 0.6 final induction with final concentration of 0.5mM IPTG, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM Tris (pH 8.1).
The solubilized TCR α and β chains were rapidly mixed in 5M Urea, 0.4M arginine, 20mM Tris (pH 8.1),3.7mM cystamine,6.6mM β -mercapoethylamine (4 ℃) at a 1:1 mass ratio to a final concentration of 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA.
Example 6 BIAcore analysis results
The method described in example 3 was used to detect the affinity of α β heterodimeric TCR and TSSELMAITR-HLA-a1101 complex incorporating high affinity CDR regions.
The invention obtains the amino acid sequences of the alpha chain and beta chain variable domains of a novel TCR, which are respectively shown in figures 7(1) - (20) and figures 8(1) - (4). Since the CDR regions of the TCR molecules determine their affinity for the corresponding pMHC complex, one skilled in the art would be able to expect that α β heterodimeric TCRs incorporating high affinity mutations also have high affinity for the TSSELMAITR-HLA-a1101 complex. The expression vector was constructed using the method described in example 4, the above α β heterodimeric TCR introduced with high affinity mutation was expressed, renatured and purified using the method described in example 5, and then its affinity to the TSSELMAITR-HLA-a1101 complex was determined using BIAcore T200, as shown in table 2 below.
TABLE 2
Figure BDA0002494521140000261
Figure BDA0002494521140000271
As can be seen from table 2 above, the affinity of the heterodimeric TCR was at least 2-fold greater than the affinity of the wild-type TCR for the TSSELMAITR-HLA-a1101 complex.
Example 7 expression, renaturation and purification of fusions of anti-CD 3 antibodies with high affinity α β heterodimeric TCRs
Fusion molecules were prepared by fusing an anti-CD 3 single chain antibody (scFv) to an α β heterodimeric TCR. The scFv of anti-CD 3 is fused to the β chain of the TCR, which β chain may comprise the β chain variable domain of any of the above-described high affinity α β heterodimeric TCRs, and the TCR α chain of the fused molecule may comprise the α chain variable domain of any of the above-described high affinity α β heterodimeric TCRs.
Construction of fusion molecule expression vectors
1. Construction of alpha chain expression vector: the target gene carrying the alpha chain of the α β heterodimeric TCR was double-digested with Nco i and Not i, and ligated to pET28a vector double-digested with Nco i and Not i. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for inversion overnight, positive clones were picked for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli Tuner (DE3) for expression.
2. Construction of anti-CD 3(scFv) -beta chain expression vector: by the overlap PCR method, primers were designed to link the anti-CD 3 scFv and the high-affinity heterodimeric TCR beta chain gene with the short linker peptide GGGGGGS (SEQ ID NO:31) in between, and the anti-CD 3 scFv was made to bear the restriction endonuclease sites NcoI (CCATGG (SEQ ID NO:32)) and Not I (GCGGCCGC (SEQ ID NO:33)) to the gene fragment of the fusion protein of the high-affinity heterodimeric TCR beta chain. The PCR amplification product was digested simultaneously with Nco I and Not I, and ligated with pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α competent cells, coated with LB plates containing kanamycin, inverted cultured overnight at 37 ℃, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli Tuner (DE3) competent cells for expression.
Expression, renaturation and purification of fusion proteins
The expression plasmids were transformed into E.coli Tuner (DE3) competent cells, respectively, and LB plates (kanamycin 50. mu.g/mL) were plated and incubated at 37 ℃ overnight. The next day, the selected clones were inoculated into 10mL LB liquid medium (kanamycin 50. mu.g/mL) for 2-3h of culture, inoculated into 1L of LB medium at a volume ratio of 1:100, cultured until OD600 became 0.5-0.8, and added with 1mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000rpm for 10 min. The cells were washed once with PBS buffer and aliquoted, and 200mL of the cells from the bacterial culture were lysed with 5mL of BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000g for 15 min. 4 detergent washes were then performed to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove the detergent and salts. Finally, inclusion bodies were dissolved in a buffer solution containing 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM Tris, pH 8.1, and the inclusion body concentration was measured, and they were stored frozen at-80 ℃ after being dispensed.
The TCR α chain and the anti-CD 3(scFv) - β chain after solubilization were separated by a 2: 5 in 5M Urea (urea),0.4M L-arginine (L-arginine),20mM Tris pH 8.1,3.7mM cystamine,6.6mM β -mer capoethylamine (4 ℃ C.), final concentrations of α chain and anti-CD 3(scFv) - β chain were 0.1mg/mL, 0.25mg/mL, respectively.
After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and after 12 hours, the deionized water was changed to a buffer (10mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. mu.M filter and then purified by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The eluted peaks contain TCR alpha chain and anti-CD 3(scFv) -beta chain dimers of which the renaturation was successful TCR alpha chain was confirmed by SDS-PAGE gel. The TCR fusion molecules were then further purified by size exclusion chromatography (S-10016/60, GE healthcare) and re-purified on an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The purity of the purified TCR fusion molecule was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA.
Example 8 activation function experiment of Effector cells transfected with high affinity TCR of the invention against short peptide-loaded T2 cells
IFN- γ is a potent immunomodulatory factor produced by activated T lymphocytes, and thus the present example examines the IFN- γ numbers by ELISPOT assays well known to those skilled in the art to verify the activation function and antigen specificity of cells transfected with the high affinity TCR of the invention. The high affinity TCRs of the invention were transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells and CD3+ T cells transfected with wild type TCRs (WT-TCRs) or other TCRs (a6) in the same volunteers as controls. The target cells used were T2-A11 loaded with AFP antigen short peptide TSSELMAITR (i.e., T2 cells transfected with HLA-A1101, the same applies below), T2-A11 loaded with other short peptides, and unloaded T2-A11.
The following experiments were carried out in two batches (I), (II) one after the other:
(I) the high affinity TCR can be learned from Table 2, respectively
Figure BDA0002494521140000291
(II) the high affinity TCR is shown in Table 2 as
Figure BDA0002494521140000292
Both batches were subjected to the following experimental procedure: first, an ELISPOT plate was prepared. ELISPOT flat plate ethanol activation coatingOvernight at 4 ℃. Day 1 of the experiment, coating was removed, washed and blocked, incubated at room temperature for two hours, blocking solution removed, and the components of the experiment were added to ELISPOT plates: target cells are 1 x 1042 x 10 effector cells per well3One well (calculated as the positive rate of antibody) and two duplicate wells were set. Adding corresponding short peptide to make the final concentration of short peptide in ELISPOT pore plate be 1X 10- 6And M. Incubation overnight (37 ℃, 5% CO)2). On day 2 of the experiment, the plates were washed and subjected to secondary detection and color development, dried, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).
Experimental results as shown in fig. 12a and 12b, the effector cells transfected with the high affinity TCR of the present invention activated more significantly than the wild-type effector cells against the target cells loaded with the AFP antigen short peptide TSSELMAITR, while the effector cells transfected with other TCRs were inactivated; meanwhile, effector cells transfected with the high affinity TCRs of the invention are not activated by other short peptide-loaded or unloaded target cells.
Example 9 functional assay for activation of Effector cells transfected with high affinity TCRs of the invention against tumor cell lines
This example again demonstrates the activation function and specificity of effector cells transfected with the high affinity TCRs of the invention using tumor cell lines. Again, detection is by ELISPOT assays well known to those skilled in the art. The high affinity TCRs of the invention were transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells and CD3+ T cells transfected with other TCRs (a6) or null transfected (NC) from the same volunteer as negative controls. The following experiments were carried out in two batches (I), (II) one after the other:
(I) the high affinity TCR can be learned from Table 2, respectively
Figure BDA0002494521140000293
The AFP positive tumor cell line used in this batch was SK-MEL-28-AFP (AFP overexpression) and the negative tumor cell line was SK-MEL-28-AFP (AFP overexpression)SNU423, HUCCT1, HepG2 and SK-MEL-28. First, an ELISPOT plate was prepared. ELISPOT plate ethanol activation coating, 4 degrees C overnight. On day 1 of the experiment, the coating was removed, washed and blocked, incubated at room temperature for two hours, the blocking solution was removed, and the components of the experiment were added to the ELISPOT plate in the following order: the target cell is 2X 104 2X 10 effector cells per well3One well (calculated as the positive rate of antibody) and two duplicate wells were set. Incubation overnight (37 ℃, 5% CO)2). On day 2 of the experiment, the plates were washed and subjected to secondary detection and color development, dried, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).
(II) the high affinity TCRs are known from Table 2 as TCR1 (alpha chain variable domain SEQ ID NO:13, beta chain variable domain SEQ ID NO:2), TCR2 (alpha chain variable domain SEQ ID NO:14, beta chain variable domain SEQ ID NO:2) and TCR3 (alpha chain variable domain SEQ ID NO:15, beta chain variable domain SEQ ID NO:2), respectively. The AFP-positive tumor cell lines used in this batch were SK-MEL-28-AFP (AFP over-expression), and the negative tumor cell lines were Huh-1, HepG2, SK-MEL-28 and HUCCT 1. The components of the assay were added to the ELISPOT plate in the following order: the target cell is 2X 104Number/well, effector cells 8X 103One well (calculated as the antibody positive rate) and two duplicate wells were set. The remaining steps were the same as batch (I).
The experimental results are shown in fig. 13a and 13b, and the effector cells transfected with the high affinity TCR of the invention can be well activated by an AFP positive tumor cell line, with significant activation effect and no non-specificity. T cells transfected with other TCRs or free transfected were not activated by AFP positive tumor cell lines.
Example 10 killing function experiment of effector cells transfected with high affinity TCR of the invention against gradient short peptide-loaded T2 cells
Lactate Dehydrogenase (LDH) is abundant in the cytoplasm, normally cannot pass through the cell membrane, and is released to the outside of the cell when the cell is damaged or dies, and the LDH activity in the cell culture solution is proportional to the number of cell death. This example demonstrates the killing function of cells transfected with a TCR of the invention by measuring LDH release by non-radioactive cytotoxicity assays well known to those skilled in the art. This test is a colorimetric substitution test for the 51 Cr-release cytotoxicity test, and quantitatively determines LDH released after cell lysis. LDH released in the medium was detected using a 30min coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader. The formula is calculated as% cytotoxicity 100% × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximal-target cell spontaneous).
This example uses CD3+ T cells isolated from the blood of healthy volunteers transfected with the high affinity TCR of the invention as effector cells and CD3+ T cells transfected with other TCRs (a6) or free-standing (NC) from the same volunteer as negative controls. Wherein the high affinity TCRs and their numbering are known from Table 2 as TCR1 (alpha chain variable domain SEQ ID NO:13, beta chain variable domain SEQ ID NO:2), TCR2 (alpha chain variable domain SEQ ID NO:14, beta chain variable domain SEQ ID NO:2) and TCR3 (alpha chain variable domain SEQ ID NO:15, beta chain variable domain SEQ ID NO:2), respectively. The target cells were T2-A11 (T2 cells transfected with HLA-A1101, the same below) loaded with TSSELMAITR peptide, T2-A11 loaded with other short peptides or unloaded.
LDH plates were prepared first, 3X 10 target cells were first aligned4Single cell/well, effector cell 3X 104Adding each cell/well (calculated according to the positive rate of the antibody) into the corresponding well, then adding AFP antigen short peptide TSSELMAITR into the experimental group, and enabling the final concentration of the short peptide in an ELISPOT pore plate to be 1 × 10 in sequence-13M to 1X 10-6M, 8 gradients in total; adding other short peptides into the control group, and making the final concentration of the short peptides to be 1 × 10-8M to 1X 10-6M,3 gradients are provided, and three multiple wells are provided. Meanwhile, an effector cell spontaneous hole, a target cell maximum hole, a volume correction control hole and a culture medium background control hole are arranged. Incubation overnight (37 ℃, 5% CO)2). On day 2 of the experiment, color development was detected, and after termination of the reaction, the absorbance was recorded at 490nm using a microplate reader (Bioteck).
The experimental results are shown in fig. 14, and the effector cells transfected with the high affinity TCR of the present invention showed strong killing effect against the target cells gradient-loaded with the AFP antigen short peptide TSSELMAITR, and reacted at the lower concentration of the specific short peptide, while the effector cells transfected with other TCRs or empty transfection had no killing effect from the beginning; meanwhile, the effector cells transfected with the high-affinity TCR have no killing effect on target cells loaded with other short peptides or unloaded, which shows that the effector cells have good specificity.
Example 11 killing function experiment of Effector cells transfected with high affinity TCR of the invention against tumor cell lines
This example also demonstrates the killing function of cells transfected with a TCR of the invention by measuring LDH release by nonradioactive cytotoxicity assays well known to those skilled in the art. The LDH assay of this example transfected the high affinity TCRs of the invention with CD3+ T cells isolated from the blood of healthy volunteers as effector cells and the same volunteers transfected with other TCRs (a6) or empty transfected (NC) CD3+ T cells as negative controls.
The following experiments were carried out in three batches (I), (II), (III) one after the other:
(I) the high affinity TCRs and their numbering are known from Table 2 as TCR6 (alpha chain variable domain SEQ ID NO:18, beta chain variable domain SEQ ID NO:2), TCR1 (alpha chain variable domain SEQ ID NO:13, beta chain variable domain SEQ ID NO:2), TCR5 (alpha chain variable domain SEQ ID NO:17, beta chain variable domain SEQ ID NO:2) and TCR3 (alpha chain variable domain SEQ ID NO:15, beta chain variable domain SEQ ID NO:2), respectively. The AFP positive tumor cell lines used in this batch were: HepG2-A11(HLA-A1101 over-expression), SK-MEL-28-AFP (AFP over-expression) and SNU423-AFP (AFP over-expression), and negative tumor cell lines HepG2, SK-MEL-28 and SNU 423. The experimental procedure was as follows: LDH plates were first prepared and the individual components of the assay were added to the plates in the following order: target cell 3X 104Single cell/well, effector cell 3X 104Individual cells/well (calculated as antibody positivity) were added to the corresponding wells and three replicate wells were set. Meanwhile, an effector cell spontaneous hole, a target cell maximum hole, a volume correction control hole and a culture medium background control hole are arranged. Incubation overnight (37 ℃, 5% CO)2). Experiment ofOn day 2, color development was detected, and after termination of the reaction, absorbance was recorded at 490nm using a microplate reader (Bioteck).
(II) the high affinity TCRs and their numbering are known from Table 2 as TCR2 (alpha chain variable domain SEQ ID NO:14, beta chain variable domain SEQ ID NO:2) and TCR13 (alpha chain variable domain SEQ ID NO:21, beta chain variable domain SEQ ID NO:2), respectively. The AFP-positive tumor cells used in this batch were SK-MEL-28-AFP (AFP over-expression), and the negative tumor cells were HepG2, HUCCT1, SK-MEL-28 and SNU 423. The experimental procedure was the same as batch (I).
(III) the high affinity TCR and its numbering is known from Table 2 as TCR4 (alpha chain variable domain SEQ ID NO:16, beta chain variable domain SEQ ID NO: 2). The positive tumor cell line used in this batch was SK-MEL-28-AFP (AFP over-expression), and the negative tumor cell line was LCLs-150909A, SK-MEL-28. LDH plates were first prepared and the individual components of the assay were added to the plates in the following order: target cell 2X 104Single cell/well, effector cell 2X 104Individual cells/well (calculated as antibody positivity) were added to the corresponding wells and three replicate wells were set. The remaining experimental procedures were the same as batch (I).
Experimental results as shown in fig. 15a, 15b and 15c, effector cells transfected with the high affinity TCRs of the invention still showed strong killing efficacy against AFP positive tumor cell lines, while effector cells transfected with other TCRs or free-standing were essentially unreactive; at the same time, the T cells transfected with the high affinity TCR of the invention have essentially no killing of the negative tumor cell line. This experiment further embodies the very good specific killing function of cells transfected with the high affinity TCR of the invention.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Guangdong Xiangxue accurate medical technology Limited
<120> a high affinity T cell receptor recognizing AFP antigen
<130> P2020-0849
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 1
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Ala Thr
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 2
<211> 116
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 2
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Leu
85 90 95
Val Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu
115
<210> 3
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 3
Ala Gln Ser Val Thr Gln Pro Asp Ile His Leu Thr Val Ser Glu Gly
1 5 10 15
Glu Ser Val Glu Ile Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Pro Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Ala Thr
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Leu Ser Val
100 105 110
Thr Pro
<210> 4
<211> 116
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 4
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Val Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu
85 90 95
Val Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Asp
115
<210> 5
<211> 342
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
gctcaatctg ttactcaacc ggacatccat ctgactgtat ctgaaggcga aagcgtggaa 60
attcgctgca actattccta cggtgccact ccgtacctgt tttggtacgt acagtctccg 120
ggtcagggtc tgcagctgct gctgaaatac ttctctggtg acaccctggt tcagggtatc 180
aaaggtttcg aggcggaatt taaacgctct cagtcttcct tcaacctgcg caagccgtct 240
gttcacccgt ccgacgctgc ggaatacttc tgtgctgtgg ttgctaccga ctcttggggt 300
aaactgcagt ttggcgccgg tactcagctg tctgttaccc ca 342
<210> 6
<211> 348
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
ggtgcaggtg tttcccagtc cccacgttac ctgtccgtga aacgtggtca ggacgttgcg 60
ctgcgttgtg atccaatctc cggtcatgtt tccctgttct ggtatcagca ggcaccgggc 120
cagggtccag aattcctgac ttacttccag aatgaagcgc agctggacaa aagcggcctg 180
ccgtccgacc gtttcttcgc tgaacgtccg gagggttccg tgtctactct gaaaatccag 240
cgcgtgcagc cggaggactc cgcagtttac ttctgcgcgt cctctctggt agctggtgca 300
cgtaccgaca cccagtactt cggtccaggc acccgtctga ccgttgat 348
<210> 7
<211> 24
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 7
Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly
1 5 10 15
Gly Gly Ser Glu Gly Gly Thr Gly
20
<210> 8
<211> 72
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
ggcggtggta gcgagggtgg cggttccgaa ggtggcggca gcgaaggcgg tggttccgag 60
ggtggcaccg gc 72
<210> 9
<211> 254
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 9
Ala Gln Ser Val Thr Gln Pro Asp Ile His Leu Thr Val Ser Glu Gly
1 5 10 15
Glu Ser Val Glu Ile Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Pro Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Ala Thr
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Leu Ser Val
100 105 110
Thr Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser
115 120 125
Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Gly Ala Gly Val Ser Gln
130 135 140
Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly Gln Asp Val Ala Leu Arg
145 150 155 160
Cys Asp Pro Ile Ser Gly His Val Ser Leu Phe Trp Tyr Gln Gln Ala
165 170 175
Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr Phe Gln Asn Glu Ala Gln
180 185 190
Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg Phe Phe Ala Glu Arg Pro
195 200 205
Glu Gly Ser Val Ser Thr Leu Lys Ile Gln Arg Val Gln Pro Glu Asp
210 215 220
Ser Ala Val Tyr Phe Cys Ala Ser Ser Leu Val Ala Gly Ala Arg Thr
225 230 235 240
Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Asp
245 250
<210> 10
<211> 762
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
gctcaatctg ttactcaacc ggacatccat ctgactgtat ctgaaggcga aagcgtggaa 60
attcgctgca actattccta cggtgccact ccgtacctgt tttggtacgt acagtctccg 120
ggtcagggtc tgcagctgct gctgaaatac ttctctggtg acaccctggt tcagggtatc 180
aaaggtttcg aggcggaatt taaacgctct cagtcttcct tcaacctgcg caagccgtct 240
gttcacccgt ccgacgctgc ggaatacttc tgtgctgtgg ttgctaccga ctcttggggt 300
aaactgcagt ttggcgccgg tactcagctg tctgttaccc caggcggtgg tagcgagggt 360
ggcggttccg aaggtggcgg cagcgaaggc ggtggttccg agggtggcac cggcggtgca 420
ggtgtttccc agtccccacg ttacctgtcc gtgaaacgtg gtcaggacgt tgcgctgcgt 480
tgtgatccaa tctccggtca tgtttccctg ttctggtatc agcaggcacc gggccagggt 540
ccagaattcc tgacttactt ccagaatgaa gcgcagctgg acaaaagcgg cctgccgtcc 600
gaccgtttct tcgctgaacg tccggagggt tccgtgtcta ctctgaaaat ccagcgcgtg 660
cagccggagg actccgcagt ttacttctgc gcgtcctctc tggtagctgg tgcacgtacc 720
gacacccagt acttcggtcc aggcacccgt ctgaccgttg at 762
<210> 11
<211> 209
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 11
Met Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu
1 5 10 15
Gly Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro
20 25 30
Tyr Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu
35 40 45
Leu Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe
50 55 60
Glu Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro
65 70 75 80
Ser Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Ala
85 90 95
Thr Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val
100 105 110
Val Thr Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg
115 120 125
Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp
130 135 140
Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr
145 150 155 160
Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser
165 170 175
Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe
180 185 190
Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Cys Ser Pro Glu Ser
195 200 205
Ser
<210> 12
<211> 247
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 12
Met Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg
1 5 10 15
Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser
20 25 30
Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr
35 40 45
Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp
50 55 60
Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile
65 70 75 80
Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser
85 90 95
Leu Val Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr
100 105 110
Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val
115 120 125
Ala Val Phe Glu Pro Ser Glu Cys Glu Ile Ser His Thr Gln Lys Ala
130 135 140
Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu
145 150 155 160
Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp
165 170 175
Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala
180 185 190
Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg
195 200 205
Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp
210 215 220
Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala
225 230 235 240
Glu Ala Trp Gly Arg Ala Asp
245
<210> 13
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 13
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Leu
85 90 95
Lys Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 14
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 14
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Met
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 15
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 15
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Met
85 90 95
Gln Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 16
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 16
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Tyr
85 90 95
Gln Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 17
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 17
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Leu
85 90 95
Ser Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 18
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 18
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Leu
85 90 95
Glu Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 19
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 19
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Ile
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 20
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 20
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Asp Leu
85 90 95
Ala Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 21
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 21
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Pro Asp Met
85 90 95
Lys Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 22
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 22
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Pro Ser Met
85 90 95
His Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 23
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 23
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Pro Ser Thr
85 90 95
Glu Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 24
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 24
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Asp Met
85 90 95
Asn Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 25
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 25
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Gly Leu
85 90 95
Pro Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 26
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 26
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Leu
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 27
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 27
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Leu
85 90 95
Gln Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 28
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 28
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Leu
85 90 95
Arg Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 29
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 29
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Met
85 90 95
Glu Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 30
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 30
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Met
85 90 95
His Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 31
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 31
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Met
85 90 95
Asn Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 32
<211> 114
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 32
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Ala Ser Met
85 90 95
Arg Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro
<210> 33
<211> 116
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 33
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Trp
85 90 95
Ile Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu
115
<210> 34
<211> 116
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 34
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Thr Met
85 90 95
Ile Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu
115
<210> 35
<211> 116
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 35
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Thr Leu
85 90 95
Leu Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu
115
<210> 36
<211> 116
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 36
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Thr Met
85 90 95
Leu Gly Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu
115
<210> 37
<211> 208
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 37
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Ala Thr
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp
115 120 125
Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser
130 135 140
Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp
145 150 155 160
Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
165 170 175
Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn
180 185 190
Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
<210> 38
<211> 246
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 38
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Leu
85 90 95
Val Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala
115 120 125
Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
130 135 140
Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser
145 150 155 160
Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro
165 170 175
Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu
180 185 190
Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn
195 200 205
His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
210 215 220
Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu
225 230 235 240
Ala Trp Gly Arg Ala Asp
245
<210> 39
<211> 255
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 39
Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val Ser Glu Gly
1 5 10 15
Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala Thr Pro Tyr
20 25 30
Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln Leu Leu Leu
35 40 45
Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys Gly Phe Glu
50 55 60
Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg Lys Pro Ser
65 70 75 80
Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Val Val Ala Thr
85 90 95
Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val Val
100 105 110
Thr Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp
115 120 125
Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser
130 135 140
Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp
145 150 155 160
Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
165 170 175
Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn
180 185 190
Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200 205
Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu
210 215 220
Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys
225 230 235 240
Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
245 250 255
<210> 40
<211> 295
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 40
Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala Lys Arg Gly
1 5 10 15
Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu
20 25 30
Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro Ser Asp Arg
50 55 60
Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu Lys Ile Gln
65 70 75 80
Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala Ser Ser Leu
85 90 95
Val Ala Gly Ala Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala
115 120 125
Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
130 135 140
Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser
145 150 155 160
Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro
165 170 175
Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu
180 185 190
Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn
195 200 205
His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
210 215 220
Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu
225 230 235 240
Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln
245 250 255
Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala
260 265 270
Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val
275 280 285
Lys Arg Lys Asp Ser Arg Gly
290 295

Claims (10)

1. A T Cell Receptor (TCR) comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, characterised in that it has the activity of binding TSSELMAITR-HLA a1101 complex and in that the amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence homology with the amino acid sequence set out in SEQ ID No. 1 and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence homology with the amino acid sequence set out in SEQ ID No. 2.
2. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in claim 1.
3. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR as claimed in claim 1, or the complement thereof.
4. A vector comprising the nucleic acid molecule of claim 3.
5. A host cell comprising the vector of claim 4 or a nucleic acid molecule of claim 3 integrated into the chromosome.
6. An isolated cell expressing a TCR as claimed in claim 1.
7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as claimed in claim 1, or a TCR complex as claimed in claim 2, or a cell as claimed in claim 6.
8. A method of treating a disease comprising administering to a subject in need thereof a TCR as claimed in claim 1, or a TCR complex as claimed in claim 2, or a cell as claimed in claim 6, or a pharmaceutical composition as claimed in claim 7, preferably wherein the disease is an AFP positive tumour, more preferably wherein the tumour is liver cancer.
9. Use of a T cell receptor as claimed in claim 1, a TCR complex as claimed in claim 2 or a cell as claimed in claim 6 for the preparation of a medicament for the treatment of a tumour, preferably wherein the tumour is an AFP positive tumour, more preferably wherein the tumour is liver cancer.
10. A method of preparing a T cell receptor according to claim 1, comprising the steps of:
(i) culturing the host cell of claim 5 so as to express the T cell receptor of claim 1;
(ii) isolating or purifying said T cells.
CN202010414690.0A 2020-05-15 2020-05-15 High-affinity T cell receptor for recognizing AFP antigen Pending CN113667008A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN202010414690.0A CN113667008A (en) 2020-05-15 2020-05-15 High-affinity T cell receptor for recognizing AFP antigen
PCT/CN2021/093947 WO2021228255A1 (en) 2020-05-15 2021-05-14 High-affinity t cell receptor capable of recognizing afp antigen
TW110117492A TW202144404A (en) 2020-05-15 2021-05-14 High-affinity t cell receptor capable of recognizing AFP antigen

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010414690.0A CN113667008A (en) 2020-05-15 2020-05-15 High-affinity T cell receptor for recognizing AFP antigen

Publications (1)

Publication Number Publication Date
CN113667008A true CN113667008A (en) 2021-11-19

Family

ID=78525994

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010414690.0A Pending CN113667008A (en) 2020-05-15 2020-05-15 High-affinity T cell receptor for recognizing AFP antigen

Country Status (3)

Country Link
CN (1) CN113667008A (en)
TW (1) TW202144404A (en)
WO (1) WO2021228255A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113321727A (en) * 2020-02-28 2021-08-31 香雪生命科学技术（广东）有限公司 T cell receptor for identifying AFP antigen short peptide and its coding sequence

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL2618835T3 (en) * 2010-09-20 2017-12-29 Biontech Cell & Gene Therapies Gmbh Antigen-specific t cell receptors and t cell epitopes
CN109879957B (en) * 2017-12-06 2022-03-18 香雪生命科学技术（广东）有限公司 High affinity T cell receptors for PRAME
CN110577590B (en) * 2018-06-08 2022-09-09 香雪生命科学技术（广东）有限公司 TCR capable of recognizing AFP antigen and encoding nucleic acid thereof
CN110938136B (en) * 2018-09-21 2022-09-09 香雪生命科学技术（广东）有限公司 High-affinity T cell receptor for recognizing AFP antigen

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113321727A (en) * 2020-02-28 2021-08-31 香雪生命科学技术（广东）有限公司 T cell receptor for identifying AFP antigen short peptide and its coding sequence
CN113321727B (en) * 2020-02-28 2024-04-09 香雪生命科学技术（广东）有限公司 T cell receptor for identifying AFP antigen short peptide and coding sequence thereof

Also Published As

Publication number Publication date
TW202144404A (en) 2021-12-01
WO2021228255A1 (en) 2021-11-18

Similar Documents

Publication Publication Date Title
CN109879957B (en) High affinity T cell receptors for PRAME
CN112759641B (en) High-affinity TCR for recognizing Kras G12V
CN110198953B (en) High affinity TCRs for NY-ESO
US20230331805A1 (en) High-affinity tcr for recognizing afp antigen
EP4026844A1 (en) High-affinity t cell receptor that recognizes ssx2
CN111187345B (en) High-affinity TCR for identifying AFP
CN111662374B (en) High-affinity TCR for identifying AFP antigen
CN110938136B (en) High-affinity T cell receptor for recognizing AFP antigen
CN112409474B (en) High-affinity TCR (T cell receptor) for identifying SSX2 antigen
CN112390875B (en) High-affinity T cell receptor for identifying AFP
CN110156889B (en) High affinity HBs T cell receptor
CN114057864B (en) High affinity TCR for identifying AFP
WO2021228255A1 (en) High-affinity t cell receptor capable of recognizing afp antigen
CN112300268B (en) High affinity T cell receptors recognizing the NY-ESO-1 antigen
CN113512124A (en) High-affinity TCR (T cell receptor) for recognizing HPV16
CN113801217A (en) High-affinity T cell receptor for recognizing HPV (human papilloma Virus) antigen
CN112940106A (en) High affinity AFPT cellular receptors
WO2023221959A1 (en) High-affinity t cell receptors for recognizing mage and use thereof
WO2022206860A1 (en) T cell receptor for afp
WO2023179768A1 (en) High-affinity tcr for identifying mage-a4 antigen, and sequence and use thereof
WO2023040946A1 (en) High-affinity tcr recognizing ssx2
CN115490773A (en) High-affinity T cell receptor aiming at AFP antigen
CN115677846A (en) High affinity T cell receptors for the antigen SSX2
CN116135879A (en) High affinity TCR against AFP
CN117659163A (en) High-affinity T cell receptor for identifying AFP and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination