WO2022166904A1 - 一种识别hpv的t细胞受体 - Google Patents

一种识别hpv的t细胞受体 Download PDF

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WO2022166904A1
WO2022166904A1 PCT/CN2022/075111 CN2022075111W WO2022166904A1 WO 2022166904 A1 WO2022166904 A1 WO 2022166904A1 CN 2022075111 W CN2022075111 W CN 2022075111W WO 2022166904 A1 WO2022166904 A1 WO 2022166904A1
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tcr
chain
amino acid
seq
exon
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French (fr)
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李懿
杨东雪
吴万里
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香雪生命科学技术（广东）有限公司
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
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    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
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    • A61K39/464838—Viral antigens
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a TCR capable of recognizing a short peptide derived from HPV16 E6 antigen and its coding sequence, the present invention also relates to HPV16 E6-specific T cells obtained by transducing the above-mentioned TCR, and their use in the prevention and treatment of HPV16 E6-related diseases use.
  • the E6 gene is one of the early region genes of the human papillomavirus (HPV) genome, located at bases 83-559 of the HPV gene, and encodes the E6 protein.
  • HPV16 The most prevalent type of cervical cancer worldwide is HPV16, accounting for 50% to 60% of detected cases (Acta Acad Med Sin, 2007, 29(5): 678-684); and E6 protein is a high-risk HPV infection of cervical One of two important tumorigenic proteins encoded by epithelial cells ([J]. Journal of Jiangsu University (Medical Edition), 2018, 28(2):135-139).
  • HPV16 E6 can also cause head and neck tumors (Chinese Journal of Otolaryngology Skull Base Surgery, 2017, 23(6):594-598), conjunctival intraepithelial neoplasia (CIN), and invasive squamous cell carcinoma (SCC) of the cornea and conjunctiva. (International Journal of Ophthalmology 2018;18(6):1047-1050) and other diseases.
  • TIHDIILECV SEQ ID NO: 9 is a short peptide derived from the HPV16 E6 protein and is a target for the treatment of HPV16 E6-related diseases.
  • T cell adoptive immunotherapy is the transfer of T cells specific for target cell antigens into the patient's body, so that they can play a role against the target cells.
  • T cell receptor TCR
  • T cell receptor is a membrane protein on the surface of T cells that can recognize short antigenic peptides on the surface of the corresponding target cells.
  • APCs antigen presenting cells
  • T cells and The other cell membrane surface molecules of the two APCs interact, causing a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities can exert immune effects on their target cells.
  • the purpose of the present invention is to provide a T cell receptor that recognizes the HPV16 E6 antigenic short peptide.
  • the first aspect of the present invention provides a T cell receptor (TCR), the TCR can bind to the TIHDIILECV-HLA A0201 complex.
  • TCR T cell receptor
  • the present invention provides an isolated or purified TCR, which can bind to the TIHDIILECV-HLA A0201 complex.
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is AMREGTGTASKLT (SEQ ID NO: 12); and/or the The amino acid sequence of CDR3 of the variable domain of the TCR beta chain is ASSPYGQGTSPLH (SEQ ID NO: 15).
  • the three complementarity determining regions (CDRs) of the variable domain of the TCR ⁇ chain are:
  • the three complementarity determining regions of the TCR ⁇ chain variable domain are:
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1
  • the TCR beta chain variable domain is an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5.
  • the TCR comprises the ⁇ chain variable domain amino acid sequence SEQ ID NO: 1.
  • the TCR comprises ⁇ chain variable domain amino acid sequence SEQ ID NO:5.
  • the TCR is an ⁇ heterodimer, which comprises a TCR ⁇ chain constant region TRAC*01 and a TCR ⁇ chain constant region TRBC1*01 or TRBC2*01.
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 3 and/or the amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 7.
  • the TCR of the present invention is of human origin.
  • the TCR is soluble.
  • the TCR is single-chain.
  • the TCR is formed by linking the ⁇ chain variable domain and the ⁇ chain variable domain through a peptide linker sequence.
  • the constant regions of the ⁇ and ⁇ chains of the TCR are the constant regions of the murine ⁇ and ⁇ chains, respectively.
  • the TCR is at the 11th, 13th, 19th, 21st, 53rd, 76th, 89th, 91st, or 94th amino acid position of the ⁇ chain variable region, and/or the reciprocal amino acid of the ⁇ chain J gene short peptide has one or more mutations in position 3, 5 from the bottom, or 7 from the bottom; and/or the TCR is at amino acids 11, 13, 19, 21, 53, 76, 89, 91 of the beta chain variable region , or position 94, and/or one or more mutations in the penultimate amino acid position 2, 4 or 6 of the ⁇ -chain J gene short peptide, wherein the amino acid positions are numbered according to IMGT (International Information on Immunogenetics) system) listed in the position number.
  • IMGT International Information on Immunogenetics
  • the amino acid sequence of the ⁇ -chain variable domain of the TCR comprises SEQ ID NO:32 and/or the amino acid sequence of the ⁇ -chain variable domain of the TCR comprises SEQ ID NO:34.
  • amino acid sequence of the TCR is SEQ ID NO:30.
  • the TCR comprises (a) all or part of the TCR ⁇ chain excluding the transmembrane domain; and (b) all or part of the TCR ⁇ chain excluding the transmembrane domain;
  • (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.
  • cysteine residues form an artificial disulfide bond between the constant domains of the ⁇ and ⁇ chains of the TCR.
  • cysteine residues that form artificial disulfide bonds in the TCR are substituted for one or more groups of sites selected from the following:
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 26 and/or the amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO: 28.
  • an artificial interchain disulfide bond is contained between the variable region of the ⁇ chain and the constant region of the ⁇ chain of the TCR.
  • cysteine residues that form artificial interchain disulfide bonds in the TCR are substituted with one or more sites selected from the following groups:
  • the TCR comprises an ⁇ -chain variable domain and a ⁇ -chain variable domain and all or part of the ⁇ -chain constant domain except the transmembrane domain, but it does not comprise an ⁇ -chain constant domain, and the TCR
  • the ⁇ chain variable domain forms a heterodimer with the ⁇ chain.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or the ⁇ chain of the TCR.
  • the conjugate bound to the T cell receptor is a detectable label, a therapeutic agent, a PK modification moiety or a combination of any of these substances; preferably, the therapeutic agent is an anti-CD3 antibody .
  • the second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.
  • the third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or a complementary sequence thereof.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 33 encoding the variable domain of the TCR ⁇ chain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 or SEQ ID NO: 35 encoding the variable domain of the TCR beta chain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO:4 encoding the TCR ⁇ chain and/or the nucleotide sequence SEQ ID NO:8 encoding the TCR ⁇ chain.
  • the fourth aspect of the present invention provides a vector, which contains the nucleic acid molecule described in the third aspect of the present invention; preferably, the vector is a viral vector; more preferably, the vector is a viral vector viral vector.
  • the fifth aspect of the present invention provides an isolated host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention or the exogenous nucleic acid molecule described in the third aspect of the present invention is integrated into the genome .
  • the sixth aspect of the present invention provides a cell transduced with the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention; preferably, the cell is a T cell, NK cells, NKT cells or stem cells.
  • a seventh aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, the present
  • the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, or the cell of the sixth aspect of the present invention is provided.
  • the eighth aspect of the present invention provides the use of the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention, for use in To prepare a medicine for treating tumor or autoimmune disease, preferably, the tumor is cervical cancer.
  • the ninth aspect of the present invention provides the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention for use in the treatment of tumors or Drugs for autoimmune diseases; preferably, the tumor is cervical cancer.
  • the tenth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the T cell receptor described in the first aspect of the present invention or the TCR complex described in the second aspect of the present invention to a subject in need of treatment , or the cells described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention; preferably, the disease is a tumor, preferably the tumor is cervical cancer, head and neck tumor.
  • Fig. 1a, Fig. 1b, Fig. 1c, Fig. 1d, Fig. 1e and Fig. 1f are respectively the amino acid sequence of TCR ⁇ chain variable domain, the nucleotide sequence of TCR ⁇ chain variable domain, the amino acid sequence of TCR ⁇ chain, the nucleotide sequence of TCR ⁇ chain, with The amino acid sequence of the TCR ⁇ chain of the leader sequence and the nucleotide sequence of the TCR ⁇ chain with the leader sequence.
  • Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the variable domain of the TCR ⁇ chain, the nucleotide sequence of the variable domain of the TCR ⁇ chain, the amino acid sequence of the TCR ⁇ chain, and the nucleotide sequence of the TCR ⁇ chain, respectively.
  • Figure 3 shows the results of CD8+ and tetramer-PE double-positive staining of monoclonal cells.
  • Figure 4a and Figure 4b show the amino acid sequence and nucleotide sequence of the soluble TCR ⁇ chain, respectively.
  • Figure 5a and Figure 5b are the amino acid sequence and nucleotide sequence of the soluble TCR beta chain, respectively.
  • Figure 6 is a gel image of the soluble TCR obtained after purification.
  • the leftmost lane is the reducing gel
  • the middle lane is the molecular weight marker
  • the rightmost lane is the non-reducing gel.
  • Figure 7a and Figure 7b show the amino acid sequence and nucleotide sequence of the single-chain TCR, respectively.
  • Figures 8a and 8b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR alpha chain, respectively.
  • Figures 9a and 9b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCR beta chain, respectively.
  • Figures 10a and 10b are the amino acid sequence and nucleotide sequence of the single-stranded TCR linker, respectively.
  • Figure 11 is a gel image of the soluble single-chain TCR obtained after purification.
  • the leftmost lane is the reducing gel
  • the middle lane is the molecular weight marker
  • the rightmost lane is the non-reducing gel.
  • Figure 12 is a BIAcore kinetic map of the binding of the soluble TCR of the present invention to the TIHDIILECV-HLA A0201 complex.
  • Figure 13 is a BIAcore kinetic map of the binding of the soluble single-chain TCR of the present invention to the TIHDIILECV-HLA A0201 complex.
  • Fig. 14 shows the results of verification of the ELISPOT activation function of the obtained T cell clones.
  • Figure 15 shows the results of ELISPOT activation function verification in effector cells transfected with the TCR of the present invention.
  • FIG. 16 shows the results of ELISPOT activation function verification of effector cells transfected with the TCR of the present invention for tumor cell lines.
  • the present invention also provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule.
  • the present invention also provides cells transduced with the TCR of the present invention.
  • MHC molecules are proteins of the immunoglobulin superfamily and can be class I or class II MHC molecules. Therefore, it is specific for antigen presentation, and different individuals have different MHCs, which can present different short peptides in a protein antigen to their respective APC cell surfaces.
  • the human MHC is often referred to as the HLA gene or HLA complex.
  • TCR The T cell receptor
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • TCR is a glycoprotein on the surface of the cell membrane that exists in the form of heterodimers of ⁇ chain/ ⁇ chain or ⁇ chain/ ⁇ chain.
  • TCR heterodimers consist of alpha and beta chains in 95% of T cells, whereas 5% of T cells have TCRs composed of gamma and delta chains.
  • a native ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain, and the ⁇ chain and the ⁇ chain constitute the subunits of the ⁇ heterodimeric TCR.
  • the alpha and beta chains each contain a variable region, a linker region, and a constant region
  • the beta chain usually also contains a short variable region between the variable region and the linker region, but the variable region is often regarded as the linker region a part of.
  • Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimerically incorporated in framework regions.
  • the CDR region determines the binding of TCR to the pMHC complex, and CDR3 is recombined from the variable region and the linker region, which is called the hypervariable region.
  • the alpha and beta chains of a TCR are generally viewed as having two "domains” each, a variable domain and a constant domain, the variable domains being composed of linked variable and linking regions.
  • the sequences of the TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT). 01" or "TRBC2*01".
  • IMGT International Immunogenetics Information System
  • the ⁇ and ⁇ chains of TCR also contain a transmembrane region and a cytoplasmic region, and the cytoplasmic region is very short.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention T cell receptor of the present invention
  • the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered sequentially from the N-terminus to the C-terminus, such as TRBC1*01 or TRBC2*01 , the 60th amino acid in the sequence from the N-terminus to the C-terminus is P (proline), then in the present invention, it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01, or it can be described as Pro60 of exon 1 of TRBC1*01 It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in TRBC1*01 or TRBC2*01, the 61st amino acid in the sequence from the N-terminus to the C-terminus is Q (glutamate).
  • the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are numbered according to the position numbers listed in IMGT. For example, for a certain amino acid in TRAV, the position number listed in IMGT is 46, then it is described as the 46th amino acid of TRAV in the present invention, and so on. In the present invention, if the sequence position numbering of other amino acids has special instructions, the special instructions are followed.
  • a first aspect of the present invention provides a TCR molecule capable of binding the TIHDIILECV-HLA A0201 complex.
  • the TCR molecule is isolated or purified.
  • the alpha and beta chains of this TCR each have three complementarity determining regions (CDRs).
  • the alpha chain of the TCR comprises a CDR having the following amino acid sequence:
  • the three complementarity determining regions of the TCR ⁇ chain variable domain are:
  • Chimeric TCRs can be prepared by inserting the above-described amino acid sequences of the CDR regions of the present invention into any suitable framework structure.
  • the framework structure is compatible with the CDR regions of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR regions disclosed in the present invention.
  • the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned alpha and/or beta chain CDR region sequences and any suitable framework structure.
  • the TCR ⁇ chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity with SEQ ID NO: 1; and/or the TCR ⁇ chain variable domain of the present invention is an amino acid sequence with SEQ ID NO: 1 NO:5 has an amino acid sequence of at least 90%, preferably 95%, more preferably 98% sequence identity.
  • the TCR molecule of the present invention is a heterodimer composed of ⁇ and ⁇ chains.
  • the ⁇ chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain
  • the amino acid sequence of the ⁇ chain variable domain comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 10) and CDR2 (SEQ ID NO: 10) of the above-mentioned ⁇ chain. ID NO: 11) and CDR3 (SEQ ID NO: 12).
  • the TCR molecule comprises the alpha chain variable domain amino acid sequence of SEQ ID NO: 1.
  • the ⁇ chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO: 1.
  • the ⁇ chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the ⁇ chain variable domain comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 13) of the above-mentioned ⁇ chain NO: 14) and CDR3 (SEQ ID NO: 15).
  • the TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the ⁇ chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO:5.
  • the TCR molecule of the present invention is a single-chain TCR molecule composed of part or all of the ⁇ chain and/or part or all of the ⁇ chain.
  • a description of single-chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. Those skilled in the art can readily construct single-chain TCR molecules comprising the CDRs regions of the present invention as described in the literature.
  • the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably linked in order from the N-terminus to the C-terminus.
  • the ⁇ chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-mentioned ⁇ chain.
  • the single-chain TCR molecule comprises the alpha chain variable domain amino acid sequence of SEQ ID NO: 1. More preferably, the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO: 1.
  • the beta chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above beta chain.
  • the single-chain TCR molecule comprises the beta chain variable domain amino acid sequence of SEQ ID NO:5. More preferably, the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule is SEQ ID NO:5.
  • the constant domain of the TCR molecule of the present invention is a human constant domain.
  • the constant domain sequence of the alpha chain of the TCR molecule of the present invention can be "TRAC*01”
  • the constant domain sequence of the beta chain of the TCR molecule can be "TRBC1*01” or "TRBC2*01”.
  • the 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is represented here as: Arg53 of exon 1 of TRAC*01, and so on.
  • the amino acid sequence of the ⁇ chain of the TCR molecule of the present invention is SEQ ID NO:3, and/or the amino acid sequence of the ⁇ chain is SEQ ID NO:7.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules need to be obtained. Soluble TCR molecules do not include their transmembrane domains. Soluble TCR has a wide range of uses, not only to study the interaction of TCR with pMHC, but also as a diagnostic tool to detect infection or as a marker for autoimmune diseases.
  • soluble TCRs can be used to deliver therapeutic agents (eg, cytotoxic or immunostimulatory compounds) to cells presenting specific antigens, and in addition, soluble TCRs can bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells so that they target cells presenting specific antigens.
  • therapeutic agents eg, cytotoxic or immunostimulatory compounds
  • soluble TCRs can bind to other molecules (eg, anti-CD3 antibodies) to redirect T cells so that they target cells presenting specific antigens.
  • the present invention also obtains a soluble TCR specific for the HPV16 E6 antigenic short peptide.
  • the TCRs of the invention may be TCRs in which artificial disulfide bonds are introduced between residues of their alpha and beta chain constant domains.
  • Cysteine residues form artificial interchain disulfide bonds between the constant domains of the alpha and beta chains of the TCR.
  • Cysteine residues can be substituted for other amino acid residues at appropriate sites in the native TCR to form artificial interchain disulfide bonds. For example, substitution of Thr48 of exon 1 of TRAC*01 and substitution of cysteine residues of Ser57 of exon 1 of TRBC1*01 or TRBC2*01 to form a disulfide bond.
  • cysteine residues are introduced to form disulfide bonds can also be: Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; exon 1 of TRAC*01 1 Tyr10 and TRBC1*01 or Ser17 of TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and Glu15 in exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 and exon 1 of TRAC*01 Ala19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.
  • cysteine residues are substituted for any set of sites in the constant domains of the alpha and beta chains described above.
  • One or more C-termini of the TCR constant domains of the invention may be truncated 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 that they do not include
  • the purpose of deleting the natural disulfide bond can be achieved by the cysteine residue, or by mutating the cysteine residue forming the natural disulfide bond into another amino acid to achieve the above purpose.
  • the TCRs of the present invention may contain artificial disulfide bonds introduced between residues of their alpha and beta chain constant domains. It should be noted that the TCRs of the invention may contain both a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced above between the constant domains.
  • the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by natural disulfide bonds present in the TCR.
  • the TCR of the present invention also includes a TCR with mutations in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation that can improve the stability of the soluble TCR of the present invention, such as in the publication No. Described in the patent document of WO2014/206304.
  • Such a TCR may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the 3rd, 5th, 7th amino acid position from the bottom of the short peptide of ⁇ -chain J gene (TRAJ), and/or the 2nd, 4th, 6th position of the penultimate amino acid position of the short peptide of ⁇ -chain J gene (TRBJ), wherein the position number of the amino acid sequence Numbered by position as listed in the International Information System on Immunogenetics (IMGT).
  • IMGT International Information System on Immunogenetics
  • the mutated TCR in the hydrophobic core region can be a stable soluble single-chain TCR composed of a flexible peptide chain linking the variable domains of the ⁇ and ⁇ chains of the TCR.
  • the flexible peptide chain in the present invention can be any peptide chain suitable for linking the variable domains of TCR ⁇ and ⁇ chain.
  • its ⁇ chain variable domain amino acid sequence is SEQ ID NO: 32, and the encoded nucleotide sequence is SEQ ID NO: 33; the ⁇ chain variable domain amino acid sequence is SEQ ID NO: 33; is SEQ ID NO:34, and the encoded nucleotide sequence is SEQ ID NO:35.
  • Patent Document 201680003540.2 also discloses that the introduction of artificial interchain disulfide bonds between the ⁇ chain variable region and the ⁇ chain constant region of TCR can significantly improve the stability of TCR. Therefore, the TCR of the present invention may further contain an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region.
  • cysteine residue that forms an artificial interchain disulfide bond between the ⁇ chain variable region and the ⁇ chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* 01 amino acid 60 of exon 1; TRAV amino acid 47 and TRBC1*01 or TRBC2*01 exon 1 amino acid 61; TRAV 46 amino acid and TRBC1*01 or TRBC2*01 exon Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
  • such a TCR may comprise (i) all or part of the TCR alpha chain excluding its transmembrane domain, and (ii) all or part of the TCR beta chain excluding its transmembrane domain, wherein (i) and (ii) ) both contain the variable domain and at least a part of the constant domain of the TCR chain, and the ⁇ chain and the ⁇ chain form a heterodimer.
  • such a TCR may contain an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except the transmembrane domain, but it does not contain the alpha chain constant domain, the alpha chain of the TCR.
  • the chain variable domains form heterodimers with beta chains.
  • the TCRs of the present invention may also be provided in the form of multivalent complexes.
  • the multivalent TCR complexes of the present invention comprise two, three, four or more multimers formed by combining the TCRs of the present invention, for example, the tetramerization domain of p53 can be used to generate tetramers, or multiple A complex formed by binding of a TCR of the present invention to another molecule.
  • the TCR complexes of the present invention can be used to track or target cells presenting specific antigens in vitro or in vivo, as well as to generate intermediates for other multivalent TCR complexes with such applications.
  • the TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the TIHDIILECV-HLA A0201 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any of the above combination or conjugation.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
  • Therapeutic agents that can be bound or conjugated to the TCR of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biotoxicity (Chaudhary et al., 1989) , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2, etc.
  • Viral particles (Peng et al, 2004, Gene Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (eg, DT-myocardial flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.
  • DTD DT-myocardial flavinase
  • BPHL biphenyl hydrolase-like protein
  • the TCRs of the present invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the TCRs of the present invention may comprise human variable domains and murine constant domains. The downside of this approach is the possibility of eliciting an immune response. Therefore, there should be a regulatory regime for immunosuppression when it is used in adoptive T cell therapy to allow engraftment of murine expressing T cells.
  • 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 portion may be one or more CDRs, variable domains of alpha and/or beta chains, and alpha chains and/or or beta chains.
  • nucleotide sequence encoding the ⁇ chain CDR region of the TCR molecule according to the first aspect of the present invention is as follows:
  • nucleotide sequence encoding the beta chain CDR region of the TCR molecule according to the first aspect of the present invention is as follows:
  • nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR alpha chains of the invention include SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the nucleotide sequences of the nucleic acid molecules of the invention encoding the TCR beta chains of the invention
  • Nucleotide sequences include SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns.
  • the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but is capable of encoding the polypeptide of the present invention, for example, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR alpha chain of the present invention includes SEQ ID NO: 2 and /or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR beta chain of the present invention comprises SEQ ID NO:6.
  • the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR alpha chain of the present invention includes SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR beta chain of the present invention includes SEQ ID NO: 33 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 present invention is SEQ ID NO:31.
  • nucleic acid sequences encoding the TCRs of the present invention may be identical or degenerate variants of the nucleic acid sequences shown in the figures of the present invention.
  • degenerate variant refers to a nucleic acid sequence that encodes the protein sequence of 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 use of specific codons. Depending on the type of cell, the codons in the sequence can be changed to increase the amount of expression. Codon usage tables for mammalian cells, as well as 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 generally be obtained by, but not limited to, PCR amplification method, recombinant method or artificial synthesis method.
  • the DNA sequences encoding the TCRs of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. DNA can be the coding or non-coding strand.
  • the present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, ie constructs capable of in vivo or in vitro expression.
  • expression 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, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
  • AAV adeno-associated virus
  • the vector can transfer the nucleotides of the invention into a cell, such as a T cell, such that the cell expresses a TCR specific for the HPV16 E6 antigen.
  • a cell such as a T cell
  • the vector should be able to express consistently high levels in T cells.
  • the present invention also relates to host cells genetically engineered with the vectors or coding sequences of the present invention.
  • the host cell contains the vector of the present invention or the nucleic acid molecule of the present invention is integrated into the chromosome.
  • the host cell is selected from: prokaryotic cells and eukaryotic cells, such as E. coli, yeast cells, CHO cells, and the like.
  • the present invention also includes isolated cells expressing the TCR of the present invention, which can be, but are not limited to, T cells, NK cells, NKT cells, especially T cells.
  • the T cells can be derived from T cells isolated from the subject, or can be part of a mixed population of cells isolated from the subject, such as a peripheral blood lymphocyte (PBL) population.
  • PBL peripheral blood lymphocyte
  • the cells can be isolated from peripheral blood mononuclear cells (PBMCs) and can be CD4+ helper T cells or CD8+ cytotoxic T cells.
  • PBMCs peripheral blood mononuclear cells
  • the cells may be in a mixed population of CD4+ helper T cells/CD8+ cytotoxic T cells.
  • the cells can be activated with antibodies (eg, anti-CD3 or anti-CD28 antibodies) to render them more receptive to transfection, eg, with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention. dye.
  • antibodies eg, anti-CD3 or anti-CD28 antibodies
  • the cells of the invention may also be or derived from stem cells, such as hematopoietic stem cells (HSCs).
  • stem cells such as hematopoietic stem cells (HSCs).
  • HSCs hematopoietic stem cells
  • T cells expressing the TCR of the present invention can be used for adoptive immunotherapy.
  • Those skilled in the art are aware of many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • the present invention also relates to a method of treating and/or preventing a disease associated with HPV16 E6 in a subject, comprising the step of adoptively transferring HPV16 E6-specific T cells to the subject.
  • the HPV16 E6-specific T cells recognized the TIHDIILECV-HLA A0201 complex.
  • HPV16 E6-specific T cells of the present invention can be used to treat any HPV16 E6-related diseases that present the HPV16 E6 antigen short peptide TIHDIILECV-HLA A0201 complex, including but not limited to tumors, such as cervical cancer, head and neck tumors, and the like.
  • Treatment can be performed by isolating T cells from patients or volunteers suffering from a disease associated with the HPV16 E6 antigen, introducing the TCR of the present invention into the above T cells, and then infusing these genetically engineered cells back into the patient. Therefore, the present invention provides a method for treating HPV16 E6-related diseases, which comprises the steps of infusing the isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself, into the patient.
  • the number of cells isolated, transfected, and reinfused can be determined by the physician.
  • the TCR of the present invention can specifically bind to the HPV16 E6 antigen short peptide complex TIHDIILECV-HLA A0201, and the effector cells transduced with the TCR of the present invention can be specifically activated.
  • Peripheral blood lymphocytes from healthy volunteers with HLA-A0201 genotype were stimulated with the synthetic short peptide TIHDIILECV (SEQ ID NO: 9; Jiangsu GenScript Biotechnology Co., Ltd.). pHLA haploids were prepared by renaturing the TIHDIILECV peptide with biotin-labeled HLA-A0201. These haploids were combined with PE-labeled streptavidin (BD) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double positive cells were sorted. Sorted cells were expanded and subjected to secondary sorting as described above, followed by monocloning by limiting dilution. Monoclonal cells were stained with tetramers, and the screened double-positive clones were shown in Figure 3. The double-positive clones obtained through layer-by-layer screening still need to meet further functional tests.
  • TIHDIILECV synthetic short peptide TIHDIILECV
  • the function and specificity of the T cell clone were further tested by ELISPOT assay.
  • Those skilled in the art are familiar with methods for detecting cell function using ELISPOT assays.
  • the effector cells used in the IFN- ⁇ ELISPOT experiment in this example are the T cell clones obtained in the present invention, the target cells are T2 cells loaded with TIHDIILECV short peptide, A375-E6 (overexpressing E6), and the control group is loaded with other short peptides. Peptide T2 cells and MCF-7.
  • the ELISPOT experimental steps are as follows: Add the components of the test to the ELISPOT plate in the following order: 20,000 target cells/well, 2000 effector cells/well, add 20 ⁇ l of the corresponding short peptides to the experimental group and the control group , the blank group was added with 20 ⁇ l of culture medium (test medium), and 2 duplicate wells were set up. It was then incubated overnight (37°C, 5% CO2 ). The plates were then washed and subjected to secondary detection and color development, the plates were dried for 1 hour, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID Corporation). The experimental results are shown in Figure 14.
  • T cell clones have obvious activation responses to T2 cells loaded with TIHDIILECV short peptide and A375-E6 overexpressing E6 antigen, while T2 cells loaded with other short peptides and negative cells have obvious activation responses.
  • Line MCF-7 basically no response.
  • RNA of the antigen short peptide TIHDLILECV-specific and HLA-A0201-restricted T cell clone screened in Example 1 was extracted with Quick-RNA TM MiniPrep (ZYMO research).
  • the cDNA was synthesized using the SMART RACE cDNA amplification kit from clontech, and the primers used were designed in the C-terminal conserved region of the human TCR gene.
  • the sequences were cloned into T vector (TAKARA) for sequencing. It should be noted that this sequence is complementary and does not contain introns. After sequencing, the chain and chain sequence structures of the TCR expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively.
  • Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e, and Figure 1f are the variable domains of the TCR ⁇ chain, respectively.
  • Figure 2a, Figure 2b , Figure 2c, Figure 2d, Figure 2e, and Figure 2f are the amino acid sequence of the variable domain of the TCR ⁇ chain, the nucleotide sequence of the variable domain of the TCR ⁇ chain, the amino acid sequence of the TCR ⁇ chain, the nucleotide sequence of the TCR ⁇ chain, and the TCR ⁇ chain with a leader sequence.
  • the alpha chain was identified as comprising CDRs with the following amino acid sequence:
  • the beta chain contains CDRs with the following amino acid sequence:
  • the full-length genes of the TCR alpha chain and beta chain were cloned into the lentiviral expression vector pLenti (addgene), respectively, by overlapping PCR.
  • the TCR ⁇ -2A-TCR ⁇ fragment was obtained by connecting the full-length genes of the TCR ⁇ chain and the TCR ⁇ chain by overlap PCR.
  • the pLenti-TRA-2A-TRB-IRES-NGFR plasmid was obtained by ligating the lentiviral expression vector and TCR ⁇ -2A-TCR ⁇ restriction enzyme.
  • the lentiviral vector pLenti-eGFP expressing eGFP was also constructed as a control. The pseudovirus was then packaged with 293T/17.
  • the ⁇ and ⁇ chains of the TCR molecule of the present invention can respectively contain only their variable domains and part of their constant domains, and a cysteine residue is introduced into the constant domains of the ⁇ and ⁇ chains respectively.
  • the amino acid sequence and nucleotide sequence of the ⁇ chain are shown in Figure 4a and Figure 4b, respectively, and the amino acid sequence and nucleotide sequence of the ⁇ chain are shown in Figure 5a and Figure 5b, respectively. .
  • the target gene sequences of the above TCR ⁇ and ⁇ chains were synthesized and inserted into the expression vector pET28a+ (Novagene ), the upstream and downstream cloning sites are NcoI and NotI, respectively. The insert was confirmed by sequencing.
  • the inclusion bodies formed later were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution. The inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA). ), 20 mM Tris (pH 8.1).
  • the dissolved TCR ⁇ and ⁇ chains were rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM ⁇ -mercapoethylamine (4°C) at a mass ratio of 1:1, the final concentration was 60mg/mL.
  • the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was replaced with a buffer (20 mM Tris, pH 8.0), and the dialysis was continued at 4°C for 12 hours.
  • variable domains of TCR ⁇ and ⁇ chains in Example 2 were constructed into a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker) by site-directed mutagenesis.
  • the amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in Figure 7a and Figure 7b, respectively.
  • the amino acid sequence and nucleotide sequence of its ⁇ chain variable domain are shown in Figure 8a and Figure 8b, respectively; the amino acid sequence and nucleotide sequence of its ⁇ chain variable domain are shown in Figure 9a and Figure 9b, respectively; its linker
  • the amino acid sequence and nucleotide sequence of the sequence are shown in Figure 10a and Figure 10b, respectively.
  • the target gene was double digested with Nco I and Not I, and ligated with the pET28a vector that was double digested with Nco I and Not I.
  • the ligation product was transformed into E.coli DH5 ⁇ , coated with LB plate containing kanamycin, and cultured overnight at 37°C by inversion. Positive clones were picked for PCR screening, and the positive recombinants were sequenced. After confirming that the sequences were correct, the recombinant plasmids were extracted and transformed. to E. coli BL21 (DE3) for expression.
  • Example 5 Expression, renaturation and purification of soluble single-chain TCR specific for HPV16 E6 antigen short peptide
  • the cell pellet was harvested by centrifugation at 5000 rpm for 15 min, the cell pellet was lysed with Bugbuster Master Mix (Merck), the inclusion body was recovered by centrifugation at 6000 rpm for 15 min, and then washed with Bugbuster (Merck) to remove cell debris and membrane components, and the inclusion body was collected by centrifugation at 6000 rpm for 15 min. body.
  • the inclusion bodies were dissolved in buffer (20mM Tris-HCl pH 8.0, 8M urea), and insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA method, and then divided into packaging and stored at -80°C for future use.
  • renaturation buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • renaturation buffer 100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM ⁇ -mercapthoethylamine, 1.87mM Cystamine
  • dialysate was replaced with 1 L of pre-cooled buffer (20mM Tris-HCl pH 8.0), and the dialysis was continued for 8 h at 4°C, and then the dialysate was replaced with the same fresh buffer and continued dialysis overnight.
  • the sample was filtered through a 0.45 ⁇ m filter membrane, degassed in vacuo and passed through an anion exchange column (HiTrap Q HP, GE Healthcare), and the protein was purified with a linear gradient of 0-1 M NaCl prepared with 20 mM Tris-HCl pH 8.0, The collected elution fractions were analyzed by SDS-PAGE, the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.
  • the eluted fractions for BIAcore analysis were further tested for purity by gel filtration.
  • the conditions are: Column Agilent Bio SEC-3 (300A, ), the mobile phase was 150 mM phosphate buffer, the flow rate was 0.5 mL/min, the column temperature was 25°C, and the UV detection wavelength was 214 nm.
  • Figure 11 shows the SDS-PAGE gel image of the soluble single-chain TCR obtained by the present invention.
  • the binding activity of the TCR molecules obtained in Example 3 and Example 5 to the TIHDIILECV-HLA A0201 complex was detected using the BIAcore T200 real-time analysis system.
  • Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was then flowed through a CM5 chip preactivated with EDC and NHS to immobilize the antibody on the chip surface , and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process with a coupling level of about 15,000RU.
  • Collect 100ml of E.coli bacteria that induces the expression of heavy or light chains centrifuge at 8000g at 4°C for 10min, wash the cells once with 10ml PBS, and then use 5ml BugBuster Master Mix Extraction Reagents (Merck) to vigorously shake the cells to resuspend the cells. Incubate with rotation at room temperature for 20 min, then centrifuge at 6000g for 15 min at 4°C, discard the supernatant, and collect the inclusion bodies.
  • the synthetic short peptide TIHDIILECV (Jiangsu GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml.
  • the inclusion bodies of light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA before renaturation.
  • TIHDIILECV peptide was added to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then added 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three times, 8h/time), and renatured at 4°C for at least 3 days To complete, SDS-PAGE test whether the renaturation is successful.
  • renaturation buffer 0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C
  • renaturation buffer by dialyzing against 10 volumes of 20 mM Tris pH 8.0, at least twice, to sufficiently reduce the ionic strength of the solution.
  • the protein solution was filtered through a 0.45 ⁇ m cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume).
  • the protein was eluted with Akta purifier (GE), 0-400mM NaCl linear gradient prepared with 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.
  • Purified pMHC molecules were concentrated with Millipore ultrafiltration tubes while buffer exchanged to 20 mM Tris pH 8.0, followed by addition of biotinylation reagents 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA Enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was checked by SDS-PAGE.
  • the biotinylated pMHC molecules were concentrated to 1 ml with a Millipore ultrafiltration tube, and the biotinylated pMHC was purified by gel filtration chromatography.
  • HiPrepTM was pre-equilibrated with filtered PBS using an Akta purifier (GE).
  • GE Akta purifier
  • a 16/60S200HR column (GE) loaded with 1 ml of concentrated biotinylated pMHC molecules, was then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules eluted as a single peak at about 55 ml.
  • the fractions containing protein were combined, concentrated with Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to store the biotinylated pMHC molecules in aliquots at -80°C.
  • the above method was used to detect the binding activity of the soluble TCR molecule of the present invention to several other irrelevant antigen short peptides and HLA complexes, and the results showed that the TCR molecule of the present invention did not bind to other irrelevant antigens.
  • Example 7 Activation experiment of effector cells transfected with TCR of the present invention for T2 cells loaded with short peptides
  • IFN- ⁇ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN- ⁇ was detected by the ELISPOT experiment well-known to those skilled in the art to verify the activation function of the cells transfected with the TCR of the present invention. antigen specificity.
  • the effector cells used in this experiment were CD3 + T cells expressing the TCR of the present invention, and CD3 + T cells transfected with other TCR (A6) from the same volunteer were used as a control group.
  • the target cells used were T2 cells loaded with the HPV16 E6 antigen short peptide TIHDIILECV, and empty T2 cells loaded with other irrelevant peptides were used as controls.
  • the components of the test were added to the ELISPOT plate: 1 ⁇ 10 4 target cells/well for target cells, 2 ⁇ 10 3 cells/well for effector cells (calculated according to the positive rate of transfection), and two duplicate wells were set.
  • TIHDIILECV short peptide was added to the corresponding well, so that the final concentration of the short peptide in the ELISPOT plate was 1 ⁇ 10 -12 M to 1 ⁇ 10 -6 M in sequence, a total of 7 gradients; -6 M for testing.
  • the well plates as follows: Dilute the anti-human IFN- ⁇ capture antibody 1:200 in 10 mL of sterile PBS per plate, then aliquot 100 ⁇ l of the diluted capture antibody to each well . Plates were incubated overnight at 4°C. After incubation, the plate was washed to remove excess capture antibody. 100 microliters/well of RPMI 1640 medium containing 10% FBS was added and the plates were incubated for 2 hours at room temperature to block the plates. The medium was then washed from the well plate and any residual wash buffer was removed by flicking and tapping the ELISPOT well plate on the paper.
  • Streptavidin-alkaline phosphatase was diluted 1:100 in PBS containing 10% FBS, 100 microliters of diluted streptavidin-alkaline phosphatase was added to each well and the plate was incubated at room temperature 1 hour. Then wash 4 times with wash buffer 2 times with PBS and tap the plate on a paper towel to remove excess wash buffer and PBS. After washing, 100 ⁇ l/well of BCIP/NBT solution provided by the kit was added for development. Cover the well plate with foil during development to protect from light and let stand for 5-15 minutes. Routinely check the spots of the developing plate during this period to determine the optimal time to terminate the reaction.
  • the experimental results are shown in Figure 15.
  • the T cells transfected with the TCR of the present invention have an obvious activation response to the target cells loaded with the TIHDIILECV short peptide, while the T cells transfected with other TCRs have no response to the corresponding target cells from the beginning; at the same time,
  • the T cells transfected with the TCR of the present invention have no activation response to the T2 cells loaded with its non-specific short peptide and the empty T2 cells.
  • Example 8 Activation function experiment of effector cells transfected with TCR of the present invention for tumor cell lines
  • the function and specificity of the TCR of the present invention in cells were also detected by ELISPOT experiment.
  • the effector cells used were CD3 + T cells expressing the HPV16 E6 antigen short peptide-specific TCR of the present invention, and CD3 + T cells transfected with other TCR (A6) from the same volunteer were used as a control group.
  • the positive tumor cell line used was A375-E6 (E6 overexpression).
  • the HPV-negative cell lines used were HCCC9810, LCLs and SK-MEL-5 as controls.
  • ELISPOT plates were ethanol activated and coated overnight at 4°C.
  • the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).
  • the effector cells transfected with the TCR of the present invention produced a good specific activation response, and the T cells transfected with other TCRs basically did not produce activation responses; while for the negative tumor cells Lines, the effector cells transfected with the TCR of the present invention are substantially inactive.

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Abstract

本发明提供了一种能够特异性结合衍生自HPV16 E6抗原的短肽TIHDIILECV的T细胞受体(TCR)，所述抗原短肽TIHDIILECV可与HLA A0201形成复合物并一起被呈递到细胞表面。本发明还提供了编码所述TCR的核酸分子以及包含所述核酸分子的载体。另外，本发明还提供了转导本发明TCR的细胞。

Description

一种识别HPV的T细胞受体 技术领域
本发明涉及能够识别源自HPV16 E6抗原短肽的TCR及其编码序列，本发明还涉及转导上述TCR来获得的HPV16 E6特异性的T细胞，及他们在预防和治疗HPV16 E6相关疾病中的用途。
背景技术
E6基因为人乳头状瘤病毒(HPV)基因组的早期区基因之一，位于HPV基因的83～559碱基上，编码E6蛋白。在世界范围内子宫颈癌中最流行的类型为HPV16,占检测病例的50％～60％(Acta Acad Med Sin,2007,29(5):678-684)；而E6蛋白是高危型HPV感染宫颈上皮细胞后编码的两种重要致瘤蛋白之一([J].江苏大学学报(医学版)，2018，28(2):135-139)。HPV16 E6还会引起头颈部肿瘤(中国耳鼻咽喉颅底外科杂志，2017，23(6):594-598)、结膜上皮内瘤变(CIN)以及角结膜侵袭性鳞状细胞癌(SCC)(国际眼科杂志2018；18(6):1047-1050)等疾病。TIHDIILECV(SEQ ID NO:9)是衍生自HPV16 E6蛋白的短肽，是HPV16 E6相关疾病治疗的一种靶标。
T细胞过继免疫治疗是将对靶细胞抗原具有特异性的反应性T细胞转入病人体内，使其针对靶细胞发挥作用。T细胞受体(TCR)是T细胞表面的一种膜蛋白，其能够识别相应的靶细胞表面的抗原短肽。在免疫系统中，通过抗原短肽特异性的TCR与短肽-主组织相容性复合体(pMHC复合物)的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，引起一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。因此，本领域技术人员致力于分离出对HPV16 E6抗原短肽具有特异性的TCR，以及将该TCR转导T细胞来获得对HPV16 E6抗原短肽具有特异性的T细胞，从而使他们在细胞免疫治疗中发挥作用。
发明内容
本发明的目的在于提供一种识别HPV16 E6抗原短肽的T细胞受体。
本发明的第一方面，提供了一种T细胞受体(TCR)，所述TCR能够与TIHDIILECV-HLA A0201复合物结合。
在另一优选例中，本发明提供了一种分离或纯化的TCR，所述TCR能够与TIHDIILECV-HLA A0201复合物结合。
在另一优选例中，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域的CDR3的氨基酸序列为AMREGTGTASKLT(SEQ ID NO:12)；和/或所述TCRβ链可变域的CDR3的氨基酸序列为ASSPYGQGTSPLH(SEQ ID NO:15)。
在另一优选例中，所述TCRα链可变域的3个互补决定区(CDR)为：
αCDR1-TSDPSYG(SEQ ID NO:10)
αCDR2-QGSYDQQN(SEQ ID NO:11)
αCDR3-AMREGTGTASKLT(SEQ ID NO:12)；和/或
所述TCRβ链可变域的3个互补决定区为：
βCDR1-SGHDN(SEQ ID NO:13)
βCDR2-FVKESK(SEQ ID NO:14)
βCDR3-ASSPYGQGTSPLH(SEQ ID NO:15)。
在另一优选例中，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域为与SEQ ID NO:1具有至少90％序列相同性的氨基酸序列；和/或所述TCRβ链可变域为与SEQ ID NO:5具有至少90％序列相同性的氨基酸序列。
在另一优选例中，所述TCR包含α链可变域氨基酸序列SEQ ID NO:1。
在另一优选例中，所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。
在另一优选例中，所述TCR为αβ异质二聚体，其包含TCRα链恒定区TRAC*01和TCRβ链恒定区TRBC1*01或TRBC2*01。
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO:3和/或所述TCR的β链氨基酸序列为SEQ ID NO:7。
在另一优选例中，本发明所述TCR是人源的。
在另一优选例中，所述TCR是可溶的。
在另一优选例中，所述TCR为单链。
在另一优选例中，所述TCR是由α链可变域与β链可变域通过肽连接序列连接而成。
在另一优选例中，所述TCR的α与β链的恒定区分别为鼠源的α与β链的恒定区。
在另一优选例中，所述TCR在α链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或α链J基因短肽氨基酸倒数第3位、倒数第5位或倒数第7位中具有一个或多个突变；和/或所述TCR在β链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或β链J基因短肽氨基酸倒数第2位、倒数第4位或倒数第6位中具有一个或多个突变，其中氨基酸位置编号按IMGT(国际免疫遗传学信息系统)中列出的位置编号。
在另一优选例中，所述TCR的α链可变域氨基酸序列包含SEQ ID NO:32和/或所述TCR的β链可变域氨基酸序列包含SEQ ID NO:34。
在另一优选例中，所述TCR的氨基酸序列为SEQ ID NO:30。
在另一优选例中，所述TCR包括(a)除跨膜结构域以外的全部或部分TCRα链；以及(b)除跨膜结构域以外的全部或部分TCRβ链；
并且(a)和(b)各自包含功能性可变结构域,或包含功能性可变结构域和所述TCR链恒定结构域的至少一部分。
在另一优选例中，半胱氨酸残基在所述TCR的α和β链恒定域之间形成人工二硫键。
在另一优选例中，在所述TCR中形成人工二硫键的半胱氨酸残基取代了选自下列的一 组或多组位点：
TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；和
TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO:26和/或所述TCR的β链氨基酸序列为SEQ ID NO:28。
在另一优选例中，所述TCR的α链可变区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，其特征在于，在所述TCR中形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；
TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或
TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。
在另一优选例中，所述TCR包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
在另一优选例中，所述TCR的α链和/或β链的C-或N-末端结合有偶联物。
在另一优选例中，与所述T细胞受体结合的偶联物为可检测标记物、治疗剂、PK修饰部分或任何这些物质的组合；优选地，所述治疗剂为抗-CD3抗体。
本发明的第二方面，提供了一种多价TCR复合物，其包含至少两个TCR分子，并且其中的至少一个TCR分子为本发明第一方面所述的TCR。
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的TCR分子的核酸序列或其互补序列。
在另一优选例中，所述核酸分子包含编码TCRα链可变域的核苷酸序列SEQ ID NO:2或SEQ ID NO:33。
在另一优选例中，所述的核酸分子包含编码TCRβ链可变域的核苷酸序列SEQ ID NO:6或SEQ ID NO:35。
在另一优选例中，所述核酸分子包含编码TCRα链的核苷酸序列SEQ ID NO:4和/或包含编码TCRβ链的核苷酸序列SEQ ID NO:8。
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。
本发明的第五方面，提供了一种分离的宿主细胞，所述的宿主细胞中含有本发明第四方面所述的载体或基因组中整合有外源的本发明第三方面所述的核酸分子。
本发明的第六方面，提供了一种细胞，所述细胞转导有本发明第三方面所述的核酸分子或本发明第四方面所述的载体；优选地，所述细胞为T细胞、NK细胞、NKT细胞或干细胞。
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的TCR、本发明第二方面所述的TCR复合物、本发明第三方面所述的核酸分子、本发明第四方面所述的载体、或本发明第六方面所述的细胞。
本发明的第八方面，提供了本发明第一方面所述的T细胞受体、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞的用途，用于制备治疗肿瘤或自身免疫疾病的药物，优选地，所述肿瘤为宫颈癌。
本发明的第九方面，提供了本发明第一方面所述的T细胞受体、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞用作治疗肿瘤或自身免疫疾病的药物；优选地，所述肿瘤为宫颈癌。
本发明的第十方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的T细胞受体、或本发明第二方面所述的TCR复合物、或本发明第六方面所述的细胞、或本发明第七方面所述的药物组合物；优选地，所述的疾病为肿瘤，优选地所述肿瘤为宫颈癌、头颈部肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1a、图1b、图1c、图1d、图1e和图1f分别为TCRα链可变域氨基酸序列、TCRα链可变域核苷酸序列、TCRα链氨基酸序列、TCRα链核苷酸序列、具有前导序列的TCRα链氨基酸序列以及具有前导序列的TCRα链核苷酸序列。
图2a、图2b、图2c、图2d、图2e和图2f分别为TCRβ链可变域氨基酸序列、TCRβ链可变域核苷酸序列、TCRβ链氨基酸序列、TCRβ链核苷酸序列、具有前导序列的TCRβ链氨基酸序列以及具有前导序列的TCRβ链核苷酸序列。
图3为单克隆细胞的CD8+及四聚体-PE双阳性染色结果。
图4a和图4b分别为可溶性TCRα链的氨基酸序列和核苷酸序列。
图5a和图5b分别为可溶性TCRβ链的氨基酸序列和核苷酸序列。
图6为纯化后得到的可溶性TCR的胶图。最左侧泳道为还原胶，中间泳道为分子量标记(marker)，最右侧泳道为非还原胶。
图7a和图7b分别为单链TCR的氨基酸序列和核苷酸序列。
图8a和图8b分别为单链TCRα链可变域的氨基酸序列和核苷酸序列。
图9a和图9b分别为单链TCRβ链可变域的氨基酸序列和核苷酸序列。
图10a和图10b分别为单链TCR连接序列(linker)的氨基酸序列和核苷酸序列。
图11为纯化后得到的可溶性单链TCR的胶图。最左侧泳道为还原胶，中间泳道为分子量标记(marker)，最右侧泳道为非还原胶。
图12为本发明可溶性TCR与TIHDIILECV-HLA A0201复合物结合的BIAcore动力学图谱。
图13为本发明可溶性单链TCR与TIHDIILECV-HLA A0201复合物结合的BIAcore动力学图谱。
图14为得到的T细胞克隆的ELISPOT激活功能验证结果。
图15为转染本发明的TCR的效应细胞的ELISPOT激活功能验证结果。
图16为针对肿瘤细胞系，转染本发明的TCR的效应细胞的ELISPOT激活功能验证结果。
具体实施方式
本发明人经过广泛而深入的研究，找到了与HPV16 E6抗原短肽TIHDIILECV(SEQ ID NO:9)能够特异性结合的TCR，所述抗原短肽TIHDIILECV可与HLA A0201形成复合物并一起被呈递到细胞表面。本发明还提供了编码所述TCR的核酸分子以及包含所述核酸分子的载体。另外，本发明还提供了转导本发明TCR的细胞。
术语
MHC分子是免疫球蛋白超家族的蛋白质，可以是I类或II类MHC分子。因此，其对于抗原的呈递具有特异性，不同的个体有不同的MHC，能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
T细胞受体(TCR)，是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T 细胞对其靶细胞发挥免疫效应。
TCR是由α链/β链或者γ链/δ链以异质二聚体形式存在的细胞膜表面的糖蛋白。在95％的T细胞中TCR异质二聚体由α和β链组成，而5％的T细胞具有由γ和δ链组成的TCR。天然αβ异质二聚TCR具有α链和β链，α链和β链构成αβ异源二聚TCR的亚单位。广义上讲，α和β各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。各可变区包含嵌合在框架结构(framework regions)中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。CDR区决定了TCR与pMHC复合物的结合，其中CDR3由可变区和连接区重组而成，被称为超变区。TCR的α和β链一般看作各有两个“结构域”即可变域和恒定域，可变域由连接的可变区和连接区构成。TCR恒定域的序列可以在国际免疫遗传学信息系统(IMGT)的公开数据库中找到，如TCR分子α链的恒定域序列为“TRAC*01”，TCR分子β链的恒定域序列为“TRBC1*01”或“TRBC2*01”。此外，TCR的α和β链还包含跨膜区和胞质区，胞质区很短。
在本发明中，术语“本发明多肽”、“本发明的TCR”、“本发明的T细胞受体”可互换使用。
天然链间二硫键与人工链间二硫键
在天然TCR的近膜区Cα与Cβ链间存在一组二硫键，本发明中称为“天然链间二硫键”。在本发明中，将人工引入的，位置与天然链间二硫键的位置不同的链间共价二硫键称为“人工链间二硫键”。
为方便描述二硫键的位置，本发明中TRAC*01与TRBC1*01或TRBC2*01氨基酸序列的位置编号按从N端到C端依次的顺序进行位置编号，如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第60个氨基酸为P(脯氨酸)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Pro60，也可将其表述为TRBC1*01或TRBC2*01外显子1的第60位氨基酸，又如TRBC1*01或TRBC2*01中，按从N端到C端依次的顺序第61个氨基酸为Q(谷氨酰胺)，则本发明中可将其描述为TRBC1*01或TRBC2*01外显子1的Gln61，也可将其表述为TRBC1*01或TRBC2*01外显子1的第61位氨基酸，其他以此类推。本发明中，可变区TRAV与TRBV的氨基酸序列的位置编号，按照IMGT中列出的位置编号。如TRAV中的某个氨基酸，IMGT中列出的位置编号为46，则本发明中将其描述为TRAV第46位氨基酸，其他以此类推。本发明中，其他氨基酸的序列位置编号有特殊说明的，则按特殊说明。
发明详述
TCR分子
在抗原加工过程中，抗原在细胞内被降解，然后通过MHC分子携带至细胞表面。T细胞受体能够识别抗原呈递细胞表面的肽-MHC复合物。因此，本发明的第一方面提供了一种能够结合TIHDIILECV-HLA A0201复合物的TCR分子。优选地，所述TCR分子是分离的或纯化的。该TCR的α和β链各具有3个互补决定区(CDR)。
在本发明的一个优选地实施方式中，所述TCR的α链包含具有以下氨基酸序列的CDR：
αCDR1-TSDPSYG(SEQ ID NO:10)
αCDR2-QGSYDQQN(SEQ ID NO:11)
αCDR3-AMREGTGTASKLT(SEQ ID NO:12)；和/或
所述TCRβ链可变域的3个互补决定区为：
βCDR1-SGHDN(SEQ ID NO:13)
βCDR2-FVKESK(SEQ ID NO:14)
βCDR3-ASSPYGQGTSPLH(SEQ ID NO:15)。
可以将上述本发明的CDR区氨基酸序列嵌入到任何适合的框架结构中来制备嵌合TCR。只要框架结构与本发明的TCR的CDR区兼容，本领域技术人员根据本发明公开的CDR区就能够设计或合成出具有相应功能的TCR分子。因此，本发明TCR分子是指包含上述α和/或β链CDR区序列及任何适合的框架结构的TCR分子。本发明TCRα链可变域为与SEQ ID NO:1具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列；和/或本发明TCRβ链可变域为与SEQ ID NO:5具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列。
在本发明的一个优选例中，本发明的TCR分子是由α与β链构成的异质二聚体。具体地，一方面所述异质二聚TCR分子的α链包含可变域和恒定域，所述α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO:10)、CDR2(SEQ ID NO:11)和CDR3(SEQ ID NO:12)。优选地，所述TCR分子包含α链可变域氨基酸序列SEQ ID NO:1。更优选地，所述TCR分子的α链可变域氨基酸序列为SEQ ID NO:1。另一方面，所述异质二聚TCR分子的β链包含可变域和恒定域，所述β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO:13)、CDR2(SEQ ID NO:14)和CDR3(SEQ ID NO:15)。优选地，所述TCR分子包含β链可变域氨基酸序列SEQ ID NO:5。更优选地，所述TCR分子的β链可变域氨基酸序列为SEQ ID NO:5。
在本发明的一个优选例中，本发明的TCR分子是由α链的部分或全部和/或β链的部分或全部组成的单链TCR分子。有关单链TCR分子的描述可以参考文献Chung et al(1994)Proc.Natl.Acad.Sci.USA 91,12654-12658。根据文献中所述，本领域技术人员能够容易地构建包含本发明CDRs区的单链TCR分子。具体地，所述单链TCR分子包含Vα、Vβ和Cβ，优选地按照从N端到C端的顺序连接。
所述单链TCR分子的α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO:10)、CDR2(SEQ ID NO:11)和CDR3(SEQ ID NO:12)。优选地，所述单链TCR分子包含α链可变域氨基酸序列SEQ ID NO:1。更优选地，所述单链TCR分子的α链可变域氨基酸序列为SEQ ID NO:1。所述单链TCR分子的β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO:13)、CDR2(SEQ ID NO:14)和CDR3(SEQ ID NO:15)。优选地，所述单链TCR分子包含β链可变域氨基酸序列SEQ ID NO:5。更优选地，所述单链TCR分子的β链可变域氨基酸序列为SEQ ID NO:5。
在本发明的一个优选例中，本发明的TCR分子的恒定域是人的恒定域。本领域技术人员知晓或可以通过查阅相关书籍或IMGT(国际免疫遗传学信息系统)的公开数据库来获得人的恒定域氨基酸序列。例如，本发明TCR分子α链的恒定域序列可以为“TRAC*01”，TCR分子β链的恒定域序列可以为“TRBC1*01”或“TRBC2*01”。IMGT的TRAC*01中给出的氨基酸序列的第53位为Arg，在此表示为：TRAC*01外显子1的Arg53，其他以此类推。优选地，本发明TCR分子α链的氨基酸序列为SEQ ID NO:3，和/或β链的氨基酸序列为SEQ ID NO:7。
天然存在的TCR是一种膜蛋白，通过其跨膜区得以稳定。如同免疫球蛋白(抗体)作为抗原识别分子一样，TCR也可以被开发应用于诊断和治疗，这时需要获得可溶性的TCR分子。可溶性的TCR分子不包括其跨膜区。可溶性TCR有很广泛的用途，它不仅可用于研究TCR与pMHC的相互作用，也可用作检测感染的诊断工具或作为自身免疫病的标志物。类似地，可溶性TCR可以被用来将治疗剂(如细胞毒素化合物或免疫刺激性化合物)输送到呈递特异性抗原的细胞，另外，可溶性TCR还可与其他分子(如，抗-CD3抗体)结合来重新定向T细胞，从而使其靶向呈递特定抗原的细胞。本发明也获得了对HPV16 E6抗原短肽具有特异性的可溶性TCR。
为获得可溶性TCR，一方面，本发明TCR可以是在其α和β链恒定域的残基之间引入人工二硫键的TCR。半胱氨酸残基在所述TCR的α和β链恒定域间形成人工链间二硫键。半胱氨酸残基可以取代在天然TCR中合适位点的其他氨基酸残基以形成人工链间二硫键。例如，取代TRAC*01外显子1的Thr48和取代TRBC1*01或TRBC2*01外显子1的Ser57的半胱氨酸残基来形成二硫键。引入半胱氨酸残基以形成二硫键的其他位点还可以是：TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；或TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。即半胱氨酸残基取代了上述α与β链恒定域中任一组位点。可在本发明TCR恒定域的一个或多个C末端截短最多50个、或最多30个、或最多15个、或最多10个、或最多8个或更少的氨基酸，以使其不包括半胱氨酸残基来达到缺失天然二硫键的目的，也可通过将形成天然二硫键的半胱氨酸残基突变为另一氨基酸来达到上述目的。
如上所述，本发明的TCR可以包含在其α和β链恒定域的残基间引入的人工二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本发明的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域序列可通过存在于TCR中的天然二硫键连接。
为获得可溶性TCR，另一方面，本发明TCR还包括在其疏水芯区域发生突变的TCR，这些疏水芯区域的突变优选为能够使本发明可溶性TCR的稳定性提高的突变，如在公开号 为WO2014/206304的专利文献中所述。这样的TCR可在其下列可变域疏水芯位置发生突变：(α和/或β链)可变区氨基酸第11、13、19、21、53、76、89、91、94位，和/或α链J基因(TRAJ)短肽氨基酸位置倒数第3、5、7位，和/或β链J基因(TRBJ)短肽氨基酸位置倒数第2、4、6位，其中氨基酸序列的位置编号按国际免疫遗传学信息系统(IMGT)中列出的位置编号。本领域技术人员知晓上述国际免疫遗传学信息系统，并可根据该数据库得到不同TCR的氨基酸残基在IMGT中的位置编号。
本发明中疏水芯区域发生突变的TCR可以是由一柔性肽链连接TCR的α与β链的可变域而构成的稳定性可溶单链TCR。应注意，本发明中柔性肽链可以是任何适合连接TCRα与β链可变域的肽链。如在本发明实施例4中构建的单链可溶性TCR，其α链可变域氨基酸序列为SEQ ID NO:32，编码的核苷酸序列为SEQ ID NO:33；β链可变域氨基酸序列为SEQ ID NO:34，编码的核苷酸序列为SEQ ID NO:35。
另外，对于稳定性而言，专利文献201680003540.2还公开了在TCR的α链可变区与β链恒定区之间引入人工链间二硫键能够使TCR的稳定性显著提高。因此，本发明的TCR的α链可变区与β链恒定区之间还可以含有人工链间二硫键。具体地，在所述TCR的α链可变区与β链恒定区之间形成人工链间二硫键的半胱氨酸残基取代了：TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。优选地，这样的TCR可以包含(i)除其跨膜结构域以外的全部或部分TCRα链，和(ii)除其跨膜结构域以外的全部或部分TCRβ链，其中(i)和(ii)均包含TCR链的可变域和至少一部分恒定域，α链与β链形成异质二聚体。更优选地，这样的TCR可以包含α链可变域和β链可变域以及除跨膜结构域以外的全部或部分β链恒定域，但其不包含α链恒定域，所述TCR的α链可变域与β链形成异质二聚体。
本发明的TCR也可以多价复合体的形式提供。本发明的多价TCR复合体包含两个、三个、四个或更多个本发明TCR相结合而形成的多聚物，如可以用p53的四聚结构域来产生四聚体，或多个本发明TCR与另一分子结合而形成的复合物。本发明的TCR复合物可用于体外或体内追踪或靶向呈递特定抗原的细胞，也可用于产生具有此类应用的其他多价TCR复合物的中间体。
本发明的TCR可以单独使用，也可与偶联物以共价或其他方式结合，优选以共价方式结合。所述偶联物包括可检测标记物(为诊断目的，其中所述TCR用于检测呈递TIHDIILECV-HLA A0201复合物的细胞的存在)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明TCR结合或偶联的治疗剂包括但不限于：1.放射性核素(Koppe等，2005，癌转移评论(Cancer metastasis reviews)24，539)；2.生物毒(Chaudhary等，1989，自然 (Nature)339，394；Epel等，2002，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51，565)；3.细胞因子如IL-2等(Gillies等，1992，美国国家科学院院刊(PNAS)89，1428；Card等，2004，癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53，345；Halin等，2003，癌症研究(Cancer Research)63，3202)；4.抗体Fc片段(Mosquera等，2005，免疫学杂志(The Journal Of Immunology)174，4381)；5.抗体scFv片段(Zhu等，1995，癌症国际期刊(International Journal of Cancer)62,319)；6.金纳米颗粒/纳米棒(Lapotko等，2005，癌症通信(Cancer letters)239，36；Huang等，2006，美国化学学会杂志(Journal of the American Chemical Society)128，2115)；7.病毒颗粒(Peng等，2004，基因治疗(Gene therapy)11，1234)；8.脂质体(Mamot等，2005，癌症研究(Cancer research)65，11631)；9.纳米磁粒；10.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；11.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
另外，本发明的TCR还可以是包含衍生自超过一种物种序列的杂合TCR。例如，有研究显示鼠科TCR在人T细胞中比人TCR能够更有效地表达。因此，本发明TCR可包含人可变域和鼠的恒定域。这一方法的缺陷是可能引发免疫应答。因此，在其用于过继性T细胞治疗时应当有调节方案来进行免疫抑制，以允许表达鼠科的T细胞的植入。
应理解，本文中氨基酸名称采用国际通用的单英文字母或三英文字母表示，氨基酸名称的单英文字母与三英文字母的对应关系如下：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
核酸分子
本发明的第二方面提供了编码本发明第一方面TCR分子或其部分的核酸分子，所述部分可以是一个或多个CDR，α和/或β链的可变域，以及α链和/或β链。
编码本发明第一方面TCR分子α链CDR区的核苷酸序列如下：
CDR1α-accagtgatccaagttatggt(SEQ ID NO:16)
CDR2α-caggggtcttatgaccagcaaaat(SEQ ID NO:17)
CDR3α-gcaatgagagagggcaccggcactgccagtaaactcacc(SEQ ID NO:18)
编码本发明第一方面TCR分子β链CDR区的核苷酸序列如下：
CDR1β-tctggacatgataat(SEQ ID NO:19)
CDR2β-tttgtgaaagagtctaaa(SEQ ID NO:20)
CDR3β-gccagcagcccgtacggacagggaacttcacccctccac(SEQ ID NO:21)
因此，编码本发明TCRα链的本发明核酸分子的核苷酸序列包括SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18，和/或编码本发明TCRβ链的本发明核酸分子的核苷酸序列包括SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21。
本发明核酸分子的核苷酸序列可以是单链或双链的，该核酸分子可以是RNA或DNA，并且可以包含或不包含内含子。优选地，本发明核酸分子的核苷酸序列不包含内含子但能够 编码本发明多肽，例如编码本发明TCRα链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:2和/或编码本发明TCRβ链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:6。或者，编码本发明TCRα链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:33和/或编码本发明TCRβ链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO:35。更优选地，本发明核酸分子的核苷酸序列包含SEQ ID NO:4和/或SEQ ID NO:8。或者，本发明核酸分子的核苷酸序列为SEQ ID NO:31。
应理解，由于遗传密码的简并，不同的核苷酸序列可以编码相同的多肽。因此，编码本发明TCR的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。以本发明中的其中一个例子来说明，“简并的变异体”是指编码具有SEQ ID NO:1的蛋白序列，但与SEQ ID NO:2的序列有差别的核酸序列。
核苷酸序列可以是经密码子优化的。不同的细胞在具体密码子的利用上是不同的，可以根据细胞的类型，改变序列中的密码子来增加表达量。哺乳动物细胞以及多种其他生物的密码子选择表是本领域技术人员公知的。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明TCR(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。DNA可以是编码链或非编码链。
载体
本发明还涉及包含本发明的核酸分子的载体，包括表达载体，即能够在体内或体外表达的构建体。常用的载体包括细菌质粒、噬菌体和动植物病毒。
病毒递送系统包括但不限于腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、逆转录病毒载体、慢病毒载体、杆状病毒载体。
优选地，载体可以将本发明的核苷酸转移至细胞中，例如T细胞中，使得该细胞表达HPV16 E6抗原特异性的TCR。理想的情况下，该载体应当能够在T细胞中持续高水平地表达。
细胞
本发明还涉及用本发明的载体或编码序列经基因工程产生的宿主细胞。所述宿主细胞中含有本发明的载体或染色体中整合有本发明的核酸分子。宿主细胞选自：原核细胞和真核细胞，例如大肠杆菌、酵母细胞、CHO细胞等。
另外，本发明还包括表达本发明的TCR的分离的细胞，可以但不仅限为T细胞、NK细胞、NKT细胞，特别是T细胞。该T细胞可衍生自从受试者分离的T细胞，或者可以是从受试者中分离的混合细胞群，诸如外周血淋巴细胞(PBL)群的一部分。如，该细胞可以分离自外周血单核细胞(PBMC)，可以是CD4+辅助T细胞或CD8+细胞毒性T细胞。该细胞可在CD4+辅助T细胞/CD8+细胞毒性T细胞的混合群中。一般地，该细胞可以用抗体(如， 抗-CD3或抗-CD28的抗体)活化，以便使它们能够更容易接受转染，例如用包含编码本发明TCR分子的核苷酸序列的载体进行转染。
备选地，本发明的细胞还可以是或衍生自干细胞，如造血干细胞(HSC)。将基因转移至HSC不会导致在细胞表面表达TCR，因为干细胞表面不表达CD3分子。然而，当干细胞分化为迁移至胸腺的淋巴前体(lymphoid precursor)时，CD3分子的表达将启动在胸腺细胞的表面表达该引入的TCR分子。
有许多方法适合于用编码本发明TCR的DNA或RNA进行T细胞转染(如，Robbins等.，(2008)J.Immunol.180:6116-6131)。表达本发明TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev Cancer 8(4):299-308)。
HPV16 E6抗原相关疾病
本发明还涉及在受试者中治疗和/或预防与HPV16 E6相关疾病的方法，其包括过继性转移HPV16 E6特异性T细胞至该受试者的步骤。该HPV16 E6特异性T细胞可识别TIHDIILECV-HLA A0201复合物。
本发明的HPV16 E6特异性的T细胞可用于治疗任何呈递HPV16 E6抗原短肽TIHDIILECV-HLA A0201复合物的HPV16 E6相关疾病，包括但不限于肿瘤，如宫颈癌、头颈部肿瘤等。
治疗方法
可以通过分离患有与HPV16 E6抗原相关疾病的病人或志愿者的T细胞，并将本发明的TCR导入上述T细胞中，随后将这些基因工程修饰的细胞回输到病人体内来进行治疗。因此，本发明提供了一种治疗HPV16 E6相关疾病的方法，包括将分离的表达本发明TCR的T细胞，优选地，该T细胞来源于病人本身，输入到病人体内。一般地，包括(1)分离病人的T细胞，(2)用本发明核酸分子或能够编码本发明TCR分子的核酸分子体外转导T细胞，(3)将基因工程修饰的T细胞输入到病人体内。分离、转染及回输的细胞的数量可以由医师决定。
本发明的主要优点：
本发明的TCR能够与HPV16 E6抗原短肽复合物TIHDIILECV-HLA A0201特异性结合，并且转导了本发明TCR的效应细胞能够被特异性激活。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明， 否则百分比和份数按重量计算。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1.克隆HPV16 E6抗原短肽特异性T细胞
利用合成短肽TIHDIILECV(SEQ ID NO:9；江苏金斯瑞生物科技有限公司)刺激来自于基因型为HLA-A0201的健康志愿者的外周血淋巴细胞(PBL)。将TIHDIILECV短肽与带有生物素标记的HLA-A0201复性来制备pHLA单倍体。这些单倍体与用PE标记的链霉亲和素(BD公司)组合成PE标记的四聚体，分选该四聚体及抗-CD8-APC双阳性细胞。扩增分选的细胞，并按上述方法进行二次分选，随后用有限稀释法进行单克隆。单克隆细胞用四聚体染色，筛选到的双阳性克隆如图3所示。经过层层筛选得到的双阳性克隆，还需要满足进一步的功能测试。
通过ELISPOT实验进一步检测该T细胞克隆的功能及特异性。本领域技术人员熟知利用ELISPOT实验检测细胞功能的方法。本实施例IFN-γELISPOT实验中所用的效应细胞为本发明中获得的T细胞克隆，靶细胞为负载了TIHDIILECV短肽的T2细胞、A375-E6(E6过表达)，对照组为负载了其他短肽的T2细胞和MCF-7。
首先准备ELISPOT平板，ELISPOT实验步骤如下：按以下顺序将试验的各个组分加入ELISPOT平板：靶细胞20,000个/孔、效应细胞2000个/孔后，在实验组和对照组加入20μl相应的短肽，空白组加入20μl培养基(试验培养基)，并设置2复孔。然后温育过夜(37℃，5％CO 2)。随后洗涤平板并进行二级检测和显色，干燥平板1小时，再利用免疫斑点平板读数计(ELISPOT READER system；AID公司)计数膜上形成的斑点。实验结果如图14所示，得到的T细胞克隆对负载了TIHDIILECV短肽的T2细胞和过表达E6抗原的A375-E6起明显的激活反应，而对负载了其他短肽的T2细胞及阴性细胞系MCF-7基本没有反应。
实施例2.获取HPV16 E6抗原短肽特异性T细胞克隆的TCR基因与载体的构建
用Quick-RNA TM MiniPrep(ZYMO research)抽提实施例1中筛选到的抗原短肽TIHDIILECV特异性、HLA-A0201限制性的T细胞克隆的总RNA。cDNA的合成采用clontech的SMART RACE cDNA扩增试剂盒，采用的引物是设计在人类TCR基因的C端保守区。将序列克隆至T载体(TAKARA)上进行测序。应注意，该序列为互补序列，不包含内含子。经测序，该双阳性克隆表达的TCR的链和链序列结构分别如图1和图2所示，图1a、图1b、图1c、图1d、图1e和图1f分别为TCRα链可变域氨基酸序列、TCRα链可变域核苷酸序列、TCRα链氨基酸序列、TCRα链核苷酸序列、具有前导序列的TCRα链氨基酸序列以及具有前导序列的TCRα链核苷酸序列；图2a、图2b、图2c、图2d、图2e和图2f分别为TCRβ链可变域氨基酸序列、TCRβ链可变域核苷酸序列、TCRβ链氨基酸序列、TCRβ链核苷酸序列、具有前导序列的TCRβ链氨基酸序列以及具有前导序列的TCRβ链核苷酸序列。
经鉴定，α链包含具有以下氨基酸序列的CDR：
αCDR1-TSDPSYG(SEQ ID NO:10)
αCDR2-QGSYDQQN(SEQ ID NO:11)
αCDR3-AMREGTGTASKLT(SEQ ID NO:12)
β链包含具有以下氨基酸序列的CDR：
βCDR1-SGHDN(SEQ ID NO:13)
βCDR2-FVKESK(SEQ ID NO:14)
βCDR3-ASSPYGQGTSPLH(SEQ ID NO:15)。
通过重叠(overlap)PCR分别将TCRα链和β链的全长基因克隆至慢病毒表达载体pLenti(addgene)。具体为：用overlap PCR将TCRα链和TCRβ链的全长基因进行连接得到TCRα-2A-TCRβ片段。将慢病毒表达载体及TCRα-2A-TCRβ酶切连接得到pLenti-TRA-2A-TRB-IRES-NGFR质粒。同时也构建表达eGFP的慢病毒载体pLenti-eGFP作为对照。之后再用293T/17包装假病毒。
实施例3.HPV16 E6抗原短肽特异性可溶TCR的表达、重折叠和纯化
为获得可溶的TCR分子，本发明的TCR分子的α和β链可以分别只包含其可变域及部分恒定域，并且α和β链的恒定域中分别引入了一个半胱氨酸残基以形成人工链间二硫键，其α链的氨基酸序列与核苷酸序列分别如图4a和图4b所示，其β链的氨基酸序列与核苷酸序列分别如图5a和图5b所示。通过《分子克隆实验室手册》(Molecular Cloning a Laboratory Manual)(第三版，Sambrook和Russell)中描述的标准方法将上述TCRα和β链的目的基因序列经合成后分别插入到表达载体pET28a+(Novagene)，上下游的克隆位点分别是NcoI和NotI。插入片段经过测序确认无误。
将TCRα和β链的表达载体分别通过化学转化法转化进入表达细菌BL21(DE3),细菌用LB培养液生长，于OD 600＝0.6时用终浓度0.5mM IPTG诱导，TCR的α和β链表达后形成的包涵体通过BugBuster Mix(Novagene)进行提取，并且经BugBuster溶液反复多次洗涤，包涵体最后溶解于6M盐酸胍、10mM二硫苏糖醇(DTT)、10mM乙二胺四乙酸(EDTA)、20mM Tris(pH 8.1)中。
溶解后的TCRα和β链以1:1的质量比快速混合于5M尿素、0.4M精氨酸、20mM Tris(pH 8.1)、3.7mM cystamine、6.6mMβ-mercapoethylamine(4℃)中，终浓度为60mg/mL。混合后将溶液置于10倍体积的去离子水中透析(4℃)，12小时后将去离子水换成缓冲液(20mM Tris,pH 8.0)继续于4℃透析12小时。透析完成后的溶液经0.45μM的滤膜过滤后，通过阴离子交换柱(HiTrap Q HP,5ml,GE Healthcare)纯化。洗脱峰含有复性成功的α和β二聚体的TCR通过SDS-PAGE胶确认。TCR随后通过凝胶过滤层析(HiPrep 16/60，Sephacryl S-100 HR，GE Healthcare)进一步纯化。纯化后的TCR纯度经过SDS-PAGE测定大于90％，浓度由BCA法确定。本发明得到的可溶性TCR的SDS-PAGE胶图如图6所示。
实施例4.HPV16 E6抗原短肽特异性的可溶性单链TCR的产生
根据专利文献WO2014/206304中所述，利用定点突变的方法将实施例2中TCRα与β 链的可变域构建成了一个以柔性短肽(linker)连接的稳定的可溶性单链TCR分子。该单链TCR分子的氨基酸序列及核苷酸序列分别如图7a和图7b所示。其α链可变域的氨基酸序列及核苷酸序列分别如图8a和图8b所示；其β链可变域的氨基酸序列及核苷酸序列分别如图9a和图9b所示；其linker序列的氨基酸序列及核苷酸序列分别如图10a和图10b所示。
将目的基因经Nco I和Not I双酶切，与经过Nco I和Not I双酶切的pET28a载体连接。连接产物转化至E.coli DH5α，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli BL21(DE3)用于表达。
实施例5.HPV16 E6抗原短肽特异性的可溶性单链TCR的表达、复性和纯化
将实施例4中制备的含有重组质粒pET28a-模板链的BL21(DE 3)菌落全部接种于含有卡那霉素的LB培养基中，37℃培养至OD 600为0.6-0.8，加入IPTG至终浓度为0.5mM，37℃继续培养4h。5000rpm离心15min收获细胞沉淀物，用Bugbuster Master Mix(Merck)裂解细胞沉淀物，6000rpm离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000rpm离心15min,收集包涵体。将包涵体溶解在缓冲液(20mM Tris-HCl pH 8.0，8M尿素)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向5mg溶解的单链TCR包涵体蛋白中，加入2.5mL缓冲液(6M Gua-HCl，50mM Tris-HCl pH 8.1，100mM NaCl，10mM EDTA)，再加入DTT至终浓度为10mM,37℃处理30min。用注射器向125mL复性缓冲液(100mM Tris-HCl pH 8.1、0.4M L-精氨酸、5M尿素、2mM EDTA、6.5mMβ-mercapthoethylamine、1.87mM Cystamine)中滴加上述处理后的单链TCR，4℃搅拌10min，然后将复性液装入截留量为4kDa的纤维素膜透析袋，透析袋置于1L预冷的水中，4℃缓慢搅拌过夜。17小时后，将透析液换成1L预冷的缓冲液(20mM Tris-HCl pH 8.0)，4℃继续透析8h，然后将透析液换成相同的新鲜缓冲液继续透析过夜。17小时后，样品经0.45μm滤膜过滤，真空脱气后通过阴离子交换柱(HiTrap Q HP,GE Healthcare)，用20mM Tris-HCl pH 8.0配制的0-1M NaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析，包含单链TCR的组分浓缩后进一步用凝胶过滤柱(Superdex75 10/300,GE Healthcare)进行纯化，目标组分也进行SDS-PAGE分析。
用于BIAcore分析的洗脱组分进一步采用凝胶过滤法测试其纯度。条件为：色谱柱Agilent Bio SEC-3(300A，
Figure PCTCN2022075111-appb-000001
)，流动相为150mM磷酸盐缓冲液，流速0.5mL/min，柱温25℃，紫外检测波长214nm。
本发明获得的可溶性单链TCR的SDS-PAGE胶图如图11所示。
实施例6.结合表征
BIAcore分析
本实施例证明了可溶性的本发明TCR分子能够与TIHDIILECV-HLA A0201复合物特 异性结合。
使用BIAcore T200实时分析系统检测实施例3和实施例5中得到的TCR分子与TIHDIILECV-HLA A0201复合物的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH 4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15,000RU。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将TIHDIILECV-HLA A0201复合物流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min，封闭链霉亲和素剩余的结合位点。
上述TIHDIILECV-HLA A0201复合物的制备过程如下：
a.纯化
收集100ml诱导表达重链或轻链的E.coli菌液，于4℃8000g离心10min后用10ml PBS洗涤菌体一次，之后用5ml BugBuster Master Mix Extraction Reagents(Merck)剧烈震荡重悬菌体，并于室温旋转孵育20min，之后于4℃，6000g离心15min，弃去上清，收集包涵体。
将上述包涵体重悬于5ml BugBuster Master Mix中，室温旋转孵育5min；加30ml稀释10倍的BugBuster，混匀，4℃6000g离心15min；弃去上清，加30ml稀释10倍的BugBuster重悬包涵体，混匀，4℃6000g离心15min，重复两次，加30ml 20mM Tris-HCl pH 8.0重悬包涵体，混匀，4℃6000g离心15min，最后用20mM Tris-HCl 8M尿素溶解包涵体，SDS-PAGE检测包涵体纯度，BCA试剂盒测浓度。
b.复性
将合成的短肽TIHDIILECV(江苏金斯瑞生物科技有限公司)溶解于DMSO至20mg/ml的浓度。轻链和重链的包涵体用8M尿素、20mM Tris pH 8.0、10mM DTT来溶解，复性前加入3M盐酸胍、10mM醋酸钠、10mM EDTA进一步变性。将TIHDIILECV肽以25mg/L(终浓度)加入复性缓冲液(0.4M L-精氨酸、100mM Tris pH 8.3、2mM EDTA、0.5mM氧化性谷胱甘肽、5mM还原型谷胱甘肽、0.2mM PMSF，冷却至4℃)，然后依次加入20mg/L的轻链和90mg/L的重链(终浓度，重链分三次加入，8h/次)，复性在4℃进行至少3天至完成，SDS-PAGE检测能否复性成功。
c.复性后纯化
用10体积的20mM Tris pH 8.0作透析来更换复性缓冲液，至少更换缓冲液两次来充分降低溶液的离子强度。透析后用0.45μm醋酸纤维素滤膜过滤蛋白质溶液，然后加载到HiTrap Q HP(GE通用电气公司)阴离子交换柱上(5ml床体积)。利用Akta纯化仪(GE通用电气公司)，20mM Tris pH 8.0配制的0-400mM NaCl线性梯度液洗脱蛋白，pMHC约在250mM NaCl处洗脱，收集诸峰组分，SDS-PAGE检测纯度。
d.生物素化
用Millipore超滤管将纯化的pMHC分子浓缩，同时将缓冲液置换为20mM Tris pH 8.0， 然后加入生物素化试剂0.05M Bicine pH 8.3、10mM ATP、10mM MgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
e.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的pMHC分子浓缩至1ml，采用凝胶过滤层析纯化生物素化的pMHC，利用Akta纯化仪(GE通用电气公司)，用过滤过的PBS预平衡HiPrepTM 16/60S200HR柱(GE通用电气公司)，加载1ml浓缩过的生物素化pMHC分子，然后用PBS以1ml/min流速洗脱。生物素化的pMHC分子在约55ml时作为单峰洗脱出现。合并含有蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，加入蛋白酶抑制剂cocktail(Roche)将生物素化的pMHC分子分装保存在-80℃。
利用BIAcore Evaluation软件计算动力学参数，得到本发明可溶性的TCR分子以及本发明构建的可溶性单链TCR分子与TIHDIILECV-HLA A0201复合物结合的动力学图谱分别如图12和图13所示。图谱显示，本发明得到的可溶性TCR分子以及可溶性单链TCR分子都能够与TIHDIILECV-HLA A0201复合物结合。同时，还利用上述方法检测了本发明可溶性的TCR分子与其他几种无关抗原短肽与HLA复合物的结合活性，结果显示本发明TCR分子与其他无关抗原均无结合。
实施例7.针对负载短肽的T2细胞，转染本发明TCR的效应细胞的激活实验
IFN-γ是活化T淋巴细胞产生的一种强有力的免疫调节因子，因此本实施例通过本领域技术人员熟知的ELISPOT实验检测IFN-γ数以验证转染本发明TCR的细胞的激活功能及抗原特异性。
本实验中所用的效应细胞是表达本发明TCR的CD3 +T细胞，并以同一志愿者转染其他TCR(A6)的CD3 +T细胞作为对照组。所用的靶细胞为负载了HPV16 E6抗原短肽TIHDIILECV的T2细胞，并以负载其他无关肽的、空载的T2细胞作为对照。将试验的各组分加入ELISPOT孔板：靶细胞1×10 4个靶细胞/孔、效应细胞2×10 3个/孔(按转染阳性率计算)，并设置两个复孔。然后在相应孔加入TIHDIILECV短肽，使短肽在ELISPOT孔板中的终浓度依次为1×10 -12M到1×10 -6M，共7个梯度；无关肽直接以最高浓度1×10 -6M进行试验。
按照生产商提供的说明书，如下所述准备孔板：以每块板10毫升无菌PBS按1:200稀释抗人IFN-γ捕捉抗体，然后将100微升的稀释捕捉抗体等分加入各孔。4℃下孵育孔板过夜。孵育后，洗涤孔板以除去多余的捕捉抗体。加入100微升/孔含有10％FBS的RPMI 1640培养基，并在室温下温育孔板2小时以封闭孔板。然后从孔板中洗去培养基，通过在纸上轻弹和轻拍ELISPOT孔板以除去任何残余的洗涤缓冲液。
然后温育孔板过夜(37℃/5％CO 2)第二天，弃培养基，用双蒸水洗涤孔板2次，再用洗涤缓冲液洗涤3次，在纸巾上轻拍以除去残余的洗涤缓冲液。然后用含有10％FBS的PBS按1:200稀释检测抗体，按100微升/孔加入各孔。室温下温育孔板2小时，再用洗涤缓冲液洗涤3次，在纸巾上轻拍孔板以除去过量的洗涤缓冲液。用含有10％FBS的PBS按1:100 稀释链霉亲和素-碱性磷酸酶，将100微升稀释的链霉亲和素-碱性磷酸酶加入各孔并在室温下温育孔板1小时。然后用洗涤缓冲液洗涤4次PBS洗涤2次，在纸巾上轻拍孔板以除去过量的洗涤缓冲液和PBS。洗涤完毕后加入试剂盒提供的BCIP/NBT溶液100微升/孔进行显影。在显影期间用锡箔纸覆盖孔板避光，静置5-15分钟。在此期间常规检测显影孔板的斑点，确定终止反应的最佳时间。去除BCIP/NBT溶液并用双蒸水冲洗孔板以中止显影反应，甩干，然后将孔板底部去除，在室温下干燥孔板直至每个孔完全干燥，再利用免疫斑点平板计数计(CTL，Cellular Technology Limited)计数孔板内底膜形成的斑点。利用graphpad prism6绘制各孔中观察到的ELSPOT斑点数量。
实验结果如图15所示，转染本发明TCR的T细胞对负载TIHDIILECV短肽的靶细胞起明显的激活反应，而转染其他TCR的T细胞对相应的靶细胞自始无反应；同时，转染本发明TCR的T细胞对负载其非特异的短肽的T2细胞及空载的T2细胞无激活反应。
实施例8.针对肿瘤细胞系，转染本发明TCR的效应细胞的激活功能实验
本实施例同样通过ELISPOT实验检测本发明TCR在细胞中的功能及特异性。所用的效应细胞是表达本发明HPV16 E6抗原短肽特异性TCR的CD3 +T细胞，并以同一志愿者转染其他TCR(A6)的CD3 +T细胞作为对照组。所用阳性肿瘤细胞系为A375-E6(E6过表达)。所用HPV阴性细胞系为HCCC9810、LCLs和SK-MEL-5，作为对照组。
首先准备ELISPOT平板。ELISPOT平板乙醇活化包被，4℃过夜。实验第1天，去掉包被液，洗涤封闭，室温下孵育两个小时，去除封闭液，将试验的各个组分加入ELISPOT平板：靶细胞为2×10 4个/孔，效应细胞为4×10 3个/孔(按转染阳性率计算)，并设置二个复孔。温育过夜(37℃，5％CO 2)。实验第2天，洗涤平板并进行二级检测和显色，干燥平板，再利用免疫斑点平板读数计(ELISPOT READER system；AID20公司)计数膜上形成的斑点。
实验结果如图16所示，针对阳性肿瘤细胞系，转染本发明TCR的效应细胞产生了很好的特异性激活反应，转染其他TCR的T细胞基本没有产生激活反应；而对阴性肿瘤细胞系，转染本发明TCR的效应细胞基本不起激活反应。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (26)

  1. 一种T细胞受体(TCR)，其特征在于，所述TCR能够与TIHDIILECV-HLA A0201复合物结合；并且，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域的3个互补决定区(CDR)为：
    αCDR1-TSDPSYG  (SEQ ID NO:10)
    αCDR2-QGSYDQQN  (SEQ ID NO:11)
    αCDR3-AMREGTGTASKLT  (SEQ ID NO:12)；和/或
    所述TCRβ链可变域的3个互补决定区为：
    βCDR1-SGHDN  (SEQ ID NO:13)
    βCDR2-FVKESK  (SEQ ID NO:14)
    βCDR3-ASSPYGQGTSPLH  (SEQ ID NO:15)。
  2. 如权利要求1所述的TCR，其特征在于，其包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域为与SEQ ID NO:1具有至少90％序列相同性的氨基酸序列；和/或所述TCRβ链可变域为与SEQ ID NO：5具有至少90％序列相同性的氨基酸序列。
  3. 如权利要求1所述的TCR，其特征在于，所述TCR包含α链可变域氨基酸序列SEQ ID NO:1；和/或所述TCR包含β链可变域氨基酸序列SEQ ID NO:5。
  4. 如权利要求1所述的TCR，其特征在于，所述TCR为αβ异质二聚体，其包含TCRα链恒定区TRAC*01和TCRβ链恒定区TRBC1*01或TRBC2*01；优选地，所述TCR的α链氨基酸序列为SEQ ID NO：3和所述TCR的β链氨基酸序列为SEQ ID NO:7。
  5. 如权利要求1所述的TCR，其特征在于，所述TCR是可溶的。
  6. 如权利要求5所述的TCR，其特征在于，所述TCR为单链；优选地，所述TCR是由α链可变域与β链可变域通过肽连接序列连接而成。
  7. 如权利要求6所述的TCR，其特征在于，所述TCR在α链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或α链J基因短肽氨基酸倒数第3位、倒数第5位或倒数第7位中具有一个或多个突变；和/或所述TCR在β链可变区氨基酸第11、13、19、21、53、76、89、91、或第94位，和/或β链J基因短肽氨基酸倒数第2位、倒数第4位或倒数第6位中具有一个或多个突变，其中氨基酸位置编号按IMGT(国际免疫遗传学信息系统)中列出的位置编号；优选地，所述TCR的α链可变域氨基酸序列包含SEQ ID NO:32和/或所述TCR的β链可变域氨基酸序列包含SEQ ID NO:34；更优选地，所述TCR的氨基酸序列为SEQ ID NO:30。
  8. 如权利要求1所述的TCR，其特征在于，所述TCR包含(ⅰ)TCRα链可变域和除跨膜结构域以外的全部或部分TCRα链恒定区；和(ⅱ)TCRβ链可变域和除跨膜结构域以外的全部或部分TCRβ链恒定区。
  9. 如权利要求8所述的TCR，其特征在于，半胱氨酸残基在所述TCR的α和β链 恒定域之间形成人工二硫键；优选地，在所述TCR中形成人工二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
    TRAC*01外显子1的Thr48和TRBC1*01或TRBC2*01外显子1的Ser57；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Ser77；
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Ser17；
    TRAC*01外显子1的Thr45和TRBC1*01或TRBC2*01外显子1的Asp59；
    TRAC*01外显子1的Ser15和TRBC1*01或TRBC2*01外显子1的Glu15；
    TRAC*01外显子1的Arg53和TRBC1*01或TRBC2*01外显子1的Ser54；
    TRAC*01外显子1的Pro89和TRBC1*01或TRBC2*01外显子1的Ala19；和
    TRAC*01外显子1的Tyr10和TRBC1*01或TRBC2*01外显子1的Glu20。
  10. 如权利要求9所述的TCR，其特征在于，所述TCR的α链氨基酸序列为SEQ ID NO:26和/或所述TCR的β链氨基酸序列为SEQ ID NO:28。
  11. 如权利要求8所述的TCR，其特征在于，所述TCR的α链可变区与β链恒定区之间含有人工链间二硫键；优选地，在所述TCR中形成人工链间二硫键的半胱氨酸残基取代了选自下列的一组或多组位点：
    TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸；
    TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的61位氨基酸；
    TRAV的第46位氨基酸和TRBC1*01或TRBC2*01外显子1的第61位氨基酸；或
    TRAV的第47位氨基酸和TRBC1*01或TRBC2*01外显子1的第60位氨基酸。
  12. 如权利要求1所述的TCR，其特征在于，所述TCR包含α链恒定区和β链恒定区，所述α链恒定区为鼠源的和/或所述β链恒定区为鼠源的。
  13. 如权利要求1所述的TCR，其特征在于，所述TCR的α链和/或β链的C-或N-末端结合有偶联物；优选地，与所述T细胞受体结合的偶联物为可检测标记物或治疗剂；更优选地，所述治疗剂为抗-CD3抗体。
  14. 如权利要求1所述的TCR，其特征在于，所述TCR是分离的或纯化的。
  15. 一种多价TCR复合物，其特征在于，包含至少两个TCR分子，并且其中的至少一个TCR分子为上述权利要求中任一项所述的TCR。
  16. 一种核酸分子，其特征在于，所述核酸分子包含编码上述任一权利要求所述的TCR分子的核酸序列或其互补序列。
  17. 如权利要求16所述的核酸分子，其特征在于，其包含编码TCRα链可变域的核苷酸序列SEQ ID NO：2或SEQ ID NO:33。
  18. 如权利要求16或17所述的核酸分子，其特征在于，其包含编码TCRβ链可变域的核苷酸序列SEQ ID NO：6或SEQ ID NO:35。
  19. 如权利要求16所述的核酸分子，其特征在于，其包含编码TCRα链的核苷酸序列SEQ ID NO：4和/或包含编码TCRβ链的核苷酸序列SEQ ID NO：8。
  20. 一种载体，其特征在于，所述的载体含有权利要求16-19中任一所述的核酸分 子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。
  21. 一种分离的宿主细胞，其特征在于，所述的宿主细胞中含有权利要求20中所述的载体或染色体中整合有外源的权利要求16-19中任一所述的核酸分子。
  22. 一种细胞，其特征在于，所述细胞中转导有权利要求16-19中任一所述的核酸分子或权利要求20中所述载体；优选地，所述细胞为T细胞或干细胞。
  23. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1-14中任一项所述的TCR、权利要求15中所述的TCR复合物、或权利要求22中所述的细胞。
  24. 权利要求1-14中任一项所述的T细胞受体、或权利要求15中所述的TCR复合物或权利要求22中所述的细胞的用途，其特征在于，用于制备治疗肿瘤或自身免疫疾病的药物；优选地，所述肿瘤为HPV阳性肿瘤。
  25. 权利要求1-14中任一项所述的T细胞受体、或权利要求15中所述的TCR复合物或权利要求22中所述的细胞，用作治疗肿瘤或自身免疫疾病的药物；优选地，所述肿瘤为HPV阳性肿瘤。
  26. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1-14中任一所述的TCR、权利要求15中所述TCR复合物、权利要求22中所述的细胞或权利要求23中所述的药物组合物；
    优选地，所述的疾病为肿瘤，更优选地所述肿瘤为HPV阳性肿瘤。
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