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WO2019091347A1 - Épitope d'identification de tcr de l'antigène de surface du virus de l'hépatite b (vhb) s183-91 et son utilisation - Google Patents

Épitope d'identification de tcr de l'antigène de surface du virus de l'hépatite b (vhb) s183-91 et son utilisation Download PDF

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WO2019091347A1
WO2019091347A1 PCT/CN2018/113882 CN2018113882W WO2019091347A1 WO 2019091347 A1 WO2019091347 A1 WO 2019091347A1 CN 2018113882 W CN2018113882 W CN 2018113882W WO 2019091347 A1 WO2019091347 A1 WO 2019091347A1
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seq
tcr
cell
cells
hbv
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许嘉峰
夏总平
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Hangzhou Sustained Complete Remission Biotechnology Co Ltd
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Hangzhou Sustained Complete Remission Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a TCR capable of recognizing a short peptide derived from the HBV S183-91 antigen, and to the S183-91-specific T cells obtained by transducing the above TCR, and their use in the prevention and treatment of diseases associated with HBV infection. .
  • Hepatitis B virus infects the liver of the superfamily (including human) of humans and causes inflammation called hepatitis. It is a DNA virus and is one of many independent viruses that cause viral hepatitis. Chronic HBV infection can eventually lead to cirrhosis and hepatocellular carcinoma (HCC), a deadly disease that responds very badly to current chemotherapy.
  • HCC hepatocellular carcinoma
  • HBV infection can only inhibit, but not eliminate, HBV. Although most HBV-infected patients respond to currently available treatments, manifested by improvements in liver histology and serum alanine aminotransferase (ALT) levels, almost all patients relapse when treatment is discontinued.
  • ALT serum alanine aminotransferase
  • more common methods of treating HBV include the use of nucleotide analog drugs such as lamivudine and adefovir. However, these drugs can cause HBV resistance in patients. At this point, the original antiviral drugs will have little effect.
  • HBV epitope-specific T cells found in some subjects enable the subject to be naturally controlled for HBV infection.
  • Past studies have shown that HBV epitope-specific T cell deletion or functional changes in patients with persistent/chronic HBV infection. Therefore, as people's awareness of the interaction between virus and host during HBV infection increases, it is speculated that therapeutic reconstruction of the immune system in patients with chronic HBV infection may abolish or even eliminate HBV symptoms.
  • other diseases usually blood system diseases
  • HBV surface antigen (HBsAg) restores serological normality.
  • HBsAg restores serological normality.
  • vaccination attempts to clear HBV by enhancing the immunity of chronic hepatitis B patients have not yet made people hope.
  • the possible reason for the failure of the therapeutic vaccine strategy is that the immune system of chronically infected HBV is different from the immune system of healthy people (such as antigen presentation efficiency).
  • sustained large-scale production of viral antigens in chronic HBV-infected individuals deletes or tolerates HBV antigen-specific T cells.
  • patients with chronic HBV infection are characterized by a low or absent response to HBV-specific CD4+ and CD8+ T cells.
  • T cell adoptive immunotherapy is the transfer of reactive T cells specific for the target cell antigen into the patient to act on the target cells.
  • the T cell receptor (TCR) is a membrane protein on the surface of T cells that recognizes antigenic short peptides on the surface of the corresponding target cells.
  • APCs antigen presenting cells
  • pMHC complex major histocompatibility antigen
  • the other cell membrane surface molecules interact with each other, causing a series of subsequent cell signaling and other physiological responses, allowing different antigen-specific T cells to exert an immune effect on their target cells.
  • the present invention is directed to solving the above problems, and in particular, the use of exogenous T cell receptor (TCR) to provide an effective and efficient treatment for HBV infection by redirecting the specificity of lymphocytes in patients with chronic HBV infection. method.
  • TCR T cell receptor
  • an HBV epitope which is HLA-A2-restricted and comprises at least one hepatitis B surface antigen; preferably, the HBV epitope is The HBs183-91 epitope consisting of SEQ ID NO:31.
  • TCR T cell receptor
  • An isolated cell comprising the T cell receptor of claim 2.
  • An isolated polynucleotide comprising at least one sequence encoding the alpha chain of the TCR of claim 2, and at least one sequence encoding the beta strand of the TCR of claim 2.
  • sequence encoding the ⁇ chain comprises at least one sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 6, at least one sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: And at least one sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 8;
  • the sequence encoding the ⁇ chain comprises at least one sequence selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 21, at least one sequence selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 22, and selected from the group consisting of At least one of SEQ ID NO: 18 and SEQ ID NO: 23.
  • sequence encoding the ⁇ chain is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 5 or SEQ ID NO: 10, and the sequence encoding the ⁇ chain is selected from the group consisting of SEQ ID NO: 19.
  • polypeptide comprises:
  • the ⁇ chain comprising SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and the ⁇ chain comprising SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • the ⁇ chain is selected from SEQ ID NO: 14 or SEQ ID NO: 15, and the ⁇ chain is selected from SEQ ID NO: 29 or SEQ ID NO: 30.
  • the polypeptide is a soluble TCR or a fragment thereof.
  • the soluble TCR or a fragment thereof has a conjugate bound to the C- or N-terminus; preferably, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a protein kinase modification A combination of some or any of these substances; preferably, the therapeutic agent is an antiviral drug or an anti-CD3 antibody.
  • a vector comprising the polynucleotide described above.
  • a cell comprising the above vector.
  • a cell comprising a T cell receptor comprising the isolated polypeptide described above.
  • the cell is a hematopoietic stem cell or a peripheral blood lymphocyte (PBL) source T cell.
  • PBL peripheral blood lymphocyte
  • a method of preparing at least one HBV epitope reactive exogenous T cell receptor comprising:
  • PBL peripheral blood lymphocytes
  • step (b) a HBV epitope-reactive T cell clone sorted by step (a) encoding a polynucleotide sequence encoding a T cell receptor alpha chain and/or a beta chain;
  • step (c) delivering the polynucleotide of step (b) to the cell;
  • the present invention also relates to the use of the above cells, vectors, polypeptides for the preparation of a medicament for the treatment of HBV infection and/or HBV-associated hepatocellular carcinoma, and a liver for detecting and/or treating HBV infection and/or HBV-associated Kit for cellular liver cancer.
  • the main advantage of the present invention is that the TCR of the present invention can bind to the HBV S183-91 antigen short peptide complex FLLLILLTI-HLA*A0201, and the cells transducing the TCR of the present invention can be specifically activated and have strong target cells. The killing effect.
  • Figure 1 is a flow chart of sorting of lymphocytes labeled with Interferon- ⁇ -PE; the horizontal axis is CD8-FITC and the vertical axis is IFN- ⁇ -PE.
  • R10 represents the cell population to be sorted.
  • Figure 2 is a cell-derived cDNA product.
  • the TCR ⁇ gene fragment containing the CDR3 region was amplified by two rounds of nested PCR, further subcloned into a vector, and 8 clones were selected. Colony PCR identified positive clones for agarose gel electrophoresis. Figure.
  • Figure 3 #1-#12 are T cells transfected with the TCR ⁇ / ⁇ gene pair 1-12.
  • Figure 3-1 is a TcR expression map; since the pHAGE plasmid carries the IRES-ZsGreen original, the transfected cells express ZsGreen.
  • Figure 3-2 is a plot of pentamer binding ability of transfected T cells; the circle strategy is: lymphocyte->ZsGreen+/mTRBC+ double positive cells->cells bound to pentamers.
  • HepG2_S183 is a target HepG2 cell in which S183 polypeptide is loaded without any effector T cells.
  • C18 indicates that the effector T cell is a target cell transfected with the C18TCR ⁇ / ⁇ gene and capable of recognizing HLA*A0201 loaded with the C18 polypeptide.
  • HepG2_C18 is a target HepG2 cell loaded with C18 polypeptide without any effector T cells.
  • Figure 6 is a graph of the ability of T cells to secrete cytokines.
  • the same #1-#12 were T cells transfected with the TCR ⁇ / ⁇ gene pair 1-12.
  • C18 indicates that the effector T cells are antigen-presenting cells transfected with the C18TCR ⁇ / ⁇ gene and capable of recognizing HLA*A0201 loaded with the C18 polypeptide.
  • 0 uM means that no polypeptide is loaded; 1 uM means that the loaded polypeptide is 1 uM (C18-loaded C18 polypeptide, #1-#12-loaded is S183 polypeptide).
  • Figure 6-1 is a IFN- ⁇ secretion map of #1-#4;
  • Figure 6-2 is a IFN- ⁇ secretion map of #5-#8;
  • Figure 6-3 is a IFN- ⁇ secretion map of #9-#12 .
  • the inventors have extensively and intensively studied and found a cell capable of binding to HLA-A2-restricted HBV S183-91 whose sequence comprises the epitope (SEQ ID NO: 31), and cloned with the epitope capable of A TCR that specifically binds.
  • the present invention also provides a nucleic acid molecule encoding the TCR and a vector comprising the same. Further, the present invention provides a cell which transduces the TCR of the present invention.
  • the term “consisting essentially of” means that the exogenous TCR polypeptides and/or polynucleotides of the invention constitute a "substantial” portion of a sequence.
  • the 5' and/or 3' ends of the exogenous TCR polypeptide and/or polynucleotide sequences of the invention may comprise additional sequences. Accordingly, a polypeptide "consisting essentially of sequence X" is novel with respect to a polypeptide which, although comprising sequence X, is not a substantial portion of the X sequence.
  • exogenous T cell receptor refers to the expression of a recombinant TCR in a cell by introduction of a foreign sequence encoding a TCR.
  • the HBV epitope-reactive TCR can be expressed in cells that are not naturally expressed or whose expression levels are too low to be sufficient to produce a response in the expressing or responding cells when the TCR binds to the ligand.
  • fragment refers to an incomplete or isolated portion of the HBV epitope-reactive exogenous TCR full-length sequence comprising an active site that confers the properties and functions of the HBV epitope-reactive exogenous TCR.
  • it can be at least one nucleotide or amino acid shorter than the full length sequence. More specifically, the fragment comprises an active site that enables the HBV epitope-reactive exogenous TCR to recognize the HBs183-91 epitope.
  • HBV epitope-reactive T cell receptor refers to a TCR that binds to a major histocompatibility complex (MHC)-binding HBV epitope to induce help in cells expressing recombinant TCR. Response or cytotoxic response.
  • MHC major histocompatibility complex
  • the HBV epitope can be HBs 183-91. More specifically, the HBV epitope may comprise the sequence of SEQ ID NO:31.
  • HBs183-91 epitope refers to an epitope capable of stimulating HLAI-like restricted T cells. It can be used interchangeably with HBs183, HBs183-91, HBs183-91 peptide and the peptide in the present invention.
  • the sequence of the epitope can be "FLLTRILTI" (SEQ ID NO: 31).
  • HBs 183-91 refers to the HBs183-91 epitope of SEQ ID NO: 31 whose sequence genotype A/D is common in Caucasians unless otherwise stated.
  • the region of the T cell receptor that binds to this epitope is called HBs183-91TCR or HBs183TCR.
  • immunotherapeutic effective amount refers to an amount that brings an immune-mediated prophylactic or therapeutic effect in a subject, ie, prevents or reduces at least 50% of the symptoms compared to before or compared to a suitable control. The amount.
  • isolated is defined herein as a biological component (such as a nucleic acid, peptide or protein) that has substantially been derived from cells of an organism in which the component is naturally produced, and other biological components (ie, other Chromosomes, extrachromosomal DNA, RNA, and proteins are isolated, prepared separately, or purified.
  • nucleic acids, peptides, and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods.
  • the term also encompasses nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.
  • mutants refers to a "mutant” or “mutant” of a TCR epitope whose amino acid sequence is substituted, deleted or added with at least one amino acid and at least one virus
  • the encoded reference sequences are different, but retain the ability to bind and activate TCRs that bind to and activate with non-mutated epitopes.
  • the mutants can be produced in a naturally occurring, recombinant or synthetic manner.
  • soluble TCR refers to a soluble (secreting) form of a membrane-bound TCR, which is a molecule that recognizes a specific antigen by a T cell. Among its soluble forms, it is similar to a monoclonal antibody except that it recognizes a peptide fragment that binds to an MHC molecule and the antibody recognizes a determinant on the entire protein.
  • the isolated polypeptides of the invention can be used to prepare soluble TCRs using any method known in the art. More specifically, the soluble TCR can bind to the HBs183-91 epitope of HBV.
  • subject refers to a vertebrate, especially a mammal, more particularly a human.
  • the subject may specifically be at least one animal model, for example, a mouse, a rat, or the like.
  • the sequences of the TCR alpha and beta chains can be selected based on the species.
  • transgenic animals expressing human MHC molecules can also be used to evaluate particular embodiments of the invention.
  • TCR is a glycoprotein on the surface of a cell membrane in the form of a heterodimer formed by an alpha chain/beta chain or a gamma chain/delta chain.
  • the TCR heterodimer consists of alpha and beta chains in 95% of T cells, while 5% of T cells have a TCR consisting of gamma and delta chains.
  • the native alpha beta heterodimer TCR has an alpha chain and a beta chain, and the alpha chain and the beta chain constitute a subunit of the alpha beta heterodimeric TCR.
  • the alpha and beta chains comprise a variable region, a joining region and a constant region, and the beta strand typically also contains a short polymorphic region between the variable region and the linking region.
  • variable region is often considered as part of the connection zone.
  • Each variable region comprises three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in framework regions.
  • the CDR regions determine the binding of the TCR to the pMHC complex, wherein the CDR3 is recombined from the variable region and the junction region and is referred to as the hypervariable region.
  • the alpha and beta chains of TCR are generally considered to have two "domains", namely a variable domain and a constant domain, and the variable domain consists of linked variable and linking regions.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the TCR molecule ⁇ chain is "TRAC*01”
  • the constant domain sequence of the TCR molecule ⁇ chain is "TRBC1*”. 01” or "TRBC2*01”.
  • the alpha and beta chains of TCR also contain a transmembrane and cytoplasmic regions with a short cytoplasmic region.
  • 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 MHC molecule is a protein of the immunoglobulin superfamily and may be a class I or class II MHC molecule. Different individuals have different MHCs and, therefore, are specific for antigen presentation and can present different short peptides of a protein antigen to the surface of their respective APC cells. Human MHC is commonly referred to as the HLA gene or the HLA complex.
  • the antigen is degraded within the cell and then presented to the cell surface along with the MHC molecule.
  • the MHC restriction of an epitope is determined by at least one specific human leukocyte antigen (HLA) expressed by an antigen presenting cell.
  • HLA human leukocyte antigen
  • Different HLA types ie, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1 limit different epitopes.
  • the HBV-reactive TCR of the invention recognizes an HLA-A2-restricted HBV epitope. About 50% of the total population express HLA-A2 (MHC class I molecule), an HLA-A serotype. Thus, HLA-A2-restricted TCRs can have universal therapeutic utility. In particular, this subtype can recognize the products of many HLA-A*02 alleles, including the gene products of HLA-A*0201, *0202, *0203, *0206, and *0207. There are significant differences between the subtypes of Caucasians and Asians.
  • HLA-A2-positive Caucasians More than 95% of HLA-A2-positive Caucasians are HLA-A0201; while HLA-A2-positive Chinese are composed of the following HLA-A2 subtypes: 23% HLA-A0201; 45% HLA-A0207; 8% HLA- A0206; 23% HLA-A0203.
  • the HBs183-91 epitope showed potential to reduce HBV surface antigen (HBsAg), and the HBsAg serology (HBsAg+ to HBsAg-) transition marked the complete elimination of HBV virus in HBV-infected individuals.
  • an isolated cell comprising an HBV epitope reactive exogenous T cell receptor (TCR) and/or a fragment thereof is used.
  • TCR T cell receptor
  • This isolated cell can be used directly in an adoptive treatment regimen after being expanded and expanded, making it a particularly effective treatment modality.
  • the isolated cells of the present invention can solve the problem of HBV-specific CD8+ and CD4+ cell deletion and dysfunction in patients with chronic HBV infection that may be present.
  • the isolated cells can be isolated using any technique known in the art. More specifically, the cells can be isolated using a cell separation kit, Ficoll-Paque density gradient centrifugation, Beckman Coulter Moflo XDP cell sorting system or the like.
  • HBV-infected individuals who have cleared viral infections can provide HBV-reactive T cells that express high-affinity TCR.
  • the HBV epitope-reactive TCR can be isolated by isolating at least one HBV-reactive T cell from an HBV-infected aviric individual.
  • the HBV-reactive TCR of the present invention recognizes the HBs183-91 epitope consisting of SEQ ID NO: 31 that binds to the HLA-A2 MHC molecule.
  • HBV-reactive TCRs can be obtained by using one or more polynucleotides encoding functional alpha and beta chains, and/or functional alpha and beta chain polypeptides (which can be assembled to form functional HBV epitopes).
  • Reactive TCR is prepared by transforming or transducing isolated cells
  • the HBV-reactive TCRs of the invention may be functional in expressing their isolated cells.
  • they can be a functional alpha and beta TCR chain heterodimer with a CD3 complex that recognizes a HBV epitope that binds to a class I or class II MHC molecule.
  • the TCR may comprise at least one alpha chain comprising at least one alpha chain comprising SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; the beta strand may be predominantly by the sequence SEQ ID Composition consisting of NO:26, SEQ ID NO:27 and SEQ ID NO:28.
  • a second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention, or a portion thereof, which may be one or more CDRs, a variable domain of an alpha and/or beta chain, and an alpha chain and/or Or beta chain.
  • nucleotide sequence encoding the CDR region of the alpha chain of the TCR molecule of the first aspect of the invention is as follows:
  • nucleotide sequence encoding the CDR region of the ⁇ chain of the TCR molecule of the first aspect of the invention is as follows:
  • sequence encoding the alpha chain is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 5 or SEQ ID NO: 10, and the sequence encoding the beta strand is selected from the group consisting of SEQ ID NO: 19.
  • the nucleotide sequence can be codon optimized. Different cells are different in the utilization of specific codons, and the number of expressions can be increased by changing the codons in the sequence depending on the type of the cell. Codon selection tables for mammalian cells as well as a variety of other organisms are well known to those skilled in the art.
  • the full length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, amplification, recombinant or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding the TCR (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The DNA can be a coding strand or a non-coding strand.
  • the TCR molecule is isolated or purified.
  • the alpha and beta chains of the 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 (CDRs) of the TCR ⁇ chain variable region are:
  • the chimeric TCR can be prepared by embedding the above-described CDR region amino acid sequences of the present invention into any suitable framework structure.
  • the framework structure is compatible with the CDR regions of the TCRs of the present invention, one skilled in the art can design or synthesize TCR molecules having corresponding functions in accordance with the CDR regions disclosed herein.
  • a TCR molecule of the invention refers to a TCR molecule comprising the above-described alpha and/or beta chain CDR region sequences and any suitable framework structure.
  • the TCR molecule of the invention is a heterodimer composed of alpha and beta chains.
  • the alpha chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain
  • the alpha chain variable domain amino acid sequence comprises the CDR1 (SEQ ID NO: 11), CDR2 of the above ⁇ chain ( SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13).
  • the beta strand of the heterodimeric TCR molecule comprises a variable domain and a constant domain, the beta strand variable domain amino acid sequence comprising the CDR1 (SEQ ID NO: 26) CDR2 of the beta strand described above (SEQ ID NO: 27) and CDR3 (SEQ ID NO: 28).
  • the alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1 (SEQ ID NO: 11), CDR2 (SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13) of the above alpha chain.
  • the ⁇ chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO: 27) and CDR3 (SEQ ID NO: 28) of the above-described ⁇ chain.
  • the constant domain of the TCR molecule of the invention is a human constant domain.
  • a human constant domain amino acid sequence by consulting a related book or a public database of IMGT (International Immunogenetics Information System).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the ⁇ chain of the TCR molecule of the present invention may be "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule may be "TRBC1*01" or "TRBC2*01”.
  • the constant domain of the TCR molecule of the invention may also be the constant domain of a mouse.
  • TCR The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane domain.
  • TCR can also be developed for diagnosis and treatment, when soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCR has a wide range of uses, not only for studying the interaction of TCR with pMHC, but also as a diagnostic tool for detecting infection or as a marker for autoimmune diseases.
  • soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells that present specific antigens.
  • soluble TCRs can also bind to other molecules (eg, anti-CD3 antibodies). To redirect T cells so that they target cells that present a particular antigen.
  • the TCR of the present invention can also be provided in the form of a multivalent complex.
  • the multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention, such as a complex formed by combining a plurality of TCRs of the present invention with another molecule.
  • the TCR complexes of the invention can be used to track or target cells that present a particular antigen in vitro or in vivo, as well as intermediates that produce other multivalent TCR complexes for such applications.
  • the TCR of the present invention may be used singly or in combination with the conjugate in a covalent or other manner, preferably in a covalent manner.
  • the conjugate comprises a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of a cell presenting the FLLTRILTI-HLA*A0201 complex), a therapeutic agent, a PK (protein kinase) modified moiety, or any of these Combination or coupling of substances.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.
  • the TCR of the invention may also be a hybrid TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCR can be expressed more efficiently in human T cells than human TCR.
  • the TCR of the invention may comprise a human variable domain and a murine constant domain. A drawback of this approach is that it may trigger an immune response.
  • amino acid names in this article are represented by the international single letter or three English letters.
  • the correspondence between the single English letters of the amino acid name and the three English letters is as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Try (W), Tyr (Y), Val (V).
  • the invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, ie, constructs that are capable of expression in vivo or in vitro.
  • 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 a nucleotide of the invention into a cell, such as a T cell, such that the cell expresses a TCR specific for the HBV S183-91 antigen.
  • the vector should be capable of sustained high levels of expression in T cells.
  • the invention also relates to host cells genetically engineered using the vectors or coding sequences of the invention.
  • the host cell contains the vector of the present invention or a nucleic acid molecule of the present invention in which the chromosome is integrated.
  • the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, such as E. coli, yeast cells, CHO cells, and the like.
  • the invention also encompasses isolated cells, particularly T cells, which express the TCR of the invention.
  • the T cell can be derived from a T cell isolated from the subject, or can be a mixed cell population isolated from the subject, such as a portion of a peripheral blood lymphocyte (PBL) population.
  • PBL peripheral blood lymphocyte
  • the cells can be isolated from peripheral blood mononuclear cells (PBMC), which can be CD4+ helper T cells or CD8+ cytotoxic T cells.
  • PBMC peripheral blood mononuclear cells
  • the cells can be in a mixed population of CD4+ helper T cells/CD8+ cytotoxic T cells.
  • the cells can be activated with antibodies (e.g., anti-CD3 or anti-CD28 antibodies) to enable them to be more readily transfected, e.g., with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention. dye.
  • antibodies e.g., anti-CD3 or anti-CD28 antibodies
  • the cells of the invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSCs). Transfer of the gene to HSC does not result in the expression of TCR on the cell surface because the stem cell surface does not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of thymocytes.
  • stem cells differentiate into lymphoid precursors that migrate to the thymus
  • CD3 molecule will initiate expression of the introduced TCR molecule on the surface of thymocytes.
  • T cell transfection with DNA or RNA encoding the TCR of the invention e.g., Robbins et al., (2008) J. Immunol. 180: 6116-6131.
  • T cells expressing the TCR of the present invention can be used in adoptive immunotherapy.
  • Those skilled in the art will be aware of many suitable methods for performing adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4): 298-308).
  • the invention is further illustrated by the following specific examples. It is to be understood that the examples are not intended to limit the scope of the invention.
  • the experimental methods in the following examples, which do not specify the specific conditions are usually in accordance with conventional conditions, for example (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Third Edition) (2001) CSHL Press) The conditions described in the above, or in accordance with the conditions recommended by the manufacturer, unless otherwise stated, the percentages and parts are by weight.
  • the experimental materials and reagents used in the following examples are available from commercially available sources unless otherwise specified.
  • Peripheral blood lymphocytes were isolated from fresh blood obtained from HBV-infected volunteers with HLA-type HLA-A*0201.
  • PBL was isolated by Ficoll-Hypaque (GE) density gradient centrifugation. The PBL was then washed with DPBS (Life), resuspended in RPMI medium (Life) (2% human Serum), and added to a final concentration of 1 uM of HBV S183-91 polypeptide (FLLTRILTI, SEQ ID NO: 31; Jin Sirui ) and 20 IU/ml of interleukin 2 (IL-2; peprotech), cultured in 24-well plates for 10 days.
  • FLLTRILTI HBV S183-91 polypeptide
  • IL-2 interleukin 2
  • CD8+ and IFN- ⁇ double positive were sorted by flow cytometry (Moflo XDP, Beckman Coulter).
  • the single cells sorting results are shown in Figure 1) were plated into 96-well PCR plates, centrifuged and placed at -80 °C for subsequent reverse transcription.
  • 293T cells were co-transfected with psPAX2, and pMD2.G3 plasmid packaging system. After 24 hours of transfection, the first lentiviral supernatants were collected; after 48 hours, a second batch of lentiviral supernatants were collected.
  • the lentivirus supernatant and polybrene (sigma, final concentration 8 ug/mL) were added 1:1, centrifuged at 1000 g for 60 minutes, and then placed. Return to the 37 ° C incubator. After 4 hours, switch back to fresh cRPMI medium.
  • the components of the cRPMI medium were 10% heat-inactivated FBS, 1X GlutaMax, 1Xsodium pyruvate, 1XHEPES (all from Gibco, Life), 50 uM 2-ME (sigma), penicilin/streptomycin (Hangzhou Branch).
  • FIG. 3-1 The results of TcR expression are shown in Figure 3-1: #1-#12 are T cells transfected with the TCR ⁇ / ⁇ gene pair 1-12. Since the pHAGE plasmid used in lentiviral packaging carries the IRES-ZsGreen original, the transfected cells express ZsGreen.
  • Figure 3-2 is a plot of pentamer binding ability of transfected T cells; the circle strategy is: lymphocyte->ZsGreen+/mTRBC+ double positive cells->cells bound to pentamers.
  • the pentameric binding assay showed 12 pairs of T cloned from HLA-A2-restricted HBVS183-91 epitope (SEQ ID NO: 31) reactive T cells isolated from peripheral blood derived from HBV-infected volunteers. In the cell receptor gene pair, two pairs of #9, #10 showed a pentameric binding ability.
  • S183-91 polypeptide was added to HepG2 cells (HLA*A0201+) stably expressing ZsGreen, placed on ice for 1 hour, then washed with PBS, trypsin digestion, 1:1 mixed with HepG2 not transfected with ZsGreen, 4X10 ⁇ 5 cells/well were seeded in 24-well plates.
  • the effector T cells for the positive control were transfected with the TCR gene capable of recognizing the C18-27 epitope of the HBV core antigen, the gene sequence was from the patent CN200980126398; the target cells for the positive control were loaded with the C18 polypeptide (FLPSDFFPSV; patent CN200980126398).
  • the transfected T cells were counted and added to a 24-well plate seeded with HepG2 cells according to different potency:target ratios. After 12 hours, the cells in the 24-well plates were collected, and the ratio of ZsGreen+ cells to ZsGreen-cells in the surviving HepG2 cells was analyzed by flow cytometry (Cytoflex S, Beckman Coulter) (effect: flow histogram with a target ratio of 10 see Figure 4): #1-#12 are 1-12 pairs of TCR ⁇ / ⁇ genes.
  • HepG2_S183 is a target HepG2 cell in which S183 polypeptide is loaded without any effector T cells.
  • C18 indicates that the effector T cell is a target cell transfected with the C18TCR ⁇ / ⁇ gene and capable of recognizing HLA*A0201 loaded with the C18 polypeptide.
  • HepG2_C18 is a target HepG2 cell loaded with C18 polypeptide without any effector T cells.
  • MCF-7 cells HLA*A0201+
  • PBS PBS
  • trypsin digested PBS
  • the positive control was loaded with C18 polypeptide (FLPSDFFPSV).
  • MCF-7 cells without any polypeptide were digested with trypsin and plated in 24-well plates at 4 ⁇ 10 ⁇ 5 cells/well.
  • the transfected T cells were counted and added to a 24-well plate seeded with MCF-7 cells at 1:1 while adding a final concentration of 1X Brefeldin A (Biolegend). After 5 hours, the cells in the 24-well plate were collected, washed with Wash buffer according to Biolegend Intracellular Staining Protocol, and then added with APC-CD8 (Biolegnd) antibody. After standing at room temperature for 20 minutes, add fixation buffer (Biolegend), avoid at room temperature. Place the light for 20 minutes.
  • Figure 6-1 is a IFN- ⁇ secretion map of #1-#4;
  • Figure 6-2 is a IFN- ⁇ secretion map of #5-#8;
  • Figure 6-3 is a IFN- ⁇ secretion map of #9-#12 .
  • Cytokine secretion assay showed 12 pairs of T cloned from HLA-A2-restricted HBV S183-91 epitope (SEQ ID NO: 31) reactive T cells isolated from peripheral blood derived from HBV-infected volunteers. In the cell receptor gene pair, #5, #6, #8, #10, #11, #12 all showed strong cytokine secretion ability similar to the positive control (C18).
  • the TcR sequence of #12 was selected. #12 expression level is higher, in addition to pentameric binding ability, cell killing toxicity and ability to secrete interferon ⁇ are superior to C18 this positive control.
  • the IMGT analysis showed that the ⁇ chain of #12 was the TRAV12-1 gene; the ⁇ chain was TRBV28.

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Abstract

L'invention concerne des procédés d'isolement d'un lymphocyte T spécifique d'un antigène et de clonage d'un gène TCR spécifique d'un antigène à partir du lymphocyte T spécifique d'un antigène. L'invention concerne également une molécule d'acide nucléique codant pour le TCR spécifique de l'antigène de surface du VHB S183-91 et un vecteur comprenant la molécule d'acide nucléique. Le TCR est capable de se lier à un complexe peptide court-antigène du VHB S183-91, FLLLILLTI-HLA*A0201, et les cellules transduites avec le TCR peuvent être spécifiquement activées et avoir un fort effet létal sur les cellules cibles.
PCT/CN2018/113882 2017-11-09 2018-11-05 Épitope d'identification de tcr de l'antigène de surface du virus de l'hépatite b (vhb) s183-91 et son utilisation Ceased WO2019091347A1 (fr)

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CN111592590A (zh) * 2020-05-22 2020-08-28 深圳市因诺转化医学研究院 识别人乙型肝炎病毒核心抗原的t细胞受体
CN111592589A (zh) * 2020-05-22 2020-08-28 深圳市因诺转化医学研究院 识别人乙型肝炎病毒核心抗原c18-27表位的特异性tcr
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CN107827959B (zh) * 2017-11-09 2018-10-30 杭州续缓生物科技有限公司 识别乙肝病毒(hbv)表面抗原s183-91表位的tcr及其用途
CN110156889B (zh) * 2018-02-14 2023-03-10 中国科学院广州生物医药与健康研究院 高亲和力HBs T细胞受体
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CN118620040A (zh) * 2022-01-05 2024-09-10 成都朗谷生物科技股份有限公司 一种针对乙型肝炎病毒的抗原肽
CN117625545A (zh) * 2022-08-29 2024-03-01 新加坡星汉德生物医药有限公司 经修饰的靶向hbv免疫细胞及其医药用途
CN117003856B (zh) * 2023-09-27 2023-12-01 南方医科大学南方医院 一种靶向乙肝表面抗原的t细胞受体工程化t细胞

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