WO2023138658A1 - 新型冠状病毒特异性t细胞受体和其用途 - Google Patents

新型冠状病毒特异性t细胞受体和其用途 Download PDF

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WO2023138658A1
WO2023138658A1 PCT/CN2023/073163 CN2023073163W WO2023138658A1 WO 2023138658 A1 WO2023138658 A1 WO 2023138658A1 CN 2023073163 W CN2023073163 W CN 2023073163W WO 2023138658 A1 WO2023138658 A1 WO 2023138658A1
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chain
cells
amino acid
tcr
seq
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钟晓松
仝帅
白玥
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卡瑞济(北京)生命科技有限公司
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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
    • C07K14/435—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • 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
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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

Definitions

  • the present invention relates generally to the field of immunology. More specifically, the present invention relates to a T cell receptor (hereinafter also abbreviated as TCR) that specifically binds to the nucleocapsid protein of the novel coronavirus, a nucleic acid encoding the TCR, a host cell containing it, and a method for preparing the host cell.
  • TCR T cell receptor
  • the present invention also relates to the use of the TCR and the host cell in preventing and/or treating novel coronavirus infection.
  • 2019-nCoV 2019 novel CoV
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the viral E protein fuses with the host cell plasma membrane and initiates a series of intracellular events, including the interaction between the M and N proteins. Therefore, these four structural proteins are the main targets for the development of anti-SARS-CoV-2 drugs or vaccines.
  • the inactivated virus vaccine is the most classic form of vaccine. It is easy to prepare and can efficiently induce humoral immune response. It is often the preferred vaccine solution for emerging infectious diseases.
  • the inactivated virus vaccine is mainly obtained by inactivating the virus by means selected from formaldehyde, ⁇ -propiolactone and ultraviolet rays, which can induce the human body to produce neutralizing antibodies against the virus.
  • formaldehyde, ⁇ -propiolactone and ultraviolet rays which can induce the human body to produce neutralizing antibodies against the virus.
  • the T cell immune response caused by the inactivated vaccine is generally weak, and some studies have proved that the inactivated vaccine against SARS cannot effectively stimulate the body to produce a cellular immune response.
  • T cell immune responses play an important role in clearing viruses and infected cells.
  • no researchers have conducted drug research on SARS-CoV-2 from the perspective of cellular immunity, and there is no report on the treatment of Coronavirus disease 2019 (COVID-19) by specific T cells.
  • SARS-CoV-2 enters the human body, it mainly attacks the immune system, causing a sharp decrease in T lymphocytes (Bertoletti A, Tan AT., Challenges of CAR-and TCR-T cell-based therapy for chronic infections, J Exp Med, 2020, 217(5): 1-11). Therefore, it is expected that increasing the number of T cells in the human body is the most effective advanced means to treat the new coronavirus.
  • This field needs to develop specific T cells targeting coronavirus-specific antigens, such as TCR-T cells, to effectively prevent and treat SARS-CoV-2 infection.
  • the present invention provides an isolated or purified T cell receptor (TCR), which specifically binds to the new coronavirus nucleocapsid protein
  • TCR comprises an ⁇ chain and a ⁇ chain, wherein each of the ⁇ chain and the ⁇ chain comprises three complementarity determining regions (CDRs), and the amino acid sequence of the CDR3 of the ⁇ chain is selected from SEQ ID NO: 4, 7 and The sequence has a variant with 1 or 2 amino acid residue changes, and the amino acid sequence of CDR3 of the ⁇ chain is selected from SEQ ID NO: 11, 14 and the variant with 1 or 2 amino acid residue changes from the sequence.
  • TCR T cell receptor
  • the TCR of the present invention comprises an alpha chain variable region sequence shown in SEQ ID NO: 18 or 22 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; , 95%, 96%, 97%, 98% or 99% identical sequences.
  • a TCR of the invention comprises an alpha chain sequence set forth in SEQ ID NO: 17 or 21 or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a beta chain sequence set forth in SEQ ID NO: 19 or 23 or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the present invention provides nucleic acids encoding TCR ⁇ chains and/or ⁇ chains of the present invention, and vectors comprising nucleic acids encoding TCR ⁇ chains and/or ⁇ chains of the present invention.
  • the vector is an expression vector, more preferably a retroviral vector.
  • the present invention provides a host cell comprising a TCR alpha chain and/or beta chain of the present invention, a nucleic acid encoding a TCR alpha chain and/or a beta chain of the present invention, or a vector comprising a nucleic acid encoding a TCR alpha chain and/or beta chain of the present invention.
  • the host cells are T cells, preferably human T cells, eg, human CD4+ helper T cells or CD8+ cytotoxic T cells, or a mixed cell population of CD4+ helper T cells and CD8+ cytotoxic T cells.
  • the host cell is a stem cell, eg, a hematopoietic stem cell (HSC).
  • the present invention provides a method for preparing a host cell of the present invention, said method comprising the steps of:
  • the present invention provides a method for identifying a novel coronavirus N protein antigenic peptide, the method comprising the following steps:
  • Figure 1 shows a schematic diagram of the structure of the virus particle of the new coronavirus.
  • Fig. 2 is a schematic diagram illustrating the acquisition of the TCR of the present invention, the production scheme of the host cell of the present invention, and its functional study.
  • Fig. 3 illustrates a schematic diagram of the HLA-A0201 full-length gene for expression in the vector.
  • FIG. 4 shows the amount of IFN- ⁇ secreted by T cells activated by the novel coronavirus N protein antigen.
  • Figure 6 shows the volcano map of differentially expressed genes in T cells after being activated by the novel coronavirus N protein antigen compared with the blank control group.
  • Figure 7 shows the in vitro verification test of the constructed TCR-T cells.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • Binding affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg TCR) and its binding partner (eg antigen).
  • binding affinity refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, TCR and antigen).
  • the affinity of a molecule X for its partner Y can generally be described in terms of a binding-dissociation equilibrium constant ( KD ). Affinity can be measured by common methods known in the art, including those known in the art and described herein.
  • polynucleotide or “nucleic acid” used interchangeably herein refers to a chain of nucleotides of any length and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence for comparison purposes.
  • Subsequent comparisons at corresponding amino acid positions or nucleotide positions amino acid residues or nucleotides When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com), which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or the PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6, the The percent identity between two amino acid sequences.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com) using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4), using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid sequences and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • APC antigen presenting cell
  • helper cells eg, B-cells, dendritic cells, etc.
  • MHC major histocompatibility complex
  • T cells can recognize these complexes using their T cell receptor (TCR).
  • TCR T cell receptor
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny are comparable in nucleic acid content to The parental cells may not be identical, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the cells screened or selected in the originally transformed cells.
  • subject and “individual” refer to animals, preferably mammals, more preferably humans, who need to alleviate, prevent and/or treat novel coronavirus infection. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. The term includes human subjects who have or are at risk of having a novel coronavirus infection.
  • TCR T cell receptor
  • SARS-CoV-2 is a close relative of SARS coronavirus (SARS-CoV).
  • the new coronavirus (2019-nCoV) is an enveloped (envelope derived from the lipid bilayer of the host cell membrane) ( Figure 1), a single-stranded positive-sense RNA virus with a size of 80-120nm, and a genome length of about 29.9kb.
  • the homology between this virus and the genome sequence of SARS-CoV belonging to the genus Betacoronavirus of the Coronaviridae family is 80%.
  • S protein of 2019-nCoV virus and SARS-CoV virus has a similarity of 75%. It is reported that the amino acid residues at positions 442, 472, 479, 487, and 491 of the complex interface between S protein and ACE2 receptor (mainly distributed in respiratory epithelial cells, lungs, heart, kidneys, and digestive tracts in humans) in multiple SARS-CoV coronavirus isolates are highly conserved.
  • the 2019-nCoV S protein Compared with the S protein of SARS-CoV, at the 5 sites, the 2019-nCoV S protein only has the same amino acid at position 491, and the other 4 amino acids are mutated (Xu X et al., Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission, Sci China Life Sci., 2 020 Mar;63(3):457-460). ⁇ , ⁇ 3D ⁇ 2019-nCOV S ⁇ ACE2 ⁇ 4 ⁇ , ⁇ SARS-CoV S ⁇ ,2019-nCoV S ⁇ (receptor binding domain,RBD) ⁇ , ⁇ 2019-nCoV S ⁇ ACE2 ⁇ , ⁇ (Wrapp D ⁇ ,Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation,Science,.2020 ⁇ 2 ⁇ 19 ⁇ , ⁇ ,pii:eabb2507.doi:10.1126/science.abb2507 ⁇ Xiaolong Tian ⁇ ,Potent binding of 2019 novel coronavirus spike protein by
  • the nucleocapsid protein (N protein) is relatively conservative and is often used as a diagnostic tool for new coronaviruses.
  • COVID-19 infection induces IgG antibodies against the N protein that are detectable in serum as early as day 4 after disease onset, with most patients seroconverted by day 14.
  • Laboratory evidence from clinical patients suggests that specific T cell responses to SARS-CoV-2 are important for recognizing and killing infected cells, especially in the lungs of infected individuals (Mohsen Rokni et al., Immune responses and pathogenesis of SARS-CoV-2 during an outbreak in Iran: Comparison with SARS and MERS, Re v Med Virol. 2020; 30:e2107, p1-6, DOI: 10.1002/rmv.2107).
  • the present inventors identified the TCR of the nucleocapsid protein (N protein) in SARS-CoV-2 for the first time and prepared SARS-CoV-2 specific TCR T cells, providing a new strategy for preventing or treating SARS-CoV-2 infection.
  • SARS-CoV-2 virus nucleocapsid protein (N protein) is processed in the cell and carried to the cell surface by major histocompatibility complex (MHC) molecules, presenting as a peptide-MHC complex.
  • MHC major histocompatibility complex
  • T cell receptors are molecules present on the surface of T cells that are responsible for recognizing peptide-MHC complexes. Specific binding of TCRs to peptide-MHC complexes triggers T cell activation through a series of biochemical events mediated by associated enzymes, co-receptors, and accessory molecules. In 95% of T cells, TCR heterodimers consist of ⁇ and ⁇ chains, whereas in 5% of T cells, TCR heterodimers consist of ⁇ and ⁇ chains.
  • Each chain of the TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane region (i.e., transmembrane region), and a short cytoplasmic tail at the C-terminus.
  • Ig immunoglobulin
  • V variable
  • C Ig constant
  • transmembrane region i.e., transmembrane region
  • short cytoplasmic tail at the C-terminus.
  • each variable domain has three hypervariable regions or complementarity determining regions (CDRs), with CDR3 in each variable domain being the major CDR responsible for recognizing processed antigen.
  • CDR2 is thought to recognize MHC molecules.
  • the constant domain of the TCR consists of a short linker sequence in which cysteine residues form disulfide bonds, creating a link between the TCR alpha and beta chains.
  • TCR and CD3 form a TCR/CD3 complex.
  • the formation of the TCR/CD3 complex usually proceeds in the following order; first, the three peptide chains of CD3 ⁇ , ⁇ , and ⁇ pass through the formation of two heterologous binary chains of ⁇ - ⁇ and ⁇ - ⁇
  • the aggregate becomes a stable core of the complex, to which TCR ⁇ (or TCR ⁇ ) binds, and then the ⁇ - ⁇ or ⁇ - ⁇ dimer binds to the TCR ⁇ (or TCR ⁇ )/CD3 ⁇ complex, and finally transfers to the T cell surface.
  • the signal is transmitted from the TCR into the cell through the TCR/CD3 complex.
  • this co-receptor is the CD4 molecule, which is specific for MHC class II, and in cytotoxic T cells, this co-receptor is CD8, which is specific for MHC class I.
  • T cell receptor has its conventional meaning in the art and is used to denote a molecule capable of recognizing a peptide presented by an MHC molecule.
  • the molecule is a heterodimer of two chains ⁇ and ⁇ (or optionally ⁇ and ⁇ ).
  • TCR T cell receptor alpha and/or beta chains.
  • a TCR of the invention may be a hybrid TCR comprising sequences derived from more than one species.
  • a TCR may comprise a human variable region and a murine constant region, given that murine TCRs are more efficiently expressed in human T cells than human TCRs.
  • the TCR of the present invention comprises an ⁇ chain and a ⁇ chain, wherein each of the ⁇ chain and the ⁇ chain comprises three complementarity determining regions (CDRs), and wherein the amino acid sequence of the TCR ⁇ chain CDR3 mainly responsible for antigen recognition is selected from SEQ ID NO:4, 7 and variants with 1 or 2 amino acid residue changes from the sequence, and the amino acid sequence of the ⁇ chain CDR3 is selected from SEQ ID NO: 11, 14 and variants with 1 or 2 amino acid residue changes from the sequence.
  • CDRs complementarity determining regions
  • the TCR of the present invention comprises an ⁇ chain and a ⁇ chain, wherein the amino acid sequence of the CDR3 of the ⁇ chain and the amino acid sequence of the CDR3 of the ⁇ chain are:
  • the TCR of the present invention comprises an ⁇ chain and a ⁇ chain
  • the amino acid sequences of the three complementarity determining regions (CDRs) included in the ⁇ chain and the amino acid sequences of the three CDRs included in the ⁇ chain are:
  • the TCR of the present invention comprises an alpha chain variable region sequence shown in SEQ ID NO: 18 or 22 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; , 95%, 96%, 97%, 98% or 99% identical sequences.
  • the TCR of the present invention further comprises a constant region, more preferably, the TCR of the present invention further comprises a mouse constant region, for example, the TCR comprises an ⁇ chain sequence shown in SEQ ID NO: 17 or 21 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith; and a ⁇ chain sequence shown in SEQ ID NO: 19 or 23 or having at least A sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the change of the amino acid residues in the TCR variant of the present invention is the substitution, addition or deletion of amino acid residues in any of the sequences of SEQ ID NO: 2-24, provided that the TCR variant still retains or improves the ability to bind to the epitope peptide-MHC complex of the new coronavirus nucleocapsid protein.
  • the substitutions are conservative substitutions. Examples of conservative substitutions are given in Table 1 below.
  • Amino acids can be grouped according to common side chain properties:
  • the invention also relates to nucleic acids encoding the TCRs of the invention or portions thereof, eg, one or more CDRs; one or more variable regions; alpha chains; or beta chains, and the like.
  • the nucleic acid can be double-stranded or single-stranded, and can be RNA or DNA.
  • the nucleic acid sequence may be codon optimized for high expression in mammalian producer cells. Codon usage in mammalian cells, as well as in a variety of other organisms, is well known in the art. Codon optimization can also include removal of mRNA instability motifs and cryptic splice sites.
  • the present invention also relates to a vector comprising the nucleic acid encoding the TCR of the present invention.
  • vector includes expression vectors, ie vectors capable of expression in vivo or in vitro/ex vivo.
  • the vector transfers the nucleic acid encoding the TCR of the present invention into cells, such as T cells, so that the cells express the novel coronavirus-specific TCR.
  • the vector consistently expresses a high level of TCR in T cells such that the introduced TCR can successfully compete with the endogenous TCR for the limited pool of CD3 molecules.
  • increasing the supply of CD3 molecules can also increase TCR expression in genetically modified cells.
  • the vector thus optionally comprises genes for CD3-gamma, CD3-delta, CD3-epsilon and/or CD3-zeta.
  • the vector contains only the gene for CD3- ⁇ .
  • one or more separate vectors encoding the CD3 gene may also be provided for co-transfer into cells with the TCR encoding vector.
  • Viral delivery systems used as vectors include, but are not limited to, retroviral vectors, lentiviral vectors, baculoviral vectors, herpesviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors.
  • retroviral vectors lentiviral vectors
  • baculoviral vectors baculoviral vectors
  • herpesviral vectors herpesviral vectors
  • adenoviral vectors adeno-associated viral (AAV) vectors.
  • AAV adeno-associated viral
  • the vector is a retroviral vector.
  • viral particles can be packaged with an amphotropic envelope or a gibbon leukemia virus envelope.
  • T Cell Receptors of the Invention eg. TCR-T
  • TCR-T T cell receptor
  • the high-affinity (K D ⁇ 400pM) TCR prepared in this study has a binding half-life of more than 3 hours, can specifically target HIV-infected cells, and recognizes all common escape variants of this cell.
  • CD8 cells transduced by TCR can produce more IL-2, IFN- ⁇ , TNF- ⁇ and other cytokines than negative control CD8 cells, effectively control the wild-type and mutant strains of HIV, and achieve the effect of T cell therapy (Varela-Rohena A, Molloy PE, Dunn SM, Li Y et al., Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor. Nat Med. 2008(12):1390-5).
  • the inventor constructed a TCR targeting the N protein antigen of the SARS-CoV-2 virus through genetic engineering technology, introduced the TCR into T cells, and prepared a large number of specific T cells targeting the N protein antigen of the SARS-CoV-2 virus, that is, TCR-T cells, providing a new method for the early prevention and treatment of diseases caused by SARS-CoV-2 virus infection and the development of the disease.
  • the cells that express human HLA alleles and have the ability to present the SARS-CoV-2 virus N protein can be, for example, TAP2-deficient lymphoblastoid T2 cells that naturally express human HLA-A0201, K562 cells that can transiently or stably express an MHC I allele (for example, a human HLA allele), and K562 cells can be transduced with a human MHC I allele (HLA-A, -B or -C).
  • the 62 cell line produced the cells.
  • Wild-type K562 cells are of human erythroleukemia origin and lack expression of endogenous MHC I and MHC II alleles (Boegel S, M, Bukur T et al Oncoimmunology. 2014;3(8):37–41). However, wild-type K562 cells express ⁇ -2 microglobulin, a ubiquitous component included in functional MHC complexes. After the transgenic MHC I ⁇ chain was introduced into and expressed in wild-type K562 cells, the cells had a fully functional antigen processing and presentation mechanism (Suhoski MM, Golovina TN, Aqui NA et al. Mol Ther. 2007; 15(5):981–8), therefore, they could function as antigen-presenting cells.
  • mouse fibroblast NIH/3T3 cells are used, which stably express a single human MHC allele, thereby obtaining endogenous presentation of epitopes in the context of human MHC complexes.
  • the MHC may be MHC II.
  • K562 cells were transfected with one MHC II allele.
  • single MHC II-expressing cells based on human RM3 (Raji) B cells can be used. T cells stimulated by MHC II expressing cells will be CD4+ T cells.
  • MHC gene-transfected K562 cells are further modified to express co-stimulatory molecules, e.g., CD40, CD40L, CD70, CD80, CD83, CD86, ICOSL, GITRL, CD137L, and/or CD252, thereby amplifying the T cell response.
  • co-stimulatory molecules e.g., CD40, CD40L, CD70, CD80, CD83, CD86, ICOSL, GITRL, CD137L, and/or CD252, thereby amplifying the T cell response.
  • K562 cells transfected with MHC genes can be further modified to express molecules that enhance antigen processing and presentation, such as HLA-DM and CD74.
  • K562 cells present epitopes of antigens on their MHC molecules after internal processing of antigens.
  • K562 cells stably express intact full-length antigen, eg, after transfection with ivtRNA encoding the antigen. Transient expressions can also be used.
  • step b the antigen-presenting K562 cells are contacted with T cells from a human subject for about 2-7 days to achieve optimal activation of antigen-specific T cells in the T cell sample.
  • the addition of cytokines is avoided during said contacting to prevent non-specific activation of T cells.
  • the T cells from the human subject of step b are in the form of PBMCs, ie the T cells in the PBMCs are not isolated. This may be beneficial because PBMCs provide a more natural environment.
  • the T cells from the human subject of step b are isolated, purified T cells from PBMCs, such as purified CD4+ cells, preferably CD8+ T cells.
  • the stimulation of T cells in step b is performed for 2-7 days, for example, for 3 days, 4 days, 5 days, 6 days or 7 days.
  • T cells carrying a TCR specific for the antigenic peptide presented by the K562 cells were activated.
  • said specifically activated T cells are selected based on activation markers (step c).
  • the specifically activated T cells upregulate activation markers, such as INF- ⁇ , CD107, CD137, CD56.
  • the activation marker can be used, for example, as a marker for detecting and isolating antigen peptide-specific T cells by FACS, ELISOPT technique, CFSE experiment and the like.
  • CD137 is used as a specific marker for isolating antigenic peptide-specific CD8+ T cells. Sorting based on CD137 expression, eg by FACS, is one method to select specific T cells.
  • specific T cells are selected based on the measurement of release of the activation marker IFNy.
  • CD8+ T cells activated by MHC class I molecule-antigen peptide complexes release vesicles with cytokines such as IFN ⁇ .
  • T cells activated by the TCR signaling pathway can be selected for IFNy by measuring IFN ⁇ by a combination of cytokine capture assays and FACS analysis.
  • nucleic acids encoding the TCR alpha chain and TCR beta chain of said T cells are isolated in step d.
  • total RNA from cells is extracted, and cDNA is generated by rapid 5' amplification (RACE) of the cDNA ends of the TCR ⁇ chain and TCR ⁇ chain genes, followed by PCR amplification.
  • RACE rapid 5' amplification
  • the PCR product was cloned into an expression plasmid and transformed into bacteria. Each bacterial colony can be considered to carry a PCR TCR ⁇ or TCR ⁇ gene segment.
  • the vector DNA of the bacterial colony is extracted and the insert (TCR ⁇ or TCR ⁇ gene fragment) in the vector DNA is sequenced.
  • the TCR sequence is determined using next generation sequencing technology.
  • the human variable region of the TCR can be combined with a murine constant region, e.g., a minimal murine constant region, i.e., the human constant region contains only a limited number of amino acids from the murine constant region and contains additional cysteine bridges, thereby increasing the preferential combining of transgenic TCR chains with each other and reducing pairing with endogenous TCR chains expressed by T cells.
  • a murine constant region e.g., a minimal murine constant region, i.e., the human constant region contains only a limited number of amino acids from the murine constant region and contains additional cysteine bridges
  • TCR-expressing allogeneic T cells can also be prepared using T cells isolated from different subjects.
  • the T cells to be introduced with nucleic acids encoding TCR ⁇ chains and TCR ⁇ chains are CD4+ helper T cells or CD8+ cytotoxic T cells, or a mixture of CD4+ helper T cells and CD8+ cytotoxic T cells. Synthetic cell population. TCR gene transfer into regulatory T cells is undesirable because the regulatory T cells may suppress the antiviral activity of genetically modified cytotoxic and helper T cells. The CD4+CD25+ cell population can therefore be removed prior to TCR gene transfer.
  • the cells into which nucleic acids encoding TCR ⁇ chain and TCR ⁇ chain are introduced are stem cells, eg, hematopoietic stem cells (HSC). Transfer of TCR genes to HSCs does not result in the expression of TCRs on the cell surface because stem cells do not express the CD3 molecule. However, when the stem cells differentiate into lymphoid precursors that migrate to the thymus, initiation of CD3 expression will result in expression of the introduced TCR on the surface of the thymocytes.
  • stem cells eg, hematopoietic stem cells (HSC).
  • HSC hematopoietic stem cells
  • TCR gene-modified stem cells are a continuous source of mature T cells with the desired antigen specificity. Therefore, TCR gene-modified stem cells produce T cells expressing the TCR of the present invention after differentiation.
  • the present invention also provides a method for identifying novel coronavirus N protein antigenic peptides that can be presented by MHC, said method comprising performing steps a-e of the above method for preparing TCR-T cells against SARS-CoV-2 virus, and identifying epitopes capable of activating said recombinant T cells.
  • the full-length sequence of the N protein of SARS-CoV-2 virus is as follows:
  • Epitope prediction can be used as part of an epitope mapping strategy, but TCR specificity does not necessarily match the data predicted from an epitope prediction algorithm.
  • the antigenic peptides that induce antigen-specific T cell responses can be confirmed by binding to antigen-specific TCRs, and the antigenic peptides can be used to develop vaccine formulations containing the antigenic peptide sequences or nucleic acid sequences expressing the antigenic peptides.
  • the novel coronavirus N protein antigenic peptide can be obtained, which can be presented as an MHC-antigen peptide complex by antigen-presenting cells after the novel coronavirus N protein is processed inside the cell, and expressed on the cell surface.
  • antigenic peptide vaccines can provide T cell immunity to patients to clear the new coronavirus or cells infected by the virus.
  • the present invention also provides a method for preventing or treating novel coronavirus infection, which comprises providing the recombinant TCR cells of the present invention or the TCR nucleic acid of the present invention to subjects in need.
  • the present invention provides a kit comprising the recombinant TCR cells of the present invention or the TCR nucleic acid of the present invention for preventing or treating novel coronavirus infection.
  • the full-length gene of HLA-A0201 was cloned, and the SFG-HLA-A0201 vector was constructed.
  • HLA-A0201, A19 the total RNA of human B lymphoblastoid cells (HLA-A0201, A19) was extracted, and restriction enzymes HindIII and SalI were added to both ends of the HLA-A locus cDNA-specific primers (upstream primer: TATAAAAGCTTATGGCCCTCATGGCGCCCC (SEQ ID NO: 25); downstream primer: GCGGCGTCGACTCACACTTTACAAGCTGTG (SEQ ID NO: 26)
  • upstream primer TATAAAAGCTTATGGCCCTCATGGCGCCCC (SEQ ID NO: 25); downstream primer: GCGGCGTCGACTCACACTTTACAAGCTGTG (SEQ ID NO: 26)
  • PCR conditions pre-denaturation at 94°C for 5 min, 94°C for 1 min, annealing at 66°C for 1 min, extension at 72°C for 1.5 min, 30 cycles, and finally extension at 72°C for 10 min) to implement RT-PCR.
  • the PCR products were digested with restriction enzymes HindIII and SalI, and the sequencing vector pBlueScriptSK(+/-) was digested with restriction enzymes HindIII and SalI, the digested products were separated by low-melting point agarose gel, the gel was cut, purified, the digested PCR products were ligated into the sequencing vector pBlueScriptSK(+/-), and transformed into DH5 ⁇ bacteria by TSS transformation method.
  • the transformed DH5 ⁇ bacteria were inoculated onto LB plates containing kanamycin antibiotic at a working concentration of 50ug/mL overnight. Pick white colonies, culture bacteria, and extract plasmid DNA.
  • Retroviral vector pMSGV1 (Addgene) was digested with restriction enzymes SalI and NotI, the target fragment HLA-A0201 was ligated into the retroviral vector pMSGV1 (in this specification, pSMGV1 is also referred to as "SFG"), transformed into DH5 ⁇ bacteria, cultured, and positive colonies were selected.
  • pSMGV1 in this specification, pSMGV1 is also referred to as "SFG”
  • a retroviral vector containing the full-length HLA-A0201 gene was identified by restriction enzyme digestion and PCR, which is also referred to as the SFG-HLA-A0201 vector in this paper, and its partial structure is shown in FIG. 3 .
  • the SFG-HLA-A0201 vector was transfected into the K562 cells by retrovirus technology, and the K562-HLA-A0201 cell line was constructed, and the cells were cultured to expand the cells in large quantities.
  • the wild-type K562 cells themselves do not express HLA class I and class II molecules on the cell surface, and the K562-HLA-A0201 cells obtained after HLA-A0201 gene transfection can play the role of antigen-presenting cells, process and present antigen peptides to HLA-A0201-restricted cytotoxic T lymphocytes.
  • FIG. 1 A schematic diagram of the structure of the virus particle of the SARS-CoV-2 virus is shown in Figure 1.
  • the K562 cells loaded with HLA-A0201 gene (also referred to as K562-HLA-A0201 cells) prepared in Example 1 are stimulated by using the N protein of SARS-CoV-2 virus (also referred to as "N protein" herein) to activate the K562-HLA-A0201 cells.
  • Cells were cultured in 1640 complete medium.
  • the specific activation operation is: inoculate 1x104 K562-HLA-A0201 cells/well in a 96-well plate, add 50 ⁇ g/ml N protein and 10 ⁇ g/ml ⁇ 2M, and activate the cells for about 12-48 hours, for example, 24 hours.
  • T2 cells (ATCC) can be stimulated and activated with N protein.
  • the activated K562-HLA-A0201 cells obtained in Example 2 were co-cultured with T cells extracted from healthy people for 7 days.
  • T2 cells stimulated and activated with N protein can be co-cultured with T cells extracted from healthy people for 7 days.
  • This experiment was divided into K562-HLA-A0201 cell group, T cell+K562-HLA-A0201 cell group, T cell+N protein+K562-HLA-A0201 cell group.
  • 5x104 target cells ie, K562-HLA-A0201 cells
  • 5x104 effector T cells ie, K562-HLA-A0201 cells
  • interferon- ⁇ (INF- ⁇ ) ELISA kit R&D Systems, DY008
  • the standard R&D Systems, DY008
  • the sample to be tested into the pre-coated transparent enzyme-coated plate, respectively, after incubation for a sufficient time, wash to remove unbound components, then add the enzyme-labeled working solution, and after incubation for a sufficient time, wash to remove unbound components.
  • Substrates A and B in the interferon- ⁇ (INF- ⁇ ) ELISA kit were added sequentially.
  • the substrate (TMB) was converted into a blue product under the catalysis of horseradish peroxidase (HRP), and turned yellow under the action of acid.
  • the OD value of the product was used to calculate the content of interferon- ⁇ (INF- ⁇ ) in the sample to be tested. The result is shown in Figure 4.
  • T cell group T cell+K562-HLA-A0201 cell group
  • T cell+K562-HLA-A0201 cell+N protein that is, SARS-CoV-2 virus N protein, also referred to as "PepN" protein in this article
  • TCR V ⁇ Repertoire Kit TUBE A-E (Beckman Coulter)
  • the expression of TCR V ⁇ subfamily in CD3-positive T lymphocytes was analyzed by setting the gate on CD3-positive cells. The result is shown in Figure 5.
  • TCR V ⁇ 13.6, TCR V ⁇ 11, and TCR V ⁇ 12 appeared Significant increase in expression. It is preliminarily shown that TCR V ⁇ 13.6, TCR V ⁇ 11, and TCR V ⁇ 12 subfamilies are dominantly used after being stimulated by the N protein of SARS-CoV-2 virus.
  • T cells were co-cultured with K562-HLA-A0201 cells and SARS-CoV-2 virus N protein using RPMI 1640 complete medium for about 7 days, the IFN- ⁇ in the culture supernatant was detected by ELISPOT technology, and the T cell subpopulation with specific killing function was screened out, and TCR V ⁇ 13.6, TCR V ⁇ 11, TCR V ⁇ 12 antibodies (1:50 dilution stock solution, BD company antibody) were used to detect by flow cytometry The TCRV ⁇ family was sorted by cytometry, and the sorting rate was 90%.
  • Methodology After the total cellular RNA sample is qualified, according to the different characteristics of mRNA, since the 3' end of eukaryotic mRNA has a polyA tail structure, magnetic beads with Oligo (dT) are selected for enrichment and purification. Add fragmentation buffer to the purified mRNA to fragment it into short fragments, then use the fragmented mRNA as a template, use six base random primers to synthesize the first strand of cDNA, and add buffer, dNTPs, RNaseH and DNA polymerase I to synthesize the second strand of cDNA, which is purified by QIAQuick PCR kit and eluted with EB buffer.
  • dT Oligo
  • the purified double-stranded cDNA was then subjected to end repair, base A addition, and sequencing adapter processing (P5 adapter sequence: AATGATACGGCGACCACCGAGATCTACAC, SEQ ID NO: 27; P7 adapter sequence: ATCTCGTATGCCGTCTTCTGCTTG, SEQ ID NO: 28), and then agarose gel electrophoresis was used to recover fragments with the desired size and perform PCR amplification (primer sequence: ACACTCTTTCCCTACACGACGC TCTTCCGATCT, SEQ ID NO: 29; AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC, SEQ ID NO: 30.
  • the PCR reaction program is as follows: pre-denaturation at 95°C for 30s, denaturation at 95°C for 10s, annealing at 60°C for 30s, extension at 72°C for 30s, 40 cycles) to complete the entire library preparation.
  • PepN sample and PepNC control sample wherein PepN represents the full length of N protein, and the N protein is dissolved with solvent DMSO, and PepNC represents a blank control, only the solvent DMSO is added.
  • Table 2 below is the statistical table of transcriptome sequencing analysis results.
  • RNAseq sequencing results showed that the TCR structures with significant expression differences between the PepN full-length sample experimental group and the PepNC blank control group were as follows: TRAV13-2, TRAV25, TRAJ29, TRBV4-1, TRBV6-5, TRBD1, TRBJ2-2P.
  • a single-cell bank (10X Genomics Chromium) was carried out on the specific T cells of Example 3 (see Elham Azizi et al., “Single-cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment", Cell, August 23, 2018, 174(5): 1293-1308; Tomonori Hosoya et al., "High-Throughput Single-Cell Sequencing of both TCR- ⁇ Alleles", J. Immunol., 2018 Dec 1, 201(11):3465-3470).
  • the specific method is as follows.
  • Chromium Single Cell 5’Library Construction Kit purchased from 10x Genomics, Inc.
  • Use a pipette to fully resuspend the cell suspension before adding the cell suspension to the MasterMix. Do not mix cells and water directly; add MasterMix, water and cell mixture, GelBeads and Partitioning Oil (10XGenomics) to the chip in turn. Minimize the time between chip loading and running; run the library construction program.
  • the library concentration on the machine is 20 pmol/ ⁇ l. Dilute the upper-machine library to the final concentration of the upper-machine library and perform sequencing on the machine to obtain multiple TCR ⁇ chains and ⁇ -chains, which have the following V region, D region, and J region sequences respectively.
  • CDR2 IRSNMDK (SEQ ID NO: 3)
  • CDR2 LVKSGEV (SEQ ID NO: 6)
  • CDR2 SVGAGI (SEQ ID NO: 13)
  • TCR ⁇ chain and ⁇ chain By pairing the TCR ⁇ chain and ⁇ chain, two TCRs as exemplified below are obtained, respectively:
  • TCR-1 TRAV13-2-J29-TRBV4-1-D1-J2-2P
  • the expression vector pMSGV1 (vector source: Addgene) was designed to express the full-length TCR ⁇ chain gene sequence and the full-length TCR ⁇ chain gene sequence.
  • the nucleic acid sequence encoding the following TCR was cloned into the vector pMSGV1 for expression.
  • the mouse C region (constant region) sequence (see Stephanie L. Goff et al., "Enhanced receptor expression and in vitro effector function of a murine-human hybrid MART-1-reactive T cell receptor following a rapid expansion", Cancer Immunol Immunother.2010 Oct; 59(10):1551–1560.) to replace the human C region sequence.
  • a gene expressing green fluorescent protein (GFP) was designed in the expression vector for evaluation of transfection efficiency.
  • TCR-T cells were prepared from the peripheral blood of healthy volunteers or patients in recovery from COVID-19. The specific method is as follows.
  • PBMCs Before TCR retrovirus transfection, 1 ml of freshly isolated PBMCs (about 1 ⁇ 10 6 cells) were activated and cultured with 50 U/ml IL-2 and 50 ng/ml OKT3 in a 24-well plate for 48 hours. 500 ⁇ l of activated PBMCs (approximately 5 ⁇ 10 5 cells) were spread evenly in a 24-well plate coated with 5 ⁇ g/well of fibronectin, and 12 wells of each sample of PBMC were spread.
  • Retrovirus-packaging 293T cells were cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum in an environment of 37°C and 5% CO 2 .
  • the culture medium of each TCR-T cell and negative control cells was RPMI 1640 complete medium, supplemented with IL-2 to maintain growth, and OKT3 was added to stimulate culture if necessary. Observe the growth of the cells every day, change the medium every 48 hours and add IL-2 (30ng/mL of OKT3, 50U/mL IL-2).
  • IL-2 (30ng/mL of OKT3, 50U/mL IL-2).
  • PBMC freezing solution was added, and quickly placed in a -80°C refrigerator.
  • When recovering take the frozen cells out of the liquid nitrogen tank or -80°C refrigerator and quickly put them in a 37°C water bath for 5 minutes. After the cells are completely thawed, wash them twice with 6-8ml RPMI 1640 complete medium before use.
  • TCR-T cells prepared in Example 6 were subjected to in vitro function research, and their safety and effectiveness were preliminarily verified.
  • 5x104 target cells ie, K562-HLA-A0201 cells
  • 5x104 target cells ie, K562-HLA-A0201 cells
  • 50 ⁇ g/ml 2019-nCoV-N protein and 10 ⁇ g/ml ⁇ 2M for 24 hours to activate the target cells.
  • Substrates A and B in the interferon- ⁇ (INF- ⁇ ) ELISA kit were added in turn, and the substrate (TMB) was converted into a blue product under the catalysis of horseradish peroxidase (HRP), and turned yellow under the action of acid.
  • - gamma (INF-gamma) content The results are shown in Figure 7, wherein the T cells expressing TCR-1 and TCR-2 in Examples 5 and 6 are respectively represented as TCR-1-T and TCR-2-T, and the content of INF- ⁇ in the supernatant of the TCR-T cell+K562-HLA-A0201 cell group was significantly higher than that of the T cell+K562-HLA-A0201 cell group. This shows that the T cells in the TCR-T cell + N protein + K562-HLA-A0201 cell group can also be activated by the N protein and have the ability to specifically target the SARS-Cov-2 virus N protein.

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Abstract

本发明涉及特异性结合新型冠状病毒核衣壳蛋白的T细胞受体（TCR）、编码所述TCR的核酸及包含其的宿主细胞，以及制备所述宿主细胞的方法。本发明还涉及所述TCR和所述宿主细胞在预防和/或治疗新型冠状病毒感染中的用途。

Description

新型冠状病毒特异性T细胞受体和其用途 技术领域
本发明总体上涉及免疫学领域。更具体地，本发明涉及特异性结合新型冠状病毒核衣壳蛋白的T细胞受体(下文中也缩写为TCR)、编码所述TCR的核酸及包含其的宿主细胞，以及制备所述宿主细胞的方法。本发明还涉及所述TCR和所述宿主细胞在预防和/或治疗新型冠状病毒感染中的用途。
背景技术
2019年12月以来，新型冠状病毒(2019 novel CoV(2019-nCoV)，也称为严重急性呼吸系统综合征冠状病毒2型(SARS-CoV-2))肆虐全球，感染2019-nCoV的病例数量一直在增加。来自世界卫生组织的数据表明，截至2021年8月，已有超过400万人死于2019年冠状病毒病(COVID-19)。目前，世界各地的确诊病例仍在攀升，多地仍处于疫情紧急状态，因此，发现高效的抗SARS-CoV-2药物以及用于SARS-CoV-2治疗的策略迫在眉睫。
SARS-CoV-2为单股正链RNA病毒，是第7个巳发现的可感染人的冠状病毒。SARS-CoV-2病毒包含4个结构蛋白，分别为棘突蛋白(Spike，S)、包膜蛋白(Envelope，E)、膜蛋白(Membrane/matrix，M)和核衣壳蛋白(Nucleocapsid，N)。S蛋白通过位于其S1亚基上的受体结合区(Receptor-binding domain,RBD)与宿主细胞上的受体血管紧张素转换酶Ⅱ(angiotensin converting enzymeⅡ，也称为ACE2)结合(Ashour HM等人，Insights into the Recent 2019 Novel Coronavirus(SARS-CoV-2)in Light of Past Human Coronavirus Outbreaks，Pathogens，2020年3月4日；9(3).pii:E186.doi:10.3390/pathogens9030186；Roujian Lu等人，Genomic characterisation and epidemiology of 2019 novel coronavirus:implications for virus origins and receptor binding，www.thelancet.com，2020年1月29日网上公开，https://doi.org/10.1016/S0140-6736(20)30251-8)。在这个初始的结合之后，病毒的E蛋白与宿主细胞质膜融合并引发一系列的细胞内事件，包括M蛋白和N蛋白之间的相互作用。因此，这四种结构蛋白是开发抗SARS-CoV-2药物或疫苗的主要靶标。
迄今为止，已经做出了许多努力来寻找一种可以阻止SARS-CoV-2感染并较少病毒载量的药物或疫苗。病毒的灭活疫苗是一种最为经典的疫苗形式，其易于制备且能高效引起体液免疫应答，往往是新发传染病的首选疫苗方案。病毒的灭活疫苗主要通过选自甲醛、β-丙内酯和紫外线的方式灭活病毒获得，能诱导人体生成针对病毒的中和抗体。但是所述灭活疫苗引起的T细胞免疫应答普遍偏弱，有研究证明针对SARS的灭活疫苗无法有效刺激机体产生细胞免疫应答。因此，即使灭活疫苗产生高滴度的血清中和抗体，其保护效力也并不令人满意，另外灭活疫苗生产过程中需要操作高浓度的活病毒，具有一定的生物安全风险，因此该疫苗策略需谨慎考虑。
T细胞免疫应答在清除病毒和感染细胞上发挥着重要的作用。目前尚未有研究者从细胞免疫的角度针对SARS-CoV-2进行药物研究，通过特异性T细胞治疗2019冠状病毒病(Coronavirus disease 2019，缩写为COVID-19)尚无任何报道。据研究报导，SARS-CoV-2进入人体后主要攻击的是免疫系统，使T淋巴细胞急剧减少(Bertoletti A,Tan AT.,Challenges of CAR-and TCR-T cell-based therapy for chronic infections,J Exp Med,2020,217(5):1-11)。因此，预期提高人体T细胞数量是治疗新冠病毒的最行之有效的先进手段，本领域需要开发出针对冠状病毒特异性抗原的特异性T细胞，例如，TCR-T细胞，来有效预防和治疗SARS-CoV-2感染。
发明内容
本发明人通过锐意研究，获得了能与新型冠状病毒核衣壳蛋白特异性结合的T细胞受体(TCR)，并使用所述T细胞受体制备了重组的表达该TCR的宿主细胞，由此，能够通过T细胞免疫应答来清除人体内的新型冠状病毒和被新型冠状病毒感染的细胞，从而满足了上述需求。
因此，在一个方面，本发明提供了分离的或纯化的T细胞受体(TCR)，其与新型冠状病毒核衣壳蛋白特异性结合，优选地，所述TCR包含α链和β链，其中所述α链和β链各包含三个互补决定区(CDR)，并且α链的CDR3的氨基酸序列选自SEQ ID NO:4、7和与 所述序列具有1个或2个氨基酸残基改变的变体，β链的CDR3的氨基酸序列选自SEQ ID NO:11、14和与所述序列具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCRα链的CDR3的氨基酸序列和β链的CDR3的氨基酸序列是：
(i)SEQ ID NO:4所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:11所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；或
(ii)SEQ ID NO:7所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:14所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCR的α链包含的三个互补决定区(CDR)的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
(i)SEQ ID NO:2、3、4所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:9、10、11所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；或
(ii)SEQ ID NO:5、6、7所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:12、13、14所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体。
又在一个实施方案中，本发明的TCR包含SEQ ID NO:18或22所示的α链可变区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:20或24所示的β链可变区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。在一些实施方案中，本发明的TCR包含SEQ ID NO:17或21所示的α链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:19或23所示的β链序 列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在第二方面，本发明提供了编码本发明TCRα链和/或β链的核酸、以及包含编码本发明TCRα链和/或β链的核酸的载体。在一个实施方案中，所述载体是表达载体，更优选地是逆转录病毒载体。
在第三方面，本发明提供了包含本发明的TCRα链和/或β链、包含编码本发明TCRα链和/或β链的核酸或包含编码本发明TCRα链和/或β链的核酸的载体的宿主细胞。在一个实施方案中，所述宿主细胞是T细胞，优选地是人T细胞，例如，人CD4+辅助T细胞或CD8+细胞毒性T细胞，或CD4+辅助T细胞和CD8+细胞毒性T细胞的混合细胞群。在一个实施方案中，所述宿主细胞是干细胞，例如，造血干细胞(HSC)。
在第四方面，本发明提供了制备本发明宿主细胞的方法，所述方法包括以下步骤：
(a)用SARS-CoV-2病毒N蛋白刺激负载HLA-A0201基因的K562细胞；
(b)将获得的表达人HLA等位基因、呈递SARS-CoV-2病毒N蛋白肽的负载HLA-A0201基因的K562细胞与人类受试者T细胞接触，
(c)选择由所述接触活化的T细胞，优选地基于由所述活化的T细胞表达的活化标志物进行所述选择；
(d)自所述活化的T细胞分离编码TCR的TCRα链和TCRβ链的核酸；和
(e)将编码TCR的TCRα链和TCRβ链的核酸导入人T细胞或人干细胞。
在第五方面，本发明提供了鉴定新型冠状病毒N蛋白抗原肽的方法，所述方法包括以下步骤：
(a)用SARS-CoV-2病毒N蛋白刺激负载HLA-A0201基因的K562细胞；
(b)使刺激后的所述细胞接触并呈递SARS-CoV-2病毒N蛋白肽至人类受试者T细胞，
(c)选择由所述接触活化的T细胞，优选地基于由所述活化的T细胞表达的活化标志物进行所述选择；
(d)自所述活化的T细胞分离编码TCR的TCRα链和TCRβ链的核酸；
(e)将编码TCR的TCRα链和TCRβ链的核酸导入人T细胞或人干细胞；和
(f)鉴定能够活化所述重组T细胞的表位。
在第六方面，本发明提供了本发明的TCR、本发明的宿主细胞在预防和/或治疗新型冠状病毒感染中的用途。
附图说明
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1显示了新型冠状病毒的病毒粒子的结构示意图。
图2例示了本发明的TCR的获得、本发明的宿主细胞的制造方案以及其功能性研究的示意图。
图3例示了用于在载体中表达的HLA-A0201全长基因的示意图。
图4显示了被新型冠状病毒N蛋白抗原活化的T细胞分泌的IFN-γ的量。
图5显示了被新型冠状病毒N蛋白抗原活化后T细胞ELIspot检测。
图6显示了被新型冠状病毒N蛋白抗原活化后T细胞对比空白对照组的差异表达基因火山图。
图7显示了构建的TCR-T细胞的体外验证试验。
发明详述
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
I.定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
“亲和力”是指分子(例如TCR)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。除非另有说明，在用于本文时，“结合亲和力”指反映结合对的成员(例如TCR与抗原)之间1∶1相互作用的内在结合亲和力。分子X对其配偶体Y的亲和力通常可用结合解离平衡常数(KD)来表述。亲和力可通过本领域知道的常用方法来测量，包括现有技术已知以及本文中所描述的那些方法。
如本领域已知，在本文中可交换使用的“多核苷酸”或“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位 置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是同一的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
术语“抗原呈递细胞”或“APC”是指其表面上呈递与主要组织相容性复合体(MHC)复合的外来抗原的免疫系统细胞，比如辅助细胞(例如，B-细胞、树突细胞等)。T细胞可以使用其T细胞受体(TCR)识别这些复合体。APC加工抗原且将抗原呈递给T细胞。
如本文所用，“载体(vector)”表示构建体，其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、质粒、粘粒或噬菌体载体。
在本发明中术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且是指已经引入外源性核酸的细胞，包括这些细胞的子代。宿主细胞包括“转化子”和“转化的细胞”，其包括原代转化细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与 亲代细胞可能不完全相同，但可能含有突变。本文包括与在初始转化的细胞中筛选或选择的细胞具有相同功能或生物学活性的突变子代。
在本文中，“受试者”、“个体”指需要缓解、预防和/或治疗新型冠状病毒感染的动物，优选哺乳动物，更优选是人。哺乳动物还包括但不限于农场动物、竞赛动物、宠物、灵长类、马、犬、猫、小鼠和大鼠。该术语包括具有新型冠状病毒感染或处于具有冠状病毒感染风险的人受试者。
II.本发明的T细胞受体(TCR)所针对的新型冠状病毒、其结构和进入宿主细胞的方式
在2020年1月6日，发布了一种新型冠状病毒的全基因组序列(GenBank登录号：MN908947)。2020年1月14日，世界卫生组织(WHO)正式将引发此轮疾病的病毒命名为2019新型冠状病毒(2019-nCoV)。在2020年2月11日国际病毒分类委员会(International Committee on Taxonomy of Viruses，ICTV)冠状病毒研究小组(Coronavirus StudyGroup)正式命名2019新型冠状病毒为严重急性呼吸综合征冠状病毒2(Severe Acute Respiratory Syndrome Coronavirus 2，SARS-CoV-2)。因此，在本文中，“2019-nCoV”和“SARS-CoV-2”可互换地使用。从冠状病毒分类学角度上讲，SARS-CoV-2属于SARS冠状病毒(SARS-CoV)的近亲。同日，世界卫生组织(World Health Organization，WHO)将由这一病毒导致的疾病正式名称为COVID-19(Coronavirus Disease 2019)(YU W B,TANG G D,ZHANG L等人，Decoding the evolution and transmissions of the novel pneumonia coronavirus using whole genomic data[J].ChinaXiv:2020,86(1):3995-4008；中国疾病预防控制中心,新型冠状病毒研究资源专栏[EB/OL],[2020-02-25]http://www.chinacdc.cn/)。
新型冠状病毒(2019-nCoV)是被包膜的(所被包膜衍生自宿主细胞膜的脂质双层)(图1)、大小为80-120nm的单股正链RNA病毒，基因组长度约为29.9kb，该病毒与同属于冠状病毒科β冠状病毒属的SARS-CoV的基因组序列之间的同源性为80％。病毒基因组的可读框(Open reading frame，ORF)ORF1a和ORF1b占基因组的2/3，表达水解酶以及与复制、转录相关的酶，例如，半胱氨酸蛋白酶(PLpro)和丝氨酸蛋白酶(3CLpro)，RNA依赖RNA聚合酶(RdRp)和解旋酶(Hel)；后面的基因组1/3区域主要负责编码病毒结构蛋白，包括刺突蛋白 (Spike，S)、包膜蛋白(Envelope，E)、膜蛋白(Membrane，M)、核衣壳蛋白(Nucleocapsid，N)等主要结构蛋白，其中S蛋白、M蛋白和E蛋白均嵌入在病毒包膜中，E蛋白和M蛋白主要参与病毒的装配过程，S蛋白则主要通过与宿主细胞受体结合介导病毒的入侵并决定病毒的宿主特异性，N蛋白包裹病毒基因组形成核蛋白复合体，位于病毒颗粒的核心，形成核衣壳。N蛋白主要负责病毒RNA的复制功能。
经序列比对，发现2019-nCoV病毒和SARS-CoV病毒的S蛋白具有75％的相似度，据报道称多株SARS-CoV冠状病毒分离株中位于S蛋白与ACE2受体(在人体中主要分布于呼吸道上皮细胞、肺脏、心脏、肾脏和消化道等位置)复合物界面的442、472、479、487和491位点的氨基酸残基是高度保守的。与SARS-CoV的S蛋白相比较，在所述5个位点处，2019-nCoV S蛋白仅第491位氨基酸相同，其它4处氨基酸都发生了突变(Xu X等人，Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission，Sci China Life Sci.,2020年3月；63(3):457-460)。尽管如此，通过蛋白质3D结构模拟预测发现虽然2019-nCOV S蛋白与ACE2受体结合的所述4个关键氨基酸都发生了替换，但是相对于SARS-CoV S蛋白，2019-nCoV S蛋白中的受体结合结构域(receptor binding domain，RBD)的三维结构几乎不变，由此2019-nCoV S蛋白与人体ACE2仍然具有较高的亲和力，最近的文章(Wrapp D等人，Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation，Science,.2020年2月19日，网上公开，pii:eabb2507.doi:10.1126/science.abb2507以及Xiaolong Tian等人，Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody,Emerging Microbes & Infections，2020,9:1,p382-385,DOI:10.1080/22221751.2020.1729069)通过Fortebio检测，2019-nCoV的S蛋白结合人类ACE2的亲和力(KD)约为15nM，与SARS-CoV的S蛋白结合人类ACE2的亲和力相当，由此可见，ACE2也是2019-nCoV感染人体进入细胞内部的受体蛋白。目前许多研究致力于产生针对冠状病毒S蛋白的、旨在阻断S蛋白与ACE2受体结合的高亲和力中和抗体，以预防和治疗新型冠状病毒感染。
与冠状病毒S蛋白相比，核衣壳蛋白(N蛋白)具有相对保守性，常作为新型冠状病毒诊断检测工具。COVID-19感染诱导产生针对N蛋白的IgG抗体，这种抗体最早可在疾病发作后第4天通过血清检测到，大多数患者在第14天发生血清转换。临床患者的实验室证据表明，针对SARS-CoV-2的特异性T细胞反应对于识别和杀死受感染的细胞非常重要，尤其是在受感染个体的肺部识别和杀死受感染的细胞非常重要(Mohsen Rokni等人,Immune responses and pathogenesis of SARS-CoV-2 during an outbreak in Iran:Comparison with SARS and MERS，Rev Med Virol.2020；30:e2107，p1－6，DOI:10.1002/rmv.2107)。
本发明人首次鉴定了SARS-CoV-2中核衣壳蛋白(N蛋白)的TCR并制备了SARS-CoV-2特异性的TCR T细胞，提供了预防或治疗SARS-CoV-2感染的新策略。
III.本发明的T细胞受体(TCR)、编码TCR的核酸和包含所述核酸的载体
SARS-CoV-2病毒核衣壳蛋白(N蛋白)在细胞中经加工，通过主要组织相容性复合物(MHC)分子携带至细胞表面，呈现为肽-MHC复合物形式。
T细胞受体(TCR)是存在于T细胞表面的分子，其负责识别肽-MHC复合物。TCR与肽-MHC复合物的特异性结合引发T细胞通过一系列由相关酶、共受体和辅助性分子介导的生化事件而活化。在95％的T细胞中，TCR异二聚体由α和β链组成，而在5％的T细胞中，TCR异二聚体由γ和δ链组成。
TCR的每一条链均属于免疫球蛋白超家族的成员，具有一个N端免疫球蛋白(Ig)可变(V)结构域、一个Ig恒定(C)结构域、跨细胞膜区域(即，跨膜区)以及在C末端的短胞质尾。
在TCRα链和β链的可变结构域中，各可变结构域具有三个高变区或互补决定区(CDR)，其中各可变结构域中的CDR3是负责识别经加工的抗原的主要CDR。认为CDR2识别MHC分子。
TCR的恒定结构域由短的连接序列组成，其中的半胱氨酸残基形成二硫键，在TCRα链和β链之间产生连接。
在T细胞成熟过程中，TCR与CD3形成TCR/CD3复合体。TCR/CD3复合体形成过程通常是按以下顺序进行的；首先CD3γ、δ和ε三种肽链通过形成γ-ε和δ-ε两种异源二 聚体成为稳定的复合物核心，TCRαβ(或TCRγδ)与之结合，随后ζ-ζ或ζ-η二聚体与TCRαβ(或TCRγδ)/CD3γεδε复合物结合，最后转移到T细胞表面。通过TCR/CD3复合体将信号从TCR传导至细胞内。
来自TCR/CD3复合体的信号通过MHC与特异性共受体的同时结合而增强。在辅助T细胞中，这一共受体是CD4分子，所述CD4分子对II类MHC是特异性的；而在细胞毒性T细胞中，这一共受体是CD8，所述CD8分子对对I类MHC是特异性的。
在本文中，术语“T细胞受体”具有本领域的常规意义，用于表示能够识别由MHC分子呈递的肽的分子。该分子是两条链α和β(或任选地γ和δ)的异二聚体。
本发明提供了分离的或纯化的T细胞受体(TCR)α链和/或β链。本发明的TCR可以是包含衍生自超过一种物种的序列的杂合TCR。例如，考虑到鼠科TCR在人T细胞中能够比人TCR更有效地表达，TCR可包含人可变区和鼠科恒定区。
在一个实施方案中，本发明的TCR包含α链和β链，其中所述α链和β链各包含三个互补决定区(CDR)，且其中主要负责抗原识别的TCRα链CDR3的氨基酸序列选自SEQ ID NO:4、7和与所述序列具有1个或2个氨基酸残基改变的变体，β链CDR3的氨基酸序列选自SEQ ID NO:11、14和与所述序列具有1个或2个氨基酸残基改变的变体。
又在一个实施方案中，本发明的TCR包含α链和β链，其中所述α链的CDR3的氨基酸序列和β链的CDR3的氨基酸序列是：
(i)SEQ ID NO:4所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:11所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；或
(ii)SEQ ID NO:7所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:14所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCR包含α链和β链，所述α链包含的三个互补决定区(CDR)的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
(i)SEQ ID NO:2、3、4所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:9、10、11所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；或
(ii)SEQ ID NO:5、6、7所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:12、13、14所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体。
在一个实施方案中，本发明的TCR包含SEQ ID NO:18或22所示的α链可变区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:20或24所示的β链可变区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。优选地，本发明的TCR还包含恒定区，更优选地，本发明的TCR还包含小鼠恒定区，例如，所述TCR包含SEQ ID NO:17或21所示的α链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:19或23所示的β链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一些实施方案中，本发明的TCR变体中所述氨基酸残基的改变是任一SEQ ID NO:2－24的序列中的氨基酸残基的取代、添加或缺失，条件是该TCR变体仍保留了或改善了与新型冠状病毒核衣壳蛋白的表位肽-MHC复合物结合的能力。在一个实施方案中，所述取代是保守性取代。下表1中给出了保守性取代的例子。
表1

氨基酸可以根据常见的侧链特性分组：
(1)疏水性：正亮氨酸、Met、Ala、Val、Leu；Ile；
(2)中性亲水：Cys、Ser、Thr、Asn；Gln；
(3)酸性：Asp、Glu；
(4)碱性：His、Lys、Arg；
(5)影响链方向的残基：Gly、Pro；
(6)芳族：Trp、Tyr、Phe。
非保守性取代将使这些分类之一的成员交换为另一个分类的成员。
本发明还涉及编码本发明TCR或其部分的核酸，所述TCR部分例如，一个或多个CDR；一个或多个可变区；α链；或β链等。该核酸可以是双链或单链的，并且可以是RNA或DNA。该核酸序列可以是经密码子优化的，以实现哺乳动物生产细胞中的高表达。哺乳动物细胞以及多种其他生物的密码子选择是本领域公知的。密码子优化还可包括移除mRNA不稳定基序和隐藏的剪接位点。
进一步地，本发明还涉及包含编码本发明TCR的核酸的载体。术语“载体”包括表达载体，即能够在体内或在体外/离体表达的载体。
载体将编码本发明TCR的核酸转移至细胞中，诸如T细胞中，使得细胞表达新型冠状病毒特异的TCR。优选地，载体在T细胞中持续高水平表达TCR，使得引入的TCR可成功地与内源TCR竞争有限的CD3分子池。备选地，增加CD3分子供应也可增加基因修饰细胞中的TCR表达。因此载体任选地包含CD3-γ、CD3-δ、CD3-ε和/或CD3-ζ的基因。在一个实施方案中，该载体仅包含CD3-ζ的基因。另外，也可提供一个或多个编码CD3基因的单独的载体用于与TCR编码载体共转移入细胞中。
用作载体的病毒递送系统包括但不限于逆转录病毒载体、慢病毒载体、杆状病毒载体、疱疹病毒载体、腺病毒载体、腺相关病毒(AAV)载体。
逆转录病毒是RNA病毒，其具有与裂解性病毒(lytic virus)不同的生命周期。具体而言，逆转录病毒是通过DNA中间体复制的传染性病毒。当逆转录病毒感染细胞时，逆转录病毒基因组经逆转录酶转化为DNA形式，然后以该DNA形式为模板产生新的病毒RNA基因组、以及产生对组装传染性病毒颗粒所需的病毒编码蛋白。逆转录病毒的详细列表参见Coffin等人，“Retroviruses”1997 Cold Spring Harbour Laboratory Press编辑:JM Coffin,SM Hughes,HE Varmus，p758-763。
在一个实施方案中，载体是逆转录病毒载体。为有效感染人细胞，病毒颗粒可以用双嗜性包膜或长臂猿白血病病毒包膜进行包装。
IV.表达本发明的T细胞受体的重组细胞(例如，TCR-T)
20世纪90年代初,临床研究人员观察到，ー些异基因造血干细胞移植后白血病复发的患者，可以通过输注捐赠者的T细胞进入缓解期。这些数据表明了T细胞介导的抗白血病作用，并提示了这种细胞治疗方法在应对病毒感染中的潜力。
在严重免疫缺陷患者中，同种异体特异性CTL对EB病毒(EBV)感染、巨细胞病毒感染、腺病毒感染、BK病毒感染和人类疱疹病毒感染的应答率非常高。
现有技术中，针对病毒的特异性CTL可用于治疗覆盖绝大多数HLA等位基因的多种病毒，因此，提供了一种适合于治疗大多数患者的方法。这些成功提高了使用病毒特异性CTL靶向病毒相关癌症的治疗前景，包括由EBV、乙型肝炎病毒等引起的癌症。由于患有病毒相关癌症的患者保留了足够的免疫排斥同种异体细胞，因此，研究者们将注意力主要集中在自体特异性CTL的过继转移或自体转基因T细胞上(Miyao K，Terakura S，Okuno S，Julamanee J等人，Introduction of Genetically Modified CD3Z Improves Proliferation and Persistence of Antigen-Specific CTLs，Cancer Immunol Res，2018(6):733-744)。
在TCR-T治疗HIV病毒方面，有研究显示利用噬菌体可增强T细胞受体(TCR)的功能，通过寻找靶抗原并建立患者来源的TCR-T细胞悬液，该细胞可解除HLA-A*02的限制，并靶向HIV的特异性肽段SLYNTVATL(SL9)。在此研究中所制备的高亲和カ(KD<400pM)TCR结合半衰期超过3小时，可特异性靶向HIV感染后的细胞，并识别该细胞的所有常见的逃逸变异体。经TCR转导的CD8细胞比阴性对照组CD8细胞可产生更多IL-2、IFN-γ、TNF-α等细胞因子，有效地控制了HIV的野生型和突变株，达到了T细胞治疗的效果(Varela-Rohena A，Molloy PE，Dunn SM，Li Y等人，Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor.Nat Med.2008(12):1390-5)。
在TCR-T治疗CMV病毒方面，有研究者利用ATAM+TCR-T靶向CMV，观察到ATAM+TCR-T细胞在体内、体外均表现出较好的增殖活性及持久性。在此研究中，通过ATAM转导而制备了多种靶向的TCR-T，从而实施了多靶向的细胞免疫治疗，由此提高了TCR-T治疗的有效性，也为TCR-T治疗病毒感染性疾病提供了新的思路(Miyao K，Terakura S，Okuno S，Julamanee J.等人，Introduction of Genetically Modified CD3ζ Improves  Proliferation and Persistence of Antigen-Specific CTLs[J].Cancer Immunol Res,2018(6):733-744)。
在TCR-T治疗EB病毒方面，TCR-T细胞疗法为治疗EB病毒相关移植后淋巴增生性疾病(PTLD)的ー种有希望的治疗方式。现有技术中的几项临床试验表明，EBV-TCR-T治疗EBV的总有效率高达80％。因此，在第六届欧洲传染病会议上，有众多专家推荐EBV-TCR-T用于EBV疾病的预防和早期治疗以及HSCT后EBV相关的PTLD的治疗(Hong J，Ni J，Ruan M，Yang M等人，LMP1-specific cytotoxic T cells for the treatment of EBV-related post-transplantation lymphoproliferative disorders[J].Int J Hematol.2020，47(12):1390-1395)。
TCR-T对于因病毒感染引起的肿瘤具有显著的治疗效果，如美国Kite Pharma公司在CAR-T疗法之外，同时也在开发TCR-T疗法。其中在研HTE-439近日发布临床I期实验的早期数据，结果表明靶向16型HPV(HPV-16)E7蛋白的HTE-439能够让部分HPV-16阳性癌症患者获得部分缓解。在I期临床的早期阶段，共有8位转移性HPV-16阳性肿瘤患者接受了HTE-439治疗(表达E7 TCR的T细胞疗法)。在最初接受TCR-T细胞疗法的6位患者中，回输的T细胞于90-99％的细胞表面表达E7 TCR。而且在患者接受治疗6周后，仍然可以在外周血液中检测到这种T细胞。这证实了E7蛋白是TCR治疗的病毒靶点。该公司计划递交使用HTE-439治疗HPV-16 E7实体瘤的新药临床试验申请(IND)。
在本申请中，本发明人通过基因工程技术构建了针对SARS-CoV-2病毒N蛋白抗原的TCR，将所述TCR导入T细胞中，制备了大量针对SARS-CoV-2病毒N蛋白抗原的特异性T细胞，即TCR-T细胞，为早期预防和治疗SARS-CoV-2病毒感染所引起的疾病及疾病的发展提供了新方法。
具体而言，本发明获得了针对SARS-CoV-2病毒N蛋白的特异性TCR，进而制备了临床级别的具有特异性杀伤新冠病毒或者病毒感染细胞的TCR-T细胞，建立了整套标准的急性感染期的病毒感染性疾病细胞治疗标准。
在一些实施方案中，本发明提供了制备针对SARS-CoV-2病毒的TCR-T细胞的方法，所述方法包括以下步骤：
(a)用SARS-CoV-2病毒N蛋白(在本文中，也称为PepN)刺激负载HLA-A0201基因的K562/或刺激T2细胞约12－48小时，例如，18小时、24小时、30小时、36小时、42小时；
(b)使刺激后的所述细胞接触并呈递SARS-CoV-2病毒N蛋白至人类受试者T细胞约2－7天，和
(c)选择由所述接触活化的T细胞，优选地基于由所述活化的T细胞表达的活化标志物进行所述选择；
(d)自所述活化的T细胞分离编码TCRα链和TCRβ链的核酸；和
(e)将编码TCRα链和TCRβ链的核酸导入T细胞或干细胞。
在步骤a中，表达人HLA等位基因、具有呈递SARS-CoV-2病毒N蛋白的能力的细胞例如可以是天然地表达人HLA-A0201的TAP2缺陷型淋巴母细胞T2细胞、可以是瞬时地或稳定地表达一种MHC I等位基因(例如，一种人HLA等位基因)的K562细胞，可通过将一种人MHC I等位基因(HLA-A、-B或-C)转导K562细胞系生成所述细胞。
野生型K562细胞具有人红白血病来源并缺乏内源性MHC I和MHC II等位基因的表达(Boegel S,M,Bukur T等Oncoimmunology.2014；3(8):37–41)。然而，野生型K562细胞表达β-2微球蛋白(所述β-2微球蛋白是包含于有功能的MHC复合物中的一种遍在组分)。将转基因MHC Iα链导入野生型K562细胞并表达后，细胞具有了完全功能的抗原加工和呈递机制(Suhoski MM,Golovina TN,Aqui NA等Mol Ther.2007；15(5):981–8)，因此，能够作为抗原呈递细胞发挥作用。
又在一个实施方案中，作为K562细胞的备选，使用小鼠成纤维细胞NIH/3T3细胞，其稳定地表达单一人MHC等位基因，从而获得在人MHC复合物的情况下表位的内源性呈递。
在一个实施方案中，MHC可以是MHC II。在这种情况下，用一个MHC II等位基因转染K562细胞。备选地，可使用基于人RM3(Raji)B细胞的单一MHC II-表达细胞。由表达MHC II的细胞刺激的T细胞将是CD4+T细胞。
在一些实施方案中，对MHC基因转染的K562细胞进一步修饰以表达共刺激分子，例如，CD40、CD40L、CD70、CD80、CD83、CD86、ICOSL、GITRL、CD137L和/或CD252，由此可放大T细胞应答。
然而，为分离高亲和力TCR，对MHC基因转染的K562细胞不修饰任何更多的共刺激分子则可能是优选的。
此外，对MHC基因转染的K562细胞可进一步修饰，以表达增强抗原加工和呈递的分子，例如HLA-DM和CD74。
K562细胞在对抗原内部加工后，在其MHC分子上呈递抗原的表位。优选地，K562细胞稳定地表达完整的全长抗原，例如，在用编码抗原的ivtRNA转染后。也可使用瞬时表达。
在步骤b中，用人类受试者的T细胞接触呈递抗原的K562细胞约2－7天，以达到T细胞样品中的抗原特异性T细胞的最佳活化。优选地，在所述接触期间避免细胞因子的加入以防止T细胞的非特异性活化。
在一个实施方案中，步骤b的来自人类受试者的T细胞呈现PBMC的形式，即不将PBMC中的T细胞分离出来。这可能是有益的，因为PBMC提供一种更天然的环境。备选地，步骤b的来自人类受试者的T细胞是自PBMC分离的、纯化的T细胞，如纯化的CD4+细胞，优选地CD8+T细胞。
步骤b中对T细胞的刺激进行2－7天，例如，进行3天、4天、5天、6天或7天。
T细胞样品中，携带对由K562细胞呈递的抗原肽有特异性的TCR的T细胞被活化。接下来，对所述被特异性活化的T细胞基于活化标志物(步骤c)进行选择。在一个实施方案中，所述被特异性活化的T细胞上调活化标志物，例如INF-γ、CD107、CD137、CD56。 所述活化标志物可用作例如通过FACS、ELISOPT技术、CFSE实验等检测和分离抗原肽特异性T细胞的标志物。
在一个实施方案中，使用CD137作为分离抗原肽特异性CD8+T细胞的特异性标志物。基于CD137表达的分选，例如通过FACS，是选择特异性T细胞的一个方法。
又在一个实施方案中，基于活化标志物IFNγ释放的测量，选择特异性T细胞。被MHC I类分子-抗原肽复合体激活的CD8+T细胞释放具有细胞因子(如IFNγ)的囊泡。可以通过细胞因子捕获测定法与FACS分析的组合来测定IFNγ，从而选择TCR信号传导通路活化的T细胞。
选择活化的T细胞后，在步骤d中分离编码所述T细胞的TCRα链和TCRβ链的核酸。在一个实施方案中，提取细胞的总RNA，通过TCRα链和TCRβ链基因的cDNA末端的5’快速扩增(RACE)生成cDNA，然后进行PCR扩增。将PCR产物克隆入表达质粒，转化细菌。可将每个细菌菌落视为携带一个PCR TCRα或TCRβ基因片段。提取细菌菌落的载体DNA，然后对载体DNA中的插入物(TCRα或TCRβ基因片段)测序。在一个实施方案中，使用二代测序技术进行TCR序列的测定。
在步骤e中，将编码TCRα链和TCRβ链的核酸导入T细胞，获得了重组表达特异性针对新型冠状病毒N蛋白的TCR的T细胞。在一个实施方案中，在将TCR导入T细胞之前，对步骤d中测得的TCRα链和β链序列进行修饰，任选地，优化密码子选择以提高重组T细胞中TCR的表达。此外，可将TCR的人可变区与鼠恒定区结合，例如，与最小的鼠恒定区结合，即，人恒定区仅含有来自鼠恒定区的有限的氨基酸且包含额外的半胱氨酸桥，由此，增加转基因TCR链彼此的优先组合并减少与T细胞表达的内源性TCR链的配对。
当步骤e制备的重组T细胞用于患者的预防或治疗时，使用患者的自身T细胞。备选地，也可以使用从不同的受试者分离T细胞来制备表达TCR的同种异体T细胞。
在一个实施方案中，所述待导入编码TCRα链和TCRβ链的核酸的T细胞是CD4+辅助T细胞或CD8+细胞毒性T细胞，或CD4+辅助T细胞和CD8+细胞毒性T细胞的混 合细胞群。TCR基因转移至调节性T细胞中是不期望的，因为所述调节性T细胞可能抑制基因修饰的细胞毒和辅助T细胞的抗病毒活性。因此可在TCR基因转移之前除去CD4+CD25+细胞群。
在一个实施方案中，所述待导入编码TCRα链和TCRβ链的核酸的细胞是是干细胞，例如，造血干细胞(HSC)。将TCR基因转移至HSC不会导致在细胞表面表达TCR，因为干细胞不表达CD3分子。然而，当干细胞分化为迁移至胸腺的淋巴前体细胞(lymphoid precursor)时，CD3表达的启动将导致在胸腺细胞的表面表达该引入的TCR。这一方法的优点是成熟T细胞一旦产生，其仅表达引入的TCR，而表达很少的或不表达内源TCR链，因为引入的TCR链的表达抑制了内源TCR基因片段重排形成功能性TCRα和β基因。这一方法的其他益处是，TCR基因修饰的干细胞是具有期望的抗原特异性的成熟T细胞的持续来源。因此，TCR基因修饰的干细胞在分化后产生表达本发明TCR的T细胞。
V.新型冠状病毒N蛋白抗原肽
本发明还提供了鉴定能够由MHC呈递的新型冠状病毒N蛋白抗原肽的方法，所述方法包括进行上述制备针对SARS-CoV-2病毒的TCR-T细胞的方法的步骤a-e，和鉴定能够活化所述重组T细胞的表位。
表位作图策略是本领域已知的。在一个实施方案中，SARS-CoV-2病毒N蛋白全长序列如下所示：
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA(SEQ ID NO:1)
表位预测可用作表位作图策略的一部分，但是TCR特异性不必然匹配从表位预测算法预测的数据。诱导抗原特异性T细胞应答的抗原肽可以通过与抗原特异性TCR的结合来证实，并且所述抗原肽可用来开发含有该抗原肽序列或者含有表达该抗原肽的核酸序列的疫苗制剂。
通过本发明的抗原肽鉴定方法，能够获得新型冠状病毒N蛋白抗原肽，其在新型冠状病毒N蛋白被细胞内部加工后能够被抗原呈递细胞呈递为MHC-抗原肽复合体，表达于细胞表面。
抗原肽疫苗的应用可提供给患者T细胞免疫以清除新型冠状病毒或该病毒感染的细胞。
VI.预防或治疗新型冠状病毒感染的方法
本发明还提供了一种预防或治疗新型冠状病毒感染的方法，其包括对有需要的受试者提供本发明的重组TCR细胞或本发明的TCR核酸。
在一个实施方案中，本发明提供了一种试剂盒，其包含本发明的重组TCR细胞或本发明的TCR核酸，用于预防或治疗新型冠状病毒感染。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
通过参考以下实施例将更容易地理解本文一般地描述的本发明，这些实施例是以举例说明的方式提供的，并且不旨在限制本发明的范围。这些实施例并不旨在表示下面的实验是全部进行了的实验或仅进行了的实验。
实施例1.构建K562-HLA-A0201细胞系
采用图2所示的方法实施TCR的获得和其功能性研究。
首先克隆了HLA-A0201全长基因，并构建了SFG-HLA-A0201载体。
具体而言，提取人B淋巴母细胞(HLA-A0201，A19)的总RNA，在HLA-A基因座cDNA特异性引物两端分别加上限制性酶HindIII、SalI的酶切序列(上游引物：TATAAAAGCTTATGGCCCTCATGGCGCCCC(SEQ ID NO:25)；下游引物：GCGGCGTCGACTCACACTTTACAAGCTGTG(SEQ ID NO:26))(PCR条件：94℃预变性5min，94℃1min，66℃退火1min，72℃延伸1.5min，循环30次，最后72℃延伸10min)实施RT-PCR。用限制性酶HindIII、SalI酶切PCR产物，并用限制性酶HindIII、SalI酶切测序载体pBlueScriptSK(+/-)，通过低熔点琼脂糖凝胶分离酶切产物，切胶、纯化、将经酶切的PCR产物连接入测序载体pBlueScriptSK(+/-)，用TSS转化法转入DH5α细菌中。将经转化的DH5α细菌接种至含50ug/mL工作浓度的卡那霉素抗生素的LB平板上，过夜。挑选白色菌落，培养细菌，提取质粒DNA。经酶切和PCR初步鉴定，再用限制性酶HinfI酶切PCR产物以区分HLA-A2和HLA-A19，送选测序，测序结果显示与GeneBank中HLA-A0201的cDNA序列(登录号：M84379)完全一致。
用限制性酶SalI和NotI酶切逆转录病毒载体pMSGV1(Addgene公司)，将目的片段HLA-A0201连接入该逆转录病毒载体pMSGV1(在本说明书中，pSMGV1也称为“SFG”)，转化至DH5α细菌中，培养，并挑选阳性菌落。经酶切和PCR方法鉴定出含HLA-A0201全长基因的逆转录病毒载体，本文中也称为SFG-HLA-A0201载体，其部分结构如图3所示。
然后，采用野生型人慢性骨髓性白血病细胞系(human chronic myelogenous leukemia cell line)K562细胞(ATCC)，通过逆转录病毒技术，将SFG-HLA-A0201载体转导入K562细胞，构建了K562-HLA-A0201细胞系，并进行细胞培养来大量扩增细胞。所述野生型K562细胞本身在细胞表面不表达HLA I类和II类分子，经HLA-A0201基因转染后获得的K562-HLA-A0201细胞能够发挥抗原呈递细胞的作用，加工并呈递抗原肽至HLA-A0201限制的细胞毒性T淋巴细胞。
实施例2.用SARS-CoV-2病毒N蛋白刺激和活化负载HLA-A0201基因的K562细 胞
SARS-CoV-2病毒的病毒粒子的结构示意图如图1所示。利用SARS-CoV-2病毒N蛋白(本文中也简称为“N蛋白”)对实施例1制备的负载HLA-A0201基因的K562细胞(也称为K562-HLA-A0201细胞)进行刺激，使K562-HLA-A0201细胞活化，其中使用的SARS-CoV-2病毒N蛋白浓度为50μg/ml，用含1％青/链霉素和10％FBS的RPMI 1640完全培养基培养细胞。具体的活化操作为：96孔板中接种入1x104个K562-HLA-A0201细胞/孔，加入50μg/ml的N蛋白和10μg/ml的β2M，活化细胞约12－48小时，例如，24小时。类似地，可以用N蛋白刺激和活化T2细胞(ATCC)。
实施例3.具有特异性杀伤功能的T细胞群的筛选
将实施例2中获得的经活化后的K562-HLA-A0201细胞与自健康人提取的T细胞共培养7天。类似地，可以将用N蛋白刺激和活化后的T2细胞与自健康人提取的T细胞共培养7天。
3.1.经N蛋白活化的K562-HLA-A0201细胞与T细胞共培养的结果：
本实验分为K562-HLA-A0201细胞组、T细胞+K562-HLA-A0201细胞组、T细胞+N蛋白+K562-HLA-A0201细胞组。
将5x104个靶细胞(即，K562-HLA-A0201细胞)加入96孔板，使用50μg/ml2019-nCoV-N蛋白和10μg/mlβ2M刺激24小时，活化靶细胞。然后，加入5x104个效应T细胞共培养7天后，用无菌15ml离心管1000rpm离心5分钟以收集上清，作为待测样本。
使用市售的干扰素-γ(INF-γ)ELISA试剂盒(R&D Systems，DY008)，按照制造商的说明书，将标准品(R&D Systems，DY008)、待测样本分别加入到预包被的透明酶标包被板中，温育足够时间后，洗涤除去未结合的成分，再加入酶标工作液，温育足够时间后，洗涤除去未结合的成分。依次加入干扰素-γ(INF-γ)ELISA试剂盒中的底物A、B，底物(TMB)在辣根过氧化物酶(HRP)催化下转化为蓝色产物，在酸的作用下变成黄色，颜色的深浅与样本中的干扰素-γ(INF-γ)浓度呈正相关，450nm波长下测定OD值，根据标准品和待测样 品的OD值，计算出待测样本中干扰素-γ(INF-γ)的含量。结果如图4所示。
由图4可见，T细胞+K562-HLA-A0201细胞组的上清液中INF-γ的含量显著高于K562-HLA-A0201细胞组，而T细胞+N蛋白+K562-HLA-A0201细胞组的上清液中IFN-γ的含量显著高于T细胞+K562-HLA-A0201细胞组。这表明，T细胞+N蛋白+K562-HLA-A0201细胞组中的T细胞已经被N蛋白活化，并已具有特异性靶向SARS-Cov-2病毒N蛋白的能力。
3.2.将呈递N蛋白抗原肽的K562-HLA-A0201细胞与T细胞共培养后的流式细胞术检测结果：
本实验分为T细胞组、T细胞+K562-HLA-A0201细胞组、T细胞+K562-HLA-A0201细胞+N蛋白(即，SARS-CoV-2病毒N蛋白，本文中也简称为“PepN”蛋白)组。
各组细胞浓度为5x105个/100μl，每组取100μl至流式细胞术真空管中，每组再分为以下亚组：不加任何抗体的阴性亚组、CD3-PC5单标亚组(BeckmanCoulter)(用于检测CD3的表达)、CD4-PE单标亚组(用于检测CD4的表达)、CD8-FITC单标亚组(用于检测CD8的表达)、CD3-PC5+实验组(所述实验组为T细胞+K562-HLA-A0201细胞+N蛋白组)，CD3-PC5+对照组(所述对照组为两组：分别为T细胞组、T细胞+K562-HLA-A0201细胞组)检测TCR Vβ亚家族表达情况。由此，通过检测T细胞标志物，用以筛除残留的K562-HLA-A0201细胞的影响。
使用市售的TCR VβRepertoire Kit TUBE A-E(BeckmanCoulter)，根据该试剂盒说明书分别向各流式细胞术真空管中加入各抗体后避光孵育30min。每管中加入3ml PBS，500g离心10min，弃上清并重复一次PBS洗涤。弃上清后加入500μl 1％多聚甲醛固定液，重悬细胞待上流式细胞仪检测。以CD3阳性细胞设门，分析CD3阳性T淋巴细胞中TCR Vβ亚家族表达情况。结果如图5所示。
由图5可见，经SARS-CoV-2病毒N蛋白刺激后的T淋巴细胞中TCR Vβ亚家族的多个家族出现了不同程度的表达增加。其中TCR Vβ13.6、TCR Vβ11、TCR Vβ12出现了 显著的表达增加。初步表明经SARS-CoV-2病毒N蛋白刺激后TCR Vβ13.6、TCR Vβ11、TCR Vβ12亚家族发生优势取用。
在T细胞与K562-HLA-A0201细胞和SARS-CoV-2病毒N蛋白使用RPMI 1640完全培养基共培养约7天后，通过ELISPOT技术检测培养上清液中的IFN-γ，筛选出具有特异性杀伤功能的T细胞亚群，并使用TCR Vβ13.6、TCR Vβ11、TCR Vβ12抗体(1:50稀释原液，BD公司抗体)，通过流式细胞术分选出TCRVβ家族，分选得率是90％。
实施例4.鉴定T细胞经SARS-CoV-2病毒N蛋白肽活化后差异表达的TCR结构
本实施例通过研究T细胞被SARS-CoV-2病毒N蛋白肽活化后时期下所有的mRNA，针对实际样品信息采用灵活的差异分析策略找到不同个体间差异表达的mRNA，再通过http://bioinformatics.sc.cn/RNAmod/网址进行功能注释，最终得到mRNA在生物体中参与生命活动的清晰生物信息图谱。
方法学：细胞总RNA样品检测合格后，根据mRNA的不同特性，由于真核生物mRNA3’末端具有polyA尾的结构，所以选用带有Oligo(dT)的磁珠进行富集纯化。向纯化得到的mRNA中加入片段化缓冲液使其片段化成为短片段，再以片段化后的mRNA为模板，用六碱基随机引物合成cDNA第一链，并加入缓冲液、dNTPs、RNaseH和DNA聚合酶I合成cDNA第二链，经过QIAQuick PCR试剂盒纯化并加EB缓冲液洗脱。洗脱纯化后的双链cDNA再进行末端修复、加碱基A、加测序接头处理(P5接头序列：AATGATACGGCGACCACCGAGATCTACAC，SEQ ID NO:27；P7接头序列：ATCTCGTATGCCGTCTTCTGCTTG，SEQ ID NO:28)，然后经琼脂糖凝胶电泳回收具有目的大小的片段并进行PCR扩增(引物序列：ACACTCTTTCCCTACACGACGCTCTTCCGATCT，SEQ ID NO:29；AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC，SEQ ID NO:30。PCR反应程序如下：95℃预变性30s，95℃变性10s，60℃退火30s，72℃延伸30s，40个循环)，从而完成整个文库制备工作。
文库构建完成后，先使用Qubit3.0进行初步定量，稀释文库至1ng/μl，随后使用Agilent2100生物分析仪对文库的插入物大小(insert size)进行检测以保证文库质量。
质量合格的文库用Illumina平台进行测序。测序策略为PE150。
本实施例中共2个样品，分别为PepN样品和PepNC对照样品，其中PepN代表N蛋白全长，用溶剂DMSO溶解N蛋白，PepNC代表空白对照，仅添加了溶剂DMSO。下表2为转录组测序分析结果统计表。
表2 样品分析结果统计表
本实施例中差异表达基因的结果如下表3所示。
表3 组间比较得到的差异表达基因数目
为了研究不同处理或者不同阶段间差异表达基因的变化情况，实施了组间特有和共有的差异基因分析。
根据PepN全长样品实验组对比PepNC空白对照组的上调基因和下调基因，绘制出图6所示的差异表达基因火山图。
从mRNA水平检测，RNAseq测序结果显示PepN全长样品实验组对比PepNC空白对照组发生明显表达差异的TCR结构如下：TRAV13-2、TRAV25、TRAJ29、TRBV4-1、TRBV6-5、TRBD1、TRBJ2-2P。
实施例5.对特异性T细胞的单细胞建库(10x Genomics Chromium)和TCR的序列测定
对实施例3的特异性T细胞进行了单细胞建库(10X Genomics Chromium)(参见Elham Azizi等人，“Single-cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment”，Cell，2018年8月23日，174(5)：1293-1308；Tomonori Hosoya等人，“High-Throughput Single-Cell Sequencing of both TCR-β Alleles”，J.Immunol.，2018年12月1日，201(11)：3465-3470)。具体方法如下。
使用Chromium Single Cell 5’Library Construction Kit(购自10x Genomics,Inc.)，取出单细胞MasterMix(10X Genomics)于冰上化冻备用，手持芯片边缘将芯片放入芯片架，将培养的TCR-T细胞用含0.04％BSA的PBS重悬，同时通过台盼蓝染色或使用细胞计数仪对细胞活性和细胞浓度进行鉴定，以使细胞浓度保持在700-1200个/μl浓度，细胞活性>80％；混合单细胞悬液、MasterMix和水。根据目标捕获细胞数和细胞浓度确定上样体积和水的体积，先向MasterMix中加入水再加入细胞悬液，细胞悬液加入MasterMix前用移液器充分重悬细胞悬液。不可直接混合细胞和水；将MasterMix、水和细胞的混合物，GelBeads以及PartitioningOil(10XGenomics)分别依次加入到芯片中。尽量减少芯片装载和运行的间隔时间；运行文库构建程序。
通过二代测序技术进行TCR的序列测定。具体方法如下。
取出Box1(illunima)中的HT1(杂交缓冲液)放在4℃冰箱中融化；取出Box1中Reagent Cartridge放入装有足量室温去离子水的水中，淹没该Reagent Cartridge底部，并勿使水位超过该Reagent Cartridge上的最高水位线。将Flow Cell(未使用的测序芯片处于高盐溶液中浸泡)从储存在2℃-8℃环境中的测序试剂盒Box2中取出，室温平衡30min以上，PR2Reagent Cartridge暂时存放于4℃冰箱待用。Box1中Reagent Cartridge解冻2小时后，取出翻转10次以混匀试剂。从底部检查Reagent Cartridge，确保试剂解冻且槽中无冰晶。轻敲夹盒以使液体掉落在Reagent Cartridge底部，且确保槽内无气泡，观察每个孔的锡纸表面是否有水滴。依照文库混合(pooling)表将待上机的杂交文库混合，完成待上机文库混 合并做好标记。取20μl 1N NaOH溶液和180μl的无核酸酶水，震荡混匀后离心，配制成0.1N的NaOH溶液。使用pH试纸检测pH范围在12-13左右。取5μl的0.1N NaOH溶液和5μl 4nmol/μl的上机文库，震荡混匀后离心，室温变性5min，此时上机文库的浓度为2nmol/μl。变性5min后，取出放在4℃冰箱中的HT1，向离心管中加入990μl的HT1，震荡混匀后离心，此时上机文库浓度为20pmol/μl。将上机文库稀释至上机文库终浓度并上机测序，获得了多个TCRα链和β链，分别具有如下V区、D区、J区序列。
TRAV13-2：
CDR1：NSASDY(SEQ ID NO:2)
CDR2:IRSNMDK(SEQ ID NO:3)
CDR3:YFCAEN(SEQ ID NO:4)
TRAV25:
CDR1:TTLSN(SEQ ID NO:5)
CDR2:LVKSGEV(SEQ ID NO:6)
CDR3:TYFCAG(SEQ ID NO:7)
TRAJ29:
NSGNTPLVFGKGTRLSVIA(SEQ ID NO:8)
TRBV4-1:
CDR1:MGHRA(SEQ ID NO:9)
CDR2:YSYEKL(SEQ ID NO:10)
CDR3:YLCASSQ(SEQ ID NO:11)
TRBV6-5:
CDR1:MNHEY(SEQ ID NO:12)
CDR2:SVGAGI(SEQ ID NO:13)
CDR3:YFCASSY(SEQ ID NO:14)
TRBD1:
GTGGAPCPGQGPPVDRGPLS(SEQ ID NO:15)
TRBJ2-2P:
LRGAAGRLGGGLLVL(SEQ ID NO:16)
通过将TCRα链和β链配对，获得了如下例示的两种TCR，分别为：
1)TCR-1：TRAV13-2-J29-TRBV4-1-D1-J2-2P
2)TCR-2：TRAV25-J29-TRBV6-5-D1-J2-2P
实施例6.TCR表达载体的设计
设计了用表达载体pMSGV1(载体来源：Addgene)表达TCRα链基因序列全长和TCRβ链基因序列全长。将编码如下TCR的核酸序列克隆入载体pMSGV1中进行表达。
1)TRAV13-2-J29-TRBV4-1-D1-J2-2P
α全长：
GESVGLHLPTLSVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDIRSNMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAENNSGNTPLVFGKGTRLSVIAIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:17)
可变区：
GESVGLHLPTLSVQEGDNSIINCAYSNSASDYFIWYKQESGKGPQFIIDIRSNMDKRQGQRVTVLLNKTVKHLSLQIAATQPGDSAVYFCAEN(SEQ ID NO:18)
β全长：
DTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTGGAPCPGQGPPVDRGPLSLRGAAGRLGGGLLVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:19)
可变区：
DTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQ(SEQ ID NO:20)
2)TRAV25-J29-TRBV6-5-D1-J2-2P
α全长：
GQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGNSGNTPLVFGKGTRLSVIAIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:21)
可变区：
GQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAG(SEQ ID NO:22)
β全长：
NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYGTGGAPCPGQGPPVDRGPLSLRGAAGRLGGGLLVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:23)
可变区：
NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSY(SEQ ID NO:24)
为了提高外源性TCRα链和β链之间的配对，避免与内源性TCRα链和β链发生错配，利用小鼠的C区(恒定区)序列(参见Stephanie L.Goff等人，“Enhanced receptor expression and in vitro effector function of a murine-human hybrid MART-1-reactive T cell receptor following a rapid expansion”，Cancer Immunol Immunother.2010 Oct；59(10):1551–1560.)代替人源的C区序列。此外，在表达载体中设计表达绿色荧光蛋白(GFP)的基因，以用于评估转染效率。
采用健康志愿者或新冠恢复期患者的外周血制备了TCR-T细胞。具体方法如下。
1)PBMC分离：
取2支灭菌的15ml离心管，在第1支离心管中加入5ml淋巴细胞分离液，第2支离心管中加入5ml新鲜全血样本和5ml无菌PBS溶液混匀。将第2支离心管中的稀释全血缓慢加入第1支离心管的Ficoll液表面。2000rpm/min离心20分钟后可观察到细胞分层。将中间的白色云雾状狭带吸取到新的15ml离心管中，加入6-8ml PBS溶液后吹打混匀，进行洗涤。1000rpm/min离心10分钟后弃去上清，按上述操作重复洗涤1次。第二次洗涤后加入1ml RPMI 1640完全培养基重悬细胞，即为PBMC悬液。
TCR逆转录病毒转染前，在24孔板中将1ml新鲜分离的PBMC(约1X106个细胞)用50U/ml的IL-2和50ng/ml的OKT3激活培养48小时。将500μl激活的PBMC(约5X105个细胞)均匀铺在用5μg/孔的纤维连接蛋白包被好的24孔板中，每个样本的PBMC铺12个孔。
在前6个孔中加入浓缩的特异性TCR逆转录病毒载体pMSGV1(由此生成的转基因T细胞即为TCR-T细胞)。每孔细胞中继续加入100U/ml的IL-2和4μg/ml的硫酸鱼精蛋白，混匀。将加好逆转录病毒的24孔板放置在水平离心机上于800g/min，32℃条件下离心感染2小时。离心感染后将24孔板放置在37℃，5％CO2的培养箱中。将感染相同病毒液的6孔细胞两两混合为3个副孔继续培养。
使用逆转录病毒体系(参见Esther Drent等人，“Combined CD28 and 4–1BB costimulation potentiates affinity-tuned Chimeric Antigen Receptor-engineered T cells”，Clin  Cancer Res.2019年7月1日，25(13)：4014–4025)将包含上述各TCR的表达载体转染入T细胞中。将逆转录病毒包装293T细胞培养于37℃、5％CO2的环境中，用含有10％热灭活胎牛血清的DMEM培养基培养。传代两次后培养至细胞合适状态时提前两天将1X 106个细胞转入10cm的培养皿中进行培养，以10mL完全培养基培养使细胞在进行转染前达到80％的融合率。
2)TCR-T细胞的培养、冻存及复苏
各TCR-T细胞及阴性对照细胞的培养基为RPMI 1640完全培养基，再辅助添加IL-2以维持生长，必要时添加OKT3刺激培养。每天观察细胞的生长状况，每隔48小时进行半量换液并添加IL-2(30ng/mL of OKT3、50U/mL IL-2)。冻存时将细胞离心弃上清后加入1ml PBMC冻存液，迅速放入-80℃冰箱。复苏时将冻存的细胞从液氮罐或-80℃冰箱取出后迅速放入37℃水浴锅中5分钟，待细胞完全融化后用6-8ml的RPMI 1640完全培养基洗涤2遍后备用。
实施例7.各TCR-T细胞的体外功能性研究
将实施例6制备的各TCR-T细胞进行了体外功能研究，初步验证了其安全性及有效性。
将5x104个靶细胞(即，K562-HLA-A0201细胞)加入96孔板，使用50μg/ml2019-nCoV-N蛋白和10μg/mlβ2M刺激24小时，活化靶细胞。然后，加入1x105个TCR-T细胞共培养24小时后收集上清后使用市售的干扰素-γ(INF-γ)ELISA试剂盒(R&D Systems，DY008)，按照制造商的说明书，将标准品(R&D Systems，DY008)、待测样本分别加入到预包被的透明酶标包被板中，温育足够时间后，洗涤除去未结合的成分，再加入酶标工作液，温育足够时间后，洗涤除去未结合的成分。依次加入干扰素-γ(INF-γ)ELISA试剂盒中的底物A、B，底物(TMB)在辣根过氧化物酶(HRP)催化下转化为蓝色产物，在酸的作用下变成黄色，颜色的深浅与样本中的干扰素-γ(INF-γ)浓度呈正相关，450nm波长下测定OD值，根据标准品和待测样品的OD值，计算出待测样本中干扰素 -γ(INF-γ)的含量。结果如图7所示，其中表达实施例5和6中的TCR-1和TCR-2的T细胞分别表示为TCR-1-T和TCR-2-T，所述TCR-T细胞+K562-HLA-A0201细胞组的上清液中INF-γ的含量显著高于T细胞+K562-HLA-A0201细胞组。这表明，TCR-T细胞+N蛋白+K562-HLA-A0201细胞组中的T细胞同样地可以被N蛋白活化，并具有特异性靶向SARS-Cov-2病毒N蛋白的能力。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。

Claims (18)

  1. 分离的或纯化的T细胞受体，所述T细胞受体也简称为TCR，其特征在于，与新型冠状病毒核衣壳蛋白特异性结合，所述TCR包含α链和β链，其中所述α链和β链各包含三个互补决定区，所述互补决定区也简称为CDR，并且α链的CDR3的氨基酸序列选自SEQ ID NO:4、7和与所述序列具有1个或2个氨基酸残基改变的变体，β链的CDR3的氨基酸序列选自SEQ ID NO:11、14和与所述序列具有1个或2个氨基酸残基改变的变体。
  2. 根据权利要求1所述的TCR，其中，所述α链的CDR3的氨基酸序列和β链的CDR3的氨基酸序列是：
    (i)SEQ ID NO:4所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:11所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；或
    (ii)SEQ ID NO:7所示的α链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:14所示的β链CDR3氨基酸序列或与所述序列具有1个或2个氨基酸残基改变的变体。
  3. 根据权利要求2所述的TCR，其中，所述α链的CDR3的氨基酸序列和β链的CDR3的氨基酸序列是：
    (i)SEQ ID NO:4所示的α链CDR3氨基酸序列和SEQ ID NO:11所示的β链CDR3氨基酸序列；
    (ii)SEQ ID NO:7所示的α链CDR3氨基酸序列和SEQ ID NO:14所示的β链CDR3氨基酸序列。
  4. 根据权利要求2所述的TCR，其中，所述α链包含的三个CDR的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
    (i)SEQ ID NO:2、3、4所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:9、10、11所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；或
    (ii)SEQ ID NO:5、6、7所示的α链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体；以及SEQ ID NO:12、13、14所示的β链CDR1、CDR2、CDR3氨基酸序列或与所述序列分别具有1个或2个氨基酸残基改变的变体。
  5. 根据权利要求4所述的TCR，其中，所述α链包含的三个CDR的氨基酸序列和β链包含的三个CDR的氨基酸序列是：
    (i)SEQ ID NO:2、3、4所示的α链CDR1、CDR2、CDR3氨基酸序列和SEQ ID NO:9、10、11所示的β链CDR1、CDR2、CDR3氨基酸序列；或
    (ii)SEQ ID NO:5、6、7所示的α链CDR1、CDR2、CDR3氨基酸序列和SEQ ID NO:12、13、14所示的β链CDR1、CDR2、CDR3氨基酸序列。
  6. 根据权利要求1至5中任一项所述的TCR，其中，所述TCR包含SEQ ID NO:18或22所示的α链可变区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:20或24所示的β链可变区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
  7. 根据权利要求6所述的TCR，其中，所述TCR还包含恒定区。
  8. 根据权利要求7所述的TCR，其中，所述恒定区是小鼠恒定区。
  9. 根据权利要求6所述的TCR，其中，所述TCR包含SEQ ID NO:17或21所示的α链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和SEQ ID NO:19或23所示的β链序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
  10. 核酸分子，其特征在于，编码权利要求1至9中任一项所述的TCR。
  11. 载体，其特征在于，包含编码权利要求10所述的核酸分子。
  12. 宿主细胞，其特征在于，包含权利要求1至9中任一项所述的TCR、权利要求10 的核酸分子或权利要求11的载体。
  13. 根据权利要求12所述的宿主细胞，其中，所述宿主细胞是T细胞或者所述宿主细胞是干细胞。
  14. 根据权利要求13所述的宿主细胞，其中，所述宿主细胞是人CD4+辅助T细胞或人CD8+细胞毒性T细胞，或者是人CD4+辅助T细胞和人CD8+细胞毒性T细胞的混合细胞群；或者，所述宿主细胞是造血干细胞。
  15. 药物组合物，其特征在于，包含权利要求12－14中任一项所述的宿主细胞。
  16. 制备权利要求12－14中任一项所述的宿主细胞的方法，其特征在于，包括以下步骤：
    (a)用SARS-CoV-2病毒N蛋白刺激来自人类受试者的T细胞；
    (b)使刺激后的所述T细胞接触表达人HLA等位基因、呈递SARS-CoV-2病毒N蛋白肽的细胞，
    (c)选择由所述接触活化的T细胞；
    (d)自所述活化的T细胞获得编码TCRα链和TCRβ链的核酸序列；和
    (e)将编码TCRα链和TCRβ链的核酸序列导入人T细胞或人干细胞。
  17. 根据权利要求16所述的方法，其中，步骤(c)是基于由所述接触活化的T细胞表达的活化标志物进行选择。
  18. 根据权利要求15的药物组合物的用途，其特征在于，用于制备预防和/或治疗新型冠状病毒感染的药物。
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