CN113321726A - T cell receptor for identifying HPV - Google Patents

T cell receptor for identifying HPV Download PDF

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Publication number
CN113321726A
CN113321726A CN202010129637.6A CN202010129637A CN113321726A CN 113321726 A CN113321726 A CN 113321726A CN 202010129637 A CN202010129637 A CN 202010129637A CN 113321726 A CN113321726 A CN 113321726A
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tcr
ser
seq
gly
leu
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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 CN202010129637.6A priority Critical patent/CN113321726A/en
Priority to PCT/CN2021/078275 priority patent/WO2021170115A1/en
Priority to TW110107149A priority patent/TW202140540A/en
Publication of CN113321726A publication Critical patent/CN113321726A/en
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Abstract

The present invention provides a T Cell Receptor (TCR) capable of specifically binding short peptide YMLDLQPET derived from HPV16E7 antigen, said antigen short peptide YMLDLQPET being capable of forming a complex with HLA a0201 and being presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.

Description

T cell receptor for identifying HPV
Technical Field
The invention relates to a TCR capable of recognizing HPV16E7 antigen short peptide and a coding sequence thereof, and also relates to an HPV16E7 specific T cell obtained by transducing the TCR, and application of the T cell in preventing and treating HPV16E7 related diseases.
Background
The HPV16E7 gene is one of the early region genes of the Human Papilloma Virus (HPV) genome, and encodes an E7 protein which is a small acidic protein containing about 100 amino acids. The most popular type of cervical cancer worldwide is HPV16, which accounts for 50% -60% of detection cases (Acta Acad Med Sin,2007,29(5):678-684), wherein the high-risk HPV16E7 protein is an important cause of HPV-induced cervical cancer. In HPV-infected head and neck tumors, E7 oncoprotein plays a role in immunosuppression, and causes canceration mainly by blocking normal P16 protein expression to cause cell cycle abnormality (China journal of otorhinolaryngology and skull base surgery, 2017, 23(6):594 598). It has been shown that HPV16E7 is also a strong oncogene in anal cancer (virology.2011Dec 20; 421(2): 114-. In addition, HPV16E7 can cause diseases such as Conjunctival Intraepithelial Neoplasia (CIN) and keratoconjunctival invasive Squamous Cell Carcinoma (SCC) (International J.Ophthalmi 2018; 18(6): 1047-. YMLDLQPET (SEQ ID NO:9) is a short peptide derived from the HPV16E7 antigen, which is a target for the treatment of HPV16E7 related diseases.
T cell adoptive immunotherapy is the transfer of reactive T cells specific for a target cell antigen into a patient to act on the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that recognizes a corresponding short peptide antigen on the surface of a target cell. In the immune system, the direct physical contact between T cells and Antigen Presenting Cells (APC) is initiated by the binding of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells. Therefore, those skilled in the art have focused on isolating TCRs specific for HPV16E7 antigen short peptides and transducing the TCRs into T cells to obtain T cells specific for HPV16E7 antigen short peptides, thereby making them useful in cellular immunotherapy.
Disclosure of Invention
The invention aims to provide a T cell receptor for recognizing HPV16E7 antigen short peptide.
In a first aspect of the invention, there is provided a T Cell Receptor (TCR) capable of binding to the 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 amino acid sequence of CDR3 of the TCR alpha chain variable domain is AVNPRYGNKLV (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain 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 alpha chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1, and a TCR beta chain variable domain; 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 beta chain variable domain amino acid sequence SEQ ID NO 5.
In another preferred embodiment, the TCR is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC1 01 or TRBC2 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 3 and/or the β 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 linking an α chain variable domain to a β chain variable domain via a peptide linker.
In another preferred embodiment, the TCR has one or more mutations in amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 of the α chain variable region, and/or in the penultimate 3-, 5-, or 7-position of the short peptide amino acid of the α chain J gene; and/or the TCR has one or more mutations in beta chain variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91, or 94 th, and/or beta chain J gene short peptide amino acid penultimate 2,4 or 6 th, wherein the amino acid position numbering is according to the position numbering listed in IMGT (international immunogenetic information system).
In another preferred embodiment, 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.
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 α chain, excluding the transmembrane domain; and (b) all or part of a TCR β chain, excluding 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 alpha and beta 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 groups of sites selected from the group consisting of:
thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;
ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;
arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;
pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and
tyr10 and TRBC1 × 01 of exon 1 of TRAC × 01 or Glu20 of exon 1 of TRBC2 × 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO 26 and/or the β chain amino acid sequence of the TCR is SEQ ID NO 28.
In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain variable region and the β chain constant region.
In another preferred embodiment, the cysteine residues that form the artificial interchain disulfide bond in the TCR replace one or more groups of 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 TRBC1 x 01 or TRBC2 x 01;
amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or
Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01.
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, excluding the transmembrane domain, but which 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 TCR has a conjugate attached to the C-or N-terminus of the alpha and/or beta chain.
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 of the TCR molecules 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 according to the first aspect of the invention, or the complement thereof.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 2 or SEQ ID NO 33 encoding the variable domain of the TCR alpha chain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 6 or SEQ ID NO 35 encoding the variable domain of the TCR β chain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 4 encoding the TCR alpha chain and/or comprises the nucleotide sequence SEQ ID NO. 8 encoding the 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 genome into which has been integrated an exogenous nucleic acid molecule according to the third aspect of the invention.
In a sixth aspect of the invention, there is provided a cell which transduces 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 cell is a T cell or a stem cell.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, 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.
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, in the manufacture of a medicament for the treatment of a tumour or an autoimmune disease, preferably wherein 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 an autoimmune disease, preferably wherein the tumour is cervical cancer.
In a ninth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a 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 to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the amino acid sequence of the TCR α chain variable domain, the nucleotide sequence of the TCR α chain variable domain, the amino acid sequence of the TCR α chain, the nucleotide sequence of the TCR α chain, the amino acid sequence of the TCR α chain with leader sequence and the nucleotide sequence of the TCR α chain with leader sequence, respectively.
Fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR β chain variable domain amino acid sequence, a TCR β chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence, a TCR β chain amino acid sequence with a leader sequence and a TCR β chain nucleotide sequence with a leader sequence, respectively.
FIG. 3 is CD8 of monoclonal cells+And tetramer-PE double positive staining results.
Fig. 4a and 4b are the amino acid and nucleotide sequences, respectively, of a soluble TCR α chain.
Fig. 5a and 5b are the amino acid and nucleotide sequences, respectively, of a soluble TCR β chain.
Fig. 6a and 6b are gel maps of the soluble TCRs obtained after purification. In FIGS. 6a and 6b, the lanes on the right side are reducing gel and non-reducing gel, respectively, and the lanes on the left side are molecular weight markers (marker).
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 variable domain of the single chain TCR α chain.
FIGS. 9a and 9b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single-chain TCR β chain.
FIGS. 10a and 10b are the amino acid and nucleotide sequences, respectively, of a single-chain TCR linker sequence (linker).
FIG. 11 is a gel diagram of the soluble single chain TCR obtained after purification. The leftmost lane is a non-reducing gel, the middle lane is a molecular weight marker (marker), and the rightmost lane is a reducing gel.
FIG. 12 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the YMLDLQPET-HLA A0201 complex.
FIG. 13 is a BIAcore kinetic profile of binding of soluble single chain TCRs of the invention to the YMLDLQPET-HLA A0201 complex.
FIG. 14 shows the results of functional verification of the ELISPOT activation of the resulting T cell clones.
FIG. 15 is a demonstration of ELISPOT activation function of effector cells transduced with the TCR of the invention (target cells are T2 cells loaded with short peptides).
FIG. 16 is a demonstration of the ELISPOT activation function of effector cells transduced with the TCRs of the invention (the target cells are tumor cell lines).
FIG. 17 shows the results of the killing function of transduced TCR effector cells of the invention on target cells.
Detailed Description
The present inventors have extensively and intensively studied to find a TCR capable of specifically binding to HPV16E7 antigen short peptide YMLDLQPET (SEQ ID NO:9), which antigen short peptide YMLDLQPET can form a complex with HLA A0201 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.
Term(s) for
MHC molecules are proteins of the immunoglobulin superfamily, which may be MHC class I or class II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.
The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.
TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have TCRs consisting of γ and δ chains. Native α β heterodimeric TCRs have an α chain and a β chain, which constitute subunits of an α β heterodimeric TCR. Broadly, each of the α and β chains comprises a variable region, a linker region and a constant region, and the β chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered to be part of the linker region. Each variable region comprises 3 CDRs (complementarity determining regions) CDR1, CDR2 and CDR3, which are chimeric in framework structures (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, where CDR3 is recombined from variable and connecting regions, referred to as hypervariable regions. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domain being made up of linked variable regions and linking regions. The sequences of TCR constant domains can be found in public databases of the international immunogenetic information system (IMGT), such as the constant domain sequence of the α chain of the TCR molecule is "TRAC 01", the constant domain sequence of the β chain of the TCR molecule is "TRBC 1 01" or "TRBC 2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very 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 interchain disulfide bond and artificial interchain disulfide bond
A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".
For convenience of description of the positions of disulfide bonds, the positions of the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are numbered in the order from the N-terminus to the C-terminus, such as in TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid in the order from the N-terminus to the C-terminus is P (proline), and thus in the present invention it can be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, and as in 737bc 3 × 01 or TRBC2 × 01, and the 61 th amino acid in the order from the N-terminus to the C-terminus is Q (glutamine), and thus in the present invention it can be described as TRBC1 × 01 or TRBC 6301 × 01, or TRBC 8501, and similarly as TRBC 8261 or glbc 891. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.
Detailed Description
TCR molecules
During antigen processing, antigens are degraded within cells 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, a first aspect of 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 amino acid sequence:
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 can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR regions disclosed herein, so long as the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are those which comprise the above-described α and/or β 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 a variant of SEQ ID NO:5, 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 α and β chains. In particular, in one aspect the α chain of the heterodimeric TCR molecules comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO:11) and CDR3(SEQ ID NO:12) of the above-described α chain. Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the amino acid sequence of the α chain variable domain 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 above-described β chain. Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO 5.
In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-. From the literature, those skilled in the art are readily able to construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.
The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises the CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO:11) and CDR3(SEQ ID NO:12) of the alpha chain described above. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the beta chain variable domain of the single chain TCR molecule comprises the CDR1(SEQ ID NO:13), CDR2(SEQ ID NO:14) and CDR3(SEQ ID NO:15) of the above-described beta chain. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 5. More preferably, the amino acid sequence of the β chain variable domain 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 human constant domain amino acid sequences are known to those skilled in the art or can be obtained by consulting published databases of relevant books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules can be "TRBC 1 01" or "TRBC 2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecule of the invention is SEQ ID NO 3 and/or the amino acid sequence of the β chain is SEQ ID NO 7.
Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections 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 particular antigen. The invention also obtained soluble TCRs specific for HPV16E7 antigen short peptides.
To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide 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 their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native disulfide bond present in the TCR.
To obtain a soluble TCR, on the other hand, the inventive TCR also comprises a TCR having a mutation in its hydrophobic core region, preferably a mutation that enables an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or positions 3,5,7 of the reciprocal amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or positions 2,4,6 of the reciprocal amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.
The TCR with the mutated hydrophobic core region of the invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The single-chain soluble TCR constructed as in example 4 of the invention has an alpha chain variable domain amino acid sequence of SEQ ID NO. 32 and an encoded 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, for stability, patent document 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 the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, 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 of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.
In addition, the TCRs of the 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, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.
It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).
Nucleic acid molecules
A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a portion thereof, which may be one or more CDRs, variable domains of the alpha and/or beta chains, and the alpha and/or beta chains.
The nucleotide sequence encoding the α chain CDR regions of the TCR molecules 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 β chain CDR regions of the TCR molecules 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 sequence of the nucleic acid molecule of the invention encoding the TCR alpha chain of the invention comprises SEQ ID NO 16, 17 and 18 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR beta chain of the invention comprises SEQ ID NO 19, 20 and 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 an intron. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID NO 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID NO 6. Alternatively, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR α chain variable domain of the invention comprises SEQ ID NO 33 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR β chain variable domain 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; alternatively, the nucleotide sequence of the nucleic acid molecule of the invention is SEQ ID NO. 31.
It will be appreciated that, due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, the nucleic acid sequence encoding the TCR of the present invention may be identical to or a degenerate variant of the nucleic acid sequences shown in the figures of the present invention. As illustrated by one of the examples herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.
The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for various 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 obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.
Carrier
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, bacteriophages and animal and plant viruses.
Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.
Preferably, the vector can transfer the nucleotide of the invention into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the HPV16E7 antigen. Ideally, the vector should be capable of sustained high level expression in T cells.
Cells
The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises a vector of the invention or has integrated into its chromosome a nucleic acid molecule of the 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 cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells 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 (PBMC), which may be CD4+Helper T cell or CD8+Cytotoxic T cells. The cell may be in CD4+Helper T cell/CD 8+A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.
Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.
There are many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the TCRs of the invention may be used for adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Rev Cancer 8(4): 299-308).
HPV16E7 antigen-related diseases
The present invention also relates to a method of treating and/or preventing a disease associated with HPV16E7 in a subject, comprising the step of adoptively transferring HPV16E 7-specific T cells to the subject. The HPV16E7 specific T cell can recognize YMLDLQPET-HLA A0201 complex.
The HPV16E7 specific T cells can be used for treating any HPV16E7 related diseases presenting HPV16E7 antigen short peptide YMLDLQPET-HLA A0201 complexes. Including but not limited to tumors such as cervical cancer, head and neck tumors, anal cancer, and the like.
Method of treatment
Treatment may be effected by isolating T cells from patients or volunteers having a disease associated with the HPV16E7 antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating a disease associated with HPV16E7, comprising infusing into a patient an isolated T cell expressing a TCR of the invention, preferably the T cell is derived from the patient per se. Generally, this involves (1) isolating T cells from the patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.
The main advantages of the invention are:
(1) the inventive TCR can be specifically combined with HPV16E7 antigen short peptide complex YMLDLQPET-HLA A0201, and simultaneously, the cells transduced with the inventive TCR can be specifically activated.
(2) The effector cells that transduce the inventive TCR have a potent killing effect against the target cells.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
EXAMPLE 1 cloning of HPV16E7 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; Baisheng Gene technologies, Inc., Beijing Sai). YMLDLQPET short peptide and HLA-A0201 with biotin label are renatured to prepare pHLA haploid. These haploids were combined with streptavidin labeled with PE (BD Co.) to form PE-labeled tetramers, which were sorted for double positive anti-CD 8-APC cells. The sorted cells were expanded and subjected to secondary sorting as described above, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer and double positive clones were selected as shown in FIG. 3. The double positive clones obtained by layer-by-layer screening also need to meet the requirement of further functional test.
The function and specificity of the T cell clone were further tested by ELISPOT assay. Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The effector cells used in the IFN-gamma ELISPOT experiment in the embodiment are T cell clones obtained in the invention, the target cells are LCLs loaded with specific short peptides, and the control group is LCLs loaded with other short peptides and unloaded LCLs.
First, ELISPOT plates were prepared and the components of the assay were added to the ELISPOT plates in the following order: after 20,000 LCLs cells/well and 2000 effector cells/well, 20. mu.l of specific short peptide was added to the experimental group, 20. mu.l of non-specific short peptide was added to the control group, and 20. mu.l of medium (test medium) was added to the blank group, and 2 wells were set. Then incubated overnight (37 ℃, 5% CO)2). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID Co.). The experimental results are shown in fig. 14, and the obtained T cell clones have obvious activation response to the LCLs cells loaded with specific short peptides, but have no response to the LCLs cells loaded with other short peptides and unloaded LCLs cells.
Example 2 construction of TCR Gene and vector for obtaining HPV16E7 antigen short peptide specific T cell clone
Using Quick-RNATMMiniPrep (ZYMO research) extracted the total RNA of the T cell clone specific to the antigen short peptide YMLDLQPET and restricted by HLA-A0201 selected in example 1. cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, using primers designed to preserve the C-terminal region of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. It should be noted that the sequence is a complementary sequence, not including introns. The alpha chain and beta chain sequence structures of TCR expressed by the double positive clone are shown in figure 1 and figure 2 respectively through sequencing, figure 1a, figure 1b and figure1c, 1d, 1e and 1f are the amino acid sequence of the TCR alpha chain variable domain, the nucleotide sequence of the TCR alpha chain variable domain, the amino acid sequence of the TCR alpha chain, the nucleotide sequence of the TCR alpha chain, the amino acid sequence of the TCR alpha chain with a leader sequence and the nucleotide sequence of the TCR alpha chain with a leader sequence, respectively; fig. 2a, fig. 2b, fig. 2c, fig. 2d, fig. 2e and fig. 2f are a TCR α 0 chain variable domain amino acid sequence, a TCR α 1 chain variable domain nucleotide sequence, a TCR β chain amino acid sequence, a TCR β chain nucleotide sequence, a TCR β chain amino acid sequence with a leader sequence and a TCR β chain nucleotide sequence with a leader sequence, respectively.
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 beta 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 for the TCR α and β chains were cloned into the lentiviral expression vector pllenti (addendum) by overlap (overlap) PCR, respectively. The method specifically comprises the following steps: the TCR alpha chain and the TCR beta chain are connected by overlap PCR to obtain the TCR alpha-2A-TCR beta segment. And (3) carrying out enzyme digestion and connection on the lentivirus expression vector and the TCR alpha-2A-TCR beta to obtain pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. The pseudovirus was then packaged again at 293T/17.
Example 3 expression, refolding and purification of HPV16E7 antigen short peptide specific soluble TCR
To obtain soluble TCR molecules, the α and β chains of the TCR molecules of the invention may comprise only the variable and part of the constant domains thereof, respectively, and a cysteine residue has been introduced into the constant domains of the α and β chains, respectively, to form artificial interchain disulfide bonds, at the positions Thr48 of exon 1 TRAC × 01 and Ser57 of exon 1 TRBC2 × 01, respectively; the amino acid sequence and the nucleotide sequence of the alpha chain are respectively shown in figure 4a and figure 4b, and the amino acid sequence and the nucleotide sequence of the beta chain are respectively shown in figure 5a and figure 5 b. The above-mentioned gene sequences of interest for the TCR alpha and beta chains were synthesized and inserted into the expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning A Laboratory Manual (third edition, Sambrook and Russell), and the upstream and downstream Cloning sites were NcoI and NotI, respectively. The insert was confirmed by sequencing without error.
The expression vectors of TCR alpha and beta chains are transformed into expression bacteria BL21(DE3) by chemical transformation method, and the bacteria are grown in LB culture solution and OD600Inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times at 0.6 final induction with final concentration of 0.5mM IPTG, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT),10mM ethylenediaminetetraacetic acid (EDTA),20mM Tris (pH 8.1).
The TCR α and β chains after lysis were separated by 1: 1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1),3.7mM cystamine,6.6mM β -mercaptamine (4 ℃) to a final concentration of 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA. The SDS-PAGE gel of the soluble TCR of the invention is shown in FIG. 6.
Example 4 Generation of soluble Single chain TCR specific for HPV16E7 antigen short peptide
According to the disclosure of WO2014/206304, the variable domains of TCR α and β chains in example 2 were constructed as a stable soluble single-chain TCR molecule linked by a short flexible peptide (linker) using site-directed mutagenesis. The amino acid sequence and the nucleotide sequence of the single-chain TCR molecule are shown in FIG. 7a and FIG. 7b, respectively. The amino acid sequence and 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 its beta-chain variable domain are shown in FIG. 9a and FIG. 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 was digested simultaneously with Nco I and Not I, and ligated with pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli BL21(DE3) for expression.
Example 5 expression, renaturation and purification of soluble Single-chain TCR specific for the HPV16E7 antigen short peptide
The BL21(DE3) colony containing the recombinant plasmid pET28 a-template strand prepared in example 4 was inoculated in its entirety into LB medium containing kanamycin, cultured at 37 ℃ to OD600 of 0.6 to 0.8, IPTG was added to a final concentration of 0.5mM, and the culture was continued at 37 ℃ for 4 hours. The cell pellet was harvested by centrifugation at 5000rpm for 15min, the cell pellet was lysed by Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and centrifuged at 6000rpm for 15min to collect the inclusion bodies. The inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0,8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantified by BCA method and split charged, and stored at-80 ℃ for further use.
To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30 min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM beta-mercaptoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4 ℃ for 10min, and then the renaturation solution was filled into a cellulose membrane dialysis bag with a cut-off of 4kDa, and the bag was placed in 1L of precooled water and stirred slowly at 4 ℃ overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20mM Tris-HCl pH 8.0), dialysis was continued at 4 ℃ for 8h, and then dialysis was continued overnight with the same fresh buffer. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare), the protein was purified using a 0-1M NaCl linear gradient eluent formulated in 20mM Tris-HCl pH 8.0, the collected fractions were subjected to SDS-PAGE analysis, the fractions containing single-stranded TCR were concentrated and then further purified using a gel filtration column (Superdex 7510/300, GE Healthcare), and the target fraction was also subjected to SDS-PAGE analysis.
The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions are as follows: the chromatography column Agilent Bio SEC-3(300A,
Figure BDA0002395440870000191
) 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 214 nm.
The SDS-PAGE gel of the soluble single-chain TCR obtained by the invention is shown in FIG. 11.
Example 6 binding characterization
BIAcore analysis
Binding activity of the TCR molecules obtained in examples 3 and 5 to the YMLDLQPET-HLA A0201 complex was measured using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10mM sodium acetate buffer, pH 4.77), and then the antibody was passed through CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process at a coupling level of about 15,000 RU.
The low concentration of streptavidin was flowed over the antibody coated chip surface, then YMLDLQPET-HLA A0201 complex was flowed over the detection channel, the other channel served as the reference channel, and 0.05mM biotin was flowed over the chip at a flow rate of 10. mu.L/min for 2min to block the remaining binding sites of streptavidin.
The YMLDLQPET-HLA A0201 complex is prepared as follows:
a. purification of
Collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.
Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5 min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15 min; discarding the supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation
The synthesized short peptide YMLDLQPET (Beijing Baisheng Gene technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 8M Urea, 20mM Tris pH 8.0, 10mM DTT and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. YMLDLQPET peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure of the renaturation.
c. Purification after renaturation
The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5ml bed volume). The protein was eluted using an Akta purifier (GE general electric) with a 0-400mM NaCl linear gradient prepared in 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, the peak fractions were collected, and the purity was checked by SDS-PAGE.
d. Biotinylation of the compound
The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while displacing the buffer to 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. mu. M D-Biotin, 100. mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine the completion of biotinylation.
e. Purification of biotinylated complexes
The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using Akta purifier (GE general electric Co., Ltd.)TM16/60S200 HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by addition of the protease inhibitor cocktail (Roche).
Kinetic parameters were calculated by BIAcore Evaluation software, and kinetic profiles of the soluble TCR molecules of the invention and the binding of the soluble single-chain TCR molecules constructed by the invention to YMLDLQPET-HLA A0201 complex were obtained as shown in FIGS. 12 and 13, respectively. The maps show that both soluble TCR molecules and soluble single-chain TCR molecules obtained by the invention can be combined with YMLDLQPET-HLA A0201 complex. Meanwhile, the binding activity of the soluble TCR molecule and other irrelevant antigen short peptides and HLA complexes is detected by the method, and the result shows that the TCR molecule is not bound with other irrelevant antigens, thereby proving that the soluble TCR molecule can be specifically bound with YMLDLQPET-HLA A0201 complexes.
Example 7 activation of Effector cells transducing TCRs of the invention (target cells are T2 cells loaded with short peptides)
Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. Constructing a lentivirus vector containing the TCR target gene, transducing T cells, and carrying out an ELISPOT test to prove the specific activation response of the T cells transduced by the TCR on target cells. IFN-. gamma.production as measured by the ELISPOT assay was used as a readout for T cell activation.
First, reagents are prepared, including test media: 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) contains all the other reagents required (capture and detection antibodies, streptavidin-alkaline phosphatase and BCIP/NBT solution).
The target cell used in the experiment is T2 cell loaded with HPV16E7 antigen short peptide YMLDLQPET, and the effector cell used is CD3 expressing TCR specific to HPV16E7 antigen short peptide+T cells and transfection of CD3 of other TCRs (A6) in the same volunteer+T cells served as a control group. T cells were stimulated with anti-CD 3/CD28 coated beads (T cell amplicons, life technologies), the gene of interest was transduced, expanded in 1640 medium containing 50IU/ml IL-2 and 10ng/ml IL-7 with 10% FBS until 9-12 days post transduction, then the cells were placed in assay medium and washed by centrifugation at 300g for 10min at RT. The cells were then resuspended in the test medium at 2 × the desired final concentration. Negative control effector cells were treated as well. Corresponding short peptides were added to the corresponding target cell (T2) experimental group at a short peptide concentration of 10-6M。
The well plate was prepared as follows according to the manufacturer's instructions: 10ml of sterile PBS per plate 1: anti-human IFN-. gamma.capture antibody was diluted at 200, and 100. mu.l of the diluted capture antibody was aliquoted into each well. The plates were incubated overnight at 4 ℃. After incubation, the well plates were washed to remove excess capture antibody. 100 μ l/well of RPMI 1640 medium containing 10% FBS was added and the well plates were incubated at room temperature for 2 hours to close the well plates. The media was then washed from the well plate, and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate on paper. The components of the assay were then added to ELISPOT well plates:
the well plates were then incubated overnight (37 ℃/5% CO)2) The next day, the medium was discarded, the well plate was washed 2 times with double distilled water and 3 times with wash buffer, and tapped on a paper towel to remove residual wash buffer. Then, the mixture was mixed with PBS containing 10% FBS at a ratio of 1: the detection antibody was diluted at 200 and added to each well at 100. mu.l/well. The well plate was incubated at room temperature for 2 hours, washed 3 times with wash buffer and the well plate was tapped on a paper towel to remove excess wash buffer. PBS containing 10% FBS was used at 1: streptavidin-alkaline phosphatase was diluted 100, 100 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the wells were incubated for 1 hour at room temperature. The plates were then washed 2 times with 4 washes of PBS and tapped on a paper towel to remove excess wash buffer and PBS. After washing, 100 microliter of BCIP/NBT solution provided by the kit is added for development. And covering the well plate with tinfoil paper in the developing period, keeping the well plate in the dark, and standing for 5-15 minutes. Spots on the developing plate were routinely detected during this period to determine the optimum time for terminating the reaction. The BCIP/NBT solution was removed and the well plate was rinsed with double-distilled water to stop the development reaction, spun-dried, then the bottom of the well plate was removed, the well plate was dried at room temperature until each well was completely dried, and then the spots formed in the bottom film of the well plate were counted using an immune spot plate counter (CTL, cell Technology Limited).
The TCR-transduced T cells of the invention were tested for IFN- γ release in response to target cells loaded with HPV16E7 antigen short peptide YMLDLQPET by ELISPOT assay (as described above). The number of ELSPOT spots observed in each well was plotted using a graphipad prism 6.
The experimental results are shown in fig. 15, the T cells transduced with the TCR of the present invention showed significant activation response to the target cells loaded with the specific short peptide, while the T cells transduced with other TCRs showed substantially no response to the corresponding target cells; meanwhile, T cells transduced with the TCR of the invention were essentially non-activating in T2 cells loaded with other short peptides and in unloaded T2 cells.
Example 8 Experimental of the activation function of the Effector cells transfected with the TCR of the invention (the target cells are tumor cell lines)
This example also demonstrates that effector cells transfected with the high affinity TCRs of the invention have good specific activation of target cells. The function and specificity of the high affinity TCR of the invention in cells was examined by ELISPOT experiments.
Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The effector cell used is CD3 expressing the TCR specific to the HPV16E7 antigen short peptide+T cells, and CD3 of the same volunteer not transfected with the TCR of the invention+T cells served as a control group. The positive tumor cell line (target cell line) used was A375-E7(HPV 16E7 over-expressed). The negative tumor cell lines used were SK-MEL-28-E7(HPV16E7 overexpression), SK-MEL-28, SK-MEL-1, SK-MEL-5, MEL526, U266B1, SHP-77, HCC 827.
First, an ELISPOT plate was prepared. ELISPOT plate ethanol activation coating, 4 degrees C overnight. Day 1 of the experiment, coating was removed, washed and blocked, incubated at room temperature for two hours, blocking solution removed, and the components of the experiment were added to ELISPOT plates: cell line is 2X 104 2X 10 effector cells per well3One well (calculated as the positive rate of antibody) and two duplicate wells were set. Incubate overnight (37 ℃, 5% CO 2). On day 2 of the experiment, the plates were washed and subjected to secondary detection and color development, dried, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).
The experimental results are shown in fig. 16, and for the positive tumor cell line, the effector cells transfected with the TCR of the present invention produced very good specific activation, while the cells transduced other TCRs produced substantially no activation; at the same time, effector cells transfected with the inventive TCR were essentially non-activating in response to the negative cell line.
Example 9 experiment of LDH killing function of Effector cells transfected with TCR of the invention
This example demonstrates the killing function of cells transduced with the TCR of the invention by measuring LDH release by non-radioactive cytotoxicity assays. This test is a colorimetric substitution test for the 51Cr release cytotoxicity test, and quantitatively determines Lactate Dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30min coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader.
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 TCR specific for the HPV16E7 antigen short peptide of the invention, and CD3+ T cells transfected with other TCRs (A6) from the same volunteer were used as a control. The positive tumor cell line (target cell line) used comprises CASKI, A375-E7(HPV 16E7 overexpression). The negative tumor cell lines used: a375 and SiHa.
LDH plates were first prepared. On experiment day 1, the individual components of the experiment were added to the plate: cell line 3X 104Single cell/well, effector cell 3X 104One cell per well and three duplicate wells were set. Meanwhile, an effector cell spontaneous hole, a target cell maximum hole, a volume correction control hole and a culture medium background control hole are arranged. Incubate overnight (37 ℃, 5% CO 2). On day 2 of the experiment, color development was detected, and after termination of the reaction, the absorbance was recorded at 490nm using a microplate reader (Bioteck).
The experimental results are shown in fig. 17, and the cells transduced the TCR of the present invention had significant killing effect against the target cell line, while the killing effect of the cells transduced other TCRs was weak; at the same time, cells transduced with the inventive TCR had no killing effect on negative tumor cell lines.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Guangdong Xiangxue accurate medical technology Limited
<120> a 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
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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
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Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly
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Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser
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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
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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
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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
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Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Pro Arg Tyr Gly
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Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys Ser Tyr
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Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser
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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
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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
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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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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
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Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly
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Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr
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Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser Asp Gly Tyr
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Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser Leu Glu Ser
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Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg
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ctgggcctac ggttgatcta ttactccttt gatgtcaaag atataaacaa aggagagatc 180
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<213> Artificial Sequence (Artificial Sequence)
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Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr Lys Thr Gly
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Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His Asp Arg Met
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Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Tyr
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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
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Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr Ser Asp Arg
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Gly Gln Gly Ala Phe Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu
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Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val
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Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu
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Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp
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Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln
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Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser
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Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
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Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp
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Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala
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Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly
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Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr
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Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys
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Arg Lys Asp Ser Arg Gly
290
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gatgctgatg ttacccagac cccaaggaat aggatcacaa agacaggaaa gaggattatg 60
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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 (10)

1. A T Cell Receptor (TCR), wherein the TCR is capable of binding to the YMLDLQPET-HLA a0201 complex; preferably, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the amino acid sequence of CDR3 of the TCR alpha chain variable domain is AVNPRYGNKLV (SEQ ID NO: 12); and/or the amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ATSDRGQGAFGEQY (SEQ ID NO: 15);
more preferably, 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)。
2. a TCR as claimed in claim 1 which comprises a TCR α chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1 and a TCR β chain variable domain; 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 wherein a conjugate is attached to the C-or N-terminus of the α and/or β chains of the TCR; preferably, 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.
4. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in any one of the preceding claims.
5. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to any preceding claim, or the complement thereof;
preferably, the nucleic acid molecule comprises the nucleotide sequence encoding the variable domain of the TCR α chain SEQ ID NO:2 or SEQ ID NO: 33; and/or
The nucleic acid molecule comprises a nucleotide sequence encoding a variable domain of a TCR β chain SEQ ID NO:6 or SEQ ID NO 35.
6. A vector comprising the nucleic acid molecule of claim 5; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
7. An isolated host cell comprising the vector of claim 6 or a nucleic acid molecule of claim 5 integrated into the chromosome.
8. A cell which transduces the nucleic acid molecule of claim 5 or the vector of claim 6; preferably, the cell is a T cell or a stem cell.
9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1-3, a TCR complex according to claim 4, a nucleic acid molecule according to claim 5, or a cell according to claim 8.
10. Use of a T cell receptor according to any one of claims 1 to 3, or a TCR complex according to claim 4 or a cell according to claim 8, for the preparation of a medicament for the treatment of a tumour or an autoimmune disease.
CN202010129637.6A 2020-02-28 2020-02-28 T cell receptor for identifying HPV Pending CN113321726A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN202010129637.6A CN113321726A (en) 2020-02-28 2020-02-28 T cell receptor for identifying HPV
PCT/CN2021/078275 WO2021170115A1 (en) 2020-02-28 2021-02-26 T-cell receptor recognizing hpv
TW110107149A TW202140540A (en) 2020-02-28 2021-02-26 T-cell receptor recognizing HPV

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010129637.6A CN113321726A (en) 2020-02-28 2020-02-28 T cell receptor for identifying HPV

Publications (1)

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