WO2022111475A1 - 一种识别hpv抗原的tcr - Google Patents

一种识别hpv抗原的tcr Download PDF

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WO2022111475A1
WO2022111475A1 PCT/CN2021/132461 CN2021132461W WO2022111475A1 WO 2022111475 A1 WO2022111475 A1 WO 2022111475A1 CN 2021132461 W CN2021132461 W CN 2021132461W WO 2022111475 A1 WO2022111475 A1 WO 2022111475A1
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tcr
amino acid
present
variable domain
exon
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PCT/CN2021/132461
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French (fr)
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李懿
杨东雪
陈少沛
孙含丽
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香雪生命科学技术（广东）有限公司
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Priority to US18/039,060 priority Critical patent/US20240002464A1/en
Priority to EP21896976.4A priority patent/EP4253408A1/en
Publication of WO2022111475A1 publication Critical patent/WO2022111475A1/zh

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Definitions

  • the present invention relates to the field of biotechnology, and more particularly, to a T cell receptor (TCR) capable of recognizing derived from HPV16 E6 protein polypeptide and its preparation and use.
  • TCR T cell receptor
  • TCR T cell receptor
  • the binding surfaces on the TCR are derived from six regions: CDR1, CDR2, and CDR3, respectively, on the TCR alpha and beta chains.
  • CDR1 and CDR2 mainly bind to relatively conservative MHC molecules
  • CDR3 mainly binds to variable antigen polypeptides. This binding mode can well ensure that TCR specifically binds to MHC molecules and recognizes variable antigens. (Chinese Journal of Cell Biology 2011, 33(9):955-963).
  • CDR3 has the largest variation, which directly determines the antigen-binding specificity of TCR.
  • T cells In the immune system, direct physical contact between T cells and antigen-presenting cells (APCs) is triggered by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact. It causes a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.
  • APCs antigen-presenting cells
  • 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. and other diseases.
  • TIHDIILECV is an epitope peptide of HPV16 E6 protein and is a target for the treatment of HPV16 E6-related diseases.
  • the present invention is devoted to developing a TCR that can bind to the TIHDIILECV-HLA A0201 complex and has high application value for tumor treatment.
  • a TCR capable of targeting this tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or be transformed into T cells so that T cells expressing the TCR can destroy tumor cells for Administered to patients during the course of treatment with adoptive immunotherapy.
  • the first aspect of the present invention provides a TCR, the TCR has the activity of binding the TIHDIILECV-HLA A0201 complex, and the ⁇ chain variable domain of the TCR is the same as the amino acid sequence shown in SEQ ID NO: 1. an amino acid sequence having at least 90% sequence homology; and the beta chain variable domain of the TCR is an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:5.
  • the ⁇ chain variable domain of the TCR comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence homology.
  • the ⁇ chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% or 100% sequence homology.
  • the TCR of the present invention is of human origin.
  • the TCR is an ⁇ heterodimeric TCR, and preferably, the TCR has the ⁇ chain constant region sequence TRAC*01 and the ⁇ chain constant region sequence TRBC1*01 or TRBC2*01.
  • the constant regions of the ⁇ and ⁇ chains of the TCR are the constant regions of the murine ⁇ and ⁇ chains, respectively.
  • the TCR is soluble.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the TCR ⁇ chain variable domain comprises 3 CDR regions, wherein the amino acid sequences of the 3 CDR regions of the TCR ⁇ chain variable domain for:
  • the ⁇ chain variable domain of the TCR is an amino acid sequence with at least 95% sequence homology to the amino acid sequence shown in SEQ ID NO: 1; and the ⁇ chain variable domain of the TCR A domain is an amino acid sequence with at least 95% sequence homology to the amino acid sequence set forth in SEQ ID NO:5.
  • amino acid sequence of the ⁇ chain variable domain of the TCR is shown in SEQ ID NO: 5.
  • the ⁇ chain variable domain of the TCR is an amino acid sequence that has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 1; and the TCR ⁇ chain variable domain
  • the amino acid sequences of the three CDR regions are:
  • the ⁇ chain variable domain of the TCR is an amino acid sequence with at least 95% sequence homology to the amino acid sequence shown in SEQ ID NO: 1; and the ⁇ chain variable domain of the TCR The amino acid sequence of the domain is shown in SEQ ID NO:5.
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the TCR ⁇ chain variable domain comprises 3 CDR regions, wherein the amino acid sequence of CDR3 ⁇ in the TCR ⁇ chain variable domain is selected from:
  • ALRAGANNLF (SEQ ID NO:22), ALRAGANLPV (SEQ ID NO:23), ALRAGANTPI (SEQ ID NO:24), ALRAGAWPMK (SEQ ID NO:25).
  • the TCR ⁇ chain variable domain comprises 3 CDR regions, and the TCR ⁇ chain variable domain comprises 3 CDR regions, wherein the amino acid sequence of CDR1 ⁇ in the TCR ⁇ chain variable domain is: TRDTTYY and the amino acid sequence of CDR2 ⁇ is: RNSFDEQN; and the amino acid sequence of CDR3 ⁇ is selected from:
  • ALRAGANNLF (SEQ ID NO:22), ALRAGANLPV (SEQ ID NO:23), ALRAGANTPI (SEQ ID NO:24), ALRAGAWPMK (SEQ ID NO:25).
  • sequences of the three CDR regions of the variable domain of the TCR ⁇ chain are as follows,
  • CDR3 ⁇ ALRAGANNLF (SEQ ID NO: 22), and CDR3 ⁇ contains at least one of the following mutations:
  • sequences of the three CDR regions of the variable domain of the TCR ⁇ chain are as follows,
  • CDR3 ⁇ ALRAGANNLF (SEQ ID NO: 22), and CDR3 ⁇ contains at least one of the following mutations:
  • amino acid sequences of the three CDR regions of the variable domain of the TCR ⁇ chain are:
  • the TCR has a CDR selected from the group consisting of:
  • amino acid sequence of the ⁇ chain variable domain of the TCR is shown in any of SEQ ID NOs: 1-4; and/or the amino acid sequence of the ⁇ chain variable domain of the TCR is shown in SEQ ID NO:5 shown.
  • the TCR is selected from the following group:
  • the TCR comprises (i) all or part of the TCR ⁇ chain excluding its transmembrane domain, and (ii) all or part of the TCR ⁇ chain excluding its transmembrane domain, wherein (i) and (ii) each comprise the variable domain and at least a portion of the constant domain of the TCR chain.
  • an artificial interchain disulfide bond is contained between the constant region of the ⁇ chain and the constant region of the ⁇ chain of the TCR.
  • cysteine residues that form artificial interchain disulfide bonds are substituted for one or more groups of sites selected from the following:
  • the TCR is a single-chain TCR.
  • the single-chain TCR is formed by linking the ⁇ -chain variable domain and the ⁇ -chain variable domain by a flexible short peptide sequence (linker).
  • amino acid sequence of the single-chain TCR is shown in SEQ ID NOs: 6-9.
  • a conjugate is bound to the C- or N-terminus of the ⁇ chain and/or the ⁇ chain of the TCR.
  • the conjugate is a detectable label, a therapeutic agent, a PK modification moiety or a combination of any of these.
  • the therapeutic agent bound to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the ⁇ or ⁇ chain of the TCR.
  • 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 described in the first aspect of the present invention or the TCR complex described in the second aspect of the present invention or a complementary sequence thereof.
  • 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 a host cell containing the vector of the fourth aspect of the present invention or the exogenous nucleic acid molecule of the third aspect of the present invention integrated into the genome.
  • the sixth aspect of the present invention provides an isolated cell, the cell is 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, or the cell expresses the first aspect of the present invention.
  • the TCR preferably, the cells are T cells, NK cells or NKT cells.
  • a seventh aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, or The cell according to the sixth aspect of the present invention.
  • 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 the treatment of tumor or autoimmune disease, preferably, the tumor is HPV positive tumor, such as HPV16 E6 positive tumor, more 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 Medicine for autoimmune diseases; preferably, the tumor is HPV-positive tumor, such as HPV16 E6-positive tumor, more 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 cell according to the sixth aspect of the present invention, or the pharmaceutical composition according to the seventh aspect of the present invention; preferably, the disease is an HPV-positive tumor, more preferably, the tumor is cervical cancer.
  • the eleventh aspect of the present invention provides a method for preparing the T cell receptor described in the first aspect of the present invention, comprising the steps of:
  • Figures 1a-1d respectively show the TCR ⁇ chain variable domain amino acid sequences of the present invention capable of specifically binding to the TIHDIILECV-HLA A0201 complex.
  • Figure 2 shows the TCR beta chain variable domain amino acid sequence of the present invention capable of specifically binding to the TIHDIILECV-HLA A0201 complex.
  • Figures 3a-3d show the amino acid sequences of the single-chain TCRs of the present invention, respectively.
  • Figures 4a-4d show the amino acid sequences of the soluble TCR ⁇ chains of the present invention, respectively, wherein the mutated cysteine residues are shown in bold letters.
  • Figure 5 is the amino acid sequence of the soluble TCR ⁇ chain of the present invention, wherein the mutated cysteine residues are represented by bold letters.
  • Figures 6a and 6b respectively show the nucleotide sequence encoding the variable domain of the TCR ⁇ chain of the present invention (amino acid sequence shown in SEQ ID NO: 1) and the variable domain encoding the TCR ⁇ chain of the present invention (amino acid sequence shown in SEQ ID NO: 5) shown) nucleotide sequence.
  • Figures 7a-7d show the binding curves of the soluble TCR1, TCR2, TCR3, TCR4 of the present invention and the TIHDIILECV-HLA A0201 complex, respectively.
  • Figure 8 shows the results of the activation function experiment of effector cells transfected with the TCR of the present invention against T2 cells loaded with short peptides.
  • Figures 9a-9b show the results of the activation function experiments of effector cells transfected with the TCR of the present invention against tumor cell lines.
  • Figure 10 shows the results of the LDH experiment on the killing function of effector cells transfected with the TCR of the present invention against T2 cells loaded with a gradient of short peptides.
  • FIG. 11 shows the results of LDH experiments on the killing function of effector cells transfected with the TCR of the present invention against tumor cell lines.
  • Figure 12 shows the results of the IncuCyte assay for the killing function of effector cells transfected with the TCR of the present invention against tumor cell lines.
  • Figure 13 is a graph showing the results of double-positive staining of tetramer and anti-CD8-APC.
  • the present invention obtains a high-affinity T cell receptor (TCR) that specifically recognizes and binds to the TIHDIILECV-HLA A0201 complex, and the amino acid sequences of the three CDR regions of its ⁇ chain variable domain as follows,
  • CDR3 ⁇ ALRAGANNLF (SEQ ID NO: 22), and CDR3 ⁇ contains at least one of the following mutations:
  • amino acid sequences of the three CDR regions of the ⁇ chain variable domain are as follows,
  • TCR T cell receptor
  • TCRs can be described using the International Information System for Immunogenetics (IMGT).
  • IMGT International Information System for Immunogenetics
  • Native ⁇ heterodimeric TCRs have ⁇ and ⁇ chains. Broadly speaking, each chain contains a variable region (V region), a linker region (J region), and a constant region (C region), and the beta chain usually also contains a short variable region (D region) between the variable region and the linker region. region), but the variable region is often considered part of the linker region.
  • the junctional region of the TCR is determined by TRAJ and TRBJ of the unique IMGT, and the constant region of the TCR is determined by the TRAC and TRBC of the IMGT.
  • the alpha chain constant domain has the following symbols: TRAC*01, where "TR” represents the T cell receptor gene; "A” represents the alpha chain gene; C represents the constant region; "*01” represents the allele Gene 1.
  • the beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR” denotes the T cell receptor gene; “B” denotes the beta chain gene; C denotes the constant region; "*01” denotes the allele 1.
  • the constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes "C1" and "C2". Those skilled in the art can obtain the constant region gene sequences of TCR ⁇ and ⁇ chains through the public IMGT database.
  • TCR alpha chain variable domain refers to linked TRAV and TRAJ regions
  • TCR beta chain variable domain refers to linked TRBV and TRBD/TRBJ regions.
  • Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimerically incorporated in a framework sequence.
  • the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , respectively; the three CDRs of the variable domain of the TCR ⁇ chain are CDR1 ⁇ , CDR2 ⁇ and CDR3 ⁇ , respectively.
  • the framework sequences of the TCR variable domains of the present invention may be of murine or human origin, preferably human.
  • the constant domains of TCRs comprise an intracellular portion, a transmembrane region and an extracellular portion.
  • TCR sequences used in the present invention are of human origin.
  • 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.
  • the 60th amino acid in the sequence from end to C end is P (proline), then it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in the present invention, and it can also be expressed as TRBC1* 01 or the 60th amino acid of TRBC2*01 exon 1, and in TRBC1*01 or TRBC2*01, the 61st amino acid is Q (glutamine) in the sequence from the N-terminus to the C-terminus, then this In the invention, it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1, and it can also be described as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, and so on.
  • 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.
  • 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.
  • the special instructions are followed.
  • tumor is meant to encompass all types of cancer cell growth or oncogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or stage of infection.
  • tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions.
  • solid tumors include: malignancies of various organ systems such as sarcomas, lung squamous cell carcinomas and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (eg: colon), and genitourinary tract (eg: kidney, epithelial cells), pharynx.
  • the T cells transduced with the TCR 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.
  • the first aspect of the present invention provides a TCR, the TCR has the activity of binding TIHDIIILECV-HLA A0201 complex, and the ⁇ chain variable domain of the TCR is the amino acid shown in SEQ ID NO: 1
  • An amino acid sequence with at least 90% sequence homology in the sequence; and/or the ⁇ chain variable domain of the TCR is an amino acid sequence with at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 5 .
  • the TCR ⁇ chain variable domain of the present invention is at least 90%, preferably 95% identical to SEQ ID NO: 1, (eg, may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%) amino acid sequence sequence identity; and/or the TCR beta chain variable domain of the present invention is at least 90%, preferably 95%, with SEQ ID NO: 5, (eg, may be at least 91%) , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) amino acid sequences of sequence identity.
  • the TCR of the present invention is of human origin.
  • the TCR of the present invention comprises an ⁇ chain variable domain amino acid sequence of one of SEQ ID NO: 1-4; and the ⁇ chain variable domain amino acid sequence of the TCR is SEQ ID NO: 5 .
  • the recognition of pMHC (antigenic peptide-major histocompatibility complex) by TCR involves two types of binding: binding of TCR to MHC molecules and binding of TCR to polypeptide antigens.
  • the binding surfaces on the TCR are derived from six regions: CDR1, CDR2, and CDR3, respectively, on the TCR alpha and beta chains.
  • CDR1 and CDR2 mainly bind to relatively conservative MHC molecules
  • CDR3 mainly binds to variable antigen polypeptides. This binding mode can well ensure that TCR specifically binds to MHC molecules and recognizes variable antigens. (Chinese Journal of Cell Biology 2011, 33(9):955-963).
  • CDR3 has the largest variation, which directly determines the antigen-binding specificity of TCR.
  • the CDR regions determined by sequencing of the TCR of the present invention are as follows:
  • 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.
  • substitution with amino acids with similar or similar properties usually does not change the function of the protein.
  • the addition of one or several amino acids to the C-terminus and/or N-terminus also generally does not alter the structure and function of the protein. Therefore, the TCR of the present invention also includes up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid (especially the amino acid outside the CDR region) of the TCR of the present invention, which are similar in nature or similar amino acids, and still retain its functional TCR.
  • the constant domain of the TCR molecule of the present invention is a human constant domain.
  • IMGT International Immunogenetics Information System
  • 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 TCR is an ⁇ heterodimeric TCR, and preferably, the TCR has the ⁇ chain constant region sequence TRAC*01 and the ⁇ chain constant region sequence TRBC1*01 or TRBC2*01.
  • 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 N-terminal to C-terminal.
  • a TCR of the present invention is a portion having at least one variable domain of the TCR ⁇ and/or TCR ⁇ chain. They usually contain both the TCR ⁇ chain variable domain and the TCR ⁇ chain variable domain. They can be alpha beta heterodimers or single-chain forms or any other form that can be stably present.
  • the full-length chain of ⁇ heterodimeric TCRs (comprising cytoplasmic and transmembrane domains) can be transfected.
  • the TCRs of the present invention can be used as targeting agents for delivering therapeutic agents to antigen-presenting cells or in combination with other molecules to produce bifunctional polypeptides to target effector cells, in which case the TCRs are preferably in a soluble form.
  • 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 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 present invention also obtains a soluble TCR specific for the HPV16E6 antigenic short peptide.
  • its alpha chain variable domain amino acid sequence is one of SEQ ID NO: 10-13
  • its beta chain variable domain amino acid sequence is SEQ ID NO: 14.
  • 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 alpha chain J gene (TRAJ) short peptide amino acid position 3,5,7 from the bottom, and/or the beta chain J gene (TRBJ) short peptide amino acid position 2,4,6 from the bottom, where 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.
  • the single-chain TCR amino acids of the present invention are selected from: SEQ ID NOs: 6-9.
  • 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.
  • Mutagenesis can be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), cloning based on restriction enzymes or ligation independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme-based cloning can be found in Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (Third Edition) CSHL publishing house. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).
  • PCR polymerase chain reaction
  • LIC ligation independent cloning
  • the method for generating mutations can be, but is not limited to, screening for TCRs that specifically bind to the TIHDIILECV-HLA A0201 complex from a diverse library of phage particles displaying such TCRs, as described in the literature (Li, et al (2005) Nature Biotech 23 (3):349-354).
  • 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 combining a TCR of the present invention with 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
  • Antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, the binding of such antibodies or fragments thereof to TCR is capable of effecting effector cells. directed to better target target cells.
  • a preferred embodiment is that the TCR of the present invention binds to an anti-CD3 antibody or a functional fragment or variant of said anti-CD3 antibody.
  • the fusion molecule of the TCR and anti-CD3 single-chain antibody of the present invention comprises one selected from the group consisting of: (1) the amino acid sequence of the variable domain of the TCR ⁇ chain is one of SEQ ID NOs: 1-4, and the ⁇ chain variable of the TCR The domain amino acid sequence is SEQ ID NO: 5; or (2) the single-chain TCR amino acid sequence is one of SEQ ID NO: 6-9.
  • the present invention also relates to nucleic acid molecules encoding the TCRs of the present invention.
  • the nucleic acid molecules of the present invention may be in the form of DNA or RNA.
  • DNA can be the coding or non-coding strand.
  • the 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 encoding a protein sequence having SEQ ID NO: 1, but with the sequence of SEQ ID NO: 15. Different nucleic acid sequences.
  • 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.
  • the present invention also relates to vectors comprising the nucleic acid molecules of the present invention, as well as host cells genetically engineered with the vectors or coding sequences of the present invention.
  • the vectors comprising the nucleic acid molecules of the present invention include expression vectors, ie constructs that can be expressed 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, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors.
  • 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.
  • the vector should be able to express consistently high levels in T cells.
  • 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 may be T cells, NK cells, NKT cells, etc., preferably 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 provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell that presents the TCR of the present invention.
  • the present invention also provides a method of treating a disease, comprising administering an appropriate amount of the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention, or the pharmaceutical composition of the present invention to a subject in need of treatment.
  • the TCRs of the present invention, TCR complexes or TCR-transfected T cells of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the TCRs, multivalent TCR complexes or cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, usually in a sealed container, as part of a kit. Such kits, but not necessarily, include instructions for use. It may comprise a plurality of such unit dosage forms.
  • TCRs of the present invention can be used alone, or in combination or conjugation with other therapeutic agents (eg, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • the term refers to pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition, and are not undue toxicity upon administration. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • therapeutic compositions can be prepared as injectables, eg, liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid carriers, prior to injection can also be prepared.
  • compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous.
  • the subject to be prevented or treated can be an animal; especially a human.
  • compositions of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various pharmaceutical compositions in different dosage forms can be adopted according to the usage. Preferably, injections, oral preparations and the like can be exemplified.
  • compositions can be formulated according to conventional methods by mixing, diluting or dissolving, with occasional addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • compositions of the present invention can also be administered in the form of sustained release formulations.
  • the TCRs of the present invention can be incorporated into pellets or microcapsules in a slow release polymer carrier, which are then surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the present invention provides a method for treating HPV16 E6-related diseases, comprising 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. Typically, this involves (1) isolating T cells from a 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 genetically engineered T cells into a patient in vivo.
  • the TCR or the TCR complex of the present invention or the cell presenting the TCR of the present invention as an active ingredient can be used according to the weight, age, sex, degree of symptoms of each patient to be treated And be reasonably determined, and ultimately by the physician to decide a reasonable dosage.
  • 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 potential to elicit 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.
  • both Pro60 or 60P represent proline at position 60.
  • the expression of the specific form of the mutation in the present invention is such as "N93D” represents the substitution of N at the 93rd position by D, and similarly, “N93D/E” represents the substitution of the N at the 93rd position by D or by E. Others and so on.
  • the TCR of the present invention also includes up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid (especially the amino acid outside the CDR region) of the TCR of the present invention, which are similar in nature or similar amino acids, and still retain its functional TCR.
  • the present invention also includes slightly modified TCRs of the present invention.
  • Modified (generally without altering the primary structure) forms include: chemically derivatized forms of the TCRs of the invention such as acetylated or carboxylated.
  • Modifications also include glycosylation, such as those TCRs resulting from glycosylation modifications in the synthesis and processing of the TCRs of the invention or in further processing steps. This modification can be accomplished by exposing the TCR to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases.
  • Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • the TCR of the present invention can specifically bind to the HPV16 E6 antigen short peptide complex TIHDIILECV-HLA A0201.
  • the effector cells transduced with the TCR of the present invention can be specifically activated, and at the same time, the effector cells transduced with the TCR of the present invention also have a strong killing function.
  • Peripheral blood lymphocytes from healthy volunteers with HLA-A0201 genotype were stimulated with the synthetic short peptide TIHDIILECV (Jiangsu GenScript Biotechnology Co., Ltd.).
  • the TIHDIILECV peptide was renatured with biotin-labeled HLA-A0201 to prepare pMHC haploids.
  • 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.
  • the double positive staining results are shown in Figure 13.
  • the present invention unexpectedly obtained a T cell receptor that can specifically bind to the antigenic short peptide complex TIHDIILECV-HLA A0201 derived from HPV16 E6, and its binding characterization map is shown in Figure 7a.
  • the effector cells transduced with the TCR of the present invention can be activated and have a strong killing function.
  • TIHDIILECV-specific, HLA-A0201-restricted TCR ⁇ chain variable domain nucleotide sequence (SEQ ID NO: 15) and TCR ⁇ chain variable domain nucleotide sequence (SEQ ID NO: 16) are shown in Figure 6a, respectively and shown in 6b.
  • the amino acid sequence of the TCR ⁇ variable domain is SEQ ID NO: 1
  • the amino acid sequence of the ⁇ chain variable domain is SEQ ID NO: 5, which is named TCR1 for the convenience of description.
  • the present invention further adopts the method of site-directed mutagenesis well known to those skilled in the art.
  • a stable monolayer consisting of a flexible short peptide (linker) connecting the above-mentioned TCR ⁇ and ⁇ chain variable domains is constructed.
  • chain TCR molecules and express, renature and purify.
  • the TCR phage display and screening methods described by Li et al. ((2005) Nature Biotech 23(3):349-354) were applied to single-chain TCR templates.
  • the present invention also unexpectedly found that, for the HPV E6 positive target cell line, the effector cells transduced with TCR2, TCR3 or TCR4 can also be specifically activated and have a strong killing function.
  • the alpha chain of TCR2 or TCR3 or TCR4 shares at least 95% sequence homology with the alpha chain of TCR1, and similarly, the beta chain of TCR2 or TCR3 or TCR4 shares at least 95% sequence homology with the beta chain of TCR1.
  • TCR1 Compared with TCR1, TCR2 or TCR3 or TCR4 has 3 or 4 mutations in the CDR3 of the ⁇ chain, and the specific CDRs are shown in Table 1 below:
  • variable domain sequences of the alpha and beta chains of the TCR of the present invention are as follows:
  • the ⁇ chain variable domain amino acid sequence of the TCR is: one of SEQ ID NO: 1-4; and/or the ⁇ chain variable domain amino acid sequence of the TCR is selected from: SEQ ID NO: 5.
  • a cysteine residue was introduced into the constant domains of the ⁇ and ⁇ chains of the TCR of the present invention, respectively, to form an artificial interchain disulfide bond, so as to obtain a stable soluble TCR molecule, so as to evaluate the complexation of TCR with The interaction between TIHDIILECV-HLA A0201.
  • the amino acid sequence of its alpha chain is one of SEQ ID NOs: 10-13, as shown in Figures 4a-4d; and the amino acid sequence of its beta chain is SEQ ID NO: 14, as shown in Figure 5.
  • 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 formed inclusion bodies 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) , 20mM 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 at a final concentration of 60 mg/mL. After mixing, 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.
  • the solution after dialysis was filtered through a 0.45 ⁇ M filter membrane and purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare).
  • the eluted peaks of TCR containing successfully renatured ⁇ and ⁇ dimers were confirmed by SDS-PAGE gel.
  • TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was more than 90% determined by SDS-PAGE, and the concentration was determined by BCA method.
  • the BIAcore T200 real-time analysis system was used to detect the binding activity of the soluble TCR molecules obtained according to Example 2 to the TIHDIILECV-HLA A0201 complex.
  • the TIHDIILECV-HLA A0201 complex was prepared using methods well known to those skilled in the art, and the main processes included purification, renaturation, repurification and biotinylation.
  • 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.
  • the kinetic maps of the binding of the soluble TCR molecule of the present invention to the TIHDIILECV-HLA A0201 complex are shown in Figures 7a-7d, respectively.
  • the map shows that the soluble TCR molecules obtained in the present invention can all be combined with the TIHDIILECV-HLA A0201 complex.
  • 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 4 Activation function experiment of effector cells transfected with TCR of the present invention for target 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 TCR of the present invention was transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteer was transfected with other TCR (A6, TCR against other antigens) CD3+ T cells as a control .
  • the numbers of the TCRs and their ⁇ and ⁇ variable domain sequences are shown in Table 2.
  • the target cells used were T2 cells loaded with HPV16 E6 antigen short peptide TIHDIILECV, loaded with other peptides or empty.
  • ELISPOT plates were ethanol activated and coated overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add the components of the test to the ELISPOT plate: target cells are 1 ⁇ 10 4 cells/well, and effector cells are 2 ⁇ 10 3 cells/well (calculated according to the positive rate of transfection), the final concentration of short peptide was 1 ⁇ 10 -6 M/well, and two duplicate wells were set. Incubate overnight (37°C, 5% CO2 ). On the second day of the experiment, 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).
  • ELISPOT READER system an immunospot plate reader
  • the effector cells transfected with the TCR of the present invention have obvious activation effects, while the effector cells transfected with other TCRs are inactive;
  • the effector cells of the TCR of the present invention are substantially unresponsive to target cells loaded with other peptides or empty.
  • Example 5 Activation function experiment of effector cells transfected with TCR of the present invention for tumor cell lines
  • TCR of the present invention was transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and CD3+ T cells transfected with other TCR (A6) from the same volunteer were used as negative controls.
  • the numbers of the TCRs and their ⁇ and ⁇ variable domain sequences are shown in Table 2.
  • HPV16 E6 positive tumor cell lines used in the first round of experiments were A375-E6 (HPV16 E6 overexpression), HK-2, and the negative tumor cell lines were A375, SiHa, and HCCC9810; the HPV16 E6 positive tumor cell lines used in the second round of experiments It is A375-E6 (HPV16 E6 overexpression), and the negative tumor cell lines are A375, KATO III, MDA-MB-231, MEL526, and LCL.
  • the experimental procedure is as shown in Example 4, wherein the components added to the ELISPOT plate are: 2 ⁇ 10 4 cells/well of target cells and 2 ⁇ 10 3 cells /well of effector cells (calculated according to the positive rate of transfection).
  • Lactate dehydrogenase is abundant in the cytoplasm and cannot pass through the cell membrane under normal conditions, but can be released to the outside of the cell when the cell is damaged or died. At this time, the activity of LDH in the cell culture medium is proportional to the number of cells dying.
  • non-radioactive cytotoxicity experiments well known to those skilled in the art are used to measure the release of LDH, thereby verifying the killing function of cells transfected with the TCR of the present invention. This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies the LDH released after cell lysis.
  • LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan.
  • the amount of red product produced is proportional to the number of cells lysed.
  • CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the TCR of the present invention as effector cells, and the same volunteer was used to transfect other TCR (A6) or empty transfected (NC) CD3 +T cells served as a control group.
  • the numbers of the TCRs and their ⁇ and ⁇ variable domain sequences are shown in Table 2.
  • the target cells used were T2 cells loaded with TIHDIILECV peptide, while other antigen-loaded and empty T2 cells were used as controls.
  • effector cell spontaneous wells were set. Incubate overnight (37°C, 5% CO2 ). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with a microplate reader (Bioteck) after the reaction was terminated.
  • the effector cells transfected with the TCR of the present invention have a strong killing function, and react when the above-mentioned short peptide concentration is low, and The effector cells transfected with other TCRs or empty-transfected have no killing effect from the beginning; meanwhile, the effector cells transfected with the TCR of the present invention have no killing effect on the target cells loaded with other short peptides.
  • tumor cell lines were used to test again the killing function and specificity of the cells transfected with the TCR of the present invention. LDH release was also measured by non-radioactive cytotoxicity assays well known to those skilled in the art.
  • CD3+T cells isolated from the blood of healthy volunteers were used to transfect the TCR of the present invention as effector cells, and CD3+T cells transfected with other TCRs (A6) from the same volunteer were used as negative controls.
  • the numbers of the TCRs and their ⁇ and ⁇ variable domain sequences are shown in Table 2.
  • the HPV16 E6 positive tumor cell lines used were A375-E6 (HPV16 E6 overexpression), HK-2, and the negative tumor cell lines were A375, HCCC9810
  • Example 6 The experimental steps are as shown in Example 6, wherein the components added to the LDH plate are: target cells 3 ⁇ 10 4 cells/well, effector cells 3 ⁇ 10 4 cells/well (calculated according to the transfection positive rate), and Set up three duplicate wells.
  • the effector cells transfected with the TCR of the present invention also showed strong killing effect, while the T cells transfected with other TCRs basically did not respond;
  • the T cells of the TCR basically do not kill the negative tumor cell lines, which further reflects the good specific killing function of the cells transfected with the TCR of the present invention.
  • Example 8 For tumor cell lines, use IncuCyte to verify the immune efficacy of effector cells transfected with the TCR molecule of the present invention
  • IncuCyte is a functional analysis system that can automatically analyze images at different time points and quantify real-time apoptosis through real-time microphotography in an incubator.
  • the TCR of the present invention was transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and CD3+ T cells empty transfected (NC) of the same volunteer were used as a control group.
  • the numbers of the TCRs and their ⁇ and ⁇ variable domain sequences are shown in Table 2. In this example, experiments were performed with positive target cells CASKI.
  • the target cells were digested and centrifuged; they were resuspended in complete medium of RPMI1640+10% FBS without phenol red, and the target cells were evenly plated in a 96-well plate: 1.5 ⁇ 10 4 cells/cell Well; put it back into the incubator at 37 degrees, 5% CO 2 and incubate overnight; the next day, the medium in the 96-well plate was discarded and replaced with phenol red-free RPMI1640+10% FBS containing dye caspase3/7 reagent medium so that the dye concentration is 2 drops/ml.
  • the cells transfected with the TCR of the present invention can play a significant killing effect in a short period of time.

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Abstract

本发明提供一种T细胞受体，其能够与HPV16 E6抗原短肽复合物TIHDIILECV-HLA A0201特异性结合。同时，转导本发明TCR的效应细胞还具有很强的杀伤功能。此类TCR可以单独使用，也可与其他治疗剂联用，还可以用于过继性细胞免疫治疗，以靶向呈递TIHDIILECV-HLA A0201复合物的肿瘤细胞。

Description

一种识别HPV抗原的TCR 技术领域
本发明涉及生物技术领域，更具体地涉及能够识别衍生自HPV16 E6蛋白多肽的T细胞受体(T cell receptor,TCR)及其制备和用途。
背景技术
仅仅有两种类型的分子能够以特异性的方式识别抗原。其中一种是免疫球蛋白或抗体；另一种是T细胞受体(TCR)。TCR是由α链/β链或者γ链/δ链以异二聚体形式存在的细胞膜表面的糖蛋白，在人类中，TCR中95％的T细胞由α链和β链组成，分别由TRA和TRB编码。TCR对pMHC(抗原肽-主要组织相容性复合体)的识别涉及到两种结合：TCR与MHC分子的结合以及TCR与多肽抗原的结合。TCR上的结合面来自6个区域:TCRα、β链上各自的CDRl、CDR2以及CDR3。CDR1与CDR2主要结合相对保守的MHC分子，CDR3则主要结合多变的抗原多肽。这种结合模式可以好地保证TCR特异地结合MHC分子，并能识别多变的抗原。(Chinese Journal of Cell Biology 2011,33(9):955-963)。其中以CDR3的变异最大，直接决定了TCR的抗原结合特异性。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。
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)等疾病。TIHDIILECV是HPV16 E6蛋白的表位肽，是HPV16 E6相关疾病治疗的一种靶标。
本发明致力于开发能够结合TIHDIILECV-HLA A0201复合物且对肿瘤的治疗具有很高的应用价值的TCR。例如，能够靶向该肿瘤细胞标记的TCR可用于将细胞毒性剂或免疫刺激剂递送到靶细胞，或被转化入T细胞，使表达该TCR的T细胞能够破坏肿瘤细胞，以便在被称为过继免疫治疗的治疗过程中给予患者。
发明内容
本发明的目的在于提供一种特异性识别并结合TIHDIILECV-HLA A0201复合物的TCR。本发明的再一目的是提供一种上述类型TCR的制备方法及上述类型TCR的 用途。
本发明的第一方面，提供了一种TCR，所述TCR具有结合TIHDIILECV-HLA A0201复合物的活性，并且所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性的氨基酸序列；和所述TCR的β链可变域为与SEQ ID NO:5所示的氨基酸序列有至少90％的序列同源性的氨基酸序列。
在另一优选例中，所述TCR的α链可变域包含与SEQ ID NO:1所示的序列有至少91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列。
在另一优选例中，所述TCR的β链可变域为与SEQ ID NO:5所示的序列有至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同源性的氨基酸序列。
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为αβ异质二聚TCR，优选地，所述TCR具有α链恒定区序列TRAC*01和β链恒定区序列TRBC1*01或TRBC2*01。
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是可溶的。
在另一优选例中，所述TCRα链可变域包含3个CDR区,和所述TCRβ链可变域包含3个CDR区，其中所述TCRβ链可变域的3个CDR区的氨基酸序列为：
βCDR1-MNHNS        (SEQ ID NO:17)
βCDR2-SASEGT        (SEQ ID NO:18)
βCDR3-ASGPWGSSGNTIY        (SEQ ID NO:19)。
在另一优选例中，所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性的氨基酸序列；和所述TCR的β链可变域为与SEQ ID NO:5所示的氨基酸序列有至少95％的序列同源性的氨基酸序列。
在另一优选例中，所述TCR的β链可变域的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性的氨基酸序列；和所述TCRβ链可变域的3个CDR区的氨基酸序列为：
βCDR1-MNHNS        (SEQ ID NO:17)
βCDR2-SASEGT        (SEQ ID NO:18)
βCDR3-ASGPWGSSGNTIY        (SEQ ID NO:19)。
在另一优选例中，所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性的氨基酸序列；和所述TCR的β链可变域的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述TCRα链可变域包含3个CDR区,和所述TCRβ链可变域 包含3个CDR区，其中所述TCRα链可变域中CDR3α的氨基酸序列选自：
ALRAGANNLF(SEQ ID NO:22)、ALRAGANLPV(SEQ ID NO:23)、ALRAGANTPI(SEQ ID NO:24)、ALRAGAWPMK(SEQ ID NO:25)。
在另一优选例中，所述TCRα链可变域包含3个CDR区,和所述TCRβ链可变域包含3个CDR区,其中所述TCRα链可变域中CDR1α的氨基酸序列为：TRDTTYY；和CDR2α的氨基酸序列为：RNSFDEQN；和CDR3α的氨基酸序列选自：
ALRAGANNLF(SEQ ID NO:22)、ALRAGANLPV(SEQ ID NO:23)、ALRAGANTPI(SEQ ID NO:24)、ALRAGAWPMK(SEQ ID NO:25)。
在另一优选例中，所述TCRα链可变域的3个CDR区的序列如下，
CDR1α：TRDTTYY        (SEQ ID NO:20)
CDR2α：RNSFDEQN        (SEQ ID NO:21)
CDR3α：ALRAGANNLF(SEQ ID NO:22)，并且CDR3α含有至少一个下列突变：
突变前的残基 突变后的残基
CDR3α的第7位N W
CDR3α的第8位N L或T或P
CDR3α的第9位L P或M
CDR3α的第10位F V或I或K。
在另一优选例中，所述TCRα链可变域的3个CDR区的序列如下，
CDR1α：TRDTTYY        (SEQ ID NO:20)
CDR2α：RNSFDEQN        (SEQ ID NO:21)
CDR3α：ALRAGANNLF(SEQ ID NO:22)，并且CDR3α含有至少一个下列突变：
突变前的残基 突变后的残基
CDR3α的第7位N W
CDR3α的第8位N L或T或P
CDR3α的第9位L P或M
CDR3α的第10位F V或I或K；
并且，所述TCRβ链可变域的3个CDR区的氨基酸序列为：
βCDR1-MNHNS        (SEQ ID NO:17)
βCDR2-SASEGT        (SEQ ID NO:18)
βCDR3-ASGPWGSSGNTIY        (SEQ ID NO:19)。
在另一优选例中，所述TCR具有选自下组的CDR:
Figure PCTCN2021132461-appb-000001
Figure PCTCN2021132461-appb-000002
在另一优选例中，所述TCR的α链可变域的氨基酸序列如SEQ ID NO:1-4任一所示；和/或所述TCR的β链可变域的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述TCR选自下组：
Figure PCTCN2021132461-appb-000003
在另一优选例中，所述TCR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。
在另一优选例中，所述TCR的α链恒定区与β链恒定区之间含有人工链间二硫键。
在另一优选例中，所述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：
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为单链TCR。
在另一优选例中，所述单链TCR是α链可变域和β链可变域由一柔性短肽序列(linker)连接而成。
在另一优选例中，所述单链TCR的氨基酸序列如SEQ ID NO:6-9所示。
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，所述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。
在另一优选例中，与所述TCR结合的治疗剂为连接于所述TCR的α或β链的C-或N-末端的抗-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或本发明第二方面所述TCR复合物的核酸序列或其互补序列。
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，提供了一种分离的细胞，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，或所述细胞表达本发明第一方面所述的TCR；优选地，所述细胞为T细胞、NK细胞或NKT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、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HPV阳性肿瘤，如HPV16 E6阳性肿瘤，更优选地，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HPV阳性肿瘤，如HPV16 E6阳性肿瘤，更优选地，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HPV阳性肿瘤，更优选地所述肿瘤为宫颈癌。
本发明的第十一方面，提供了制备本发明第一方面所述的T细胞受体的方法，包括步骤：
(i)培养本发明第五方面所述的宿主细胞，从而表达本发明第一方面所述的T细胞受体；
(ii)分离或纯化出所述的T细胞受体。
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-1d分别显示了能够特异性结合TIHDIILECV-HLA A0201复合物的本发明 TCRα链可变域氨基酸序列。
图2显示了能够特异性结合TIHDIILECV-HLA A0201复合物的本发明TCRβ链可变域氨基酸序列。
图3a-3d分别显示了本发明单链TCR的氨基酸序列。
图4a-4d分别显示了本发明可溶性TCRα链的氨基酸序列，其中突变后的半胱氨酸残基以加粗字母表示。
图5分别为本发明可溶性TCRβ链的氨基酸序列，其中突变后的半胱氨酸残基以加粗字母表示。
图6a和6b分别显示了编码本发明TCRα链可变域(氨基酸序列如SEQ ID NO:1所示)的核苷酸序列和编码本发明TCRβ链可变域(氨基酸序列如SEQ ID NO:5所示)的核苷酸序列。
图7a-7d分别显示了本发明的可溶性的TCR1、TCR2、TCR3、TCR4与TIHDIILECV-HLA A0201复合物的结合曲线。
图8显示了针对负载短肽的T2细胞，转染本发明TCR的效应细胞的激活功能实验结果。
图9a-图9b显示了针对肿瘤细胞系，转染本发明TCR的效应细胞的激活功能实验结果。
图10显示了针对梯度负载短肽的T2细胞，转染本发明TCR的效应细胞的杀伤功能LDH实验结果。
图11显示了针对肿瘤细胞系，转染本发明TCR的效应细胞的杀伤功能LDH实验结果。
图12显示了针对肿瘤细胞系，转染本发明TCR的效应细胞的杀伤功能IncuCyte实验结果。
图13为四聚体和抗CD8-APC双阳染色结果图。
具体实施方式
本发明通过广泛而深入的研究，获得一种特异性识别并结合TIHDIILECV-HLA A0201复合物的高亲和性T细胞受体(TCR)，其α链可变域的3个CDR区的氨基酸序列如下，
CDR1α：TRDTTYY        (SEQ ID NO:20)
CDR2α：RNSFDEQN        (SEQ ID NO:21)
CDR3α：ALRAGANNLF(SEQ ID NO:22)，并且CDR3α含有至少一个下列突变：
突变前的残基 突变后的残基
CDR3α的第7位N W
CDR3α的第8位N L或T或P
CDR3α的第9位L P或M
CDR3α的第10位F V或I或K。
其β链可变域的3个CDR区的氨基酸序列如下，
βCDR1-MNHNS        (SEQ ID NO:17)
βCDR2-SASEGT        (SEQ ID NO:18)
βCDR3-ASGPWGSSGNTIY        (SEQ ID NO:19)。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且其意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
术语
T细胞受体(T cell receptor,TCR)
可以采用国际免疫遗传学信息系统(IMGT)来描述TCR。天然αβ异源二聚TCR具有α链和β链。广义上讲，各链包含可变区(V区)、连接区(J区)和恒定区(C区)，β链通常还在可变区和连接区之间含有短的多变区(D区)，但该多变区常视作连接区的一部分。通过独特的IMGT的TRAJ和TRBJ确定TCR的连接区，通过IMGT的TRAC和TRBC确定TCR的恒定区。
在IMGT命名法中，TRAV和TRBV的不同编号分别指代不同Vα类型和Vβ的类型。在IMGT系统中，α链恒定结构域具有以下的符号：TRAC*01，其中“TR”表示T细胞受体基因；“A”表示α链基因；C表示恒定区；“*01”表示等位基因1。β链恒定结构域具有以下的符号：TRBC1*01或TRBC2*01，其中“TR”表示T细胞受体基因；“B”表示β链基因；C表示恒定区；“*01”表示等位基因1。α链的恒定区是唯一确定的，在β链的形式中，存在两个可能的恒定区基因“C1”和“C2”。本领域技术人员通过公开的IMGT数据库可以获得TCRα与β链的恒定区基因序列。
TCR的α和β链一般看作各有两个“结构域”即可变域和恒定结构域。可变域由连接的可变区和连接区构成。因此，在本申请的说明书和权利要求书中，“TCRα链可变域”指连接的TRAV和TRAJ区，同样地，“TCRβ链可变域”指连接的TRBV和TRBD/TRBJ区。
各可变区包含嵌合在框架序列中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。TCRα链可变域的3个CDR分别为CDR1α、CDR2α和CDR3α；TCRβ链可变域的3个CDR分别为CDR1β、CDR2β和CDR3β。本发明TCR可变域的框架序列可以为鼠源的或人源的，优选为人源的。TCR的恒定结构域包含胞内部分、跨膜区和胞外部分。
本发明中所用的TCR序列为人源的。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，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。
肿瘤
术语“肿瘤”指包括所有类型的癌细胞生长或致癌过程，转移性组织或恶性转化细胞、组织或器官，不管病理类型或侵染的阶段。肿瘤的实施例非限制性地包括：实体瘤，软组织瘤，和转移性病灶。实体瘤的实施例包括：不同器官系统的恶性肿瘤，例如肉瘤，肺鳞癌和癌症。例如：感染的前列腺，肺，乳房，淋巴，肠胃(例如：结肠)，和生殖泌尿道(例如：肾脏，上皮细胞)，咽头。转导本发明的TCR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。
发明详述
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TCR，所述TCR具有结合TIHDIILECV-HLA A0201复合物的活性，并且所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性的氨基酸序列；和/或所述TCR的β链可变域为与SEQ ID NO:5所示的氨基酸序列有至少90％的序列同源性的氨基酸序列。
本发明TCRα链可变域为与SEQ ID NO:1具有至少90％，优选地95％，(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％)的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％，(如，可以是至少91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列相同性的氨基酸序列。在另一优选例中，本发明所述TCR是人源的。
在本发明的一个优选例中，本发明的TCR包含α链可变域氨基酸序列为SEQ ID NO:1-4之一；和所述TCR的β链可变域氨基酸序列为SEQ ID NO:5。
TCR对pMHC(抗原肽-主要组织相容性复合体)的识别涉及到两种结合：TCR与MHC分子的结合以及TCR与多肽抗原的结合。TCR上的结合面来自6个区域:TCRα、β链上各自的CDRl、CDR2以及CDR3。CDR1与CDR2主要结合相对保守的MHC分子，CDR3则主要结合多变的抗原多肽。这种结合模式可以好地保证TCR特异地结合MHC分子，并能识别多变的抗原。(Chinese Journal of Cell Biology 2011,33(9):955-963)。其中以CDR3的变异最大，直接决定了TCR的抗原结合特异性。本发明TCR经测序确定的CDR区如下：
Figure PCTCN2021132461-appb-000004
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。在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。因此，本发明TCR还包括本发明TCR的至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸(尤其是位于CDR区之外的氨基酸)，被性质相似或相近的氨基酸所替换，并仍能够保持其功能性的TCR。
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。
在另一优选例中，所述TCR为αβ异质二聚TCR，优选地，所述TCR具有α链恒定区序列TRAC*01和β链恒定区序列TRBC1*01或TRBC2*01。
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。
基于本发明的目的，本发明TCR是具有至少一个TCRα和/或TCRβ链可变域的部分。它们通常同时包含TCRα链可变域和TCRβ链可变域。它们可以是αβ异源二聚体或是单链形式或是其他任何能够稳定存在的形式。在过继性免疫治疗中，可将αβ异源二聚TCR的全长链(包含胞质和跨膜结构域)进行转染。本发明TCR可用作将治疗剂递送至抗原呈递细胞的靶向剂或与其他分子结合制备双功能多肽来定向效应细胞，此时TCR优选为可溶形式。
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。
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。本发明也获得了对HPV16E6抗原短肽具有特异性的可溶性TCR。如在本发明实施例2中构建的可溶性TCR，其α链可变域氨基酸序列为SEQ ID NO:10-13之一，和β链可变域氨基酸序列为SEQ ID NO:14。
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。在本发明的一个优选例中，本发明的单链TCR氨基酸选自：SEQ ID NO:6-9。
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。
突变可采用任何合适的方法进行，包括但不限于依据聚合酶链式反应(PCR)的那些、依据限制性酶的克隆或不依赖连接的克隆(LIC)方法。许多标准分子生物学教材详述了这些方法。聚合酶链式反应(PCR)诱变和依据限制性酶的克隆的更多细节可参见Sambrook和Russell,(2001)分子克隆-实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)CSHL出版社。LIC方法的更多信息可见(Rashtchian,(1995)Curr Opin Biotechnol 6(1):30-6)。
产生突变的方法可以是但不限于从展示此类TCR的噬菌体颗粒的多样性文库中筛选出对TIHDIILECV-HLA A0201复合物特异性结合的TCR，如文献(Li,et al(2005)Nature Biotech 23(3):349-354)中所述。
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。
与本发明TCR结合的抗体或其片段包括抗-T细胞或NK-细胞决定抗体，如抗-CD3或抗-CD28或抗-CD16抗体，上述抗体或其片段与TCR的结合能够对效应细胞进行定向来更好地靶向靶细胞。一个优选的实施方式是本发明TCR与抗-CD3抗体或所述抗-CD3抗体的功能片段或变体结合。具体地，本发明的TCR与抗CD3单链抗体的融合分子包括选自：(1)TCRα链可变域氨基酸序列为SEQ ID NO:1-4之一，和所述TCR的β链可变域氨基酸序列为SEQ ID NO:5；或(2)单链TCR氨基酸序列为SEQ ID NO:6-9之一。
本发明还涉及编码本发明TCR的核酸分子。本发明的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，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。举例说明“简并的变异体”的含义，如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO:1 的蛋白序列，但与SEQ ID NO:15的序列有差别的核酸序列。
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。
本发明也涉及包含本发明的核酸分子的载体，以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。
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，可以为T细胞、NK细胞、NKT细胞等，优选是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)。
本发明还提供一种药物组合物，所述药物组合物含有药学上可接受的载体以及本发明TCR、或本发明TCR复合物、或呈递本发明TCR的细胞。
本发明还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TCR、或本发明TCR复合物、或呈递本发明TCR的细胞、或本发明的药物组合物。
本发明的TCR、TCR复合物或本发明TCR转染的T细胞可与药学上可接受的载体一起在药物组合物中提供。本发明的TCR、多价TCR复合物或细胞通常作为无菌 药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。该药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。
此外，本发明的TCR可以单用，也可与其他治疗剂结合或偶联在一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在雷明顿药物科学(Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991))中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼内、肌内、静脉内、皮下、皮内、或局部给药，优选为胃肠外包括皮下、肌肉内或静脉内。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有针剂、口服剂等。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明TCR可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
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。当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明TCR或TCR复合物或呈递本发明 TCR的细胞，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定，最终由医师决定合理的用量。
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)。
本发明中，Pro60或者60P均表示第60位脯氨酸。另外，本发明中所述突变的具体形式的表述方式如“N93D”代表第93位的N被D取代，同理，“N93D/E”代表第93位的N被D取代或被E取代。其他以此类推。
在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的结构和功能。因此，本发明TCR还包括本发明TCR的至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸(尤其是位于CDR区之外的氨基酸)，被性质相似或相近的氨基酸所替换，并仍能够保持其功能性的TCR。
本发明还包括对本发明TCR略作修饰后的TCR。修饰(通常不改变一级结构)形式包括：本发明TCR的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在本发明TCR的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的TCR。这种修饰可以通过将TCR暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的TCR。
本发明的主要优点在于：
(1)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。
(2)针对阳性靶细胞，转导本发明TCR的效应细胞能够被特异性激活，同时，转导本发明TCR的效应细胞还具有很强的杀伤功能。
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。另外，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分 实施例，而不是全部。
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。其中，E.coli DH5α购自Tiangen、E.coli BL21(DE3)购自Tiangen、E.coli Tuner(DE3)购自Novagen、质粒pET28a购自Novagen。
实施例1 获取HPV16 E6抗原特异性TCR
利用合成短肽TIHDIILECV(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,制备pMHC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。双阳性染色结果如图13所示。经过多轮成功的扩增筛选，本发明意外获得了能够与源自于HPV16 E6的抗原短肽复合物TIHDIILECV-HLA A0201特异性结合的T细胞受体，其结合表征图谱如图7a所示。同时，针对HPV E6阳性靶细胞系，转导了本发明TCR的效应细胞能够被激活并具有很强的杀伤功能。经测序，TIHDIILECV特异性、HLA-A0201限制性的TCRα链可变域核苷酸序列(SEQ ID NO:15)和TCRβ链可变域核苷酸序列(SEQ ID NO:16)分别如图6a和6b所示。TCRα可变域的氨基酸序列为SEQ ID NO:1，β链可变域的氨基酸序列为SEQ ID NO:5，为方便描述，将其命名为TCR1。
本发明进一步采用本领域技术人员熟知的定点突变的方法，根据专利文献WO2014/206304中所述，构建了以一个柔性短肽(linker)连接上述TCRα与β链可变域而构成的稳定性单链TCR分子，并进行表达、复性及纯化。将Li等((2005)Nature Biotech 23(3):349-354)描述的TCR噬菌体展示和筛选方法应用于单链TCR模板。经过几轮淘选后，获得同样与源自于HPV16 E6的抗原短肽复合物TIHDIILECV-HLA A0201能够特异性结合的单链TCR，将其CDR区突变引入到αβ异质二聚TCR的可变域的相应位点中，获得TCR2,TCR3和TCR4。利用BIAcore的结合表征可知，上述TCR2，TCR3和TCR4同样能够与复合物TIHDIILECV-HLA A0201特异性结合，其结合表征图谱分别如图7b,7c和7d所示。另外，本发明还意外地发现，针对HPV E6阳性靶细胞系，转导了TCR2或TCR3或TCR4的效应细胞同样能够被特异性激活并具有很强的杀伤功能。TCR2或TCR3或TCR4的α链与TCR1的α链有至少95％的序列同源性，同样地，TCR2或TCR3或TCR4的β链与TCR1的β链有至少95％的序列同源性。
具体地，相较于TCR1，TCR2或TCR3或TCR4在α链的CDR3中有3个或4个突变，具体的CDR如下表1所示：
表1
Figure PCTCN2021132461-appb-000005
更具体地，本发明TCR的α和β链的可变域序列如下：
表2
Figure PCTCN2021132461-appb-000006
所述TCR的α链可变域氨基酸序列为：SEQ ID NO:1-4之一；和/或所述TCR的β链可变域氨基酸序列选自：SEQ ID NO:5。
实施例2 可溶TCR的表达、重折叠和纯化
本实施例在本发明TCR的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，从而获得稳定的可溶的TCR分子，以便评估TCR与复合物TIHDIILECV-HLA A0201之间的相互作用。其α链的氨基酸序列为SEQ ID NO:10-13之一，如图4a-4d所示；和其β链的氨基酸序列为SEQ ID NO:14，如图5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OD600＝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,pH8.0)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-100HR，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。
实施例3 结合表征
本实施例使用BIAcore T200实时分析系统检测根据实施例2获得的可溶TCR分子与TIHDIILECV-HLA A0201复合物的结合活性。利用本领域技术人员所熟知的方法制备TIHDIILECV-HLA A0201复合物，主要过程包括纯化、复性、再纯化和生物素化。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，pH4.77)，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。
利用BIAcore Evaluation软件计算动力学参数，得到本发明可溶性的TCR分子与TIHDIILECV-HLA A0201复合物结合的动力学图谱分别如图7a-7d所示。图谱显示，本发明得到的可溶性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4 针对负载短肽的靶细胞，转染本发明TCR的效应细胞的激活功能实验
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。将本发明TCR转染至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者转染其他TCR(A6，针对其他抗原的TCR)的CD3+T细胞作为对照。所述TCR的编号及其α、β可变域序列如表2所示。所用的靶细胞为负载HPV16 E6抗原短肽TIHDIILECV的、负载其他肽的或空载的T2细胞。
实验步骤：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1×10 4个/孔，效应细胞为2×10 3个/孔(按转染的阳性率计算)，短肽终浓度为1×10 -6M/孔，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8所示，针对负载了HPV16 E6抗原短肽TIHDIILECV的靶细胞，转染本发明TCR的效应细胞有明显的激活效应，而转染其他TCR的效应细胞无活性；同时，转染本发明TCR的效应细胞对于负载其他肽或空载的靶细胞基本无反应。
实施例5 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
为再次验证转染本发明TCR的效应细胞的激活功能及特异性，本实施例利用肿瘤细胞系进行两轮ELISPOT实验。将本发明TCR转染至从健康志愿者的血液中分离到的CD3+T细胞作为效应细胞，并以同一志愿者转染其他TCR(A6)的CD3+T细胞作为阴性对照。所述TCR的编号及其α、β可变域序列如表2所示。第一轮实验所用的HPV16 E6阳性肿瘤细胞系为A375-E6(HPV16 E6过表达)、HK-2，阴性肿瘤细胞系为A375、SiHa、HCCC9810；第二轮实验所用的HPV16 E6阳性肿瘤细胞系为A375-E6(HPV16 E6过表达)，阴性肿瘤细胞系为A375、KATOⅢ、MDA-MB-231、MEL526、LCL。
实验步骤如实施例4所示，其中加入ELISPOT平板的各个组分为：靶细胞为2×10 4个/孔，效应细胞为2×10 3个/孔(按转染的阳性率计算)。
两轮实验结果如图9a和9b所示，针对HPV16 E6阳性肿瘤细胞系，转染本发明TCR的效应细胞起明显的激活效应，而转染其他TCR的效应细胞无活性；同时，转染本发明TCR的效应细胞对HPV16 E6阴性肿瘤细胞系基本无活性。
实施例6 针对梯度负载短肽的T2细胞，转染本发明TCR的效应细胞的杀伤功能LDH实验
乳酸脱氢酶(LDH)在胞浆内含量丰富，正常时不能通过细胞膜，当细胞受损伤或死亡时可释放到细胞外，此时细胞培养液中LDH活性与细胞死亡数目成正比。本实施例通过本领域技术人员熟知的非放射性细胞毒性实验，测定LDH的释放，从而验证转染本发明TCR的细胞的杀伤功能。该试验是51Cr释放细胞毒性试验的比色替代试验，定量测定细胞裂解后释放的LDH。采用30分钟偶联的酶反应来检测释放在培养基中的LDH，在酶反应中LDH可使一种四唑盐(INT)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。可以用标准的96孔读板计收集490nm可见光吸光值数据。计算公式:％细胞毒性＝100％×(实验-效应细胞自发-靶细胞自发)/(靶细胞最大-靶细胞自发)。
本实施例LDH实验用从健康志愿者的血液中分离到的CD3+T细胞转染本发明TCR作为效应细胞，并以同一志愿者转染其他TCR(A6)或空转染(NC)的CD3+T细胞作为对照组。所述TCR的编号及其α、β可变域序列如表2所示。所用靶细胞为负载TIHDIILECV肽的T2细胞，同时以负载其他抗原和空载的T2细胞作为对照。
实验步骤：首先准备LDH平板，先按靶细胞3*10 4个细胞/孔、效应细胞3*10 4 个细胞/孔(按转染的阳性率计算)加入对应孔中，然后在实验组加入HPV16 E6抗原短肽TIHDIILECV，且使其短肽在LDH孔板中的终浓度依次为1×10 -14M到1×10 -6M，共9个梯度；在对照组加入其他短肽，且使其短肽终浓度为依次为1×10 -8M到1×10 -6M，共3个梯度，并设置三个复孔。同时设置效应细胞自发孔，靶细胞自发孔，靶细胞最大孔，体积校正对照孔及培养基背景对照孔。温育过夜(37℃，5％CO 2)。实验第2天，检测显色，终止反应后用酶标仪(Bioteck)在490nm记录吸光值。
实验结果如图10所示，针对梯度负载HPV16 E6抗原短肽TIHDIILECV的靶细胞，转染本发明TCR的效应细胞有很强的杀伤功能，且在上述短肽浓度较低时即起反应，而转染其他TCR或空转染的效应细胞自始无杀伤效应；同时，转染本发明TCR的效应细胞对负载其他短肽的靶细胞无杀伤作用。
实施例7 针对肿瘤细胞系，转染本发明TCR的效应细胞的杀伤功能LDH实验
本实施例利用肿瘤细胞系再次检测转染本发明TCR的细胞的杀伤功能及特异性。同样通过本领域技术人员熟知的非放射性细胞毒性实验，测定LDH的释放。用从健康志愿者的血液中分离到的CD3+T细胞转染本发明TCR作为效应细胞，并以同一志愿者转染其他TCR(A6)的CD3+T细胞作为阴性对照。所述TCR的编号及其α、β可变域序列如表2所示。所用的HPV16 E6阳性肿瘤细胞系为A375-E6(HPV16 E6过表达)、HK-2，阴性肿瘤细胞系为A375、HCCC9810
实验步骤如实施例6所示，其中加入LDH平板的各个组分为：靶细胞3×10 4个细胞/孔、效应细胞3×10 4个细胞/孔(按转染阳性率计算)，并设置三个复孔。
实验结果如图11所示，针对HPV16 E6阳性肿瘤细胞系，转染本发明TCR的效应细胞同样表现出强杀伤效力，而转染其他TCR的T细胞基本不起反应；同时，转染本发明TCR的T细胞对阴性肿瘤细胞系基本无杀伤，进一步体现了转染本发明TCR的细胞的很好的特异性杀伤功能。
实施例8 针对肿瘤细胞系，应用IncuCyte验证转染本发明TCR分子的效应细胞的免疫效力
本实施例通过本领域技术人员熟知利用IncuCyte实验进一步验证了转染本发明TCR的效应细胞对靶细胞有很好的免疫效力。IncuCyte是在培养箱中通过实时显微拍摄，能对不同时间点图像进行自动分析，量化实时的细胞凋亡数的功能分析系统。
将本发明TCR转染至从健康志愿者的血液中分离到的CD3+T细胞，作为效应细胞，并以同一志愿者空转染(NC)的CD3+T细胞作为对照组。所述TCR的编号及其α、β可变域序列如表2所示。本实施例用阳性靶细胞CASKI进行实验。
实验第一天，将靶细胞进行消化处理，离心；用无酚红的RPMI1640+10％FBS的完全培养基重悬，将靶细胞均匀的平铺在96孔板中：1.5×10 4个/孔；放回37度，5％CO 2的培养箱中，孵育过夜；第二天将96孔板中培养基弃掉，换成含有染料 caspase3/7 reagent的无酚红的RPMI1640+10％FBS培养基，使染料浓度为2滴/ml。弃去旧的培养基，更换新的无酚红的RPMI1640+10％FBS的培养基，将效应细胞1.5×10 4个/孔(按转染的阳性率计算)和已铺有靶细胞的实验组进行共孵育；将板子放至Incucyte检测专用的实时动态活细胞成像分析仪-IncuCyte ZooM中，孵育半小时后；开始实时观察并拍照；采用IncuCyte ZooM 2016A对检测结果进行处理和数据分析、导出。
实验结果如图12所示，针对阳性肿瘤细胞系，转染本发明TCR的细胞能够在短期内起到明显的杀伤作用。
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 (23)

  1. 一种T细胞受体(TCR)，其特征在于，其具有结合TIHDIILECV-HLA A0201复合物的活性，并且所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少90％的序列同源性的氨基酸序列；和所述TCR的β链可变域为与SEQ ID NO:5所示的氨基酸序列有至少90％的序列同源性的氨基酸序列。
  2. 如权利要求1所述的TCR，其特征在于，所述TCR为αβ异质二聚TCR，优选地，所述TCR具有α链恒定区序列TRAC*01和β链恒定区序列TRBC1*01或TRBC2*01。
  3. 如权利要求1所述的TCR，其特征在于，所述TCR是可溶的。
  4. 如权利要求1所述的TCR，其中，所述TCRα链可变域包含3个CDR区,和所述TCRβ链可变域包含3个CDR区,其特征在于，所述TCRβ链可变域的3个CDR区的氨基酸序列为：
    βCDR1-MNHNS
    βCDR2-SASEGT
    βCDR3-ASGPWGSSGNTIY。
  5. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性的氨基酸序列；和所述TCR的β链可变域为与SEQ ID NO:5所示的氨基酸序列有至少95％的序列同源性的氨基酸序列。
  6. 如权利要求1所述的TCR，其特征在于，所述TCR的β链可变域氨基酸序列为SEQ ID NO:5。
  7. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域为与SEQ ID NO:1所示的氨基酸序列有至少95％的序列同源性的氨基酸序列；和所述TCRβ链可变域的3个CDR区的氨基酸序列为：
    βCDR1-MNHNS
    βCDR2-SASEGT
    βCDR3-ASGPWGSSGNTIY。
  8. 如权利要求1所述的TCR，其特征在于所述TCR具有选自下组的CDR:
    Figure PCTCN2021132461-appb-100001
  9. 如权利要求1所述的TCR，其特征在于，所述TCR的α链可变域氨基酸 序列为：SEQ ID NO:1-4之一；和所述TCR的β链可变域氨基酸序列为：SEQ ID NO:5。
  10. 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。
  11. 如权利要求1所述的TCR，其特征在于，所述TCR包含α链恒定区与β链恒定区，并且α链恒定区与β链恒定区之间含有人工链间二硫键，优选地，所述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：
    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。
  12. 如权利要求1所述的TCR，其特征在于，所述TCR为单链TCR，优选地，所述单链TCR是α链可变域和β链可变域由一柔性短肽序列(linker)连接而成。
  13. 如以上任一权利要求所述的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，所述偶联物为抗-CD3抗体。
  14. 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分子为上述任一权利要求中所述的TCR。
  15. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1中所述的TCR的核酸序列或其互补序列。
  16. 一种载体，其特征在于，所述的载体含有权利要求15中所述的核酸分子。
  17. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求16中所述的载体或染色体中整合有外源的权利要求15中所述的核酸分子。
  18. 一种分离的细胞，其特征在于，所述细胞表达权利要求1中所述的TCR。优选地，所述细胞为T细胞。
  19. 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中所述的TCR、或权利要求14中所述的TCR复合物、或权利要求18中所述的细胞。
  20. 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权利要求1中所述的TCR、或权利要求14中所述的TCR复合物、或权利要求18中所述的细胞、或权利要求19中所述的药物组合物。
  21. 权利要求1中所述的T细胞受体、权利要求14中所述的TCR复合物或 权利要求18中所述细胞的用途，其特征在于，用于制备治疗肿瘤的药物。优选地，所述肿瘤为HPV16 E6阳性肿瘤。
  22. 权利要求1中所述的T细胞受体、权利要求14中所述的TCR复合物或权利要求18中所述的细胞用作治疗肿瘤的药物。优选地，所述肿瘤为HPV16 E6阳性肿瘤。
  23. 一种制备权利要求1中所述的T细胞受体的方法，其特征在于，包括步骤：
    (i)培养权利要求17中所述的宿主细胞，从而表达权利要求1中所述的T细胞受体；
    (ii)分离或纯化出所述的T细胞受体。
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