WO2023030312A1 - 一种用于艾滋病病毒感染基因治疗的基因序列构建体 - Google Patents

一种用于艾滋病病毒感染基因治疗的基因序列构建体 Download PDF

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WO2023030312A1
WO2023030312A1 PCT/CN2022/115822 CN2022115822W WO2023030312A1 WO 2023030312 A1 WO2023030312 A1 WO 2023030312A1 CN 2022115822 W CN2022115822 W CN 2022115822W WO 2023030312 A1 WO2023030312 A1 WO 2023030312A1
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gene
hiv infection
hiv
sequence
linker
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French (fr)
Chinese (zh)
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吴昊泉
孙保贞
党颖
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Kanglin Biotechnology Hangzhou Co Ltd
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Kanglin Biotechnology Hangzhou Co Ltd
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Priority to JP2024513388A priority Critical patent/JP2024532426A/ja
Priority to EP22863447.3A priority patent/EP4397748A4/en
Priority to CA3230492A priority patent/CA3230492A1/en
Priority to CN202280056356.XA priority patent/CN117980465A/zh
Priority to AU2022338817A priority patent/AU2022338817A1/en
Priority to US18/687,681 priority patent/US20240391981A1/en
Publication of WO2023030312A1 publication Critical patent/WO2023030312A1/zh
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • C07K16/10—RNA viruses
    • C07K16/112—Retroviridae (F), e.g. leukemia viruses
    • C07K16/114—Lentivirus (G), e.g. human immunodeficiency virus [HIV], feline immunodeficiency virus [FIV] or simian immunodeficiency virus [SIV]
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    • C07K16/10—RNA viruses
    • C07K16/112—Retroviridae (F), e.g. leukemia viruses
    • C07K16/114—Lentivirus (G), e.g. human immunodeficiency virus [HIV], feline immunodeficiency virus [FIV] or simian immunodeficiency virus [SIV]
    • C07K16/1145—Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

  • the invention belongs to the technical field of gene therapy/biomedicine, and in particular relates to a gene sequence construct used for gene therapy of AIDS virus (HIV) infection.
  • the gene sequence construct can be used for gene therapy against HIV infection.
  • the gene sequence construct can be used in vivo and in vitro to express polytropic neutralizing antibody proteins with broad-spectrum and high-efficiency neutralization of HIV virus activity, and can be used for clinical research on gene therapy drugs for HIV infection delivered by recombinant viruses or non-viral vectors and new drug development.
  • AIDS immunodeficiency syndrome
  • human immunodeficiency virus human immunodeficiency virus
  • HIV human immunodeficiency virus
  • the present invention creates a series of anti-HIV gene therapy constructs based on recombinant viral vectors. For example, a variety of broad-spectrum neutralizing antibodies against HIV and single-chain antibody variable region fragments (scFv) of neutralizing antibodies against human CD4 receptors are combined with Fc fragments in the constant region of human antibodies and HIV membrane fusion inhibition The combination of peptides is a simple and efficient expression framework.
  • scFv single-chain antibody variable region fragments
  • these gene sequence constructs for HIV infection gene therapy comprise one or more gene coding sequences of antibody molecules with the ability to inhibit HIV infection and one or more polypeptides with the ability to inhibit HIV infection (composed of 2-50 amino acid residues base) gene coding sequence to achieve the expression of a fusion protein molecule encoded by a single gene comprising an anti-HIV infection antibody molecule and a polypeptide, which has two or more targets.
  • said antibody molecule comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.
  • said light chain constant region comprises a light chain constant region of a kappa or lambda light chain.
  • said light chain variable region comprises a light chain variable region of a kappa or lambda light chain.
  • the above-mentioned gene sequence construct comprises two or more gene coding sequences of antibody molecules capable of inhibiting HIV infection.
  • the above-mentioned gene sequence construct may contain two or more gene coding sequences of polypeptides capable of inhibiting HIV infection. These polypeptides consist of 2-50 amino acid residues.
  • the fusion protein molecule encoded by a single gene and comprising an anti-HIV infection antibody molecule and a polypeptide has four or more action targets.
  • the fusion protein molecule encoded by a single gene and comprising an anti-HIV infection antibody molecule and a polypeptide has five or more action targets.
  • the fusion protein molecule encoded by a single gene and comprising an anti-HIV infection antibody molecule and a polypeptide has six or more action targets.
  • the gene sequence construct described in any one of the above items comprises two or more gene coding sequences of antibody molecules capable of inhibiting HIV infection and one or more gene coding sequences of polypeptides capable of inhibiting HIV infection.
  • the fusion protein molecule encoded by a single gene comprising an antibody molecule against HIV infection and a polypeptide has three or more action targets.
  • the coding sequence of the linker polypeptide is directly or indirectly concatenated.
  • the gene sequence construct described in any one of the above, wherein the gene coding sequence of one or more antibody molecules with the ability to inhibit HIV infection comprises anti-HIV-1-gp160 (including its cleavage products gp120 and gp41)
  • the genetic coding sequence of an antibody molecule comprises anti-HIV-1-gp160 (including its cleavage products gp120 and gp41)
  • the gene sequence construct according to any one of the above, wherein the gene coding sequence of one or more antibody molecules with the ability to inhibit HIV infection comprises the gene coding sequence of an antibody molecule binding to the human CD4 receptor site.
  • the gene sequence construct according to any one of the above, wherein the one or more gene coding sequences of polypeptides capable of inhibiting HIV infection include gene coding sequences of polypeptides inhibiting fusion of HIV and CD4+ T cell membranes.
  • the gene sequence construct described in any one of the above which comprises two or more gene coding sequences of antibody molecules with the ability to inhibit HIV infection and one or more gene coding sequences of polypeptides with the ability to inhibit HIV infection, wherein
  • the gene coding sequences of the two or more antibody molecules with the ability to inhibit HIV infection comprise the gene coding sequences of antibody molecules against HIV-1-gp160 (including its cleavage products gp120 and gp41) and the binding human CD4 receptor
  • the gene coding sequence of the antibody molecule at the body site, and the gene coding sequence of one or more polypeptides with the ability to inhibit HIV infection include the gene coding sequence of a polypeptide that inhibits fusion between HIV and CD4+ T cell membrane.
  • the gene sequence construct described in any one of the above which includes (i) light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of anti-HIV-1-gp160 (comprising its cleavage products gp120 and gp41) monoclonal antibody and Gene coding sequences of heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), (ii) light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) and heavy chain complementarity determining regions of monoclonal antibodies that bind to the human CD4 receptor site (HCDR1, HCDR2 and HCDR3), (iii) the gene coding sequence of the Fc fragment of the human IgG constant region, and (iv) the gene coding sequence of a short peptide that inhibits the fusion of HIV and CD4+ T cell membranes, wherein the antibody light
  • the coding sequences of the chain and the heavy chain can be directly or indirectly concatenated through the coding sequence of
  • the gene sequence construct described in any one of the above which includes (i) light chain variable region (VL) and heavy chain variable region (VL) of anti-HIV-1-gp160 (including its cleavage products gp120 and gp41) monoclonal antibody Region (VH) gene coding sequence, (ii) light chain variable region (VL) and heavy chain variable region (VH) gene coding sequence of monoclonal antibody binding to human CD4 receptor site, (iii) human The gene coding sequence of the Fc fragment of the IgG constant region, and (iv) the gene coding sequence of a short peptide that inhibits fusion between HIV and the CD4+ T cell membrane, wherein the light chain variable region (VL) and heavy chain variable region (VH) of the antibody
  • the coding sequence of can be directly or indirectly concatenated through the coding sequence of the linker polypeptide in no particular order.
  • the gene sequence construct according to any one of the above, further comprising a promoter located upstream of the gene coding sequence of the antibody molecule with the ability to inhibit HIV infection and the gene coding sequence of the polypeptide with the ability to inhibit HIV infection.
  • the gene sequence construct described in any one of the above which additionally comprises a secretory signal peptide coding sequence located upstream of the gene coding sequence of the antibody molecule with the ability to inhibit HIV infection and the gene coding sequence of the polypeptide with the ability to inhibit HIV infection.
  • the gene sequence construct described in any one of the above, which comprises the gene coding sequence of the first antibody molecule having the ability to inhibit HIV infection, the second gene coding sequence of the antibody molecule having the ability to inhibit HIV infection, and the gene coding sequence having the ability to inhibit HIV infection The gene coding sequence of the polypeptide of infectivity, it is selected from:
  • VL2 and VH2 are the variable region fragments of the light chain and heavy chain of the first antibody molecule respectively;
  • VL1 and VH1 are the variable region fragments of the light chain and heavy chain of the second antibody molecule respectively;
  • CH2-CH3 are human
  • the Fc fragment of the IgG constant region, linker is a linker polypeptide;
  • peptide inhibitor is a polypeptide that inhibits HIV infection (for example, a polypeptide that inhibits the fusion of HIV and CD4+ T cell membranes).
  • the above gene sequence construct which is VL2-linker-VH2-linker-VL1-linker-VH1-linker-CH2-CH3-linker-peptide inhibitor, or the combination of VL2 and VH2 or VL1 and VH1 according to different Constructs arranged in sequence.
  • linker polypeptide selected from the following amino acid sequences: GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS) 4 , (GGGGS) 5 , (GGGGS) 6 , and (GGGGS) 7 , or other optional linker polypeptide sequences.
  • the polypeptide (peptide inhibitor) that inhibits HIV and CD4+ T cell membrane fusion can be selected from membrane fusion inhibitory polypeptides P52, C34, T20 and the like.
  • the sequence of the membrane fusion inhibitory polypeptide P52 includes SEQ ID NO: 5 or its homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical; the polypeptide sequence of C34 Comprising SEQ ID NO: 6 or having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical homologous sequences thereto; the polypeptide sequence of T20 comprising SEQ ID NO: 7 or having therewith Homologous sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical.
  • the present invention also provides a viral vector genome comprising any of the aforementioned constructs and a corresponding viral vector system comprising the genome.
  • the above-mentioned viral vector system may be a lentiviral vector system or an adeno-associated virus vector system.
  • the lentiviral vector system can include the viral vector genome of any of the above-mentioned constructs and other nucleotide sequences encoding and expressing the packaging components required for the production of lentiviruses, which will be introduced into production cells to produce The lentiviral particle of the construct genome.
  • the adeno-associated virus vector system can comprise the viral vector genome of any of the above-mentioned constructs and other nucleotide sequences encoding and expressing the packaging components required for the production of adeno-associated virus, which will be introduced into production cells to produce Adeno-associated virus particles of the construct genome described above.
  • Any virus particle produced as described above which comprises the genome of any of the constructs described in the foregoing, can express anti-HIV neutralizing antibody molecules after transduction of cells, and can be used for administration to patients to inhibit or Prevent HIV infection.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned virus particle, and a pharmaceutically acceptable carrier or diluent, or cells transduced by the above-mentioned lentivirus particle in vitro, These include, but are not limited to, transduced muscle cells, liver cells, or CD4+ T cells.
  • One of the above virus particles or the pharmaceutical composition comprising the above virus particles can be used for injection into the body to express two or more target antibody molecule proteins, and the mature molecules can pass two Sulfur bonds form dimers, which can effectively and broadly block the infection process of HIV to human CD4+ T cells by binding to multiple binding sites involved in different steps of HIV infection of human CD4+ T cells, for use in Gene therapy for HIV infection, so as to achieve long-term treatment for HIV-infected people.
  • the present invention provides a method for inhibiting HIV infection, comprising administering the above-mentioned virus particle or pharmaceutical composition to cells.
  • the cells mentioned therein include muscle cells, liver cells, or CD4+T cells and the like.
  • the above-mentioned cells can be transduced in vitro or in vivo with one of the above-mentioned virus particles or pharmaceutical composition.
  • the present invention provides a method of treating HIV infection in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a viral particle or pharmaceutical composition as described above.
  • the subjects mentioned therein include HIV early-infected persons or HIV-infected persons who are receiving cocktail drug therapy or HIV-infected persons who are resistant to cocktail drug therapy.
  • the method of administering the drug to the above-mentioned subject is through intramuscular injection of one of the above-mentioned virus particles or the pharmaceutical composition.
  • one of the viral particles or pharmaceutical compositions described above or CD4+ T cells transduced therethrough is injected intravenously.
  • Virosomes and pharmaceutical compositions injected into the body by the method described above can express anti-HIV protein molecules with multiple targets and be secreted into the blood, can act on multiple nodes of HIV infection, and can It effectively blocks the HIV infection path and effectively avoids the loss of the ability to inhibit HIV infection due to HIV escape mutations, so as to achieve long-term or even permanent therapeutic effect on HIV infection with a single injection.
  • Antibody-like molecules composed of multiple single-chain antibody variable region fragments (scFv) based on the above-mentioned expression framework were delivered to mice by lentivirus and adeno-associated virus vectors, showing effective blood drug concentrations and strong In vitro cytology HIV-1 virus neutralizing activity, as well as the broad-spectrum neutralizing ability to CXCR4 and CCR5 two tropic viruses at the same time, is a technical route for the development of anti-HIV broad-spectrum neutralizing antibody gene therapy drugs with great potential.
  • scFv single-chain antibody variable region fragments
  • the scope of application of the present invention includes various forms of anti-HIV gene therapy based on the genetic expression of broad-spectrum neutralizing antibodies.
  • Figure 1 Schematic representation of the mature molecular structure of the expected tritropic anti-HIV neutralizing antibody.
  • Fig. 2 Schematic diagram of gene sequence composition of tritropic anti-HIV neutralizing antibody.
  • Figure 3 Schematic representation of the mature molecular structure of the expected amphotropic anti-HIV neutralizing class antibody.
  • FIG. 1 Schematic diagram of the gene sequence composition of the amphotropic anti-HIV neutralizing antibody.
  • Fig. 5 Schematic diagram of the gene construct in which the gene sequence of amphitropic (KL-BsHIV01) or tritropic (KL-BsHIV01-003) anti-HIV neutralizing antibody is cloned into a lentiviral vector.
  • FIG. 7 Map of the latest generation adeno-associated virus vector pAAV-MCS-CMV-EGFP (reverse) used in the present invention.
  • Fig. 8 Schematic diagram of gene constructs in which gene sequences of amphitropic (KL-BsHIV01) or tritropic (KL-BsHIV01-003) anti-HIV neutralizing antibodies are cloned into adeno-associated virus vectors.
  • FIG. 9 Western blot analysis to detect the expression of anti-HIV neutralizing antibodies in 293T cells. After transfecting 293T cells with the gene sequence of the anti-HIV neutralizing antibody cloned into the gene construct of the adeno-associated virus vector, the same volume of 293T cell culture supernatant was taken as a sample for Western blot analysis under non-reducing conditions , Anti-HIV neutralizing antibody protein was detected with goat anti-human IgG1 Fc fragment antibody. M, prestained protein size marker; 1, blank plasmid transfection control; 2, KL-BsHIV01; 3, KL-BsHIV01-003; 4, KL-BsHIV01-C34; 5, KL-BsHIV01-T20. The macromolecular bands (much larger than 180kDa) in Western blot were predicted anti-HIV neutralizing antibody molecules in the form of dimers (under non-reducing conditions).
  • FIG. 10 Western blot analysis to detect the expression of anti-HIV neutralizing antibodies in C2C12 myotube cells.
  • the same volume of C2C12 cell culture supernatant was taken as a sample for Western blot analysis under non-reducing conditions, and the protein of anti-HIV neutralizing antibody was detected with goat anti-human IgG1 Fc fragment antibody.
  • M prestained protein size markers; 1, KL-BsHIV01; 2, KL-BsHIV01-003; 3, KL-BsHIV01-C34; 4, KL-BsHIV01-T20.
  • the macromolecular bands (much larger than 180kDa) in Western blot were predicted anti-HIV neutralizing antibody molecules in the form of dimers (under non-reducing conditions).
  • the band corresponding to 180kDa is a non-specific band.
  • FIG. 1 Neutralizing activity of 293T cell culture supernatant transduced with HIV neutralizing antibody lentiviral gene therapy vector to HIV virus.
  • VCN copy number of lentiviral vectors after transduction of 293T cells.
  • FIG. 13 Expression of anti-HIV neutralizing antibody adeno-associated virus gene therapy vector in BALB/c mice after intramuscular injection. The levels of anti-HIV neutralizing antibodies secreted into mouse sera were determined by ELISA method.
  • Figure 14 Neutralizing activity of amphitropic and tritropic HIV neutralizing antibodies secreted into mouse serum at different concentrations against HIV strains. The data in the figure are the mean ⁇ SD of three parallel determinations for each sample. This experiment has been repeated more than two times.
  • bNAb Broadly neutralizing antibodies
  • bNAb Broadly neutralizing antibodies
  • CD4+ T cells CD4+ T cells
  • the infectious activity of the virus on CD4+ T cells represents a promising approach to prevent or combat HIV-1 infection.
  • the specific mechanism of its production is not clear.
  • Most HIV-1 infected persons can only produce non-neutralizing antibodies.
  • no research has successfully induced the production of broad-spectrum neutralizing antibodies in healthy subjects through standard immunization methods. .
  • recombinantly derived broadly neutralizing antibodies against the HIV-1 virus can effectively reduce the viral load in a patient and help control, if not completely eliminate HIV from the body. The infection develops into AIDS.
  • neutralizing antibodies from recombinant sources can also greatly reduce toxic and side effects, and at the same time increase patients' compliance with medication.
  • recombinant broad-spectrum neutralizing antibodies can be used not only as vaccine replacement products under specific circumstances to prevent the occurrence of HIV infection, but also as antiviral drugs in different disease stages.
  • Anti-HIV infection gene therapy drugs delivered by recombinant viruses or non-viral vectors can stably express neutralizing antibodies or antibody-like macromolecules in the body for a long time in the form of genome integration or non-integration, and can be effective for a long time or even for a lifetime after one treatment, greatly reducing the Production and use costs of macromolecular antibody drugs.
  • the present invention adopts a multiphilic antibody molecular structure, that is, the antigen binding region is composed of two or more single-chain variable region fragments (scFv) of monoclonal broad-spectrum neutralizing antibodies connected in series by a linker polypeptide (linker) .
  • scFv single-chain variable region fragments
  • linker linker polypeptide
  • the specific embodiment includes constructing a series of antibody molecular gene constructs comprising amphotropic/tritropic antibody single-chain variable region fragments (scFv), and cloning them into recombinant adeno-associated virus and recombinant lentiviral vectors. Then package the corresponding adeno-associated virus and lentivirus in 293T cells, and infect 293T cells and differentiated and undifferentiated muscle cell lines with a certain biological titer virus. The antibody molecules produced in the cell supernatant are subjected to quantitative and qualitative detection, and their neutralizing activity against the HIV-1 wild virus strain is tested in the infection activity analysis of the HIV-1 wild virus strain and the virus-sensitive reporter gene cell line TZM-bl .
  • scFv amphotropic/tritropic antibody single-chain variable region fragments
  • Antibody Can be an immunoglobulin, antigen-binding fragment, or protein molecule derived therefrom that can specifically recognize and bind an antigen (eg, HIV-1 gp41 antigen).
  • Antibody in the present invention is a broad definition covering various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, polytropic antibodies (e.g., amphiphilic antibodies, triphilic antibodies), and Antibody fragments, so long as they have specific antigen-binding activity. Specific examples of antibodies include intact immunoglobulins as well as antibody variants and fragments that retain binding affinity for the antigen.
  • antibody fragments include, but are not limited to, variable region fragments (Fv), antigen-binding fragments (e.g., Fab, Fab', Fab'-SH, or F(ab') 2 produced after proteolysis), single-chain antibody Molecules (eg, scFv), diabodies, nanobodies, and polytropic antibodies formed from combinations of antibody fragments.
  • Fv variable region fragments
  • Antibody fragments include antigen-binding fragments produced by modification of intact antibodies or antigen-binding fragments synthesized de novo using recombinant DNA techniques.
  • Single-chain antibody is a molecule obtained by genetic engineering, which contains one or more antibody light chain variable regions (VL) and heavy chain variable regions (VH), and the fragments are connected in series by a suitable linker polypeptide become a fused single-chain molecule.
  • VL antibody light chain variable regions
  • VH heavy chain variable regions
  • the tandem sequence of VL and VH in the single-chain antibody molecule usually does not affect its antigen-binding function, so the single-chain antibody composed of two tandem methods (VL-VH or VH-VL) will be used.
  • An antibody can have one or more antigen combining sites. When there is more than one antigen binding site, these binding sites may be the same or different. For example, a naturally occurring immunoglobulin has two identical antigen-binding sites, while Fab fragments produced from papain hydrolysis of immunoglobulins have only one antigen-binding site, and amphotropic single-chain antibodies (scFv) Has two distinct antigen-binding sites.
  • scFv amphotropic single-chain antibodies
  • immunoglobulin Normally, a naturally occurring immunoglobulin consists of light and heavy chains linked by disulfide bonds. Immunoglobulin genes include gamma, alpha, delta, epsilon, mu, lambda, and kappa constant region genes as well as a myriad of immunoglobulin variable region genes. There are two types of light chains, lambda and kappa. There are five main types of heavy chains ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), which determine the functional classification of antibody molecules, namely IgG, IgA, IgD, IgE, and IgM.
  • Each heavy and light chain contains a constant region and a variable region.
  • VH denotes the variable region of an antibody heavy chain, including the heavy chain variable region of an antigen-binding fragment Fv, scFv, or Fab.
  • VL denotes the variable region of an antibody light chain, including the light chain variable region of an antigen-binding fragment Fv, scFv, or Fab. In the following examples, VH and VL work together to specifically recognize and bind antigens.
  • VH and VL comprise three spaced hypervariable regions, also called complementarity determining regions (CDRs), as well as a framework region.
  • CDRs complementarity determining regions
  • the sequences of the framework regions of different light and heavy chains are relatively conserved within the same species.
  • the framework regions of an antibody determine the positions of the CDRs in the three-dimensional structure.
  • Complementarity determining regions are primarily responsible for binding to the epitope of an antigen.
  • the three complementary determining regions on the light chain are designated LCDR1, LCDR2, and LCDR3 from N-terminus to C-terminus, respectively.
  • the three complementary determining regions on the heavy chain are designated HCDR1, HCDR2, and HCDR3 from N-terminus to C-terminus, respectively.
  • the protein sequences of VH and VL in the present invention include not only the sequences disclosed in the following examples, but also any other sequences carrying their functional fragments, or at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical homologous sequences.
  • the constant region fragment of an antibody is a relatively stable region of the amino acid sequence of the immunoglobulin except the variable region with a large change in the amino acid sequence near the N-terminus, including the constant domain in the antigen-binding fragment (located at the N-terminus of the hinge region, including light chain constant domain and heavy chain constant domain) and the constant domain of the crystallizable fragment of the heavy chain (called the Fc fragment, located C-terminal to the hinge region).
  • the Fc fragment generally refers to the last two constant region domains of immunoglobulins IgA, IgD, and IgG or the last three constant region domains of IgE and IgM.
  • the Fc fragment may also include part or all of the hinge region sequence at its N-terminus.
  • Anti-HIV-1 neutralizing antibody or antigen-binding fragment specifically binds HIV-1 envelope proteins (eg, to gp41), thereby inhibiting HIV-1 envelope-associated biological Function (eg, ability to bind target receptor).
  • HIV-1 envelope proteins eg, to gp41
  • HIV-1 envelope-associated biological Function eg, ability to bind target receptor.
  • anti-HIV-1 neutralizing antibodies or antigen-binding fragments reduce the infectious titers of HIV-1 strains of different tropisms on cells.
  • Amphotropic or multiphilic antibody This type of antibody is a recombinant molecule composed of two or more different antigen-binding domains, so it can bind two or more different antigenic determinants.
  • Amphotropic or multiphilic antibodies include molecules composed of two or more different antigen-binding domains linked by chemical synthesis or genetic engineering. Antigen binding domains can be linked via a linker polypeptide. The antigen binding domain can be a monoclonal antibody, an antigen binding fragment (eg, scFv or Fab), or a combination of antigen binding domains of different origin.
  • Linker polypeptide used to connect two protein molecules or fragments into a continuous single fusion molecule, for example, in the following examples, two or more antibody molecules or antigen-binding fragments (for example, scFv) are connected into two or a multiphilic antibody molecule with multiple antigen-binding sites; or just connect the light chain variable region (VL) and heavy chain variable region (VH) of an antibody into a single-chain antigen-binding sequence; or Antibody molecules or antigen-binding fragments are linked together with other effector molecules, for example, antigen-binding fragment scFv is linked with HIV-1 membrane fusion inhibitory polypeptide to form a fusion protein.
  • VL light chain variable region
  • VH heavy chain variable region
  • Linker polypeptides are usually rich in glycine (Gly or G) to increase the flexibility of the linker, and serine (Ser or S) or threonine (Thr or T) to increase solubility, for example, as used in some of the examples below (GGGGS) n connectors of different lengths, n can be 1 or more. But the linker polypeptide sequence used in the embodiment is not limited to this, and also includes other optional linker polypeptide sequences.
  • Antigenic determinant A specific chemical group or polypeptide sequence on a molecule, which is antigenic, that is, it can stimulate a host-specific immune response.
  • An antibody specifically binds to a specific epitope on a polypeptide, such as an antibody that specifically binds to an epitope on gp41 in the examples below.
  • HIV-1 envelope protein The HIV-1 envelope protein is first synthesized as a precursor protein of 845-870 amino acid residues in size, called HIV gp160. gp160 forms a homotrimer in the host cell, is glycosylated, and undergoes cleavage to remove the signal peptide, and then is cleaved by a protease in the cell at the position of 511/512 amino acid residues to generate gp120 and gp41 polypeptide chains. Gp120 and gp41 remain associated in a homotrimer as a gp120/gp41 protomer.
  • Mature gp120 consists of amino acid residues 31-511 of the HIV-1 envelope protein and is a highly N-glycosylated protein that constitutes the domain of the HIV-1 envelope protein trimer exposed on the envelope surface the vast majority. gp120 is responsible for binding to the human CD4 cell receptor as well as coreceptors such as the chemokine receptors CCR5 or CXCR4. gp41 is composed of 512-860 amino acid residues of the HIV-1 envelope protein, including an envelope inner domain, a transmembrane domain, and an envelope outer domain. The extra-envelope domain of gp41 includes 512-644 amino acid residues, and it combines with gp120 to form a protomer, which together constitutes a homotrimer of HIV-1 envelope protein.
  • the protruding extraenvelope domain of the HIV-1 envelope protein trimer undergoes several structural rearrangements, from a closed structure that escapes antibody recognition before fusion with the host cell membrane to binding to human CD4 cell receptors and coreceptors.
  • HIV Membrane Fusion Inhibiting Polypeptide Membrane fusion between virus and host cell is a key step for HIV to infect cells. After the glycoprotein gp120/gp41 homotrimer on the HIV envelope binds to the human CD4 cell receptor and co-receptor, it undergoes several steps of structural changes, culminating in the insertion of the integrated gp41 trimer on the HIV envelope into the host cell membrane , to complete the fusion of the virus and host cell membranes, and the genetic material of HIV enters the cell.
  • the HIV membrane fusion inhibitory polypeptide binds to the envelope protein of the virus, thereby preventing it from undergoing structural changes necessary for the fusion of the virus and the host CD4 cell membrane, thereby preventing HIV from infecting the CD4 cell.
  • HIV membrane fusion inhibitory polypeptides used in the present invention include the P52, C34, T20 sequences disclosed in the following examples or have at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical homolog
  • Gene sequence construct a vector composed of a recombinant polynucleotide sequence, which is formed by linking an expression control sequence with a nucleic acid sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements, and the other expression elements are provided by the host cell.
  • Expression vectors include all vectors of the present invention, such as plasmids and viruses (eg, recombinant lentiviruses and recombinant adeno-associated viruses) carrying integrated recombinant polynucleotide sequences.
  • the vector carries nucleic acid sequences (DNA or RNA) that allow it to replicate in a host cell, such as an origin of replication, one or more selectable marker genes, and other genetic structures disclosed in the present invention.
  • Viral vectors are recombinant nucleic acid vectors that carry at least part of the nucleic acid sequences from one or more viruses.
  • the viral vector comprises one or more nucleic acid sequences encoding the disclosed antibody or antigen-binding fragment that specifically binds HIV-1 gp160 to neutralize HIV-1.
  • the viral vector may be a recombinant adeno-associated virus (AAV) vector or a recombinant lentiviral vector.
  • AAV adeno-associated virus
  • viral vectors are replication-deficient vectors, which require other auxiliary plasmids or vectors carrying gene functions or components necessary for viral replication to be amplified and packaged in cells to produce virus particles. The purified and prepared virus vector or virus particle will not be replicated and amplified in the host cell during the treatment of the patient.
  • HIV is a virus that attacks the body's immune system. It takes the most important CD4 T lymphocytes in the human immune system as the main attack target, destroys the cells in large quantities, and makes the human body lose its immune function. Many patients with acute HIV infection experience flu-like symptoms 2-4 weeks after infection, which may last from days to weeks. There is a large amount of HIV in the blood of patients in the acute infection stage, which is highly infectious. Then enter the asymptomatic chronic infection period, also known as the HIV incubation period. However, the HIV in the patient's body is still active and continues to proliferate, which will spread HIV. The average incubation period of HIV in the human body is 8 to 9 years.
  • HIV-infected persons can live and work for many years without any symptoms. However, if HIV-infected persons do not receive any anti-HIV infection treatment, they will develop into the most serious HIV infection period after the incubation period, that is, the AIDS stage. AIDS patients have a very high viral load and can easily infect HIV to other people. The immune system is severely damaged, and the human body is prone to various diseases and malignant tumors, with a high mortality rate.
  • the goals of treatment at this stage are: to maximize and sustainably reduce the viral load; to obtain immune function reconstruction and maintenance of immune function; to improve the quality of life; to reduce HIV-related morbidity and mortality.
  • the currently commonly used anti-HIV infection method is antiretroviral combination therapy (ie, cocktail therapy), which greatly improves the curative effect of anti-HIV, and significantly improves the quality of life and prognosis of patients.
  • cocktail therapy antiretroviral combination therapy
  • cocktail therapy such as the need to maintain long-term medication compliance, and the gradual emergence of HIV drug-resistant strains still cause long-term economic and social burdens to the majority of AIDS patients, and the obvious decline in quality of life and obvious pain.
  • a broad-spectrum neutralizing antibody with HIV-1 neutralizing activity isolated from a small number of elite infected individuals can efficiently and broadly bind to the surface glycoprotein of HIV virus and neutralize the infection activity of HIV virus on human CD4+ T cells , represents a promising approach to prevent or combat HIV-1 infection.
  • neutralizing antibodies from recombinant sources can also greatly reduce toxic and side effects, and at the same time increase patients' compliance with medication.
  • the only anti-HIV neutralizing antibody approved for clinical use is Ibalizumab targeting the CD4 receptor on the surface of human T cells.
  • the infectious activity of +T cells has shown excellent curative effect on multi-drug resistant patients with HIV infection.
  • the specific broad-spectrum neutralizing antibody only targets a single epitope of a single viral protein (HIV-gp160), and the escape phenomenon caused by virus mutation is still unavoidable. Much higher than the small molecule anti-HIV drugs, the combined drug has no advantage.
  • the present invention aims to develop amphotropic and polytropic neutralizing antibodies or antibody-like macromolecular drugs, cover multiple targets to avoid escape from virus mutations, and genotype protein broad-spectrum neutralizing antibodies or antibody-like macromolecular drugs. A breakthrough in the development of anti-HIV broad-spectrum neutralizing antibody drugs.
  • Anti-HIV infection gene therapy drugs delivered by recombinant viruses or non-viral vectors long-term stable expression of neutralizing antibodies or antibody-like macromolecules in the body in a genome-integrated or non-integrated manner, and a long-term treatment (for example, the drug effect lasts for one year) or several years) or even lifetime, greatly reducing the cost of production and use of macromolecular antibody drugs.
  • the structure of the anti-HIV neutralizing antibody and antibody-like molecule used in the present invention is a triphilic anti-HIV neutralizing antibody molecule: from the N-terminus to the C-terminus of the protein molecule are respectively-signal peptide-anti-HIV-gp41 broad spectrum and antibody single chain variable region fragment (VH-(ggggs) n linker-VL)-(ggggs) n linker-single chain variable region fragment of anti-human CD4 antibody (VH-(ggggs) n linker-VL)-human IgG1 CH2-CH3-(ggggs) n linker-HIV membrane fusion inhibitor short peptide.
  • the coding sequence of the indicated gene is expressed in the cell and translated into protein to form a dimer and be secreted out of the cell.
  • Figure 1 shows the mature molecular structure of the expected anti-HIV neutralizing antibody
  • Figure 2 shows the gene structure.
  • the anti-HIV-gp41 broad-spectrum neutralizing antibody single-chain variable fragment (scFv) sequences are: signal peptide (see SEQ ID NO: 1 for the protein sequence); anti-HIV-1-gp41-MPER monoclonal antibody (10E8v4- 5R+100cF)-scFv (see SEQ ID NO:2 for protein sequence); single-chain variable region fragment scFv (see SEQ ID NO:3 for protein sequence) of anti-human CD4 monoclonal antibody (Ibalizumab), human IgG1 Fc fragment ( See SEQ ID NO:4 for the protein sequence, HIV membrane fusion inhibitory short peptide P52 (see SEQ ID NO:5 for the protein sequence), HIV membrane fusion inhibitory short peptide C34 (see SEQ ID NO:6 for the protein sequence), HIV membrane fusion inhibitory Short peptide T20 (see SEQ ID NO: 7 for the protein sequence).
  • signal peptide see SEQ ID NO: 1 for the protein sequence
  • Anti-HIV amphotropic neutralizing antibody molecule KL-BsHIV01 comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF–scFv (see SEQ ID NO: 8 for the protein sequence) (see SEQ ID NO: 9 for the DNA sequence) ( As shown in Figure 3 and Figure 4);
  • Anti-HIV triphilic neutralizing antibody molecule KL-BsHIV01-003 comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF–scFv (see SEQ ID NO:10 for the protein sequence) (see SEQ ID NO:11 for the DNA sequence ) (as shown in Figure 1 and Figure 2);
  • the anti-HIV triphilic neutralizing antibody molecule KL-BsHIV01-C34 comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF–scFv see SEQ ID NO:12 for the protein sequence
  • SEQ ID NO:13 for the DNA sequence see SEQ ID NO:13 for the DNA sequence
  • Anti-HIV tritropic neutralizing antibody molecule KL-BsHIV01-T20 comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF-scFv (see SEQ ID NO:14 for the protein sequence) (see SEQ ID NO:15 for the DNA sequence ) (as shown in Figure 1 and Figure 2).
  • the above monoclonal antibody molecular gene expression framework was cloned into the latest generation of lentiviral vector pKL-kan-lenti-EF1 ⁇ -WPRE ( Figure 6) (see SEQ ID NO:16).
  • the lentiviral vector includes: 5' LTR, wherein the promoter region of LTR is replaced with CMV promoter; ⁇ packaging signal; retroviral export element RRE; cPPT; promoter CBH; Polynucleotides; post-transcriptional regulatory elements are WPRE; PPT; ⁇ U3 3'LTR; and poly(A) signal.
  • the neutralizing antibody gene expression framework (Fig. 4) designed in this example was synthesized by Nanjing GenScript Biotechnology Co., Ltd.
  • the HIV neutralizing antibody gene expression frameworks CBH-KL-BsHIV01, CBH-KL-BsHIV01-003, CBH-KL-BsHIV01-C34, CBH-KL-BsHIV01-T20 and WPRE were respectively cloned into polyclones of the latest generation adeno-associated virus vector pAAV-MCS-CMV-EGFP (reverse) (see SEQ ID NO: 20 for the sequence) in the form of multi-segment recombination connection Between the site MluI/SalI (see Figure 7).
  • the adeno-associated virus vector includes: AAV2 ITR; promoter CBH; polynucleotide encoding HIV neutralizing antibody fragment; WPRE and SV40 poly(A) signal; AAV2 ITR.
  • Plasmid is named as pAAV-CBH-KL-BsHIV01-WPRE (sequence sees SEQ ID NO: 21), pAAV-CBH-KL-BsHIV01-003-WPRE (sequence sees SEQ ID NO: 22), pAAV-CBH-KL-BsHIV01 -C34-WPRE (see SEQ ID NO: 23 for the sequence) pAAV-CBH-KL-BsHIV01-T20-WPRE (see SEQ ID NO: 24 for the sequence) (Fig. 8).
  • the lentiviral vectors for antibody gene therapy were packaged in 293T cell line with lentiviral vectors (pKL-Kan-lenti-CBH-KL-BsHIV01, pKL-Kan-lenti-CBH-KL-BsHIV01-003).
  • the antibody gene lentiviral vector (pKL-Kan-lenti-CBH-KL-BsHIV01, pKL-Kan-lenti-CBH-KL-BsHIV01-003, envelope plasmid (pKL-Kan-Vsvg, its nucleus Nucleotide sequence is shown in SEQ ID NO:25) and packaging plasmid (pKL-Kan-Rev, its nucleotide sequence is shown in SEQ ID NO:26; pKL-Kan-GagPol, its nucleotide sequence is shown in SEQ ID NO: 27) mixed and co-transfected 293T cells (purchased from the American Type Culture Collection (ATCC), ATCC preservation number is CRL-3216) at the same time after mixing, and HIV neutralizing antibody gene therapy was carried out slowly in the 293T cell line.
  • ATCC American Type Culture Collection
  • the packaging of virus is the transient transfection of eukaryotic cells mediated by PEI cationic polymer
  • PEI cationic polymer is the PEI-Max transfection reagent (purchased from Polysciences, catalog number: 24765-1) purchased from Polysciences, The transfection operation is carried out according to the standard operation recommended by the manufacturer, and the transfection scale is a 15cm cell culture dish. After 48 hours of transfection, the lentiviral vector (transfected cell culture supernatant) is harvested, and first centrifuged at 4000 rpm at room temperature on a desktop hanging bucket centrifuge Remove cell debris for 5 minutes, then centrifuge at 10,000g at 4°C for 4 hours to obtain the pellet of virus particles.
  • DMEM complete medium After removing the centrifuged supernatant, add 1 mL of DMEM complete medium to the pellet of virus particles, resuspend the virus particles with a micro-injector, and put the prepared The virus resuspension was aliquoted and frozen at -80°C for later use.
  • AAV expression vector (pAAV-CBH-KL-BsHIV01-WPRE, pAAV-CBH-KL-BsHIV01-003-WPRE, pAAV-CBH-KL-BsHIV01-C34-WPRE, pAAV-CBH-KL-BsHIV01-T20-WPRE ) Packaging of antibody gene therapy AAV vectors in 293T cell line.
  • the antibody gene AAV vector, capsid plasmid (AAV2/8, whose nucleotide sequence is shown in SEQ ID NO: 28) and packaging plasmid (pHelper, whose nucleotide sequence is shown in SEQ ID NO: 29) will be constructed in the embodiment shown) were mixed and co-transfected 293T cells at the same time, and the HIV neutralizing antibody gene therapy vector AAV was packaged in the 293T cell line.
  • the transfection method is the transient transfection of eukaryotic cells mediated by PEI cationic polymer
  • the PEI cationic polymer is the PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), and the transfection operation refers to the production
  • the standard operation recommended by the manufacturer was carried out, and the transfection scale was 15cm cell culture dish. 7 hours after transfection, the supernatant was discarded and replaced with 25ml of toxin-producing medium.
  • the packaged lentiviral vectors pKL-Kan-lenti-CBH-KL-BsHIV01 and pKL-Kan-lenti-CBH-BsHIV01-003 were used to infect 293T cells according to different MOI. After 48 hours, the supernatant and some cells were collected. The copy number of lentiviral vector infection was detected by probe method.
  • VCN copy number
  • TZM-bl cells at 2E4 cells/well. Take 50 ⁇ L of cell antibody expression supernatant with VCN of 0.05 and 0.25, mix with 50 ⁇ L of HIV virus pAD-8 or pNL4-3, incubate at 37°C for 30 minutes, add to TZM-bl cells, add only pAD-8 or pNL4-3 The wells in which the HIV virus was not added are blank. At 24 hours, discard the supernatant, add 100 ⁇ L of cell lysate, collect the lysed cells after 10 minutes, and centrifuge at 8000 rpm for 5 minutes. Add 100 ⁇ L of firefly luciferase assay reagent to 50 ⁇ L of lysed cells.
  • the packaged adeno-associated virus pAAV-CBH-KL-BsHIV01, pAAV-CBH-KL-BsHIV0-003, pAAV-CBH-KL-BsHIV01-C34, pAAV-CBH-KL-BsHIV01-T20 were infected and differentiated according to different MOI Good C2C12 cells. The supernatant was collected after 96 hours.
  • TZM-bl cells at 2E4 cells/well. Take the cell expression supernatant containing the same amount of antibody and mix them with 50 ⁇ L HIV virus pAD-8 or pNL4-3, incubate at 37°C for 30 minutes, add them to TZM-bl cells, and add only pAD-8 or pNL4-3 to the wells Negative control, the well without HIV virus is blank. At 24 hours, discard the supernatant, add 100 ⁇ L of cell lysate, collect the lysed cells after 10 minutes, and centrifuge at 8000 rpm for 5 minutes. Add 100 ⁇ L of firefly luciferase assay reagent to 50 ⁇ L of lysed cells.
  • the AAV gene therapy vector (pAAV-CBH-KL-BsHIV01-WPRE, pAAV-CBH-KL-BsHIV01-003-WPRE) was injected intramuscularly into the thigh muscle of the mouse hindlimb. Blood was drawn every 1 week, serum was separated, and the concentration of antibodies expressed in serum was detected by ELISA.
  • TZM-bl cells Plate TZM-bl cells at 2E4 cells/well. Mix serum dilutions containing the same amount of antibody with 50 ⁇ L of HIV virus pAD-8 or pNL4-3, incubate at 37°C for 30 minutes, and add to TZM-bl cells. The wells with only pAD-8 or pNL4-3 are negative As a control, the wells without HIV virus were blank. After culturing overnight, discard the supernatant, add 100 ⁇ L of cell lysate, collect the lysed cells after 10 min, and centrifuge at 8000 rpm for 5 min. Add 100 ⁇ L of firefly luciferase assay reagent to 50 ⁇ L of lysed cells.

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