CN113321726B - T cell receptor for recognizing HPV - Google Patents

T cell receptor for recognizing HPV Download PDF

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CN113321726B
CN113321726B CN202010129637.6A CN202010129637A CN113321726B CN 113321726 B CN113321726 B CN 113321726B CN 202010129637 A CN202010129637 A CN 202010129637A CN 113321726 B CN113321726 B CN 113321726B
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tcr
amino acid
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chain
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CN113321726A (en
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李懿
胡静
李俊
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Xiangxue Life Science Technology Guangdong Co ltd
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Xiangxue Life Science Technology Guangdong Co ltd
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Priority to PCT/CN2021/078275 priority patent/WO2021170115A1/en
Priority to TW110107149A priority patent/TW202140540A/en
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Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding to a short peptide YMLDLQPET derived from HPV 16E 7 antigen, which antigen short peptide YMLDLQPET can form a complex with HLA a0201 and be presented on the cell surface together. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells transduced with the TCRs of the invention.

Description

T cell receptor for recognizing HPV
Technical Field
The present invention relates to TCRs capable of recognizing HPV 16E 7 antigen-derived short peptides and coding sequences thereof, HPV 16E 7-specific T cells obtained by transduction of the above TCRs, and their use in the prevention and treatment of HPV 16E 7-related diseases.
Background
The HPV 16E 7 gene is one of the early region genes of the Human Papillomavirus (HPV) genome, which encodes an E7 protein that is a small acidic protein of about 100 amino acids. The most prevalent type in cervical cancer worldwide is HPV16, accounting for 50% -60% of detected cases (ACTA ACAD MED SIN,2007,29 (5): 678-684), with high-risk HPV 16E 7 protein being one of the major causes of HPV-induced cervical cancer. In HPV-infected head and neck tumors, E7 oncoproteins act as immunosuppression, causing cell cycle abnormalities mainly by blocking normal P16 protein expression leading to canceration (journal of china ear, nose, throat, skull base surgery, 2017, 23 (6): 594-598). There are studies showing that HPV 16E 7 is also a stronger oncogene in induced anal cancers (virology.201110ec 20;421 (2): 114-118.). In addition, HPV 16E 7 causes diseases such as Conjunctival Intraepithelial Neoplasia (CIN) and keratoconjunctival invasive Squamous Cell Carcinoma (SCC) (J.International patent application No. 2018;18 (6): 1047-1050). YMLDLQPET (SEQ ID NO: 9) is a short peptide derived from the HPV 16E 7 antigen, a target for HPV 16E 7-related disease treatment.
T cell adoptive immunotherapy involves transferring reactive T cells specific for a target cell antigen into a patient to act against the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that is capable of recognizing the corresponding antigenic short peptide on the surface of target cells. In the immune system, the direct physical contact of T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-short peptide specific TCRs to the short peptide-major histocompatibility complex (pMHC complex), and then the interaction of T cells and other cell membrane surface molecules of both APCs occurs, causing 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. Accordingly, those skilled in the art have focused on isolating TCRs specific for HPV16E7 antigen peptides and transducing the TCRs into T cells to obtain T cells specific for HPV16E7 antigen peptides, thereby allowing them to play a role in cellular immunotherapy.
Disclosure of Invention
The invention aims at providing a T cell receptor for recognizing HPV 16E 7 antigen short peptide.
In a first aspect of the invention there is provided a T Cell Receptor (TCR) capable of binding to YMLDLQPET-HLA a0201 complex.
In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain having an amino acid sequence of CDR3 of AVNPRYGNKLV (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the TCR β chain variable domain is ATSDRGQGAFGEQY (SEQ ID NO: 15).
In another preferred embodiment, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
αCDR1-DRVSQS (SEQ ID NO:10)
αCDR2-IYSNGD (SEQ ID NO:11)
Alpha CDR3-AVNPRYGNKLV (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-KGHDR (SEQ ID NO:13)
βCDR2-SFDVKD (SEQ ID NO:14)
βCDR3-ATSDRGQGAFGEQY (SEQ ID NO:15)。
In another preferred embodiment, the TCR comprises a TCR a chain variable domain that is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain is identical to SEQ ID NO:5, an amino acid sequence having at least 90% sequence identity.
In another preferred embodiment, the TCR comprises the alpha chain variable domain amino acid sequence SEQ ID NO. 1.
In another preferred embodiment, the TCR comprises the β chain variable domain amino acid sequence SEQ ID NO. 5.
In another preferred embodiment, the TCR is an αβ heterodimer comprising a TCR α chain constant region TRAC x 01 and a TCR β chain constant region TRBC1 x 01 or TRBC2 x 01.
In another preferred embodiment, the alpha chain amino acid sequence of the TCR is SEQ ID NO 3 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO 7.
In another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is single chain.
In another preferred embodiment, the TCR is formed by a linkage of an alpha chain variable domain and a beta chain variable domain via a peptide linker sequence.
In another preferred embodiment, the TCR has one or more mutations in the alpha chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, or 94, and/or the alpha chain J gene short peptide amino acid position 3, 5, or 7; and/or the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the β chain variable region, and/or the β chain J gene short peptide amino acid position 2, 4, or 6, wherein the amino acid position numbers are numbered as listed in IMGT (international immunogenetic information system).
In another preferred embodiment, the alpha chain variable domain amino acid sequence of the TCR comprises SEQ ID NO:32 and/or the beta chain variable domain amino acid sequence of the TCR comprises SEQ ID NO:34.
In another preferred embodiment, the amino acid sequence of the TCR is SEQ ID NO. 30.
In another preferred embodiment, the TCR comprises (a) all or part of a TCR a chain other than a transmembrane domain; and (b) all or part of the TCR β chain except the transmembrane domain;
And (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of the TCR chain constant domain.
In another preferred embodiment, the cysteine residues form an artificial disulfide bond between the α and β chain constant domains of the TCR.
In another preferred embodiment, the cysteine residues forming the artificial disulfide bond in the TCR are substituted at one or more of the sets of sites selected from:
Thr48 of tranc x 01 exon 1 and Ser57 of TRBC1 x 01 or TRBC2 x 01 exon 1;
Thr45 of tranc x 01 exon 1 and Ser77 of TRBC1 x 01 or TRBC2 x 01 exon 1;
tyr10 of TRAC x 01 exon 1 and Ser17 of TRBC1 x 01 or TRBC2 x 01 exon 1;
Thr45 of TRAC x 01 exon 1 and Asp59 of TRBC1 x 01 or TRBC2 x 01 exon 1;
ser15 of TRAC x 01 exon 1 and Glu15 of TRBC1 x 01 or TRBC2 x 01 exon 1;
arg53 of TRAC x 01 exon 1 and Ser54 of TRBC1 x 01 or TRBC2 x 01 exon 1;
TRAC.01 exon 1 Pro89 and TRBC 1.01 or TRBC 2.01 exon 1 Ala19; and
Tyr10 of TRAC x 01 exon 1 and Glu20 of TRBC1 x 01 or TRBC2 x 01 exon 1.
In another preferred embodiment, the alpha chain amino acid sequence of the TCR is SEQ ID NO 26 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO 28.
In another preferred embodiment, the TCR has an artificial interchain disulfide linkage between the α chain variable region and the β chain constant region.
In another preferred embodiment, the cysteine residues forming the artificial interchain disulfide bond in the TCR are substituted at one or more of the sites selected from the group consisting of:
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 TRBC 1x 01 or TRBC2 x 01;
Amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; or (b)
Amino acid 47 of TRAV and amino acid 60 of TRBC1 x 01 or TRBC2 x 01 exon 1.
In another preferred embodiment, the TCR comprises an alpha chain variable domain and a beta chain variable domain, and all or part of the beta chain constant domain, except the transmembrane domain, but does not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.
In another preferred embodiment, the C-or N-terminus of the alpha and/or beta chain of the TCR is conjugated to a conjugate.
In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these. Preferably, the therapeutic agent is an anti-CD 3 antibody.
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 according to the first aspect of the invention.
In a third aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule of the first aspect of the invention or a complement thereof.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 2 or SEQ ID NO. 33 encoding a TCR alpha chain variable domain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 6 or SEQ ID NO. 35 encoding a TCR.beta.chain variable domain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 4 encoding a TCR alpha chain and/or comprises the nucleotide sequence SEQ ID NO. 8 encoding a TCR beta chain.
In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
In a fifth aspect of the invention there is provided an isolated host cell comprising a vector according to the fourth aspect of the invention or a nucleic acid molecule according to the third aspect of the invention integrated into the genome.
In a sixth aspect of the invention, there is provided a cell transduced with a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; preferably, the cells are T cells or stem cells.
In a seventh aspect of the invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of the first aspect of the invention, a TCR complex of the second aspect of the invention, a nucleic acid molecule of the third aspect of the invention, a carrier of the fourth aspect of the invention, or a cell of the sixth aspect of the invention.
In an eighth aspect of the invention there is provided the use of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumour or autoimmune disease, preferably the tumour is cervical cancer.
In an eighth aspect of the invention there is provided a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention, for use in the manufacture of a medicament for the treatment of a tumour or autoimmune disease, preferably the tumour is cervical cancer.
In a ninth aspect, the invention provides a method of treating a disease comprising administering to a subject in need thereof an appropriate amount of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth 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 a tumor, preferably the tumor is cervical cancer, head and neck cancer, anal cancer.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIGS. 1a, 1b, 1c, 1d, 1e and 1f are, respectively, TCR alpha chain variable domain amino acid sequence, TCR alpha chain variable domain nucleotide sequence, TCR alpha chain amino acid sequence, TCR alpha chain nucleotide sequence, TCR alpha chain amino acid sequence with a leader sequence, and TCR alpha chain nucleotide sequence with a leader sequence.
FIGS. 2a, 2b, 2c, 2d, 2e and 2f are, respectively, TCR β chain variable domain amino acid sequence, TCR β chain variable domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, TCR β chain amino acid sequence with a leader sequence, and TCR β chain nucleotide sequence with a leader sequence.
FIG. 3 shows the results of double cationic staining of CD8 + and tetramer-PE of monoclonal cells.
FIGS. 4a and 4b are the amino acid and nucleotide sequences, respectively, of a soluble TCR alpha chain.
FIGS. 5a and 5b are the amino acid and nucleotide sequences, respectively, of a soluble TCR β chain.
FIGS. 6a and 6b are gel diagrams of soluble TCR obtained after purification. The right lanes in fig. 6a and 6b are respectively a reducing gel and a non-reducing gel, and the left lanes are molecular weight markers.
FIGS. 7a and 7b are the amino acid and nucleotide sequences, respectively, of a single chain TCR.
FIGS. 8a and 8b are the amino acid and nucleotide sequences, respectively, of the single chain TCR alpha chain variable domain.
FIGS. 9a and 9b are the amino acid and nucleotide sequences, respectively, of the single chain TCR β chain variable domain.
FIGS. 10a and 10b are the amino acid and nucleotide sequences, respectively, of a single chain TCR linkage sequence (linker).
FIG. 11 is a gel diagram of the soluble single chain TCR obtained after purification. The leftmost lane is non-reducing gel, the middle lane is molecular weight marker, and the rightmost lane is reducing gel.
FIG. 12 is a chart of BIAcore kinetics of binding of soluble TCR of the invention to YMLDLQPET-HLA A A0201 complex.
FIG. 13 is a chart of BIAcore kinetics of binding of the soluble single chain TCR of the invention to YMLDLQPET-HLA A A0201 complex.
FIG. 14 shows the results of ELISPOT activation function verification of the resulting T cell clones.
FIG. 15 shows the results of ELISPOT activation function validation of effector cells transduced with TCRs of the invention (target cells are short peptide-loaded T2 cells).
FIG. 16 shows the results of ELISPOT activation function validation of effector cells transduced with TCRs of the invention (target cells are tumor cell lines).
FIG. 17 is a graph showing the results of transduction of the killing function of target cells by TCR effector cells of the invention.
Detailed Description
The present inventors have conducted extensive and intensive studies to find a TCR capable of specifically binding to HPV 16E 7 antigen oligopeptide YMLDLQPET (SEQ ID NO: 9), which antigen oligopeptide YMLDLQPET can form a complex with HLA A0201 and be presented on the cell surface together. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells transduced with the TCRs of the invention.
Terminology
The MHC molecules are proteins of the immunoglobulin superfamily, which may be class I or class II MHC molecules. Thus, it is specific for antigen presentation, and different individuals have different MHCs, which are capable of presenting different short peptides of a single protein antigen to the respective APC cell surfaces. Human MHC is commonly referred to as an HLA gene or HLA complex.
T Cell Receptor (TCR), the only receptor for specific antigenic peptides presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact of T cells with Antigen Presenting Cells (APCs) is initiated by binding of antigen-specific TCRs to pMHC complexes, and then interaction of T cells with other cell membrane surface molecules of both APCs occurs, 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.
TCRs are glycoproteins on the surface of cell membranes that exist as heterodimers from either the alpha/beta or gamma/delta chain. TCR heterodimers consist of alpha and beta chains in 95% of T cells, while 5% of T cells have TCRs consisting of gamma and delta chains. The native αβ heterodimeric TCR has an α chain and a β chain, which constitute subunits of the αβ heterodimeric TCR. In a broad sense, each of the α and β chains comprises a variable region, a linking region, and a constant region, and the β chain also typically comprises a short variable region between the variable region and the linking region, but the variable region is often considered part of the linking region. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in a framework structure (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, wherein CDR3 is recombined from the variable region and the linking region, known as the hypervariable region. The α and β chains of TCRs are generally regarded as having two "domains" each, i.e., a variable domain and a constant domain, the variable domain being composed of linked variable and linking regions. The sequence of the TCR constant domain can be found in published databases of the international immunogenetic information system (IMGT), for example the constant domain sequence of the α chain of a TCR molecule is "TRAC x 01" and the constant domain sequence of the β chain of a TCR molecule is "TRBC1 x 01" or "TRBC2 x 01". In addition, the α and β chains of TCRs also contain transmembrane and cytoplasmic regions, which are short.
In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Natural inter-chain disulfide bonds and artificial inter-chain disulfide bonds
A set of disulfide bonds exist between the near membrane regions cα and cβ of a native TCR, referred to herein as "native interchain disulfide bonds". In the present invention, an inter-chain covalent disulfide bond, which is artificially introduced at a position different from that of a natural inter-chain disulfide bond, is referred to as an "artificial inter-chain disulfide bond".
For convenience of description of disulfide bond positions, TRAC.sub.01 and TRBC.sub.1.sub.01 or TRBC.sub.2.sub.01 amino acid sequences are sequentially numbered from N-terminal to C-terminal, for example, TRBC.sub.1.sub.01 or TRBC.sub.2.sub.01 is P (proline) as the 60 th amino acid in the sequence from N-terminal to C-terminal, and can be described as Pro60 of TRBC.sub.1.sub.01 or TRBC.sub.2.sub.01 exon 1, it may also be expressed as amino acid 60 of exon 1 TRBC1 x 01 or TRBC2 x 01, and as in TRBC1 x 01 or TRBC2 x 01, amino acid 61 in the order from N-terminal to C-terminal is Q (glutamine), and it may be expressed as Gln61 of exon 1 TRBC1 x 01 or TRBC2 x 01, or as amino acid 61 of exon 1 TRBC1 x 01 or TRBC2 x 01, and so on. In the present invention, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers listed in IMGT. If an amino acid in TRAV is numbered 46 in IMGT, it is described in the present invention as TRAV amino acid 46, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described, and are specifically described.
Detailed Description
TCR molecules
During antigen processing, the antigen is degraded inside the cell and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, in a first aspect the invention provides a TCR molecule capable of binding YMLDLQPET-HLA a0201 complex. Preferably, the TCR molecule is isolated or purified. The α and β chains of the TCR each have 3 Complementarity Determining Regions (CDRs).
In a preferred embodiment of the invention, the α chain of the TCR comprises CDRs having the following amino acid sequences:
αCDR1-DRVSQS (SEQ ID NO:10)
αCDR2-IYSNGD (SEQ ID NO:11)
Alpha CDR3-AVNPRYGNKLV (SEQ ID NO: 12); and/or
The 3 complementarity determining regions of the TCR β chain variable domain are:
βCDR1-KGHDR (SEQ ID NO:13)
βCDR2-SFDVKD (SEQ ID NO:14)
βCDR3-ATSDRGQGAFGEQY (SEQ ID NO:15)。
Chimeric TCRs may be prepared by embedding the CDR region amino acid sequences of the invention described above into any suitable framework structure. As long as the framework structure is compatible with the CDR regions of the TCRs of the present invention, one skilled in the art will be able to design or synthesize TCR molecules having corresponding functions based on the CDR regions disclosed herein. Accordingly, a TCR molecule of the invention refers to a TCR molecule comprising the above-described alpha and/or beta chain CDR region sequences, and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is identical to SEQ ID NO:5 has an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity.
In a preferred embodiment of the invention, the TCR molecules of the invention are heterodimers consisting of alpha and beta chains. Specifically, in one aspect the alpha chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the alpha chain variable domain amino acid sequence comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above alpha chain. Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO. 1. More preferably, the alpha chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO. 1. In another aspect, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the β chain described above. Preferably, the TCR molecule comprises the β chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the β chain variable domain amino acid sequence of the TCR molecule is SEQ ID No. 5.
In a preferred embodiment of the invention, the TCR molecule of the invention is a single chain TCR molecule consisting of part or all of the alpha chain and/or part or all of the beta chain. For descriptions of single chain TCR molecules, reference may be made to Chung et al (1994) Proc.Natl. Acad.Sci.USA 91,12654-12658. From the literature, one skilled in the art can readily construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises vα, vβ and cβ, preferably linked in order from the N-terminus to the C-terminus.
The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above alpha chain. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO. 1. More preferably, the alpha chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO. 1. The β chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the β chain described above. Preferably, the single chain TCR molecule comprises the β chain variable domain amino acid sequence SEQ ID NO. 5. More preferably, the β chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID No. 5.
In a preferred embodiment of the invention, the constant domain of the TCR molecules of the invention is a human constant domain. The person skilled in the art knows or can obtain the human constant domain amino acid sequence by consulting the public database of related books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecule of the invention may be "TRAC x 01", and the constant domain sequence of the β chain of the TCR molecule may be "TRBC1 x 01" or "TRBC2 x 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, denoted herein as: TRAC.01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the alpha chain of the TCR molecule of the invention is SEQ ID NO. 3 and/or the amino acid sequence of the beta chain is SEQ ID NO. 7.
A naturally occurring TCR is a membrane protein, which is stabilised by its transmembrane region. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs may also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are desired. Soluble TCR molecules do not include their transmembrane region. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pMHC, but also as diagnostic tools for detecting infection or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a specific antigen. The invention also provides soluble TCRs specific for HPV 16E 7 antigen short peptides.
To obtain a soluble TCR, in one aspect, the TCR of the invention may be a TCR in which an artificial disulfide bond is introduced between residues of its alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the α and β chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at suitable sites in the native TCR to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of TRAC x 01 exon 1 and substituting cysteine residue of Ser57 of TRBC1 x 01 or TRBC2 x 01 exon 1. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of tranc x 01 exon 1 and Ser77 of TRBC1 x 01 or TRBC2 x 01 exon 1; tyr10 of TRAC x 01 exon 1 and Ser17 of TRBC1 x 01 or TRBC2 x 01 exon 1; thr45 of TRAC x 01 exon 1 and Asp59 of TRBC1 x 01 or TRBC2 x 01 exon 1; ser15 of TRAC x 01 exon 1 and Glu15 of TRBC1 x 01 or TRBC2 x 01 exon 1; arg53 of TRAC x 01 exon 1 and Ser54 of TRBC1 x 01 or TRBC2 x 01 exon 1; TRAC.01 exon 1 Pro89 and TRBC 1.01 or TRBC 2.01 exon 1 Ala19; or Tyr10 of TRAC x 01 exon 1 and Glu20 of TRBC1 x 01 or TRBC2 x 01 exon 1. I.e., a cysteine residue replaces any of the set of sites in the constant domains of the alpha and beta chains described above. The deletion of the native disulfide bond may be achieved by truncating up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or less amino acids at one or more of the C-termini of the TCR constant domains of the present invention, such that they do not include a cysteine residue, or by mutating the cysteine residue forming the native disulfide bond to another amino acid.
As described above, the TCRs of the invention may comprise artificial disulfide bonds introduced between residues of the constant domains of the alpha and beta chains thereof. It should be noted that the TCRs of the invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the introduced artificial disulfide bond as described above. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequence of the TCR can be linked by a native disulfide bond present in the TCR.
To obtain a soluble TCR, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that result in an improved stability of the inventive soluble TCRs, as described in the patent publication No. WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (α and/or β chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or the α chain J gene (TRAJ) short peptide amino acid position reciprocal 3,5,7, and/or the β chain J gene (TRBJ) short peptide amino acid position reciprocal 2,4,6, wherein the position numbering of the amino acid sequences is as set forth in the international immunogenetic information system (IMGT). The person skilled in the art is aware of the above-mentioned international immunogenetic information system and can derive the position numbers of amino acid residues of different TCRs in IMGT from this database.
The TCRs of the invention in which the hydrophobic core region is mutated may be stable soluble single chain TCRs formed by a flexible peptide chain linking the variable domains of the α and β chains of the TCRs. It should be noted that the flexible peptide chain of the present invention may be any peptide chain suitable for linking the variable domains of the TCR alpha and beta chains. The single chain soluble TCR as constructed in example 4 of the invention has an alpha chain variable domain amino acid sequence of SEQ ID NO. 32 and a coding nucleotide sequence of SEQ ID NO. 33; the amino acid sequence of the beta chain variable domain is SEQ ID NO. 34, and the coded nucleotide sequence is SEQ ID NO. 35.
In addition, patent literature 201680003540.2 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of a TCR can significantly improve the stability of the TCR with respect to stability. Thus, the high affinity TCRs of the present invention may also contain artificial interchain disulfide bonds between the α chain variable and β chain constant regions. 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: 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 exon 1 of TRBC1 x 01 or TRBC2 x 01; or amino acid 47 of TRAV and amino acid 60 of TRBC1 x 01 or TRBC2 x 01 exon 1. Preferably, such TCRs may comprise (i) all or part of the TCR a chain except for its transmembrane domain, and (ii) all or part of the TCR β chain except for its transmembrane domain, wherein (i) and (ii) each comprise a variable domain and at least part of a constant domain of the TCR chain, the a chain forming a heterodimer with the β chain. More preferably, such TCRs may comprise an alpha chain variable domain and a beta chain variable domain and all or part of a beta chain constant domain other than the transmembrane domain, but they do not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.
The TCRs of the present invention may also be provided in the form of multivalent complexes. The multivalent TCR complexes of the invention comprise a multimer of two, three, four or more TCRs of the invention bound, e.g., a tetramer may be generated using the tetramer domain of p53, or a complex of a plurality of TCRs of the invention bound to 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, as well as to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the present invention may be used alone or may be covalently or otherwise bound to the conjugate, preferably covalently. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the YMLDLQPET-HLA a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination or coupling of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Therapeutic agents that may be conjugated or coupled to a TCR of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (CANCER METASTASIS REVIEWS) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, nature 339, 394; epel et al, 2002, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 et al (Gillies et al, 1992, proc. Natl. Acad. Sci. USA (PNAS) 89, 1428; card et al, 2004, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; halin et al, 2003, cancer research (CANCER RESEARCH) 63, 3202); 4. antibody Fc fragments (Mosquera et al, 2005, journal of immunology (The Journal Of Immunology) 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, J.cancer International (International Journal of Cancer) 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communication (CANCER LETTERS) 239, 36; huang et al, 2006, journal of the american Society of chemistry (Journal of THE AMERICAN CHEMICAL Society) 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy (GENE THERAPY) 11, 1234); 8. liposomes (Mamot et al, 2005, cancer research (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 any form of nanoparticle, and the like.
In addition, the TCRs of the present invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, TCRs of the invention may comprise a human variable domain and a murine constant domain. The disadvantage of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regime for immunosuppression when it is used in adoptive T cell therapy to allow implantation of T cells expressing murine species.
It should be understood that the amino acid names herein are expressed by international single english letters or three english letters, and the correspondence between the single english letters and the three english letters of the amino acid names is as follows ：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V).
Nucleic acid molecules
In a second aspect the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a portion thereof, which portion may be one or more CDRs, a variable domain of an alpha and/or beta chain, and an alpha chain and/or a beta chain.
The nucleotide sequence encoding the CDR regions of the α chain of the TCR molecule of the first aspect of the invention is as follows:
αCDR1-gaccgagtttcccagtcc(SEQ ID NO:16)
αCDR2-atatactccaatggtgac(SEQ ID NO:17)
αCDR3-gccgtgaacccccggtatggaaacaagctggtc(SEQ ID NO:18)
the nucleotide sequence encoding the CDR region of the β chain of the TCR molecule of the first aspect of the invention is as follows:
βCDR1-aagggtcatgataga(SEQ ID NO:19)
βCDR2-tcctttgatgtcaaagat(SEQ ID NO:20)
βCDR3-gccaccagtgaccgaggacagggggcttttggcgagcagtac(SEQ ID NO:21)
Thus, the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR alpha chain of the invention include SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, and/or the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR beta chain of the invention include SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21.
The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not comprise introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding the polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable domain of the TCR alpha chain of the invention comprises SEQ ID NO.2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable domain of the TCR beta chain of the invention comprises SEQ ID NO. 6. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable domain of the TCR alpha chain of the invention comprises SEQ ID NO 33 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable domain of the TCR beta chain of the invention comprises SEQ ID NO 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO. 4 and/or SEQ ID NO. 8; or the nucleotide sequence of the nucleic acid molecule is SEQ ID NO. 31.
It is understood that different nucleotide sequences may encode the same polypeptide due to the degeneracy of the genetic code. Thus, the nucleic acid sequence encoding a TCR of the invention may be identical to or degenerate from the nucleic acid sequences shown in the drawings of the invention. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein having the sequence of SEQ ID NO. 1, but differing from the sequence of SEQ ID NO. 2.
The nucleotide sequence may be codon optimized. Different cells differ in the use of specific codons, and the amount of expression can be increased by changing codons in the sequence depending on the cell type. Codon usage tables for mammalian cells and a variety of other organisms are well known to those skilled in the art.
The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be generally obtained by, but not limited to, PCR amplification, recombinant methods or artificial synthesis. At present, it is already possible to obtain the DNA sequence encoding the TCR of the invention (or a fragment or derivative thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be a coding strand or a non-coding strand.
Carrier body
The invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, phages and animal and plant viruses.
Viral delivery systems include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, baculovirus vectors.
Preferably, the vector may transfer the nucleotide of the invention into a cell, e.g. a T cell, such that the cell expresses a TCR specific for HPV 16E 7 antigen. Ideally, the vector should be capable of sustained high level expression in T cells.
Cells
The invention also relates to host cells genetically engineered with the vectors or coding sequences of the invention. The host cell contains the vector or chromosome of the present invention integrated with the nucleic acid molecule of the present invention. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.
In addition, the invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. The T cells may be derived from T cells isolated from a subject, or may be part of a mixed cell population isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMCs) and may be CD4 + helper T cells or CD8 + cytotoxic T cells. The cells may be in a mixed population of CD4 + helper T cells/CD 8 + cytotoxic T cells. Generally, the cells will be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more susceptible to transfection, for example, with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.
Alternatively, the cells of the invention may also be or be derived from stem cells, such as Hematopoietic Stem Cells (HSCs). Gene transfer to HSCs does not result in TCR expression on the cell surface, as the stem cell surface does not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus (lymphoid precursor), expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.
There are a number of methods suitable for T cell transfection with DNA or RNA encoding a TCR of the invention (e.g., robbins et al, (2008) J. Immunol. 180:6116-6131). T cells expressing the TCRs of the invention may be used in adoptive immunotherapy. Those skilled in the art will be aware of many suitable methods of performing adoptive therapy (e.g., rosenberg et al, (2008) NAT REV CANCER (4): 299-308).
HPV 16E 7 antigen-related diseases
The invention also relates to a method of treating and/or preventing a disease associated with HPV 16E 7 in a subject, comprising the step of adoptively transferring HPV 16E 7-specific T cells to the subject. The HPV 16E 7-specific T cell recognizes YMLDLQPET-HLA A A0201 complex.
The HPV 16E 7-specific T cells of the invention are useful in the treatment of any HPV 16E 7-related disease presenting HPV 16E 7 antigen oligopeptide YMLDLQPET-HLA A0201 complex. Including but not limited to tumors such as cervical cancer, head and neck tumors, anal cancers, and the like.
Therapeutic method
Treatment may be performed by isolating T cells from a patient or volunteer suffering from a disease associated with HPV 16E 7 antigen and introducing the TCR of the invention into the T cells described above, followed by reinfusion of these genetically modified cells into the patient. Accordingly, the present invention provides a method of treating a HPV 16E 7-associated disease comprising the step of introducing into the patient an isolated T cell expressing a TCR of the invention, preferably derived from the patient itself. Generally, this involves (1) isolating T cells from a patient, (2) transducing T cells outside the patient with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) introducing genetically modified T cells into the patient. The number of isolated, transfected and reinfused cells can be determined by the physician.
The invention has the main advantages that:
(1) The TCR of the invention can specifically bind to HPV 16E 7 antigen short peptide complex YMLDLQPET-HLA A0201, and meanwhile, aiming at target cells, cells transduced with the TCR of the invention can be specifically activated.
(2) Aiming at target cells, effector cells transduced with the TCR of the invention have a strong killing function.
The following specific examples further illustrate the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, for example those described in the laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL press, or by the manufacturer's recommendations (Sambrook and Russell et al, molecular cloning). Percentages and parts are by weight unless otherwise indicated. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
EXAMPLE 1 cloning of HPV 16E 7 antigen short peptide specific T cells
Peripheral Blood Lymphocytes (PBLs) from healthy volunteers of genotype HLA-A0201 were stimulated with synthetic short peptide YMLDLQPET (SEQ ID NO: 9; beijing Severe Gene technology Co., ltd.). The YMLDLQPET short peptide was renatured with biotin-labeled HLA-A0201 to prepare pHLA haploids. These haploids are combined with PE-labeled streptavidin (BD company) to form PE-labeled tetramers, which are sorted together with anti-CD 8-APC biscationic cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonal by limiting dilution. Monoclonal cells were stained with tetramers and the selected biscationic clones are shown in FIG. 3. The double-positive clones obtained by layer-by-layer screening are also required to meet further functional tests.
The function and specificity of the T cell clone was further examined by ELISPOT experiments. Methods for detecting cellular function using ELISPOT assays are well known to those skilled in the art. The effector cells used in the IFN-. Gamma.ELISPOT experiments of this example were T cell clones obtained in the present invention, target cells were LCLs cells loaded with specific short peptides, and control groups were LCLs cells loaded with other short peptides and LCLs cells empty.
First, an ELISPOT plate was prepared, and the respective components for the test were added to the ELISPOT plate in the following order: following LCLs cells 20,000 cells/well, effector cells 2000/well, the experimental group was charged with 20. Mu.l of specific short peptide, the control group with 20. Mu.l of non-specific short peptide, the blank group with 20. Mu.l of medium (test medium), and 2 wells were set. Then incubated overnight (37 ℃,5% co 2). The plates were then washed and subjected to secondary detection and development, and the plates were dried for 1 hour, and spots formed on the films were counted using an immunoblotter plate reader (ELISPOT READER SYSTEM; AID Co.). As shown in FIG. 14, the obtained T cell clone showed a significant activation reaction on LCLs cells loaded with the specific short peptide, but showed substantially no reaction on LCLs cells loaded with other short peptides and LCLs cells unloaded.
EXAMPLE 2 construction of TCR Gene and vector to obtain HPV 16E 7 antigen short peptide-specific T cell clones
Total RNA from antigen short peptide YMLDLQPET specific, HLA-A0201 restricted T cell clone selected in example 1 was extracted with Quick-RNA TM MiniPrep (ZYMO research). The cDNA is synthesized by adopting a clontech SMART RACE CDNA amplification kit, and the adopted primer is designed in the C-terminal conservation region of the human TCR gene. The sequences were cloned into a T vector (TAKARA) for sequencing. It should be noted that the sequence is a complementary sequence and does not contain an intron. The alpha chain and beta chain sequence structures of the double-positive clone expressed TCR are respectively shown in the figure 1 and the figure 2, and the figure 1a, the figure 1b, the figure 1c, the figure 1d, the figure 1e and the figure 1f are respectively TCR alpha chain variable domain amino acid sequences, TCR alpha chain variable domain nucleotide sequences, TCR alpha chain amino acid sequences, TCR alpha chain nucleotide sequences, TCR alpha chain amino acid sequences with leader sequences and TCR alpha chain nucleotide sequences with leader sequences; FIGS. 2a, 2b, 2c, 2d, 2e and 2f are, respectively, TCR β chain variable domain amino acid sequence, TCR β chain variable domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, TCR β chain amino acid sequence with a leader sequence, and TCR β chain nucleotide sequence with a leader sequence.
The alpha chain was identified to comprise CDRs with the following amino acid sequences:
αCDR1-DRVSQS (SEQ ID NO:10)
αCDR2-IYSNGD (SEQ ID NO:11)
αCDR3-AVNPRYGNKLV (SEQ ID NO:12)
the β chain comprises CDRs having the following amino acid sequences:
βCDR1-KGHDR (SEQ ID NO:13)
βCDR2-SFDVKD (SEQ ID NO:14)
βCDR3-ATSDRGQGAFGEQY (SEQ ID NO:15)。
The full length genes of the TCR alpha and beta chains, respectively, were cloned into lentiviral expression vectors pLenti (addgene) by overlap (PCR). The method comprises the following steps: the full length genes of the TCR alpha and TCR beta chains were ligated using overlap PCR to give TCR alpha-2A-TCR beta fragments. The lentiviral expression vector and TCR alpha-2A-TCR beta are subjected to enzyme digestion and connection to obtain the pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, the lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The pseudovirus is then packaged again with 293T/17.
EXAMPLE 3 expression, refolding and purification of HPV16 E7 antigen-short peptide specific soluble TCR
To obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the invention may comprise only their variable domain and part of the constant domain, respectively, and a cysteine residue is introduced in the constant domain of the α and β chains to form an artificial interchain disulfide bond, the positions of the introduced cysteine residues being Thr48 of TRAC x 01 exon 1 and Ser57 of TRBC2 x 01 exon 1, respectively; the amino acid sequence and nucleotide sequence of the alpha chain are shown in fig. 4a and 4b, respectively, and the amino acid sequence and nucleotide sequence of the beta chain are shown in fig. 5a and 5b, respectively. The target gene sequences of the above TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a+ (Novagene) by standard methods described in molecular cloning laboratory Manual (Molecular Cloning a Laboratory Manual) (third edition, sambrook and Russell), the cloning sites upstream and downstream being NcoI and NotI, respectively. The insert was confirmed by sequencing to be error-free.
Expression vectors of TCR α and β chains were transformed into expression bacteria BL21 (DE 3) by chemical transformation, respectively, the bacteria were grown in LB medium, induced with IPTG at a final concentration of 0.5mM at OD 600 = 0.6, inclusion bodies formed after expression of TCR α and β chains were extracted by BugBuster Mix (Novagene), and repeatedly washed with BugBuster solution, and finally the inclusion bodies were dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediamine tetraacetic acid (EDTA), 20mM Tris (pH 8.1).
The TCR alpha and beta chains after dissolution were found to be 1:1 in a mass ratio of 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM β -mercapoethylamine (4 ℃) at a final concentration of 60mg/mL. After mixing the solution was dialyzed (4 ℃) in 10 volumes of deionized water, after 12 hours the deionized water was changed to buffer (20 mM 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 μm filter and purified by an anion exchange column (HITRAP Q HP,5ML,GE HEALTHCARE). The elution peak contains the successfully renatured alpha and beta dimer TCR as confirmed by SDS-PAGE gel. The TCR was then 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. The SDS-PAGE gel of the soluble TCR obtained in the present invention is shown in FIG. 6.
Example 4 production of soluble single chain TCR specific for HPV16 E7 antigen short peptide
The variable domains of the tcra and β chains of example 2 were constructed as a stable soluble single chain TCR molecule linked by flexible short peptides (linker) using site-directed mutagenesis, as described in WO 2014/206304. The amino acid sequence and nucleotide sequence of the single chain TCR molecule are shown in figures 7a and 7b, respectively. The amino acid sequence and the nucleotide sequence of the alpha chain variable domain are shown in FIG. 8a and FIG. 8b respectively; the amino acid sequence and nucleotide sequence of the beta-chain variable domain are shown in fig. 9a and 9b respectively; the amino acid sequence and the nucleotide sequence of the linker sequence are shown in FIG. 10a and FIG. 10b, respectively.
The target gene is subjected to double cleavage by NcoI and NotI, and is connected with a pET28a vector subjected to double cleavage by NcoI and NotI. The ligation product was transformed into E.coli DH 5. Alpha. And the ligation product was spread on LB plates containing kanamycin, incubated at 37℃overnight in an inverted position, positive clones were picked up for PCR screening, positive recombinants were sequenced, and after the correct sequence was confirmed, the recombinant plasmid was extracted and transformed into E.coli BL21 (DE 3) for expression.
EXAMPLE 5 expression, renaturation and purification of soluble single chain TCR specific for HPV16 E7 antigen short peptide
BL21 (DE 3) colonies prepared in example 4 and containing the recombinant plasmid pET28 a-template strand were all inoculated into LB medium containing kanamycin, cultured at 37℃until OD600 was 0.6-0.8, added with IPTG to a final concentration of 0.5mM, and cultured at 37℃for 4 hours. Cell pellet was harvested by centrifugation at 5000rpm for 15min, cell pellet was lysed with 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 collected by centrifugation at 6000rpm for 15 min. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0,8M urea), high-speed centrifuged to remove insoluble material, and the supernatant was quantified by BCA method and then sub-packaged 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 pH8.1, 100mM NaCl,10mM EDTA) was added, followed by addition of DTT to a final concentration of 10mM and treatment at 37℃for 30min. The single-chain TCR after the treatment was added dropwise to 125mL of renaturation buffer (100 mM Tris-HCl pH8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM beta-mercapthoethylamine, 1.87mM Cystamine) with a syringe, stirred for 10min at 4℃and then the renaturation solution was packed into a cellulose membrane dialysis bag with a retention of 4kDa, and the dialysis bag was placed in 1L of pre-chilled water and stirred slowly overnight at 4 ℃. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued for 8 hours at 4℃and then the dialysate was changed to the same fresh buffer and dialysis continued overnight. After 17 hours, the sample was filtered through a 0.45 μm filter, vacuum degassed and passed through an anion exchange column (HITRAP Q HP, GE HEALTHCARE), the protein was purified by a linear gradient of 0-1M NaCl from 20mM Tris-HCl pH 8.0, the collected eluate fractions were subjected to SDS-PAGE analysis, the fractions containing single chain TCR were concentrated and further purified by a gel filtration column (Superdex 75/300,GE Healthcare), and the target fractions were also subjected to SDS-PAGE analysis.
The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are as follows: the column Agilent Bio SEC-3 (300A,) The mobile phase is 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214nm.
An SDS-PAGE gel of the soluble single chain TCR obtained according to the invention is shown in FIG. 11.
Example 6 characterization in combination
BIAcore analysis
The binding activity of the TCR molecules obtained in example 3 and example 5 to YMLDLQPET-HLA a0201 complex was examined using a BIAcore T200 real time assay system. The coupling process was completed by adding anti-streptavidin antibody (GenScript) to coupling buffer (10 mM sodium acetate buffer, pH 4.77), then flowing the antibody through CM5 chips previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally blocking the unreacted activated surface with ethanolamine in hydrochloric acid solution at a coupling level of about 15,000 RU.
The low concentration of streptavidin was allowed to flow over the surface of the antibody-coated chip, then YMLDLQPET-HLA A A0201 complex was allowed to flow over the detection channel, the other channel was used as a reference channel, and 0.05mM biotin was allowed to flow over the chip at a flow rate of 10. Mu.L/min for 2min, blocking the remaining binding sites for streptavidin.
The preparation process of the YMLDLQPET-HLA A0201 complex is as follows:
a. Purification
100Ml of E.coli bacterial liquid for inducing expression of heavy chains or light chains is collected, centrifugation is carried out at 8000g at 4 ℃ for 10min, then the bacterial cells are washed once by 10ml of PBS, then the bacterial cells are resuspended by intense shaking of 5ml BugBuster Master Mix Extraction Reagents (Merck), and are incubated for 20min at room temperature in a rotating way, and then centrifugation is carried out at 6000g at 4 ℃ for 15min, the supernatant is discarded, and the inclusion bodies are collected.
The inclusion body is resuspended in 5ml BugBuster Master Mix and incubated for 5min at room temperature; adding 30ml of BugBuster diluted 10 times, mixing, and centrifuging at 4deg.C for 15min at 6000 g; removing the supernatant, adding 30ml of BugBuster diluted 10 times, mixing, centrifuging at 4 ℃ for 15min, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0, mixing, centrifuging at 4 ℃ for 15min, dissolving the inclusion body with 20mM Tris-HCl 8M urea, detecting purity of the inclusion body by SDS-PAGE, and detecting concentration by BCA kit.
B. Renaturation
Synthetic short peptide YMLDLQPET (Beijing Saint Gene technology Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light and heavy chains were solubilized with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. YMLDLQPET peptide was added to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by sequential addition of 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain added in three portions, 8 h/times), renaturation was performed at 4℃for at least 3 days to completion, and SDS-PAGE was examined for success of renaturation.
C. Purification after renaturation
The renaturation buffer was exchanged with 10 volumes of 20mM Tris pH 8.0 for dialysis, at least twice to sufficiently reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto HITRAP Q HP (GE general electric company) anion exchange column (5 ml bed volume). Using an Akta purifier (GE general electric), proteins were eluted with a linear gradient of 0-400mM NaCl in 20mM Tris pH 8.0, pMHC eluted at about 250mM NaCl, and fractions were collected for SDS-PAGE to check purity.
D. Biotinylation
Purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while buffer was replaced with 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 whether biotinylation was complete.
E. purification of biotinylated complexes
Biotinylated pMHC molecules were concentrated to 1ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified using gel filtration chromatography, a HiPrep TM/60 s200 HR column (GE general electric company) was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric company), 1ml of concentrated biotinylated pMHC molecules were loaded, and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a single peak elution at about 55 ml. The protein-containing fractions were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80℃with the addition of protease inhibitors cocktail (Roche).
Kinetic parameters were calculated using BIAcore Evaluation software, and the kinetic profiles of the soluble TCR molecules of the invention and the soluble single chain TCR molecules constructed according to the invention binding to the YMLDLQPET-HLA a0201 complex are shown in figures 12 and 13, respectively. The pattern shows that the soluble TCR molecules obtained by the invention and the soluble single chain TCR molecules can be combined with YMLDLQPET-HLA A A0201 complex. Meanwhile, the binding activity of the soluble TCR molecule of the invention and other irrelevant antigens of short peptides and HLA complexes is detected by using the method, and the result shows that the TCR molecule of the invention has no binding with other irrelevant antigens, and the soluble TCR molecule of the invention can be proved to be specifically bound with YMLDLQPET-HLA A0201 complexes.
Example 7 activation experiments of effector cells transduced with TCRs of the invention (target cells are T2 cells loaded with short peptides)
Methods for detecting cellular function using ELISPOT assays are well known to those skilled in the art. Lentiviral vectors, transduced T cells, comprising the TCR genes of interest of the invention were constructed and ELISPOT assays were performed to demonstrate the activation response of TCR transduced T cells of the invention specific for target cells. IFN-gamma production as measured by the ELISPOT assay was used as a readout of T cell activation.
First, reagents are prepared, including test medium: 10% FBS (Gibco, # 16000-044), RPMI 1640 (Gibco, # C11875500 BT); wash buffer (PBST): 0.01M PBS (Gibco, #C10010500 BT)/0.05% Tween 20; PVDF ELISPOT 96-well plate (Merck Millipore, # MSIPS 4510); the human IFN-. Gamma.ELISPOT PVDF-enzyme kit (BD) was loaded with all other reagents required (capture and detection antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solution).
The target cells used in this experiment were T2 cells loaded with HPV 16E 7 antigen oligopeptide YMLDLQPET, the effector cells used were CD3 + T cells expressing the HPV 16E 7 antigen oligopeptide-specific TCR of the invention, and CD3 + T cells of the other TCR (A6) transfected from the same volunteer were used as control. T cells were stimulated with anti-CD 3/CD28 coated beads (T cell amplificate, life technologies), the gene of interest transduced, amplified in 1640 medium containing 10% FBS containing 50IU/ml IL-2 and 10ng/ml IL-7 until 9-12 days post transduction, then the cells were placed in test medium and centrifuged at 300g for 10 minutes at room temperature for washing. The cells were then resuspended in assay medium at 2X the desired final concentration. Negative control effector cells were treated as well. The corresponding short peptide was added to the corresponding target cell (T2) experimental group, wherein the concentration of the short peptide was 10 -6 M.
The well plate was prepared as follows according to the instructions provided by the manufacturer: 10ml of sterile PBS per plate was used at 1:200 dilution of anti-human IFN-gamma capture antibody, then 100 microliters of diluted capture antibody was aliquoted into each well. The well plate was incubated overnight at 4 ℃. After incubation, the well plate is washed to remove excess capture antibody. 100 microliters/well of RPMI 1640 medium containing 10% FBS was added and the well plate incubated at room temperature for 2 hours to block the well plate. The medium was then washed from the well plate and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate over paper. The components of the assay were then added to an ELISPOT well plate:
The plates were then incubated overnight (37 ℃ C./5% CO 2) the next day, the medium was discarded, the plates were washed 2 times with double distilled water, then 3 times with wash buffer, and tapped on paper towels to remove residual wash buffer. Then 1 in PBS containing 10% FBS: the detection antibody was diluted 200 and wells were added 100 μl/well. The well plate was incubated at room temperature for 2 hours, washed 3 more times with wash buffer, and the well plate was tapped on paper towels to remove excess wash buffer. PBS containing 10% fbs at 1:100 dilution of streptavidin-alkaline phosphatase 100 microliter of diluted streptavidin-alkaline phosphatase was added to each well and the well plate was incubated for 1 hour at room temperature. The plates were then tapped on paper towels to remove excess wash buffer and PBS, followed by washing 4 times with wash buffer and 2 times with PBS. After washing, 100. Mu.l/well of BCIP/NBT solution provided by the kit was added for development. The plate was covered with tinfoil during development in the dark and left to stand for 5-15 minutes. During this period the spots of the developed well plate were routinely examined and the optimal time for termination of the reaction was determined. The BCIP/NBT solution was removed and the well plate was rinsed with double distilled water to stop the development reaction, spun dry, then the bottom of the well plate was removed, the well plate was dried at room temperature until each well was completely dried, and spots formed in the bottom of the well plate were counted using an immunospot plate counter (CTL, cell technologies limited (Cellular Technology Limited)).
The TCR-transduced T cells of the invention were tested for IFN- γ release in response to target cells bearing HPV16E7 antigen oligopeptide YMLDLQPET by ELISPOT experiments (described above). The number of ELSPOT spots observed in each well was plotted using GRAPHPAD PRISM.
The experimental results are shown in FIG. 15, in which T cells transduced with TCRs of the invention had a significant activation response to target cells loaded with specific short peptides, while T cells transduced with other TCRs had substantially no response to the corresponding target cells; meanwhile, the T cells transduced with the TCR of the invention have substantially no activation response to T2 cells loaded with other short peptides and to unloaded T2 cells.
Example 8 experiment of activation function of effector cells transfected with TCR of the invention (target cells are tumor cell lines)
This example also demonstrates that effector cells transfected with the high affinity TCRs of the invention have very good specific activation on target cells. The function and specificity of the high affinity TCRs of the invention in cells were tested by ELISPOT experiments.
Methods for detecting cellular function using ELISPOT assays are well known to those skilled in the art. The effector cells used were CD3 + T cells expressing the HPV16E7 antigen-short peptide-specific TCR of the invention, and CD3 + T cells of the TCR of the invention were not transfected by the same volunteer as a control group. The positive tumor cell line used (target cell line) was A375-E7 (HPV 16E7 over-expression). The negative tumor cell lines used were SK-MEL-28-E7 (HPV 16E7 over-expression), SK-MEL-28, SK-MEL-1, SK-MEL-5, MEL526, U266B1, SHP-77, HCC827.
First, an ELISPOT plate was prepared. ELISPOT plate ethanol activation coating, 4 ℃ overnight. On day 1 of the experiment, the coating was removed, the block was washed, incubated at room temperature for two hours, the block was removed, and the individual components of the experiment were added to the ELISPOT plate: cell lines were 2×10 4/well, effector cells were 2×10 3/well (calculated as positive rate of antibody), and two duplicate wells were set. Incubation was carried out overnight (37 ℃,5% co 2). On day 2 of the experiment, the plates were washed and subjected to secondary detection and development, the plates were dried, and spots formed on the films were counted using an immunoblotter plate reader (ELISPOT READER SYSTEM; AID 20).
As shown in fig. 16, the effector cells transfected with the TCRs of the present invention produced very good specific activation against positive tumor cell lines, whereas cells transduced with other TCRs produced essentially no activation; meanwhile, effector cells transfected with the TCRs of the present invention have substantially no activation response to negative cell lines.
Example 9 LDH killing function experiment of effector cells transfected with TCRs of the invention
This example demonstrates the killing function of cells transduced with TCRs of the present invention by measuring LDH release through a non-radioactive cytotoxicity assay. The assay is a colorimetric surrogate assay for the 51 Cr-release cytotoxicity assay, quantitatively determining Lactate Dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30 minute coupled enzyme reaction in which LDH converts one tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. The 490nm absorbance data can be collected using a standard 96-well plate reader.
Methods for detecting cell function using LDH release assays are well known to those skilled in the art. The effector cells used were cd3+ T cells expressing the HPV 16E 7 antigen-short peptide-specific TCRs of the invention, and cd3+ T cells of the other TCRs (A6) were transfected in the same volunteer as a control group. Positive tumor cell lines (target cell lines) were used, CASKI, A375-E7 (HPV 16E 7 over-expression). Negative tumor cell lines used: a375, siHa.
LDH plates were first prepared. On day 1 of the experiment, each component of the experiment was added to the plate: cell lines 3×10 4 cells/well, effector cells 3×10 4 cells/well, and three multiplex wells were set. And simultaneously setting an effector cell spontaneous pore, a target cell maximum pore, a volume correction control pore and a culture medium background control pore. Incubation was carried out overnight (37 ℃,5% co 2). On experiment day 2, the color development was detected and absorbance was recorded at 490nm with an enzyme-labeled instrument (Bioteck) after termination of the reaction.
The experimental results are shown in FIG. 17, in which cells transduced with the TCR of the invention had a significant killing effect against the target cell line, while cells transduced with other TCRs had a very weak killing effect; meanwhile, cells transduced with the TCRs of the present invention have no killing effect on negative tumor cell lines.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Guangdong Xiangxue medical technology Co., ltd
<120> A T cell receptor recognizing HPV
<130> P2020-0213
<160> 37
<170> SIPOSequenceListing 1.0
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Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly
85 90 95
Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser
100 105 110
<210> 2
<211> 333
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
ctcaactgca cttacagtga ccgagtttcc cagtccttct tctggtacag acaatattct 120
gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gtgaaccccc ggtatggaaa caagctggtc 300
tttggcgcag gaaccattct gagagtcaag tcc 333
<210> 3
<211> 252
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
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Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly
85 90 95
Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser Tyr
100 105 110
Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser
115 120 125
Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn
130 135 140
Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val
145 150 155 160
Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp
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Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile
180 185 190
Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val
195 200 205
Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln
210 215 220
Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly
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Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser
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<210> 4
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cagaaggagg tggagcagaa ttctggaccc ctcagtgttc cagagggagc cattgcctct 60
ctcaactgca cttacagtga ccgagtttcc cagtccttct tctggtacag acaatattct 120
gggaaaagcc ctgagttgat aatgtccata tactccaatg gtgacaaaga agatggaagg 180
tttacagcac agctcaataa agccagccag tatgtttctc tgctcatcag agactcccag 240
cccagtgatt cagccaccta cctctgtgcc gtgaaccccc ggtatggaaa caagctggtc 300
tttggcgcag gaaccattct gagagtcaag tcctatatcc agaaccctga ccctgccgtg 360
taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 420
tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 480
ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 540
gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 600
agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 660
ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 720
tttaatctgc tcatgacgct gcggctgtgg tccagc 756
<210> 5
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Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly
1 5 10 15
Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met
20 25 30
Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr
35 40 45
Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr
50 55 60
Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser
65 70 75 80
Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg
85 90 95
Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110
Thr Val Thr
115
<210> 6
<211> 345
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 6
gatgctgatg ttacccagac cccaaggaat aggatcacaa agacaggaaa gaggattatg 60
ctggaatgtt ctcagactaa gggtcatgat agaatgtact ggtatcgaca agacccagga 120
ctgggcctac ggttgatcta ttactccttt gatgtcaaag atataaacaa aggagagatc 180
tctgatggat acagtgtctc tcgacaggca caggctaaat tctccctgtc cctagagtct 240
gccatcccca accagacagc tctttacttc tgtgccacca gtgaccgagg acagggggct 300
tttggcgagc agtacttcgg gccgggcacc aggctcacgg tcaca 345
<210> 7
<211> 294
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 7
Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly
1 5 10 15
Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met
20 25 30
Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr
35 40 45
Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr
50 55 60
Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser
65 70 75 80
Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg
85 90 95
Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110
Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
115 120 125
Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
130 135 140
Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
145 150 155 160
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
165 170 175
Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
180 185 190
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
195 200 205
Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
210 215 220
Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
225 230 235 240
Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly
245 250 255
Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
260 265 270
Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
275 280 285
Arg Lys Asp Ser Arg Gly
290
<210> 8
<211> 882
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 8
gatgctgatg ttacccagac cccaaggaat aggatcacaa agacaggaaa gaggattatg 60
ctggaatgtt ctcagactaa gggtcatgat agaatgtact ggtatcgaca agacccagga 120
ctgggcctac ggttgatcta ttactccttt gatgtcaaag atataaacaa aggagagatc 180
tctgatggat acagtgtctc tcgacaggca caggctaaat tctccctgtc cctagagtct 240
gccatcccca accagacagc tctttacttc tgtgccacca gtgaccgagg acagggggct 300
tttggcgagc agtacttcgg gccgggcacc aggctcacgg tcacagagga cctgaaaaac 360
gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 420
aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 480
tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 540
cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 600
ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 660
aatgacgagt ggacccagga tagggccaaa cctgtcaccc agatcgtcag cgccgaggcc 720
tggggtagag cagactgtgg cttcacctcc gagtcttacc agcaaggggt cctgtctgcc 780
accatcctct atgagatctt gctagggaag gccaccttgt atgccgtgct ggtcagtgcc 840
ctcgtgctga tggccatggt caagagaaag gattccagag gc 882
<210> 9
<211> 9
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 9
Tyr Met Leu Asp Leu Gln Pro Glu Thr
1 5
<210> 10
<211> 6
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 10
Asp Arg Val Ser Gln Ser
1 5
<210> 11
<211> 6
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 11
Ile Tyr Ser Asn Gly Asp
1 5
<210> 12
<211> 11
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 12
Ala Val Asn Pro Arg Tyr Gly Asn Lys Leu Val
1 5 10
<210> 13
<211> 5
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 13
Lys Gly His Asp Arg
1 5
<210> 14
<211> 6
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 14
Ser Phe Asp Val Lys Asp
1 5
<210> 15
<211> 14
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 15
Ala Thr Ser Asp Arg Gly Gln Gly Ala Phe Gly Glu Gln Tyr
1 5 10
<210> 16
<211> 18
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 16
gaccgagttt cccagtcc 18
<210> 17
<211> 18
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 17
atatactcca atggtgac 18
<210> 18
<211> 33
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 18
gccgtgaacc cccggtatgg aaacaagctg gtc 33
<210> 19
<211> 15
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 19
aagggtcatg ataga 15
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 20
tcctttgatg tcaaagat 18
<210> 21
<211> 42
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 21
gccaccagtg accgaggaca gggggctttt ggcgagcagt ac 42
<210> 22
<211> 273
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 22
Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser
1 5 10 15
Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu
20 25 30
Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp
35 40 45
Arg Val Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser
50 55 60
Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly
65 70 75 80
Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu
85 90 95
Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val
100 105 110
Asn Pro Arg Tyr Gly Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu
115 120 125
Arg Val Lys Ser Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu
130 135 140
Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe
145 150 155 160
Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile
165 170 175
Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn
180 185 190
Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala
195 200 205
Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu
210 215 220
Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr
225 230 235 240
Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu
245 250 255
Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser
260 265 270
Ser
<210> 23
<211> 819
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 23
atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60
caacagaagg aggtggagca gaattctgga cccctcagtg ttccagaggg agccattgcc 120
tctctcaact gcacttacag tgaccgagtt tcccagtcct tcttctggta cagacaatat 180
tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 240
aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 300
cagcccagtg attcagccac ctacctctgt gccgtgaacc cccggtatgg aaacaagctg 360
gtctttggcg caggaaccat tctgagagtc aagtcctata tccagaaccc tgaccctgcc 420
gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480
gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540
gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600
tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660
cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720
aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780
gggtttaatc tgctcatgac gctgcggctg tggtccagc 819
<210> 24
<211> 313
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 24
Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr
1 5 10 15
Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr
20 25 30
Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His
35 40 45
Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu
50 55 60
Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser
65 70 75 80
Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser
85 90 95
Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr
100 105 110
Ser Asp Arg Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly
115 120 125
Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu
130 135 140
Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys
145 150 155 160
Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu
165 170 175
Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr
180 185 190
Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr
195 200 205
Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro
210 215 220
Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn
225 230 235 240
Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser
245 250 255
Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr
260 265 270
Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly
275 280 285
Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala
290 295 300
Met Val Lys Arg Lys Asp Ser Arg Gly
305 310
<210> 25
<211> 939
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 25
atggcctccc tgctcttctt ctgtggggcc ttttatctcc tgggaacagg gtccatggat 60
gctgatgtta cccagacccc aaggaatagg atcacaaaga caggaaagag gattatgctg 120
gaatgttctc agactaaggg tcatgataga atgtactggt atcgacaaga cccaggactg 180
ggcctacggt tgatctatta ctcctttgat gtcaaagata taaacaaagg agagatctct 240
gatggataca gtgtctctcg acaggcacag gctaaattct ccctgtccct agagtctgcc 300
atccccaacc agacagctct ttacttctgt gccaccagtg accgaggaca gggggctttt 360
ggcgagcagt acttcgggcc gggcaccagg ctcacggtca cagaggacct gaaaaacgtg 420
ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480
gccacactgg tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540
gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600
cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660
tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720
gacgagtgga cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg 780
ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840
atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900
gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 939
<210> 26
<211> 206
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 26
Met Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu
1 5 10 15
Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln
20 25 30
Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile
35 40 45
Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala
50 55 60
Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser
65 70 75 80
Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr
85 90 95
Gly Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser
100 105 110
Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys
115 120 125
Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr
130 135 140
Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr
145 150 155 160
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala
165 170 175
Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser
180 185 190
Ile Ile Pro Glu Asp Thr Phe Phe Cys Ser Pro Glu Ser Ser
195 200 205
<210> 27
<211> 618
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 27
atgcagaaag aagtggaaca gaattctgga cccctcagtg ttccagaggg agccattgcc 60
tctctcaact gcacttacag tgaccgagtt tcccagtcct tcttctggta cagacaatat 120
tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 180
aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 240
cagcccagtg attcagccac ctacctctgt gccgtgaacc cccggtatgg aaacaagctg 300
gtctttggcg caggaaccat tctgagagtc aagtcctata tccagaaccc tgaccctgcc 360
gtttatcagc tgcgtgatag caaaagcagc gataaaagcg tgtgcctgtt caccgatttt 420
gatagccaga ccaacgtgag ccagagcaaa gatagcgatg tgtacatcac cgataaaacc 480
gtgctggata tgcgcagcat ggatttcaaa agcaatagcg cggttgcgtg gagcaacaaa 540
agcgattttg cgtgcgcgaa cgcgtttaac aacagcatca tcccggaaga tacgttcttc 600
tgcagcccag aaagttcc 618
<210> 28
<211> 246
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 28
Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr
1 5 10 15
Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg
20 25 30
Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr
35 40 45
Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly
50 55 60
Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu
65 70 75 80
Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp
85 90 95
Arg Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg
100 105 110
Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala
115 120 125
Val Phe Glu Pro Ser Glu Cys 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 Ala 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> 29
<211> 738
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 29
atggatgctg atgtgaccca gaccccaagg aataggatca caaagacagg aaagaggatt 60
atgctggaat gttctcagac taagggtcat gatagaatgt actggtatcg acaagaccca 120
ggactgggcc tacggttgat ctattactcc tttgatgtca aagatataaa caaaggagag 180
atctctgatg gatacagtgt ctctcgacag gcacaggcta aattctccct gtccctagag 240
tctgccatcc ccaaccagac agctctttac ttctgtgcca ccagtgaccg aggacagggg 300
gcttttggcg agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 360
aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aatgcgaaat tagccatacc 420
cagaaagcga ccctggtttg tctggcgacc ggtttttatc cggatcatgt ggaactgtct 480
tggtgggtga acggcaaaga agtgcatagc ggtgtttcta ccgatccgca gccgctgaaa 540
gaacagccgg cgctgaatga tagccgttat gcgctgtcta gccgtctgcg tgttagcgcg 600
accttttggc aaaatccgcg taaccatttt cgttgccagg tgcagtttta tggcctgagc 660
gaaaacgatg aatggaccca ggatcgtgcg aagccggtta cccagattgt tagcgcggaa 720
gcctggggcc gcgcagat 738
<210> 30
<211> 250
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 30
Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Glu Asn Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Val Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Asn Pro Arg Tyr Gly
85 90 95
Asn Lys Leu Val Phe Gly Ala Gly Thr Lys Leu Arg Val Lys Ser Gly
100 105 110
Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly
115 120 125
Gly Ser Glu Gly Gly Thr Gly Asp Ala Asp Val Thr Gln Thr Pro Arg
130 135 140
Asn Leu Ser Val Lys Thr Gly Lys Arg Val Thr Leu Glu Cys Ser Gln
145 150 155 160
Thr Lys Gly His Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln
165 170 175
Gly Leu Arg Leu Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys
180 185 190
Gly Glu Ile Ser Asp Arg Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys
195 200 205
Phe Ser Leu Ser Ile Glu Ser Val Glu Pro Asn Asp Thr Ala Leu Tyr
210 215 220
Phe Cys Ala Thr Ser Asp Arg Gly Gln Gly Ala Phe Gly Glu Gln Tyr
225 230 235 240
Phe Gly Pro Gly Thr Arg Leu Thr Val Thr
245 250
<210> 31
<211> 749
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 31
caaaaagaag ttgaacagaa tagtggcccg ctgagtgtgc cggaaggtga aaatgtgagt 60
attaattgta cctatagcga tcgcgttagt cagagctttt tctggtatcg tcagtatagc 120
ggtaaaagcc cggaactgat tatgagtatc tatagcaatg gcgataaaga agatggccgc 180
tttaccgcac agctgaataa ggcaagccag tatgtgagcc tgctgattcg cgatgtgcag 240
ccgagtgata gtgcaaccta tttttgtgca gtgaatccgc gttatggcaa taagctggtt 300
tttggtgccg gcacaaactg cgcgttaaaa gcggtggcgg tagcgaaggt ggcggtagtg 360
aaggtggcgg cagtgaaggt ggtggcagcg aaggtggtac cggtgacgca gatgttaccc 420
agaccccgcg taatctgagc gtgaaaaccg gcaaacgcgt gaccctggaa tgcagtcaga 480
ccaaaggcca tgatcgcatg tattggtatc gtcaagatcc gggtcagggc ctgcgtctga 540
tctattatag ctttgatgtt aaagacatca acaagggcga aattagtgat cgttatagcg 600
ttagtcgtca ggcccaggcc aaattttcac tgagtattga aagcgttgaa ccgaatgata 660
ccgccctgta tttttgcgca accagcgatc gcggccaggg tgcctttggc gaacagtatt 720
ttggcccggg tacccgcctg accgttacc 749
<210> 32
<211> 111
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 32
Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly
1 5 10 15
Glu Asn Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Val Ser Gln Ser
20 25 30
Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met
35 40 45
Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln
50 55 60
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Val Gln
65 70 75 80
Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Asn Pro Arg Tyr Gly
85 90 95
Asn Lys Leu Val Phe Gly Ala Gly Thr Lys Leu Arg Val Lys Ser
100 105 110
<210> 33
<211> 333
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 33
caaaaagaag ttgaacagaa tagtggcccg ctgagtgtgc cggaaggtga aaatgtgagt 60
attaattgta cctatagcga tcgcgttagt cagagctttt tctggtatcg tcagtatagc 120
ggtaaaagcc cggaactgat tatgagtatc tatagcaatg gcgataaaga agatggccgc 180
tttaccgcac agctgaataa ggcaagccag tatgtgagcc tgctgattcg cgatgtgcag 240
ccgagtgata gtgcaaccta tttttgtgca gtgaatccgc gttatggcaa taagctggtt 300
tttggtgccg gcaccaaact gcgcgttaaa agc 333
<210> 34
<211> 115
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 34
Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Leu Ser Val Lys Thr Gly
1 5 10 15
Lys Arg Val Thr Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met
20 25 30
Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr
35 40 45
Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Arg Tyr
50 55 60
Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Ile Glu Ser
65 70 75 80
Val Glu Pro Asn Asp Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg
85 90 95
Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu
100 105 110
Thr Val Thr
115
<210> 35
<211> 345
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 35
gacgcagatg ttacccagac cccgcgtaat ctgagcgtga aaaccggcaa acgcgtgacc 60
ctggaatgca gtcagaccaa aggccatgat cgcatgtatt ggtatcgtca agatccgggt 120
cagggcctgc gtctgatcta ttatagcttt gatgttaaag acatcaacaa gggcgaaatt 180
agtgatcgtt atagcgttag tcgtcaggcc caggccaaat tttcactgag tattgaaagc 240
gttgaaccga atgataccgc cctgtatttt tgcgcaacca gcgatcgcgg ccagggtgcc 300
tttggcgaac agtattttgg cccgggtacc cgcctgaccg ttacc 345
<210> 36
<211> 24
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 36
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> 37
<211> 72
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 37
ggtggcggta gcgaaggtgg cggtagtgaa ggtggcggca gtgaaggtgg tggcagcgaa 60
ggtggtaccg gt 72

Claims (32)

1. A T Cell Receptor (TCR), wherein the TCR is capable of binding to YMLDLQPET-HLA a0201 complex; and, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain being:
α CDR1- DRVSQS (SEQ ID NO: 10)
α CDR2- IYSNGD (SEQ ID NO: 11)
Alpha CDR 3-AVNPRYGNKLV (SEQ ID NO: 12); and
The 3 complementarity determining regions of the TCR β chain variable domain are:
β CDR1- KGHDR (SEQ ID NO: 13)
β CDR2- SFDVKD (SEQ ID NO: 14)
β CDR3- ATSDRGQGAFGEQY (SEQ ID NO: 15)。
2. A TCR as claimed in claim 1 comprising a TCR α chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain is identical to SEQ ID NO:5, an amino acid sequence having at least 90% sequence identity.
3. A TCR as claimed in claim 1 comprising the alpha chain variable domain amino acid sequence SEQ ID No. 1.
4. A TCR as claimed in claim 1 comprising the β chain variable domain amino acid sequence SEQ ID No. 5.
5. A TCR as claimed in claim 1 wherein the TCR is an αβ heterodimer comprising a TCR α chain constant region TRAC x 01 and a TCR β chain constant region TRBC1 x 01 or TRBC2 x 01.
6. A TCR as claimed in claim 5 wherein the amino acid sequence of the α chain of the TCR is SEQ ID NO:3 and the beta chain amino acid sequence of the TCR is SEQ ID NO: 7.
7. A TCR as claimed in claim 1 wherein the TCR is soluble.
8. A TCR as claimed in claim 7 which is single chain.
9. A TCR as claimed in claim 8 which is formed by the linkage of an α chain variable domain and a β chain variable domain via a peptide linker sequence.
10. A TCR as claimed in claim 9 having one or more mutations in the α chain variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, or 94, and/or the α chain J gene short peptide amino acid position 3, 5 or 7; and/or the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the β chain variable region, and/or the β chain J gene short peptide amino acid position 2, 4, or 6, wherein the amino acid position numbers are numbered as listed in IMGT (international immunogenetic information system).
11. A TCR as claimed in claim 9 wherein the α chain variable domain amino acid sequence of the TCR comprises SEQ ID No. 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID No. 34.
12. A TCR as claimed in claim 11 wherein the amino acid sequence of the TCR is SEQ ID No. 30.
13. A TCR as claimed in claim 1 comprising (i) a TCR α chain variable domain and all or part of a TCR α chain constant region other than a transmembrane domain; and (ii) a TCR β chain variable domain and all or part of a TCR β chain constant region other than the transmembrane domain.
14. A TCR as claimed in claim 13 wherein the cysteine residues form an artificial disulphide bond between the α and β chain constant domains of the TCR.
15. A TCR as claimed in claim 14 wherein the cysteine residues forming an artificial disulphide bond in the TCR are substituted at one or more of the sites selected from:
Thr48 of tranc x 01 exon 1 and Ser57 of TRBC1 x 01 or TRBC2 x 01 exon 1;
Thr45 of tranc x 01 exon 1 and Ser77 of TRBC1 x 01 or TRBC2 x 01 exon 1;
tyr10 of TRAC x 01 exon 1 and Ser17 of TRBC1 x 01 or TRBC2 x 01 exon 1;
Thr45 of TRAC x 01 exon 1 and Asp59 of TRBC1 x 01 or TRBC2 x 01 exon 1;
ser15 of TRAC x 01 exon 1 and Glu15 of TRBC1 x 01 or TRBC2 x 01 exon 1;
arg53 of TRAC x 01 exon 1 and Ser54 of TRBC1 x 01 or TRBC2 x 01 exon 1;
TRAC.01 exon 1 Pro89 and TRBC 1.01 or TRBC 2.01 exon 1 Ala19; and
Tyr10 for TRAC x 01 exon 1 and Glu20 for TRBC1 x 01 or TRBC2 x 01 exon 1;
the position numbers of the TRAC-01 and TRBC-1-01 or TRBC-2-01 amino acid sequences are sequentially numbered from the N end to the C end.
16. A TCR as claimed in claim 15 wherein the alpha chain amino acid sequence of the TCR is SEQ ID No. 26 and/or the beta chain amino acid sequence of the TCR is SEQ ID No. 28.
17. A TCR as claimed in claim 13 wherein the TCR has an artificial interchain disulphide bond between the α chain variable region and the β chain constant region.
18. A TCR as claimed in claim 17 wherein the cysteine residues forming artificial interchain disulphide bonds in the TCR are substituted at one or more of the groups selected from:
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 TRBC 1x 01 or TRBC2 x 01;
Amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; or (b)
Amino acid 47 of TRAV and amino acid 60 of TRBC1 x 01 or TRBC2 x 01 exon 1;
The position numbers of the amino acid sequences of the variable region TRAV are numbered according to the position numbers listed in IMGT, and the position numbers of the amino acid sequences TRBC 1.times.01 or TRBC 2.times.01 are numbered according to the sequence from the N end to the C end.
19. A TCR as claimed in claim 18 comprising an α chain variable domain and a β chain variable domain and all or part of a β chain constant domain other than the transmembrane domain, but which does not comprise an α chain constant domain, the α chain variable domain of the TCR forming a heterodimer with the β chain.
20. 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 claims 1 to 19.
21. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to any one of claims 1 to 19 or a complement thereof.
22. A nucleic acid molecule according to claim 21, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO. 33.
23. A nucleic acid molecule according to claim 21 or 22, comprising the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO. 35.
24. A nucleic acid molecule according to claim 21, comprising the nucleotide sequence of SEQ ID NO:4 and/or comprises the nucleotide sequence of SEQ ID NO:8.
25. A vector comprising the nucleic acid molecule of any one of claims 21-24.
26. The vector of claim 25, wherein said vector is a viral vector.
27. The vector of claim 26, wherein the vector is a lentiviral vector.
28. An isolated host cell comprising the vector of any one of claims 25-27 or the nucleic acid molecule of any one of claims 21-24 integrated into a chromosome.
29. A cell, wherein the cell transduces the nucleic acid molecule of any one of claims 21-24 or the vector of any one of claims 25-27.
30. The cell of claim 29, wherein the cell is a T cell or a stem cell.
31. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as claimed in any one of claims 1 to 19, a TCR complex as claimed in claim 20 or a cell as claimed in claim 29 or 30.
32. Use of the T cell receptor of any one of claims 1 to 19, or the TCR complex of claim 20, or the cell of claim 29 or 30, for the manufacture of a medicament for the treatment of cervical cancer, anal cancer, conjunctival Intraepithelial Neoplasia (CIN), or keratoconjunctival invasive Squamous Cell Carcinoma (SCC).
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