WO2023030315A1 - 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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WO2023030315A1
WO2023030315A1 PCT/CN2022/115831 CN2022115831W WO2023030315A1 WO 2023030315 A1 WO2023030315 A1 WO 2023030315A1 CN 2022115831 W CN2022115831 W CN 2022115831W WO 2023030315 A1 WO2023030315 A1 WO 2023030315A1
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gene
hiv
hiv infection
sequence
linker
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French (fr)
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吴昊泉
孙保贞
党颖
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康霖生物科技（杭州）有限公司
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Priority to CA3230810A priority patent/CA3230810A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • 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
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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 infection gene therapy constructs based on recombinant viral vectors. For example, a variety of broad-spectrum neutralizing antibodies against HIV, single-chain antibody variable fragments (scFv) of neutralizing antibodies against human CD4 receptors, and HIV membrane fusion inhibitory polypeptides are combined into a simple and efficient expression framework.
  • scFv single-chain antibody variable fragments
  • these gene sequence constructs for HIV infection gene therapy include one or more gene coding sequences of single-chain antibody molecules (including nanobodies) without constant regions that have the ability to inhibit HIV infection and one or more genes that have the ability to inhibit HIV infection.
  • the single-chain antibody molecule of the above-mentioned gene sequence construct comprises a heavy chain variable region and/or a light chain variable region.
  • 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 single-chain antibody molecules without constant regions that have the ability to inhibit 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 single-chain antibody molecule without a constant region and a polypeptide has four or more action targets.
  • the fusion protein molecule encoded by a single gene and comprising an anti-HIV infection single-chain antibody molecule without a constant region and a polypeptide has five or more action targets.
  • the fusion protein molecule encoded by a single gene and comprising an anti-HIV infection single-chain antibody molecule without a constant region and a polypeptide has six or more action targets.
  • the fusion protein molecule encoded by a single gene and comprising an anti-HIV infection single-chain antibody molecule without a constant region 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 which comprises two or more gene coding sequences of single-chain antibody molecules without constant regions that have the ability to inhibit HIV infection, wherein the gene coding sequences of the antibody molecules It is directly or indirectly connected in series through the coding sequence of the linker polypeptide.
  • the gene sequence construct described in any one of the above, wherein the gene coding sequence of one or more single-chain antibody molecules without constant regions that has the ability to inhibit HIV infection comprises anti-HIV-1-gp160 (or its splicing Gene coding sequences of antibody molecules that cut products gp120 and gp41).
  • the gene sequence construct described in any one of the above, wherein the gene coding sequence of one or more single-chain antibody molecules without constant regions with the ability to inhibit HIV infection comprises antibody molecules that bind to human CD4 receptor sites gene coding sequence.
  • 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 single-chain antibody molecules without constant regions that have the ability to inhibit HIV infection and one or more single-chain antibody molecules that have the ability to inhibit HIV infection The gene coding sequence of the polypeptide, wherein the gene coding sequences of the two or more single-chain antibody molecules without constant regions that have the ability to inhibit HIV infection include anti-HIV-1-gp160 (or its spliced products gp120 and The gene coding sequence of the antibody molecule of gp41) and the gene coding sequence of the antibody molecule binding to the human CD4 receptor site, and the gene coding sequence of one or more polypeptides with the ability to inhibit HIV infection include the ability to inhibit HIV and CD4 + Gene coding sequence of polypeptide for T cell membrane fusion.
  • 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 (or 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) gene coding sequence, (iii) gene coding sequence of a polypeptide that inhibits fusion between HIV and CD4+ T cell membrane, wherein the coding sequences of light chain and heavy chain of the antibody can be passed through the linker in no particular order
  • the coding sequence of the polypeptide is directly or indirectly concatenated.
  • 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 (or its cleavage products gp120 and gp41) monoclonal antibody region (VH), (ii) the gene coding sequence of the light chain variable region (VL) and the heavy chain variable region (VH) of the monoclonal antibody binding to the human CD4 receptor site, (iii) the inhibitory
  • the gene sequence construct described in any one of the above which additionally comprises a promoter located in the gene coding sequence of the single-chain antibody molecule without the constant region having the ability to inhibit HIV infection and the gene coding sequence of the polypeptide having the ability to inhibit HIV infection upstream.
  • the gene sequence construct described in any one of the above which additionally comprises a secretory signal peptide coding sequence located in the gene coding sequence of a single-chain antibody molecule without a constant region with the ability to inhibit HIV infection and a gene with a polypeptide capable of inhibiting HIV infection upstream of the coding sequence.
  • the gene sequence construct described in any one of the above, which comprises the gene coding sequence of the first single-chain antibody molecule without the constant region with the ability to inhibit HIV infection, and the second one without the constant region with the ability to inhibit HIV infection which are 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;
  • linker is the linker Polypeptide;
  • peptide inhibitor is a polypeptide that inhibits HIV infection (eg, a polypeptide that inhibits fusion of HIV with CD4+ T cell membranes).
  • VL2-linker-VH2-linker-VL1-linker-VH1 is VL2-linker-VH2-linker-VL1-linker-VH1, or a construct formed by arranging the combinations of VL2 and VH2 or VL1 and VH1 in different sequences.
  • the above gene sequence construct is VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor, or the combination of VL2 and VH2 or VL1 and VH1 is arranged in different order of constructs.
  • 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 that inhibits the membrane fusion of HIV and CD4+ T cells can be selected from the 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 molecular proteins, which can infect humans by binding to HIV
  • the multiple binding sites involved in different steps of CD4+ T cells can efficiently and broadly block HIV's infection process of human CD4+ T cells, so as to be used for gene therapy of HIV infection, so as to achieve the goal of treating HIV-infected patients long-term treatment.
  • 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 Effectively block the route of HIV infection and effectively avoid the loss of the ability to inhibit HIV infection due to escape mutations in HIV, so as to achieve long-term (for example, drug effects lasting one year or several years) against HIV infection with a single injection ) and even permanent healing effects.
  • 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 a tritropic anti-HIV neutralizing antibody expected to exist in a monomeric form.
  • Fig. 2 Schematic diagram of gene sequence composition of tritropic anti-HIV neutralizing antibody.
  • Figure 3 Schematic representation of the mature molecular structure of amphotropic anti-HIV neutralizing antibodies expected to exist in monomeric form.
  • FIG. 1 Schematic diagram of the gene sequence composition of the amphotropic anti-HIV neutralizing antibody.
  • Fig. 5 Schematic diagram of gene constructs in which the gene sequences of amphitropic (KL-BsHIV01-02) or tritropic (KL-BsHIV01-003-02) anti-HIV neutralizing antibodies are cloned into lentiviral vectors.
  • Figure 7 Schematic diagram of the gene construct in which the gene sequence of amphitropic (KL-BsHIV01-02) or tritropic (KL-BsHIV01-003-02) anti-HIV neutralizing antibody is cloned into an adeno-associated virus vector.
  • FIG. 8 Map of the latest generation of adeno-associated virus vector pAAV-MCS-CBH-WPRE used in the present invention.
  • Figure 9 Detection of anti-HIV neutralizing antibodies by Western blot analysis.
  • M prestained protein size marker; 1 and 6, purified Flag-KL-BsHIV01-003-02; 2 and 7, purified KL-BsHIV01-003-02; 3-5 and 8-10, purified Flag-KL-BsHIV01-003-02 was digested with enterokinase under different conditions.
  • the primary antibody is rabbit Anti-KL-BsHIV01-003-02 polyclonal antibody; 6-10, the primary antibody is mouse Anti-DYKDDDDK (Flag-tag).
  • FIG. 11 Expression of different doses 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.
  • FIG. 12 Expression of different doses of anti-HIV neutralizing antibody adeno-associated virus gene therapy vector in BALB/c mice after intramuscular injection. Neutralizing activity of anti-HIV neutralizing class antibodies secreted into mouse serum.
  • 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 in specific situations 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 a 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. Examples of specific 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, the ones used in some of the examples below (GGGGS) n connectors of different lengths, n can be 1 or more.
  • 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 jointly 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 used in the present invention and class antibody molecule is:
  • Triphilic anti-HIV neutralizing antibody scFv molecule from the N-terminal to the C-terminal of the protein molecule are respectively - signal peptide - anti-HIV-gp41 broad-spectrum neutralizing antibody single-chain variable region fragment (VL-(ggggs) n linker-VH)-(ggggs) n linker-single-chain variable region fragment of anti-human CD4 antibody (VL-(ggggs) n linker-VH)-(ggggs) n linker—HIV membrane fusion inhibiting short peptide.
  • the coding sequence of the indicated gene is expressed in the cell and then translated into protein, which is then 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.
  • Amphotropic anti-HIV neutralizing antibody scFv molecule from the N-terminal to the C-terminal of the protein molecule are respectively - signal peptide - anti-HIV-gp41 broad-spectrum neutralizing antibody single-chain variable region fragment (VL-(ggggs) n linker-VH)-(ggggs) n linker-single-chain variable region fragment of anti-human CD4 antibody (VL-(ggggs) n linker-VH).
  • VL-(ggggs) n linker-VH broad-spectrum neutralizing antibody single-chain variable region fragment
  • VL-(ggggs) n linker-VH broad-spectrum neutralizing antibody single-chain variable region fragment
  • VL-(ggggs) n linker-VH broad-spectrum neutralizing antibody single-chain variable region fragment
  • VL-(ggggs) n linker-VH broad-spectrum neutralizing antibody single-chain variable region fragment
  • the anti-HIV-gp41 broad-spectrum neutralizing antibody single-chain variable region fragment (scFv) sequences are: signal peptide (see SEQ ID NO: 1 for the protein sequence); anti-HIV-1-gp41-MPER antibody (10E8v4-5R+ 100cF)-scFv (see SEQ ID NO:2 for the protein sequence); the single-chain variable region fragment scFv (see SEQ ID NO:3 for the protein sequence) of anti-human CD4 antibody (Ibalizumab), HIV membrane fusion inhibitory short peptide P52 (protein The sequence is shown in SEQ ID NO: 4), HIV membrane fusion inhibitory short peptide C34 (protein sequence is shown in SEQ ID NO: 5), HIV membrane fusion inhibitory short peptide T20 (protein sequence is shown in SEQ ID NO: 6).
  • Anti-HIV amphotropic neutralizing antibody molecule KL-BsHIV01-02 comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF–scFv (see SEQ ID NO: 7 for the protein sequence) (see SEQ ID NO: 8 for the DNA sequence )( Figure 4).
  • Anti-HIV triphilic neutralizing antibody molecule KL-BsHIV01-003-02 (see SEQ ID NO: 9 for protein sequence) (see SEQ ID NO for DNA sequence) comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF–scFv :10) (Fig. 2).
  • the lentiviral vector comprises: 5' LTR, wherein the promoter region of LTR is replaced with CMV promoter; ⁇ packaging signal; retroviral export element RRE; cPPT; promoter CBH; nucleotides; post-transcriptional regulatory elements are WPRE; PPT; ⁇ U3 3'LTR; and poly(A) signal.
  • the neutralizing antibody gene expression framework ( Figure 2 and Figure 4) designed in this example was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and the CBH promoter (see SEQ ID NO: 22 for the sequence), through the well-known methods in the art.
  • the method of homologous recombination was cloned into the multi-cloning site AgeI/EcoRV on the lentiviral vector backbone pKL-kan-lenti-CBH-WPRE ( Figure 6).
  • the sequence information was confirmed by sequencing, and the plasmids were named pKL -Kan-lenti-CBH-KL-BsHIV01-02 (see SEQ ID NO: 12 for the sequence), pKL-Kan-lenti-CBH-KL-BsHIV01-003-02 (see SEQ ID NO: 13 for the sequence).
  • the HIV neutralizing antibody gene expression framework CBH-KL-BsHIV01-02 and CBH-KL-BsHIV01-003-02 present in pKL-Kan-lenti-CBH-KL-BsHIV01 were divided into multi-fragment
  • the method of recombinant connection is cloned between the multiple cloning sites AgeI/SalI of the latest generation of adeno-associated virus vector pAAV-MCS-CBH-WPRE (see SEQ ID NO: 14) currently used (see Figure 8).
  • the adeno-associated virus vector includes: AAV2 ITR; promoter CBH; polynucleotide encoding HIV neutralizing antibody fragment; WPRE and SV40 poly(A) signal; AAV2 ITR.
  • the plasmids were named pAAV-CBH-KL-BsHIV01-02-WPRE (see SEQ ID NO: 15 for the sequence) and pAAV-CBH-KL-BsHIV01-003-02-WPRE (see SEQ ID NO: 16 for the sequence).
  • HIV neutralizing antibody gene therapy lentiviral vectors in 293T cell line package of.
  • the lentiviral vector transfected cell culture supernatant
  • the lentiviral vector was harvested, and firstly in the benchtop On a hanging bucket centrifuge, centrifuge at 4000rpm at room temperature for 5 minutes to remove cell debris, and then centrifuge at 10000g at 4°C for 4 hours to obtain a pellet of virus particles. After removing the centrifuged supernatant, add 1 mL of DMEM complete medium to the pellet of virus particles, and resuspend with a micro-injector Virus particles, and the prepared virus resuspension was subpackaged and frozen at -80°C for later use.
  • the HIV neutralizing antibody gene therapy AAV vector was packaged in 293T cell line with AAV expression vector (pAAV-CBH-KL-BsHIV01-02-WPRE, pAAV-CBH-KL-BsHIV01-003-02-WPRE).
  • HIV neutralizing antibody gene therapy AAV vector, capsid plasmid (AAV2/8, its nucleotide sequence shown in SEQ ID NO:20) and packaging plasmid (pHelper, its nucleotide sequence shown in SEQ ID NO:20) with the HIV neutralizing antibody gene therapy that constructs in the embodiment ID NO: 21) 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-BsHIV01-02 and pKL-Kan-lenti-CBH-BsHIV01-003-02 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 antibody expression supernatants from different cells with a VCN of 1, dilute different times, mix with 50 ⁇ L HIV virus pAD-8 or pNL4-3, incubate at 37°C for 30 min, add to TZM-bl cells, add only pAD-8 or pNL4-3 The wells of pNL4-3 were negative control (NC), and the wells without HIV virus were 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.
  • TZM-bl cells were plated at 2E4 cells/well. Take the cell expression supernatants of KL-BsHIV01, KL-BsHIV01-02, KL-BsHIV01-003, and KL-BsHIV01-003-02 with the same MOI of 8E4vg, and take the amphotropic antibody KL-BsHIV01 containing the Fc segment respectively The cell expression supernatant of amphotropic antibody KL-BsHIV01-02 and triptropic antibody KL-BsHIV01-003-02 without the Fc segment in the same volume as triptropic antibody KL-BsHIV01-003, and 50 ⁇ L HIV virus pAD After mixing -8 or pNL4-3, incubate at 37°C for 30 minutes, and add to TZM-bl cells.
  • Wells with only pAD-8 or pNL4-3 are negative controls (NC), and wells without HIV virus are Blank.
  • N negative controls
  • discard the supernatant add 100 ⁇ L of cell lysate, collect the lysed cells after 10 minutes, and centrifuge at 8000 rpm for 5 minutes.
  • RLU relative light unit
  • the HIV neutralizing antibody AAV gene therapy vector (pAAV-CBH-KL-BsHIV01-02-WPRE, pAAV-CBH-KL-BsHIV01-003-02-WPRE) was injected intramuscularly into the hind limbs of mice at a dose of 2E11vg/mouse respectively. thigh muscles. Blood was drawn every 1 week, and serum was separated.
  • TZM-bl cells were plated at 2E4 cells/well. Take the same volume of serum dilution and mix them with 50 ⁇ L of HIV virus pAD-8 or pNL4-3, incubate at 37°C for 30 minutes, and add them to TZM-bl cells.
  • the wells with only pAD-8 or pNL4-3 are negative controls ( NC), the well without HIV virus is Blank.
  • NC negative controls
  • the well without HIV virus is Blank.
  • 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 lentiviral plasmid carrying Flag-KL-BsHIV01-003-02 was transiently transfected into 293T cells, and the supernatant was collected after 48 hours, and the Flag was purified with anti-DYKDDDDK (Flag-tag) G1 Affinity Resin (GenScript, L00432-10) - KL-BsHIV01-003-02, the resulting Flag-KL-BsHIV01-003-02 was digested with enterokinase (nearshore, PE001-01A).
  • Flag-KL-BsHIV01-003-02 before and after digestion was identified by Western blotting with rabbit Anti-KL-BsHIV01-003-02 or mouse Anti-DYKDDDDK (GenScript, A00187-100) ( Figure 9).
  • the results of Western blotting confirmed that the HIV neutralizing antibody KL-BsHIV01-003-02 carrying Flag-tag or not carrying Flag-tag was expressed in the cells and secreted into the supernatant, and had a molecular weight consistent with the prediction.
  • TZM-bl cells were plated at 2E4 cells/well. Mix the purified antibody dilution with 50 ⁇ L of HIV 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 controls. Wells without HIV virus were blank. After culturing for 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.
  • AAV gene therapy vector (pAAV-CBH-KL-BsHIV01-003--02WPRE) were injected intramuscularly into the femoral muscle of the mouse hindlimb. Blood was drawn at regular intervals to separate the serum.
  • ELISA was used to detect the concentration of antibodies expressed in serum. Coat the ELISA plate (Corning, Cat. No.: 42592) with the synthetic HIV MPER polypeptide, express the supernatant and purify and quantify the KL-BsHIV01-003-02 standard as the primary antibody, and use the rabbit anti-HIV membrane fusion inhibition short peptide
  • the polyclonal antibody was used as the secondary antibody, and HRP-labeled goat anti-rabbit IgG (KPL, 5220-0283) was used as the tertiary antibody. The color was developed with TMB, and the OD value at 450nm was detected by a microplate reader.
  • the results of ELISA ( FIG. 11 ) show that the anti-HIV neutralizing antibody adeno-associated virus vector can continuously and effectively express HIV neutralizing antibody in mice.
  • TZM-bl cells at 2E4 cells/well. Mix the 200-fold serum dilution with 50 ⁇ L of HIV virus strain pNL4-3, incubate at 37°C for 30 minutes, and add to TZM-bl cells. The wells with only pNL4-3 are negative controls, and the wells without HIV virus For 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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Abstract

一种用于艾滋病病毒(HIV)感染基因治疗的基因序列构建体。通过把针对HIV感染人CD4+ T细胞不同步骤中涉及的不同结合位点抗原的单克隆抗体的轻链和重链的各自单链可变区(scFv)的基因编码序列、结合CD4受体位点的单克隆抗体的轻链和重链的各自单链可变区(scFv)的基因编码序列、以及抑制HIV与CD4+ T细胞膜融合的多肽的基因编码序列依次通过连接头多肽的编码序列连接，置于启动子以及分泌信号肽编码序列下游构建成基因序列构建体，以表达单基因编码的分泌型类抗体蛋白分子。该重组的单基因构建体可以很方便地通过病毒载体导入目标组织细胞，且表达分泌的抗体或类抗体蛋白分子具有多抗原嗜性，能通过结合HIV感染人CD4+ T细胞不同步骤中涉及的多个结合位点从而有效和广谱地阻断HIV对人CD4+ T细胞的感染进程，并有效地避免由于HIV发生逃逸突变而失去对HIV感染的抑制能力，从而达到单次注射给药即具有对HIV感染的长期甚至永久的治疗效果。

Description

一种用于艾滋病病毒感染基因治疗的基因序列构建体
本申请要求于2021年8月30日递交的PCT专利申请第PCT/CN2021/115422号的优先权，在此全文引用上述PCT专利申请的内容以作为本申请的一部分。
技术领域
本发明属于基因治疗/生物医药技术领域，具体涉及到一种用于艾滋病病毒(HIV)感染基因治疗的基因序列构建体。该基因序列构建体可用于针对HIV感染的基因治疗。该基因序列构建体在体内外均可用于表达具有广谱及高效中和HIV病毒活性的多嗜性中和类抗体蛋白，可用于重组病毒或非病毒载体递送的HIV感染基因治疗药物的临床研究及新药研发。
背景技术
由人类免疫缺陷病毒(human immunodeficiency virus，HIV)感染造成的艾滋病(acquired immunodeficiency syndrome，AIDS)，是危害当今世界最大的感染性疾病之一。全世界3,790万名HIV感染者中每年大约有77万人因艾滋病而死。随着抗艾滋病药物的发展，经积极治疗的艾滋病毒感染者的预期生存期已得到很大程度的延长，但鸡尾酒疗法的副作用及局限性，以及逐渐浮现的HIV耐药毒株仍然给广大艾滋病患者造成了长期的经济和社会负担、生活品质的明显下降及明显的痛苦。
所以尽管抗艾滋病领域已经取得了进展，但仍然有需要开发用于治疗艾滋病病毒感染的新的药物和方法。
发明内容
本发明创造了一系列基于重组病毒载体的抗艾滋病病毒感染的基因治疗构建体。例如，将多种针对HIV的广谱中和抗体、针对人CD4受体的中和抗体的单链抗体可变区片段(scFv)，结合HIV膜融合抑制多肽组合为一个简单高效的表达框架。
首先，这些用于HIV感染基因治疗的基因序列构建体包含一个或多个具有抑制HIV感染能力的不带恒定区的单链抗体分子(包括纳米抗体)的基因编码序列和一个或多个具有抑制HIV感染能力的多肽(由2-50氨基酸残基组成)的基因编码序列，以达到表达一种由单基因编码的包含抗HIV感染的抗体分子和多肽的融合蛋白分子，其具有两种或两种以上作用靶点。
具体地讲，以上所述的基因序列构建体的单链抗体分子包含重链可变区域和/或轻链可变区域。
而其中所述轻链可变区域包含κ或λ轻链的轻链可变区域。
以上所述的基因序列构建体，包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列。
或者包含三个或三个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列。
或者包含四个或四个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列。
另外，以上所述的基因序列构建体，或可包含二个或二个以上具有抑制HIV感染能力的多肽的基因编码序列。这些多肽由2-50氨基酸残基组成。
或者包含三个或三个以上具有抑制HIV感染能力的多肽的基因编码序列。
或者包含四个或四个以上具有抑制HIV感染能力的多肽的基因编码序列。
以上任一项所述的基因序列构建体，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有三种或三种以上作用靶点。
或者，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有四种或四种以上作用靶点。
或者，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有五种或五种以上作用靶点。
或者，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有六种或六种以上作用靶点。
以上任一项所述的基因序列构建体，包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和一个或多个具有抑制HIV感染能力的多肽的基因编码序列。
以上所述的基因序列构建体，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有三种或三种以上作用靶点。
以上任一项所述的基因序列构建体，其中所述的具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和具有抑制HIV感染能力的多肽的基因编码序列之间通过连接头多肽的编码序列直接或间接串联而成。
以上任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列，其中所述的抗体分子的基因编码序列之间通过连接头多肽的编码序列直接或间接串联而成。
以上任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的多肽的基因编码序列，其中所述的多肽的基因编码序列之间通过连接头多肽的编码序列直接或间接串联而成。
以上任一项所述的基因序列构建体，其中所述的一个或多个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列包含抗HIV-1-gp160(或者其剪切产物gp120和gp41)的抗体分子的基因编码序列。
以上任一项所述的基因序列构建体，其中所述的一个或多个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列包含结合人CD4受体位点的抗体分子的基因编码序列。
以上任一项所述的基因序列构建体，其中所述的一个或多个具有抑制HIV感染能力的多肽的基因编码序列包含抑制HIV与CD4+T细胞膜融合的多肽的基因编码序列。
以上任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和一个或多个具有抑制HIV感染能力的多肽的基因编码序列，其中所述的二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列包含抗HIV-1-gp160(或者其剪切产物gp120和gp41)的抗体分子的基因编码序列和结合人CD4受体位点的抗体分子的基因编码序列，和其中所述的一个或多个具有抑制HIV感染能力的多肽的基因编码序列包含抑制HIV与CD4+T细胞膜融合的多肽的基因编码序列。
以上任一项所述的基因序列构建体，其包括(i)抗HIV-1-gp160(或者其剪切产物gp120和gp41)单克隆抗体的轻链互补决定区(LCDR1,LCDR2和LCDR3)和重链互补决定区(HCDR1,HCDR2和HCDR3)的基因编码序列、(ii)结合人CD4受体位点的单克隆抗体的轻链互补决定区(LCDR1,LCDR2和LCDR3)和重链互补决定区(HCDR1,HCDR2和HCDR3)的基因编码序列、(iii)抑制HIV与CD4+T细胞膜融合的多肽的基因编码序列，其中抗体的轻链和重链的编码序列可以不分先后的次序通过连接头多肽的编码序列直接或间接串联而成。
以上任一项所述的基因序列构建体，其包括(i)抗HIV-1-gp160(或者其剪切产物gp120和gp41)单克隆抗体的轻链可变区(VL)和重链可变区(VH)的基因编码序列、(ii)结合人CD4受体位点的单克隆抗体的轻链可变区(VL)和重链可变区(VH)的基因编码序列、(iii)抑制HIV与CD4+T细胞膜融合的多肽的基因编码序列，其中抗体的轻链可变区(VL)和重链可变区(VH)的编码序列可以不分先后的次序通过连接头多肽的编码序列直接或间接串联而成。
以上任一项所述的基因序列构建体，其另外包含启动子位于具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和具有抑制HIV感染能力的多肽的基因编码序列的上游。
以上任一项所述的基因序列构建体，其另外包含分泌信号肽编码序列位于具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和具有抑制HIV感染能力的多肽的基因编码序列的上游。
以上任一项所述的基因序列构建体，其包含第一个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列，第二个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列，和具有抑制HIV感染能力的多肽的基因编码序列，其选自：
VL2-linker-VH2-linker-VL1-linker-VH1-linker；或
VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor；或
其它把VL2和VH2或VL1和VH1的组合在构建体中按不同的先后次序排列而成的构建体。
其中，VL2和VH2分别为第一个抗体分子的轻链和重链的可变区片段；VL1和VH1分别为第二个抗体分子的轻链和重链的可变区片段；linker为连接头多肽；peptide inhibitor为抑制HIV感染的多肽(例如，抑制HIV与CD4+T细胞膜融合的多肽)。
以上所述的基因序列构建体，其为VL2-linker-VH2-linker-VL1-linker-VH1，或把其中VL2和VH2或VL1和VH1的组合按不同的先后次序排列而成的构建体。
以上所述的基因序列构建体，其为VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor，或把其中VL2和VH2或VL1和VH1的组合按不同的先后次序排列而成的构建体。
以上所述的基因序列构建体，其中VL2和VH2的蛋白序列包括SEQ ID NO:2,或其功能性片段，或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
以上所述的基因序列构建体，其中VL1和VH1的蛋白序列包括SEQ ID NO:3，或其功能性片段，或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
以上所述的基因序列构建体，其中连接头多肽(linker)的序列选自以下的氨基酸序列：GGGGS、(GGGGS) 2、(GGGGS) 3、(GGGGS) 4、(GGGGS) 5、(GGGGS) 6、和(GGGGS) 7，或其它可选的连接头多肽序列。
以上所述的基因序列构建体，其中抑制HIV与CD4+T细胞膜融合的多肽可选自膜融合抑制多肽P52、C34、T20等。
其中，膜融合抑制多肽P52的序列包括SEQ ID NO:5或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列；C34的多肽序列包括SEQ ID NO:6或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列；T20的多肽序列包括SEQ ID NO:7或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
更进一步，本发明也提供了一种包含前述任一项的构建体的病毒载体基因组以及相应的包含所述基因组的病毒载体系统。
以上所述的病毒载体系统可以是慢病毒载体系统或腺相关病毒载体系统。
其中，慢病毒载体系统，可以包含以上所述的任何一种构建体的病毒载体基因组以及其它编码和表达生产慢病毒所需的包装组件的核苷酸序列，其会被导入生产细胞生产包含以上所述的构建体基因组的慢病毒颗粒。
而腺相关病毒载体系统，可以包含以上所述的任何一种构建体的病毒载体基因组以及其它编码和表达生产腺相关病毒所需的包装组件的核苷酸序列，其会被导入生产细胞生产包含以上所述的构建体基因组的腺相关病毒颗粒。
以上所述生产的任何一种病毒颗粒，其包含前述的任一项所述的构建体的基因组，转导细胞后可以表达抗HIV中和类抗体分子，可以用于给病人给药以抑制或预防HIV感染。
本发明也提供了一种药物组合物，其包含以上所述的一种病毒颗粒，以及药学上可接受的载体或稀释剂，或者在体外被以上所述的慢病毒颗粒所转导的细胞，其包括但不仅限于被转导的肌肉细胞，肝脏细胞，或CD4+T细胞。
以上所述的一种病毒颗粒或所述的包含以上病毒颗粒的药物组合物，可以用于注射到体内以表达两种或两种以上作用靶点的抗体分子蛋白，其可通过结合HIV感染人CD4+T细胞不同步骤中涉及的多个结合位点从而高效和广谱地阻断HIV对人CD4+T细胞的感染进程，以用于对HIV感染的基因治疗，从而达到对艾滋病病毒感染者的长期治疗。
本发明提供了一种抑制HIV感染的方法，包括向细胞施用以上所述的一种病毒颗粒或药物组合物。
其中所述的细胞包含肌肉细胞，肝脏细胞，或CD4+T细胞等。
而用以上所述的一种病毒颗粒或药物组合物对以上所述的细胞可以在体外或体内进行转导。
本发明提供了一种在需要治疗的受试者中治疗HIV感染的方法，所述方法包括向该受试者施用治疗有效剂量的以上所述的一种病毒颗粒或药物组合物。
其中所述的受试者包含HIV早期感染者或已经在接受鸡尾酒药物疗法的HIV感染者或对鸡尾酒药物疗法具有抗性的HIV感染者。
其中，给以上所述的受试者施用药物的方式为通过肌肉注射以上所述的一种病毒颗粒或药物组合物。
或者，通过静脉注射以上所述的一种病毒颗粒或药物组合物或经其转导的CD4+T细胞。
通过以上所述的方法注射到体内的病毒颗粒和药物组合物，其能表达具有多作用靶点的抗HIV的蛋白分子并分泌到血液中，其能作用于多个HIV感染的节点，而能有效地阻断HIV感染路径并有效地避免由于HIV发生逃逸突变而失去对HIV感染的抑制能力，从而达到单次注射给药即具有对HIV感染的长期(例如，药效持续一年或数年)甚至永久的治疗效果。
基于上述表达框架的由多个单链抗体可变区片段(scFv)组合成的类抗体分子经慢病毒以及腺相关病毒载体递送至小鼠体内后显示出有效的血药浓度，具有很强的体外细胞学HIV-1病毒中和活性，以及同时对 CXCR4和CCR5两种嗜性病毒的广谱中和能力，为极具潜力的抗艾滋病病毒广谱中和抗体基因治疗药物开发技术路线。
本发明的应用范围包括各种形式基于广谱中和抗体基因化表达的抗艾滋病病毒的基因治疗。
附图说明
对本发明创造的附图详细说明。
图1.预期三嗜性抗HIV中和类抗体以单体形式存在的成熟分子结构示意图。
图2.三嗜性抗HIV中和类抗体的基因序列构成示意图。
图3.预期双嗜性抗HIV中和类抗体以单体形式存在的成熟分子结构示意图。
图4.双嗜性抗HIV中和类抗体的基因序列构成示意图。
图5.双嗜性(KL-BsHIV01-02)或三嗜性(KL-BsHIV01-003-02)抗HIV中和类抗体的基因序列克隆到慢病毒载体中的基因构建体的示意图。
图6.本发明应用的最新一代慢病毒载体pKL-kan-lenti-CBH-WPRE的图谱。
图7.双嗜性(KL-BsHIV01-02)或三嗜性(KL-BsHIV01-003-02)抗HIV中和类抗体的基因序列克隆到腺相关病毒载体中的基因构建体的示意图。
图8.本发明应用的最新一代腺相关病毒载体pAAV-MCS-CBH-WPRE的图谱。
图9.Western印迹分析检测抗HIV中和类抗体。M，预染的蛋白大小标记物；1和6，纯化的Flag-KL-BsHIV01-003-02；2和7，纯化的KL-BsHIV01-003-02；3-5和8-10,纯化的Flag-KL-BsHIV01-003-02用肠激酶在不同条件下酶切后产物。1-5，一抗为兔Anti-KL-BsHIV01-003-02多抗；6-10，一抗为鼠Anti-DYKDDDDK(Flag-tag)。
图10.三嗜性KL-BsHIV01-003(带Fc片段)和KL-BsHIV01-003-02(不带Fc片段)抗HIV中和类抗体的中和活性。
图11.不同剂量的抗HIV中和抗体腺相关病毒基因治疗载体肌肉注射后在BALB/c小鼠中的表达。分泌到小鼠血清中的抗HIV中和类抗体的水平由ELISA方法进行测定。
图12.不同剂量的抗HIV中和抗体腺相关病毒基因治疗载体肌肉注射后在BALB/c小鼠中的表达。分泌到小鼠血清中的抗HIV中和类抗体的中和活性。
具体实施方式
小部分精英感染者体内，经数年时间产生的具有HIV-1中和活性的广谱中和抗体(broadly neutralizing antibodies，bNAb)，能够高效及广谱的结合HIV病毒表面糖蛋白，中和HIV病毒对CD4+T细胞的感染活性，代表了一种很有前景的预防或抗HIV-1感染的方法。但其产生的具体机制并不明了，大多数HIV-1感染者体内仅能产生非中和抗体，目前尚未有研究能成功通过标准的免疫方法诱导健康受试者体内广谱中和抗体的产生。
不希望受理论束缚，据信在一些实施例中，重组来源的针对HIV-1病毒的广谱中和抗体能有效降低患者体内病毒载量，即使不能彻底清除体内HIV，也能帮助控制从HIV感染发展成艾滋病。与小分子抗艾滋病病毒药物相比，重组来源的中和抗体也能极大地减少毒副作用，同时增加病患用药的依从性。在临床上，重组广谱中和抗体既可以用于特定情况下的疫苗替代产品预防艾滋病病毒感染的发生，也可以用作不同疾病阶段的抗病毒药物。
然而与高效抗艾滋病病毒小分子药物相同的是，特定的广谱中和抗体只针对单一病毒蛋白(HIV-gp160)的某一个单一表位，由病毒突变产生的逃逸现象仍然难以避免，并且由于广谱中和抗体研制、生产、使用成本均远高于小分子抗艾滋病病毒药物，联合用药不具优势，所以大量的在研抗艾滋病病毒广谱中和抗体药物至今难以用于临床。
针对以上问题，抗艾滋病病毒广谱中和抗体药物研发主要需要在以下两个方面取得突破：
1.研发双嗜性、多嗜性中和抗体或类抗体大分子药物，以多靶点覆盖避免病毒突变产生的逃逸。
2.蛋白广谱中和抗体或类抗体大分子药物基因化。以重组病毒或非病毒载体递送的抗艾滋病病毒感染基因治疗药物，以基因组整合或非整合的方式在机体内长期稳定表达中和抗体或类抗体大分子，一次治疗长期甚至终身有效，极大地减少大分子抗体药物的生产和使用成本。
本发明采用了一种多嗜性的抗体分子结构，即由连接头多肽(linker)串联两个或两个以上单克隆广谱中和抗体的单链可变区片段(scFv)组成抗原结合区域。
具体实施方式包括构建了一系列包含双嗜性/三嗜性抗体单链可变区片段(scFv)的抗体分子基因构建体，并将其克隆到重组腺相关病毒和重组慢病毒载体中。然后在293T细胞中包装相应的腺相关病毒和慢病毒，以一定生物学滴度病毒感染293T细胞以及分化及未分化肌肉细胞系。细胞上清中产生的抗体分子接受定量、定性检测，同时在HIV-1野生病毒株与病毒敏感报告基因细胞系TZM-bl的感染活性分析中检验其对HIV-1野生病毒株的中和活性。
下面结合实施例对本发明的抗HIV中和抗体结构、基因序列构建体组成、以及所涉及的专用术语做进一步的详细说明。
抗体：可以是一个免疫球蛋白、抗原结合片段、或由此衍生的能够特异性地识别和结合一个抗原(例如，HIV-1gp41抗原)的蛋白分子。本发明中的“抗体”是一个广义的定义，涵盖各种抗体结构，包括但不仅限于单克隆抗体、多克隆抗体、多嗜性抗体(例如，双嗜性抗体、三嗜性抗体)、以及抗体片段，只要它们具有特异性的抗原结合活性。具体的抗体的例子包括完整的免疫球蛋白以及保留对抗原结合亲和性的抗体变体和片段。抗体片段的例子包括但不仅限于可变区片段(Fv)、抗原结合片段(例如，经蛋白酶水解后产生的Fab，Fab'，Fab'-SH，或F(ab') 2)、单链抗体分子(例如，scFv)、双价抗体(diabodies)、纳米抗体、以及由抗体片段组合形成的多嗜性抗体。 抗体片段包括由完整抗体修饰产生的抗原结合片段或是利用重组DNA技术从头合成的抗原结合片段。
单链抗体(scFv)是通过基因工程得到的分子，包含一个或多个抗体的轻链可变区(VL)和重链可变区(VH)，各片段之间由合适的连接头多肽串联成为一个融合的单链分子。VL和VH在单链抗体分子中的串联次序的先后通常不影响其抗原结合功能，因此由两种串联方式(VL-VH或VH-VL)构成的单链抗体都会被使用。
一个抗体可以有一个或者多个抗原结合位点。在具有一个以上的抗原结合位点的情况下，这些结合位点之间可以是相同的或是不同的。比如说，一个自然产生的免疫球蛋白具有两个相同的抗原结合位点，而从免疫球蛋白经木瓜蛋白酶水解产生的Fab片段只有一个抗原结合位点，而双嗜性单链抗体(scFv)具有两个不同的抗原结合位点。
通常情况下，一个自然产生的免疫球蛋白由轻链和重链组成，链之间通过二硫键连接。免疫球蛋白的基因包括γ、α、δ、ε、μ、λ、和κ恒定区基因以及无数的免疫球蛋白可变区基因。轻链具有λ和κ两种类型。重链具有五种主要的类型(γ、α、δ、ε、μ)，决定了抗体分子的功能分型，分别为IgG、IgA、IgD、IgE、和IgM。
每一条重链和轻链包含一个恒定区和一个可变区。VH表示抗体重链的可变区，包括抗原结合片段Fv、scFv、或Fab的重链可变区。VL表示抗体轻链的可变区，包括抗原结合片段Fv、scFv、或Fab的轻链可变区。在以下的实施例中，VH和VL共同起到特异性地识别和结合抗原的作用。
VH和VL包含三个被分隔开的高度可变区(也称作互补决定区(CDRs))以及骨架区。不同轻链和重链的骨架区的序列在同一种属内相对保守。一个抗体的骨架区决定了互补决定区在三维结构中的位置。互补决定区主要负责与一个抗原的抗原决定簇结合。轻链上的三个互补决定区从N-末端到C-末端分别标作为LCDR1、LCDR2、和LCDR3。重链上的三个互补决定区从N-末端到C-末端分别标作为HCDR1、HCDR2、和HCDR3。
本发明中VH和VL的蛋白序列包括以下实施例中公开的序列外，也包括其它任何携带其功能性片段的序列，或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
抗体的恒定区片段：为免疫球蛋白除了近N端的氨基酸序列变化较大的可变区之外的氨基酸序列相对稳定的区域，包括抗原结合片段中的恒定结构域(位于铰链区N端，包括轻链恒定结构域和重链恒定结构域)以及重链的可结晶片段的恒定结构域(称作Fc片段，位于铰链区C端)。Fc片段通常指的是免疫球蛋白IgA，IgD，和IgG的最后两个恒定区结构域或是IgE和IgM的最后三个恒定区结构域。Fc片段也可能包括位于其N端的部分或全部铰链区序列。
抗HIV-1中和抗体或抗原结合片段：该中和抗体或抗原结合片段特异性地结合HIV-1包膜蛋白(例如，结合到gp41)，从而抑制与HIV-1包膜相关的生物学功能(例如，结合目标受体的能力)。在以下实施例中，抗HIV-1中和抗体或抗原结合片段降低了不同嗜性的HIV-1毒株对细胞的感染滴度。
双嗜性或多嗜性抗体：该类抗体为由两种或两种以上不同的抗原结合域组成的重组分子，因而能结合两种或两种以上不同的抗原决定簇。双嗜性或多嗜性抗体包括由两种或两种以上不同的抗原结合域通过化学合成方式或基因工程方式连接而成的分子。抗原结合域可以通过连接头多肽连接。抗原结合域可以是单克隆抗体、抗原结合片段(例如，scFv或Fab)、或不同来源的抗原结合域的组合。
连接头多肽：用来把两个蛋白分子或片段连接成为一个连续的单一融合分子，例如，以下实施例中把两个或多个抗体分子或抗原结合片段(例如，scFv)连接成为具有两个或多个抗原结合位点的多嗜性抗体分子；或者仅是把一个抗体的轻链可变区(VL)和重链可变区(VH)连接成为一个单链的抗原结合序列；或者把抗体分子或抗原结合片段与其它效应分子连接在一起，例如，抗原结合片段scFv与HIV-1膜融合抑制多肽的连接成为一个融合蛋白。连接头多肽通常富含甘氨酸(Gly或G)以提高连接头的灵活度，以及丝氨酸(Ser或S)或苏氨酸(Thr或T)以提高可溶性,例如，以下某些实施例中使用的 不同长度的(GGGGS) n连接头，n可以是1或1以上。但实施例中使用的连接头多肽序列不仅限于此，也包括其它可选的连接头多肽序列。
抗原决定簇：为一个分子上面的特定的化学基团或多肽序列，其具有抗原性，即可以激发宿主特异的免疫反应。一个抗体特异性地结合一个多肽上的特定的抗原决定簇，比如在以下实施例中，能特异性地结合gp41上抗原决定簇的抗体。
HIV-1包膜蛋白：HIV-1包膜蛋白先是以一个大小为845-870个氨基酸残基的前体蛋白形式合成的，称作HIV gp160。gp160在宿主细胞内形成同源三聚体，被糖基化，经过剪切去除信号肽，继而被细胞内的一个蛋白酶在511/512氨基酸残基的位置切割产生gp120和gp41多肽链。gp120和gp41在同源三聚体中以gp120/gp41原聚体的形式保持结合在一起。成熟的gp120由HIV-1包膜蛋白的31-511氨基酸残基组成，是一个高度N-糖基化的蛋白，构成了HIV-1包膜蛋白三聚体暴露在包膜表面的结构域的绝大部分。gp120负责与人CD4细胞受体以及辅助受体(例如，细胞趋化因子受体CCR5或CXCR4)结合。gp41由HIV-1包膜蛋白的512-860氨基酸残基组成，包括包膜内结构域、跨膜结构域、和包膜外结构域。gp41的包膜外结构域包括512-644氨基酸残基，与gp120结合在一起形成原聚体，共同构成HIV-1包膜蛋白同源三聚体。HIV-1包膜蛋白三聚体的突出包膜外的结构域会发生几次结构重排，从可以逃避抗体识别的与宿主细胞膜融合前的封闭结构，以及结合人CD4细胞受体和辅助受体的中间结构，到膜融合后结构。
HIV膜融合抑制多肽：病毒和宿主细胞的膜融合是HIV对细胞感染的关键步骤。HIV包膜上的糖蛋白gp120/gp41同源三聚体与人CD4细胞受体和辅助受体结合后，经历几步的结构变化，最后是HIV包膜上整合的gp41三聚体插入宿主细胞膜，完成病毒和宿主细胞膜的融合，HIV的遗传物质进入细胞。HIV膜融合抑制多肽通过结合病毒的包膜蛋白，从而阻止其发生病毒和宿主CD4细胞膜融合必须的结构变化，因而阻止了HIV对CD4细胞的感染。本发明中使用的HIV膜融合抑制多肽包括以下实施例中披露的P52、C34、T20序 列或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
基因序列构建体：为一个由重组的多核苷酸序列组成的载体，由表达调控序列与待表达的核酸序列相连而成。表达载体包含足够的顺式作用元件，其它的表达元件由宿主细胞提供。表达载体包括本发明中所有的载体，例如携带整合了重组多核苷酸序列的质粒和病毒(例如，重组慢病毒和重组腺相关病毒)。载体上携带可以使其在宿主细胞中复制的核酸序列(DNA或RNA)，例如复制起始点，一个或者多个选择性标记基因，以及本发明中公开的其它基因结构。病毒载体为重组核酸载体，至少携带部分来自一个或者多个病毒的核酸序列。在以下某些实施例中，病毒载体包含编码所公开的能特异性结合HIV-1 gp160从而中和HIV-1的抗体或者抗原结合片段的一个或多个核酸序列。在有些实施例中，病毒载体可以是重组腺相关病毒(AAV)载体或是重组慢病毒载体。这些病毒载体为复制缺陷型载体，需要其它的携带病毒复制必须的基因功能或组分的辅组质粒或载体在细胞中进行扩增和包装产生病毒颗粒。纯化制备后的病毒载体或病毒颗粒用于对病人进行治疗的过程中不会在宿主细胞中复制扩增。
HIV感染的治疗：HIV是一种能攻击人体免疫系统的病毒。它把人体免疫系统中最重要的CD4T淋巴细胞作为主要攻击目标，大量破坏该细胞，使人体丧失免疫功能。许多急性HIV感染期的病人在感染后2-4周会有类似流感的症状，症状可能持续几天到几周的时间。急性感染期的病人的血液中有大量的HIV，具有很强的感染性。之后进入无症状的慢性感染期，也称作HIV潜伏期。但病人体内的HIV依然活跃，在持续地增殖，会传播HIV。HIV在人体内的潜伏期平均为8～9年，在HIV病毒潜伏期内，HIV感染者可以没有任何症状地生活和工作多年。但是HIV感染者如果不进行任何抗HIV感染治疗，经过潜伏期后会发展进入最严重的HIV感染期，即为，艾滋病阶段。艾滋病病人具有很高的病毒载量，很容易传染HIV给其他人，免疫系统严重受损，人体易于感染各种疾病，并发生恶性肿瘤，病死率较高。
目前在全世界范围内仍缺乏根治HIV感染的有效药物。现阶段的治疗目标是：最大限度和持久的降低病毒载量；获得免疫功能重建和维持免疫功能；提高生活质量；降低HIV相关的发病率和死亡率。目前普遍使用的抗HIV感染的方法为抗逆转录病毒联合疗法(即，鸡尾酒疗法)，大大提高了抗HIV的疗效，显著改善了患者的生活质量和预后。鸡尾酒疗法开始得越早越好，比如在HIV感染的急性期，即感染后的前几个月，在确诊HIV感染后即开始治疗。但鸡尾酒疗法的副作用及局限性，比如需要保持长期持续服药的依从性，以及逐渐浮现的HIV耐药毒株仍然给广大艾滋病患者造成了长期的经济和社会负担、生活品质的明显下降及明显的痛苦。
从小部分精英感染者体内鉴定分离出来的具有HIV-1中和活性的广谱中和抗体，能够高效及广谱的结合HIV病毒表面糖蛋白，中和HIV病毒对人CD4+T细胞的感染活性，代表了一种很有前景的预防或抗HIV-1感染的方法。与小分子抗HIV药物相比，重组来源的中和抗体也能极大的减少毒副作用，同时增加病患用药的依从性。目前唯一获批临床使用的抗HIV中和抗体是针对人T细胞表面的CD4受体的Ibalizumab，它通过结合CD4受体干扰HIV病毒表面糖蛋白与CD4受体的结合，从而中和病毒对CD4+T细胞的感染活性，对艾滋病病毒感染多重耐药病人显示出优秀的疗效。
但特定的广谱中和抗体只针对单一病毒蛋白(HIV-gp160)的某一个单一表位，由病毒突变产生的逃逸现象仍然难以避免，并且由于广谱中和抗体研制、生产、使用成本均远高于小分子抗艾滋病病毒药物，联合用药不具优势。本发明以研发双嗜性、多嗜性中和抗体或类抗体大分子药物，以多靶点覆盖避免病毒突变产生的逃逸，以及把蛋白广谱中和抗体或类抗体大分子药物基因化作为抗HIV广谱中和抗体药物研发的突破。以重组病毒或非病毒载体递送的抗HIV感染基因治疗药物，以基因组整合或非整合的方式在机体内长期稳定表达中和抗体或类抗体大分子，一次治疗长期(例如，药效持续一年或数年)甚至终身有效，极大地减少大分子抗体药物的生产和使用成本。
以下实施例是对本发明的解释而本发明并不仅局限于以下实施例。
实施例
一、表达HIV中和抗体的结构设计
本发明中应用的抗HIV中和抗体和类抗体分子的结构为:
1.三嗜性抗HIV中和类抗体scFv分子：由蛋白分子的N端到C端分别是—信号肽-抗HIV-gp41广谱中和抗体单链可变区片段(VL-(ggggs) nlinker-VH)-(ggggs) nlinker-抗人CD4抗体的单链可变区片段(VL-(ggggs) nlinker-VH)-(ggggs) nlinker—HIV膜融合抑制短肽。所示基因编码序列在细胞中表达被翻译成蛋白后被分泌出细胞外。预期抗HIV中和类抗体的成熟分子结构如图1显示,基因结构如图2显示。
2.双嗜性抗HIV中和类抗体scFv分子：由蛋白分子的N端到C端分别是—信号肽-抗HIV-gp41广谱中和抗体单链可变区片段(VL-(ggggs) nlinker-VH)-(ggggs) nlinker-抗人CD4抗体的单链可变区片段(VL-(ggggs) nlinker-VH)。所示基因编码序列在细胞中表达被翻译成蛋白后被分泌出细胞外。预期抗HIV中和类抗体的成熟分子结构如图3显示,基因结构如图4显示。
二、表达HIV中和抗体的基因序列
其中抗HIV-gp41广谱中和抗体单链可变区片段(scFv)序列分别为：信号肽(蛋白序列见SEQ ID NO:1)；抗HIV-1-gp41-MPER抗体(10E8v4-5R+100cF)-scFv(蛋白序列见SEQ ID NO:2)；抗人CD4抗体(Ibalizumab)的单链可变区片段scFv(蛋白序列见SEQ ID NO:3)，HIV膜融合抑制短肽P52(蛋白序列见SEQ ID NO:4),HIV膜融合抑制短肽C34(蛋白序列见SEQ ID NO:5),HIV膜融合抑制短肽T20(蛋白序列见SEQ ID NO:6)。
三、共构建抗HIV中和抗体基因表达框架2个(如图2和图4)，分别是：
1.包含抗HIV中和抗体10E8v4-5R+100cF–scFv的抗HIV双嗜性中和类抗体分子KL-BsHIV01-02(蛋白序列见SEQ ID NO:7)(DNA 序列见SEQ ID NO:8)(图4)。
2.包含抗HIV中和抗体10E8v4-5R+100cF–scFv的抗HIV三嗜性中和类抗体分子KL-BsHIV01-003-02(蛋白序列见SEQ ID NO:9)(DNA序列见SEQ ID NO:10)(图2)。
四、HIV中和抗体的基因治疗载体构建体示例
如图5所示，将上述单抗分子基因表达框架以多片段重组连接的方式克隆到当前应用的最新一代慢病毒载体pKL-kan-lenti-CBH-WPRE(图6)(DNA序列见SEQ ID NO:11)中。所述慢病毒载体包括：5′LTR，其中用CMV启动子替换LTR的启动子区域；ψ包装信号；逆转录病毒输出元件RRE；cPPT；启动子CBH；编码HIV中和抗体片段的多肽的多核苷酸；转录后调节元件为WPRE；PPT；ΔU3 3’LTR；以及poly(A)信号。本实施例设计的中和抗体基因表达框架(图2和图4)，经南京金斯瑞生物技术有限公司合成后和CBH启动子(序列见SEQ ID NO：22)，通过本领域熟知的以同源重组的方法克隆至慢病毒载体骨架pKL-kan-lenti-CBH-WPRE上的多克隆位点AgeI/EcoRV之间(图6)，克隆完成后以测序确认序列信息，质粒分别命名为pKL-Kan-lenti-CBH-KL-BsHIV01-02(序列见SEQ ID NO：12),pKL-Kan-lenti-CBH-KL-BsHIV01-003-02(序列见SEQ ID NO：13)。
如图7所示，将存在于pKL-Kan-lenti-CBH-KL-BsHIV01的HIV中和抗体基因表达框架CBH-KL-BsHIV01-02，CBH-KL-BsHIV01-003-02，分别以多片段重组连接的方式克隆到当前应用的最新一代腺相关病毒载体pAAV-MCS-CBH-WPRE(序列见SEQ ID NO：14)的多克隆位点AgeI/SalI之间(见图8)。所述腺相关病毒载体包括：AAV2 ITR；启动子CBH；编码HIV中和抗体片段的多核苷酸；WPRE及SV40 poly(A)信号；AAV2 ITR。质粒命名为pAAV-CBH-KL-BsHIV01-02-WPRE(序列见SEQ ID NO：15)和pAAV-CBH-KL-BsHIV01-003-02-WPRE(序列见SEQ ID NO：16)。
五、表达HIV中和抗体的病毒包装及纯化
以慢病毒载体(pKL-Kan-lenti-CBH-KL-BsHIV01-02、pKL-Kan-lenti-CBH-KL-BsHIV01-003-02)在293T细胞系中进行HIV中和抗体基因治疗慢病毒载体的包装。将实施例中构建的HIV中和抗体基因治疗慢病毒载体(pKL-Kan-lenti-CBH-KL-BsHIV01-02、pKL-Kan-lenti-CBH-KL-BsHIV01-003-02、包膜质粒(pKL-Kan-Vsvg，其核苷酸序列如SEQ ID NO:17所示)和包装质粒(pKL-Kan-Rev，其核苷酸序列如SEQ ID NO:18所示；pKL-Kan-GagPol，其核苷酸序列如SEQ ID NO:19所示)混合后同时共转染293T细胞(购自美国模式菌种收集中心(ATCC)，ATCC保藏号为CRL-3216)，在该293T细胞系中进行HIV中和抗体基因治疗慢病毒的包装。转染方法为PEI阳离子聚合物介导的真核细胞瞬时转染，PEI阳离子聚合物为购自Polysciences的PEI-Max转染试剂(购自Polysciences，货号：24765-1)，转染操作参照生产商推荐标准化操作进行,转染规模为15cm细胞培养皿。转染完成48小时后，收获慢病毒载体(转染细胞培养上清)，首先在台式吊桶式离心机上，室温4000rpm离心5分钟去除细胞碎片，再4℃10000g离心4小时获得病毒颗粒沉淀，去除离心上清后，以1mL DMEM完全培养基加入病毒颗粒沉淀中，以微量进样器重悬病毒颗粒，并将制备好的病毒重悬液分装冻存于-80℃备用。
以AAV表达载体(pAAV-CBH-KL-BsHIV01-02-WPRE，pAAV-CBH-KL-BsHIV01-003-02-WPRE)在293T细胞系中进行HIV中和抗体基因治疗AAV载体的包装。将实施例中构建的HIV中和抗体基因治疗AAV载体、衣壳质粒(AAV2/8，其核苷酸序列如SEQ ID NO:20所示)和包装质粒(pHelper，其核苷酸序列如SEQ ID NO:21所示)混合后同时共转染293T细胞，在该293T细胞系中进行HIV中和抗体基因治疗载体AAV的包装。转染方法为PEI阳离子聚合物介导的真核细胞瞬时转染，PEI阳离子聚合物为购自Polysciences的PEI-Max转染试剂(购自Polysciences，货号：24765-1)，转染操作参照生产商推荐标准化操作进行,转染规模为15cm细胞培养皿。转染后7h吸弃上清， 更换为25ml产毒培养基。转染完成120小时后，收集上清和细胞，4200rpm离心10min，离心完成后分离上清跟细胞，细胞加裂解液跟核酶，裂解消化1h，10000g离心10min取裂解液上清。将裂解液上清及培养基上清经亲和层析纯化后分装冻存于-80℃备用。
六、慢病毒基因治疗载体转导细胞上清表达的HIV中和抗体的功能验证
将包装好的慢病毒载体pKL-Kan-lenti-CBH-KL-BsHIV01-02、pKL-Kan-lenti-CBH-BsHIV01-003-02按照不同的MOI感染293T细胞。48小时后收集上清和部分细胞。通过探针法检测慢病毒载体感染拷贝数。
1.收集慢病毒载体感染293T细胞，用PBS清洗细胞后，4200rpm,5min离心收集细胞，重悬以20μl快速抽提溶液(QE DNA Extraction Solution)并用PCR仪运行以下程序以裂解细胞和抽提总DNA。
裂解细胞PCR程序：
温度 时间
65℃ 15min
68℃ 15min
95℃ 10min
通过本领域熟知的方法，以定量PCR计算出293T细胞感染慢病毒拷贝数(VCN)。
2.将TZM-bl细胞按2E4cells/孔铺板。分别取50μL VCN为1的不同细胞抗体表达上清稀释不同倍数后与50μL HIV病毒pAD-8或pNL4-3混合后，37℃孵育30min，加入到TZM-bl细胞中，只加pAD-8或pNL4-3的孔为阴性对照(NC)，不加HIV病毒的孔为Blank。在第24h时，弃上清，加入100μL细胞裂解液，10min后收集裂解后的细胞，8000rpm离心5min。在50μL裂解的细胞加入100μL萤火虫萤光素酶检测试剂。用化学发光功能的多功能酶标仪检测测定RLU(relative light unit)。结果显示，在体外细胞试验中，在相同的感染拷贝数时，HIV中和抗体慢病毒基因治疗载体转导293T细胞培养上清中存在可以 中和HIV病毒的抗体，且双嗜性中和抗体KL-BsHIV01-02中和效果显著优于KL-BsHIV01(表1)，而三嗜性中和抗体KL-BsHIV01-003-02中和效果显著优于KL-BsHIV01-003(表2)。
表1.HIV中和抗体慢病毒基因治疗载体转导的293T细胞培养上清对病毒的中和百分比
Figure PCTCN2022115831-appb-000001
表2.HIV中和抗体慢病毒基因治疗载体转导的293T细胞培养上清对病毒的中和百分比
Figure PCTCN2022115831-appb-000002
七、腺相关病毒基因治疗载体转导细胞上清表达的HIV中和抗体的功能验证
C2C12成肌细胞24孔细胞培养板，每孔1E5cells。用2％马血清培养基分化成肌管细胞。将包装好的腺相关病毒pAAV-CBH-KL-BsHIV01、pAAV-CBH-KL-BsHIV01-02、pAAV-CBH-KL-BsHIV01-003、pAAV-CBH-KL-BsHIV01-003-02按照不同的MOI感染分化好的C2C12细胞。96小时后收集上清。
将TZM-bl细胞按2E4cells/孔铺板。取MOI同为8E4vg的KL-BsHIV01,KL-BsHIV01-02,KL-BsHIV01-003，和KL-BsHIV01-003-02的细胞表达上清，分别取与含Fc段的双嗜性抗体KL-BsHIV01及三嗜性抗体KL-BsHIV01-003相同体积的不含Fc段的双嗜性抗体KL-BsHIV01-02和三嗜性抗体KL-BsHIV01-003-02的细胞表达上清，与50μL HIV病毒pAD-8或pNL4-3混合后，37℃孵育30min，加入到TZM-bl细胞中，只加pAD-8或pNL4-3的孔为阴性对照(NC)，不加HIV病毒的孔为Blank。在第24h时，弃上清，加入100μL细胞裂解液，10min后收集裂解后的细胞，8000rpm离心5min。在50μL裂解的细胞加入100μL萤火虫萤光素酶检测试剂。用化学发光功能的多功能酶标仪检测测定RLU(relative light unit)。结果显示(表3)，在体外细胞试验中，在MOI相同的情况下，不含Fc段的scFv单体KL-BsHIV01-02和KL-BsHIV01-003-02对HIV病毒的中和效果要显著优于带Fc段的KL-BsHIV01和KL-BsHIV01-003。
表3.HIV中和抗体腺相关病毒基因治疗载体转导的C2C12细胞培养上清对病毒的中和百分比
Figure PCTCN2022115831-appb-000003
Figure PCTCN2022115831-appb-000004
八、腺相关病毒基因治疗载体肌肉注射后在BALB/c小鼠中的表达
分别按2E11vg/只的剂量，将HIV中和抗体AAV基因治疗载体(pAAV-CBH-KL-BsHIV01-02-WPRE，pAAV-CBH-KL-BsHIV01-003-02-WPRE)通过肌肉注射小鼠后肢股部肌肉。每隔1周取一次血，分离血清。
将TZM-bl细胞按2E4cells/孔铺板。分别取相同体积的血清稀释液与50μL HIV病毒pAD-8或pNL4-3混合后，37℃孵育30min，加入到TZM-bl细胞中，只加pAD-8或pNL4-3的孔为阴性对照(NC)，不加HIV病毒的孔为Blank。在第24h时，弃上清，加入100μL细胞裂解液，10min后收集裂解后的细胞，8000rpm离心5min。在50μL裂解的细胞加入100μL萤火虫萤光素酶检测试剂。用化学发光功能的多功能酶标仪检测测定RLU(relative light unit)。结果显示(表4)，在体外细胞试验中，加入相同体积的HIV中和抗体后，不含IgG1恒定区Fc段的scFv单体KL-BsHIV01-02和KL-BsHIV01-003-02对HIV病毒的中和效果要显著优于带Fc段的KL-BsHIV01和KL-BsHIV01-003(其为我司另外一个专利申请中的抗HIV中和类抗体的构建体)。
表4.HIV中和抗体腺相关病毒基因治疗载体小鼠肌肉注射血清对病毒的中和百分比
Figure PCTCN2022115831-appb-000005
Figure PCTCN2022115831-appb-000006
九、细胞表达HIV中和抗体的鉴定
将携带Flag-KL-BsHIV01-003-02的慢病毒质粒瞬转293T细胞，48h后收集上清，用anti-DYKDDDDK(即Flag-tag)G1 Affinity Resin(金斯瑞，L00432-10)纯化Flag-KL-BsHIV01-003-02，得到的Flag-KL-BsHIV01-003-02用肠激酶(近岸，PE001-01A)酶切。将酶切前后的Flag-KL-BsHIV01-003-02用兔Anti-KL-BsHIV01-003-02或鼠Anti-DYKDDDDK(金斯瑞，A00187-100)进行Western blotting鉴定(图9)。Western blotting结果确认了携带Flag-tag或不携带Flag-tag的HIV中和抗体KL-BsHIV01-003-02在细胞中的表达并分泌到上清中，且具有与预计相符合的分子量。
十、细胞中表达纯化的HIV中和抗体的活性鉴定
将TZM-bl细胞按2E4cells/孔铺板。分别取纯化的抗体的稀释液与50μL HIV病毒pAD-8或pNL4-3混合后，37℃孵育30min，加入到TZM-bl细胞中，只加pAD-8或pNL4-3的孔为阴性对照，不加HIV病毒的孔为Blank。培养24h后，弃上清，加入100μL细胞裂解液，10min后收集裂解后的细胞，8000rpm离心5min。在50μL裂解的细胞加入100μL萤火虫萤光素酶检测试剂。用化学发光功能的多功能酶标仪检测测定RLU(relative light unit)。结果显示，在细胞试验中，不带Fc片段的抗HIV三嗜性中和抗体(KL-BsHIV01-003-02)与带Fc片段的三嗜性中和抗体(KL-BsHIV01-003)相似，都具有很强的中和HIV的活性(图10)，两者中和HIV-1毒株pAD-8(CCR5嗜性)的IC50分别为3.1pM和1.2pM，而两者中和HIV-1毒株pNL4-3(CXCR4嗜性)的IC50分别为0.2pM和0.6pM。我司发明的这两种HIV中和抗体比已经报道的HIV广谱中和抗体具有更强的对HIV的中和活性。
十一、腺相关病毒基因治疗载体肌肉注射后在BALB/c小鼠中的表达
将不同剂量的AAV基因治疗载体(pAAV-CBH-KL-BsHIV01-003--02WPRE)通过肌肉注射小鼠后肢股部肌肉。每隔一段时间取一次血，分离血清。
1.ELISA检测血清中表达的抗体浓度。用合成的HIV MPER多肽包被酶标板(Corning，货号：42592)，表达上清及纯化定量的KL-BsHIV01-003-02标准品为1级抗体，以兔anti-HIV膜融合抑制短肽的多抗作为2级抗体，以HRP标记的羊抗兔IgG(KPL，5220-0283)为3级抗体，用TMB显色，酶标仪检测450nm的OD值。ELISA结果(图11)显示抗HIV中和抗体腺相关病毒载体在小鼠体内能持续有效地表达HIV中和抗体。
2.将TZM-bl细胞按2E4cells/孔铺板。分别取稀释200倍的血清稀释液与50μL HIV病毒株pNL4-3混合后，37℃孵育30min，加入到TZM-bl细胞中，只加pNL4-3的孔为阴性对照，不加HIV病毒的孔为Blank。过夜培养后，弃上清，加入100μL细胞裂解液，10min后收集裂解后的细胞，8000rpm离心5min。在50μL裂解的细胞加入100μL萤火虫萤光素酶检测试剂。用化学发光功能的多功能酶标仪检测测定RLU(relative light unit)。结果显示，在细胞试验中，小鼠血清中表达的HIV中和抗体在稀释200倍后对HIV毒株pNL4-3有优异的中和活性，且活性随剂量提高而增加(图12)。
Figure PCTCN2022115831-appb-000007
Figure PCTCN2022115831-appb-000008
Figure PCTCN2022115831-appb-000009
Figure PCTCN2022115831-appb-000010
Figure PCTCN2022115831-appb-000011
Figure PCTCN2022115831-appb-000012
Figure PCTCN2022115831-appb-000013
Figure PCTCN2022115831-appb-000014
Figure PCTCN2022115831-appb-000015
Figure PCTCN2022115831-appb-000016
Figure PCTCN2022115831-appb-000017
Figure PCTCN2022115831-appb-000018
Figure PCTCN2022115831-appb-000019
Figure PCTCN2022115831-appb-000020
Figure PCTCN2022115831-appb-000021
Figure PCTCN2022115831-appb-000022
Figure PCTCN2022115831-appb-000023
Figure PCTCN2022115831-appb-000024
Figure PCTCN2022115831-appb-000025
Figure PCTCN2022115831-appb-000026
Figure PCTCN2022115831-appb-000027
Figure PCTCN2022115831-appb-000028
Figure PCTCN2022115831-appb-000029
Figure PCTCN2022115831-appb-000030
Figure PCTCN2022115831-appb-000031
Figure PCTCN2022115831-appb-000032
Figure PCTCN2022115831-appb-000033
Figure PCTCN2022115831-appb-000034
Figure PCTCN2022115831-appb-000035
Figure PCTCN2022115831-appb-000036
Figure PCTCN2022115831-appb-000037
Figure PCTCN2022115831-appb-000038
Figure PCTCN2022115831-appb-000039
Figure PCTCN2022115831-appb-000040
Figure PCTCN2022115831-appb-000041

Claims (50)

  1. 一种用于艾滋病病毒(HIV)感染基因治疗的基因序列构建体,其包含一个或多个具有抑制HIV感染能力的不带恒定区的单链抗体分子(包括纳米抗体)的基因编码序列和一个或多个具有抑制HIV感染能力的多肽(由2-50氨基酸残基组成)的基因编码序列，以达到表达一种由单基因编码的包含抗HIV感染的抗体分子和多肽的融合蛋白分子，其具有两种或两种以上作用靶点。
  2. 根据权利要求1所述的基因序列构建体，其中所述单链抗体分子包含重链可变区域和/或轻链可变区域。
  3. 根据权利要求2所述的基因序列构建体，其中所述轻链可变区域包含κ或λ轻链的轻链可变区域。
  4. 根据权利要求1-3中任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列。
  5. 根据权利要求1-4中任一项所述的基因序列构建体，其包含三个或三个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列。
  6. 根据权利要求1-5中任一项所述的基因序列构建体，其包含四个或四个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列。
  7. 根据权利要求1-6中任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的多肽的基因编码序列。
  8. 根据权利要求1-7中任一项所述的基因序列构建体，其包含三个或三个以上具有抑制HIV感染能力的多肽的基因编码序列。
  9. 根据权利要求1-8中任一项所述的基因序列构建体，其包含四个或四个以上具有抑制HIV感染能力的多肽的基因编码序列。
  10. 根据权利要求1-9中任一项所述的基因序列构建体，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有三种或三种以上作用靶点。
  11. 根据权利要求1-10中任一项所述的基因序列构建体，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有四种或四种以上作用靶点。
  12. 根据权利要求1-11中任一项所述的基因序列构建体，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有五种或五种以上作用靶点。
  13. 根据权利要求1-12中任一项所述的基因序列构建体，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有六种或六种以上作用靶点。
  14. 根据权利要求1-13中任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和一个或多个具有抑制HIV感染能力的多肽的基因编码序列。
  15. 根据权利要求14所述的基因序列构建体，其中由单基因编码的包含抗HIV感染的不带恒定区的单链抗体分子和多肽的融合蛋白分子具有三种或三种以上作用靶点。
  16. 根据权利要求1-15中任一项所述的基因序列构建体，其中所述的具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和具有抑制HIV感染能力的多肽的基因编码序列之间通过连接头多肽的编码序列直接或间接串联而成。
  17. 根据权利要求1-16中任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列，其中所述的抗体分子的基因编码序列之间通过连接头多肽的编码序列直接或间接串联而成。
  18. 根据权利要求1-17中任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的多肽的基因编码序列，其中所述的多肽的基因编码序列之间通过连接头多肽的编码序列直接或间接串联而成。
  19. 根据权利要求1-18中任一项所述的基因序列构建体，其中所述的一个或多个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列 包含抗HIV-1-gp160(或者其剪切产物gp120和gp41)的抗体分子的基因编码序列。
  20. 根据权利要求1-19中任一项所述的基因序列构建体，其中所述的一个或多个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列包含结合人CD4受体位点的抗体分子的基因编码序列。
  21. 根据权利要求1-20中任一项所述的基因序列构建体，其中所述的一个或多个具有抑制HIV感染能力的多肽的基因编码序列包含抑制HIV与CD4+T细胞膜融合的多肽的基因编码序列。
  22. 根据权利要求1-21中任一项所述的基因序列构建体，其包含二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和一个或多个具有抑制HIV感染能力的多肽的基因编码序列，其中所述的二个或二个以上具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列包含抗HIV-1-gp160(或者其剪切产物gp120和gp41)的抗体分子的基因编码序列和结合人CD4受体位点的抗体分子的基因编码序列，和其中所述的一个或多个具有抑制HIV感染能力的多肽的基因编码序列包含抑制HIV与CD4+T细胞膜融合的多肽的基因编码序列。
  23. 根据权利要求1-22中任一项所述的基因序列构建体，其包括(i)抗HIV-1-gp160(或者其剪切产物gp120和gp41)单克隆抗体的轻链互补决定区(LCDR1,LCDR2和LCDR3)和重链互补决定区(HCDR1,HCDR2和HCDR3)的基因编码序列、(ii)结合人CD4受体位点的单克隆抗体的轻链互补决定区(LCDR1,LCDR2和LCDR3)和重链互补决定区(HCDR1,HCDR2和HCDR3)的基因编码序列、(iii)抑制HIV与CD4+T细胞膜融合的多肽的基因编码序列，其中抗体的轻链和重链的编码序列可以不分先后的次序通过连接头多肽的编码序列直接或间接串联而成。
  24. 根据权利要求1-23中任一项所述的基因序列构建体，其包括(i)抗HIV-1-gp160(或者其剪切产物gp120和gp41)单克隆抗体的轻链可变区(VL)和重链可变区(VH)的基因编码序列、(ii)结合人CD4受体位点的单克隆抗体的轻链可变区(VL)和重链可变区(VH)的基因编码序列、(iii)抑制HIV与 CD4+T细胞膜融合的多肽的基因编码序列，其中抗体的轻链可变区(VL)和重链可变区(VH)的编码序列可以不分先后的次序通过连接头多肽的编码序列直接或间接串联而成。
  25. 根据权利要求1-24中任一项所述的基因序列构建体，其另外包含启动子位于具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和具有抑制HIV感染能力的多肽的基因编码序列的上游。
  26. 根据权利要求1-25中任一项所述的基因序列构建体，其另外包含分泌信号肽编码序列位于具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列和具有抑制HIV感染能力的多肽的基因编码序列的上游。
  27. 根据权利要求1-26中任一项所述的基因序列构建体，其包含第一个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列，第二个具有抑制HIV感染能力的不带恒定区的单链抗体分子的基因编码序列，和具有抑制HIV感染能力的多肽的基因编码序列，其选自：
    VL2-linker-VH2-linker-VL1-linker-VH1-linker；或
    VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor；或
    其它把VL2和VH2或VL1和VH1的组合在构建体中按不同的先后次序排列而成的构建体；
    其中，VL2和VH2分别为第一个抗体分子的轻链和重链的可变区片段；VL1和VH1分别为第二个抗体分子的轻链和重链的可变区片段；linker为连接头多肽；peptide inhibitor为抑制HIV感染的多肽(例如，抑制HIV与CD4+T细胞膜融合的多肽)。
  28. 根据权利要求27所述的基因序列构建体，其为VL2-linker-VH2-linker-VL1-linker-VH1，或把其中VL2和VH2或VL1和VH1的组合按不同的先后次序排列而成的构建体。
  29. 根据权利要求27所述的基因序列构建体，其为VL2-linker-VH2-linker-VL1-linker-VH1-linker-peptide inhibitor，或把其中VL2和VH2或VL1和VH1的组合按不同的先后次序排列而成的构建体。
  30. 根据权利要求27,28、和29所述的基因序列构建体，其中VL2和VH2的蛋白序列包括SEQ ID NO:2,或其功能性片段，或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
  31. 根据权利要求27,28、和29所述的基因序列构建体，其中VL1和VH1的蛋白序列包括SEQ ID NO:3，或其功能性片段，或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
  32. 根据权利要求27,28、和29所述的基因序列构建体，其中连接头多肽(linker)的序列选自以下的氨基酸序列：GGGGS、(GGGGS) 2、(GGGGS) 3、(GGGGS) 4、(GGGGS) 5、(GGGGS) 6、和(GGGGS) 7，或其它可选的连接头多肽序列。
  33. 根据权利要求27和29所述的基因序列构建体，其中抑制HIV与CD4+T细胞膜融合的多肽可选自膜融合抑制多肽P52、C34、T20等。
  34. 根据权利要求33所述的基因序列构建体，其中，膜融合抑制多肽P52的序列包括SEQ ID NO:5或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列；C34的多肽序列包括SEQ ID NO:6或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列；T20的多肽序列包括SEQ ID NO:7或与其具有至少75％，80％，85％，90％，95％，98％，或99％相同的同源序列。
  35. 病毒载体基因组，其包含前述权利要求中任一项的构建体。
  36. 病毒载体系统，其包含根据权利要求35的基因组。
  37. 根据权利要求36的病毒载体系统，其为慢病毒载体系统或腺相关病毒载体系统。
  38. 根据权利要求37的慢病毒载体系统，其包含权利要求35的基因组以及其它编码和表达生产慢病毒所需的包装组件的核苷酸序列，其会被导入生产细胞生产包含权利要求35的基因组的慢病毒颗粒。
  39. 根据权利要求37的腺相关病毒载体系统，其包含权利要求35的基因组以及其它编码和表达生产腺相关病毒所需的包装组件的核苷酸序列，其会被导入生产细胞生产包含权利要求35的基因组的腺相关病毒颗粒。
  40. 一种病毒颗粒，其包含根据权利要求1-34中任一项所述的构建体的基因组。
  41. 药物组合物，其包含根据权利要求40所述的一种病毒颗粒，以及药学上可接受的载体或稀释剂，或者在体外被根据权利要求38所述的慢病毒颗粒所转导的细胞，其包括但不仅限于被转导的肌肉细胞，肝脏细胞，或CD4+T细胞。
  42. 根据权利要求38-40所述的一种病毒颗粒或根据权利要求41所述的药物组合物，其用于注射到体内以表达两种或两种以上作用靶点的抗体分子蛋白，其可通过结合HIV感染人CD4+T细胞不同步骤中涉及的多个结合位点从而高效和广谱地阻断HIV对人CD4+T细胞的感染进程，以用于对HIV感染的基因治疗，从而达到对艾滋病病毒感染者的长期治疗。
  43. 一种抑制HIV感染的方法，包括向细胞施用权利要求38-41所述的一种病毒颗粒或药物组合物。
  44. 根据权利要求43所述的方法，其中所述的细胞包含肌肉细胞，肝脏细胞，或CD4+T细胞等。
  45. 根据权利要求43所述的方法，其方法包括使用根据权利要求38-41所述的一种病毒颗粒或药物组合物在体外或体内对权利要求44所述的细胞进行转导。
  46. 一种在需要治疗的受试者中治疗HIV感染的方法，所述方法包括向该受试者施用治疗有效剂量的权利要求38-41所述的一种病毒颗粒或药物组合物。
  47. 根据权利要求46所述的方法，其中所述的受试者包含HIV早期感染者或已经在接受鸡尾酒药物疗法的HIV感染者或对鸡尾酒药物疗法具有抗性的HIV感染者。
  48. 根据权利要求42所述的方法，通过肌肉注射根据权利要求38-41所述的一种病毒颗粒或药物组合物。
  49. 根据权利要求42所述的方法，通过静脉注射根据权利要求38-41所述的一种病毒颗粒或药物组合物或经其转导的CD4+T细胞。
  50. 根据权利要求48和49所述的方法注射到体内的病毒颗粒和药物组合物，其能表达具有多作用靶点的抗HIV的蛋白分子并分泌到血液中，其能作用于多个HIV感染的节点，而能有效地阻断HIV感染路径并有效地避免由于HIV发生逃逸突变而失去对HIV感染的抑制能力，从而达到单次注射给药即具有对HIV感染的长期(例如，药效持续一年或数年)甚至永久的治疗效果。
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