Gene sequence constructs for gene therapy of AIDS virus infection.

Abstract

The present invention relates to a gene sequence construct for gene therapy of AIDS virus infection. A gene sequence construct is constructed by connecting the gene coding sequences of the variable regions scFv of the light and heavy chains of a monoclonal antibody against different binding site antigens involved in different steps of HIV infection of human CD4+ T cells, the gene coding sequence of the variable regions scFv of the light and heavy chains of a monoclonal antibody binding to a human CD4 receptor site, the gene coding sequence of an Fc fragment in the constant region of human IgG, and the gene coding sequence of a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane in order via a linker polypeptide coding sequence, and arranging them downstream of a promoter and a secretory signal peptide coding sequence to express a secretory antibody-like protein molecule encoded by a single gene. This recombinant single gene construct can be easily introduced into target tissue cells via a viral vector, and the expressed and secreted antibody-like protein molecule has polyantigenophilicity and binds to multiple binding sites involved in different steps of HIV infection of human CD4+ T cells, thereby efficiently and broadly blocking the progression of HIV infection in human CD4+ T cells and effectively avoiding the loss of suppressive ability against HIV infection due to the occurrence of HIV escape mutations, thereby achieving long-term and even permanent therapeutic effects against HIV infection with a single injection.

Description

[Technical field]

[0001] This application claims priority to PCT Patent Application No. PCT / CN2021 / 115420, filed on August 30, 2021, the contents of which are incorporated in their entirety as part of this application.

[0002] The present invention belongs to the field of gene therapy / biomedicine technology, specifically relates to a gene sequence construct for gene therapy of AIDS virus (HIV) infection. The gene sequence construct can be used in gene therapy for HIV infection. The gene sequence construct can be used to express a polyphilic neutralizing antibody-like protein that has a broad spectrum and highly efficient neutralization of HIV virus activity both in vivo and in vitro, and can be used in clinical research of gene therapy drugs for HIV infection delivered from recombinant virus or non-virus vectors and in research and development of new drugs. [Background technology]

[0003] AIDS, caused by infection with the human immunodeficiency virus (HIV), is one of the world's most widespread infectious diseases. Of the 37.9 million people infected with HIV worldwide, approximately 770,000 die of AIDS each year. With the development of anti-AIDS drugs, the expected survival time of AIDS virus-infected people who are actively treated has been significantly extended, but the side effects and limitations of cocktail therapy, as well as the gradually emerging drug-resistant strains of HIV, still cause long-term economic and social burdens, significant decline in quality of life, and significant suffering for the vast majority of AIDS patients.

[0004] Therefore, despite the advances already made in the anti-AIDS field, there remains a need for the development of new drugs and methods for treating AIDS virus infection. Summary of the Invention

[0005] The present invention creates a series of recombinant viral vector-based anti-AIDS virus gene therapy constructs, such as single-chain antibody variable fragments (scFv) of broadly neutralizing antibodies against multiple HIV species and neutralizing antibodies against human CD4 receptor, combined with Fc fragments of human antibody constant regions and HIV membrane fusion inhibitor polypeptides, into a simple and efficient expression framework.

[0006] First, these gene sequence constructs for gene therapy of HIV infection contain gene coding sequences for one or more antibody molecules capable of suppressing HIV infection and one or more polypeptides (consisting of 2 to 50 amino acid residues) capable of suppressing HIV infection, so as to express a fusion protein molecule containing an anti-HIV antibody molecule and a polypeptide, encoded by a single gene, and the fusion protein molecule has two or more types of action targets.

[0007] In particular, the antibody molecule comprises a heavy chain constant region and / or a light chain constant region.

[0008] Here, the heavy chain constant region comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

[0009] wherein said light chain constant region comprises a light chain constant region of a κ or λ light chain.

[0010] wherein the light chain variable region comprises a light chain variable region of a κ or λ light chain.

[0011] The gene sequence construct comprises two or more gene coding sequences for antibody molecules capable of inhibiting HIV infection.

[0012] Or, it contains three or more gene coding sequences for antibody molecules capable of suppressing HIV infection.

[0013] Or, it contains four or more gene coding sequences for antibody molecules capable of suppressing HIV infection.

[0014] The gene sequence construct may also contain two or more gene coding sequences for polypeptides capable of inhibiting HIV infection, the polypeptides consisting of 2-50 amino acid residues.

[0015] Or, it contains three or more gene coding sequences for polypeptides having the ability to inhibit HIV infection.

[0016] Or, it contains four or more gene coding sequences for polypeptides having the ability to inhibit HIV infection.

[0017] In any one of the gene sequence constructs described above, the fusion protein molecule comprising the anti-HIV infection antibody molecule and the polypeptide, encoded by a single gene, has three or more action targets.

[0018] Alternatively, a fusion protein molecule comprising an anti-HIV infection antibody molecule and a polypeptide, encoded by a single gene, has four or more targets of action.

[0019] Alternatively, a fusion protein molecule comprising an anti-HIV infection antibody molecule and a polypeptide, encoded by a single gene, has five or more targets of action.

[0020] Alternatively, a fusion protein molecule comprising an anti-HIV infection antibody molecule and a polypeptide, encoded by a single gene, has six or more targets of action.

[0021] The gene sequence construct described in any one of the above comprises gene coding sequences for two or more antibody molecules capable of inhibiting HIV infection, and gene coding sequences for one or more polypeptides capable of inhibiting HIV infection.

[0022] In the above gene sequence construct, the fusion protein molecule containing the anti-HIV antibody molecule and the polypeptide, encoded by a single gene, has three or more action targets.

[0023] In the gene sequence construct described in any one of the above, the gene coding sequence for the single-chain antibody molecule having the ability to inhibit HIV infection and having no constant region and the gene coding sequence for the polypeptide having the ability to inhibit HIV infection are directly or indirectly connected in series via the coding sequence for a linker polypeptide.

[0024] The gene sequence construct described in any one of the above contains two or more gene coding sequences for antibody molecules capable of suppressing HIV infection, and the gene coding sequences for the antibody molecules are directly or indirectly connected in series between each other via a coding sequence for a linker polypeptide.

[0025] The gene sequence construct described in any one of the above contains two or more gene coding sequences for polypeptides capable of suppressing HIV infection, and the gene coding sequences for the polypeptides are directly or indirectly connected in series between each other via a coding sequence for a linker polypeptide.

[0026] In the gene sequence construct described in any one of the above, the gene coding sequence for one or more antibody molecules capable of inhibiting HIV infection includes a gene coding sequence for an antibody molecule of anti-HIV-1-gp160 (including its cleavage products gp120 and gp41).

[0027] In the gene sequence construct according to any one of the above, the gene coding sequence for one or more antibody molecules capable of inhibiting HIV infection comprises a gene coding sequence for an antibody molecule that binds to the human CD4 receptor site.

[0028] In the gene sequence construct described in any one of the above, the gene coding sequence for one or more polypeptides capable of suppressing HIV infection includes a gene coding sequence for a polypeptide that inhibits the fusion of HIV with a CD4+ T cell membrane.

[0029] The gene sequence construct described in any one of the above comprises gene coding sequences of two or more antibody molecules having the ability to inhibit HIV infection, and gene coding sequences of one or more polypeptides having the ability to inhibit HIV infection, wherein the gene coding sequences of the two or more antibody molecules having the ability to inhibit HIV infection include a gene coding sequence of an anti-HIV-1-gp160 antibody molecule (including its cleavage products gp120 and gp41) and a gene coding sequence of an antibody molecule that binds to the human CD4 receptor site, and the gene coding sequence of the one or more polypeptides having the ability to inhibit HIV infection includes a gene coding sequence of a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane.

[0030] The gene sequence construct according to any one of the above comprises (i) gene coding sequences of light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of an anti-HIV-1-gp160 (including its cleavage products gp120 and gp41) monoclonal antibody, (ii) gene coding sequences of light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of a monoclonal antibody that binds to the human CD4 receptor site, (iii) gene coding sequence of a human IgG constant region Fc fragment, and (iv) gene coding sequence of a short peptide that inhibits fusion of HIV with the CD4+ T cell membrane, and the coding sequences of the antibody light and heavy chains may be directly or indirectly connected in tandem via a coding sequence of a linker polypeptide in any order.

[0031] The gene sequence construct according to any one of the above includes (i) a gene coding sequence for the light chain variable region (VL) and heavy chain variable region (VH) of an anti-HIV-1-gp160 (including its cleavage products gp120 and gp41) monoclonal antibody, (ii) a gene coding sequence for the light chain variable region (VL) and heavy chain variable region (VH) of a monoclonal antibody that binds to the human CD4 receptor site, (iii) a gene coding sequence for a human IgG constant region Fc fragment, and (iv) a gene coding sequence for a short peptide that inhibits fusion of HIV with the CD4+ T cell membrane, and the coding sequences for the antibody light chain variable region (VL) and heavy chain variable region (VH) may be directly or indirectly connected in tandem via a coding sequence for a linker polypeptide in any order.

[0032] The gene sequence construct according to any one of the above further comprises a promoter located upstream of the gene coding sequence for the antibody molecule having HIV infection suppressing ability and the gene coding sequence for the polypeptide having HIV infection suppressing ability.

[0033] The gene sequence construct according to any one of the above further comprises a secretory signal peptide coding sequence located upstream of the gene coding sequence for an antibody molecule having HIV infection suppressing ability and the gene coding sequence for a polypeptide having HIV infection suppressing ability.

[0034] The gene sequence construct according to any one of the above aspects comprises a gene coding sequence for a first antibody molecule having an ability to suppress HIV infection, a gene coding sequence for a second antibody molecule having an ability to suppress HIV infection, and a gene coding sequence for a polypeptide having an ability to suppress HIV infection, The gene sequence construct comprises: VL2-linker-VH2-linker-VL1-linker-VH1-linker-CH2-CH3; or VL2-linker-VH2-linker-VL1-linker-VH1-linker-CH2-CH3-linker-peptide inhibitor; or other combinations of VL2 and VH2 or VL1 and VH1 arranged in a different order in the construct.

[0035] Here, VL2 and VH2 are respectively the light chain and heavy chain variable region fragments of a first antibody molecule, VL1 and VH1 are respectively the light chain and heavy chain variable region fragments of a second antibody molecule, CH2-CH3 is an Fc fragment of a human IgG constant region, linker is a linker polypeptide, and peptide inhibitor is a polypeptide that suppresses HIV infection (e.g., a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane).

[0036] The gene sequence construct is VL2-linker-VH2-linker-VL1-linker-VH1-linker-CH2-CH3, or a construct in which the combination of VL2 and VH2 or VL1 and VH1 are arranged in a different order.

[0037] The gene sequence construct is VL2-linker-VH2-linker-VL1-linker-VH1-linker-CH2-CH3-linker-peptide inhibitor, or a construct in which the combination of VL2 and VH2 or VL1 and VH1 are arranged in a different order.

[0038] In said gene sequence construct, the protein sequences of VL2 and VH2 comprise SEQ ID NO:2, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0039] In said gene sequence construct, the protein sequence of VL1 and VH1 comprises SEQ ID NO:3, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0040] In the gene sequence construct, the sequence of the linker polypeptide is GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS) 4 , (GGGGS) 5 , (GGGGS) 6 , and (GGGGS) 7 , or other alternative linker polypeptide sequences.

[0041] In the above gene sequence construct, the peptide inhibitor that inhibits the fusion of HIV with the CD4+ T cell membrane can be selected from membrane fusion inhibitor polypeptides such as P52, C34, and T20.

[0042] Here, the sequence of the membrane fusion-inhibiting polypeptide P52 comprises SEQ ID NO:5 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto, the polypeptide sequence of C34 comprises SEQ ID NO:6 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto, and the polypeptide sequence of T20 comprises SEQ ID NO:7 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0043] Furthermore, the present invention also provides a viral vector genome comprising any of the above constructs, and a corresponding viral vector system comprising said genome.

[0044] The viral vector system may be a lentiviral vector system or an adeno-associated viral vector system.

[0045] Here, the lentiviral vector system can contain the viral vector genome of any of the above constructs and other nucleotide sequences that encode and express a packaging kit necessary for lentivirus production, and is introduced into a producer cell to produce lentiviral particles containing the genome of the above construct.

[0046] The adeno-associated virus vector system can contain the viral vector genome of any of the above constructs and other nucleotide sequences that encode and express the packaging kit necessary for the production of adeno-associated virus, and is introduced into a producer cell to produce adeno-associated virus particles containing the genome of the above construct.

[0047] Any of the viral particles produced contain the genome of any of the constructs and are capable of expressing anti-HIV neutralizing antibody-like molecules after transduction of cells and can be administered to patients to suppress or prevent HIV infection.

[0048] The present invention also provides a pharmaceutical composition comprising said viral particles and a pharma- ceutically acceptable carrier or diluent, or cells transduced in vitro with said lentiviral particles, said transduced cells including, but not limited to, muscle cells, liver cells, or CD4+ T cells.

[0049] The virus particles or pharmaceutical compositions containing the virus particles can be injected into the body to express antibody molecule proteins having two or more action targets, and the mature molecules can form dimers through disulfide bonds, so as to be used in gene therapy for HIV infection, thereby achieving long-term treatment for AIDS virus-infected individuals, and efficiently and broadly block the infection progression of HIV to human CD4+ T cells by binding to multiple binding sites involved in different steps of HIV infection to human CD4+ T cells.

[0050] The present invention provides a method for inhibiting HIV infection, comprising administering the above-mentioned viral particle or pharmaceutical composition to a cell.

[0051] Here, the cells include muscle cells, liver cells, CD4+ T cells, and the like.

[0052] Said viral particle or pharmaceutical composition may transduce said cells in vitro or in vivo.

[0053] The present invention provides a method of treating HIV infection in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a viral particle or pharmaceutical composition as described above.

[0054] Here, the subject includes an HIV-infected individual with early stage HIV infection, or an HIV-infected individual undergoing cocktail drug therapy, or an HIV-infected individual with resistance to cocktail drug therapy.

[0055] The method of administering the drug to the subject is intramuscular injection of the viral particles or pharmaceutical composition.

[0056] Alternatively, the viral particles or pharmaceutical composition or CD4+ T cells transduced therewith are injected intravenously.

[0057] The viral particles and pharmaceutical composition injected into the body by the above-mentioned method can express and secrete into the blood anti-HIV protein molecules with multiple action targets, act on multiple HIV infection nodes, efficiently block the HIV infection pathway, and effectively avoid the loss of inhibitory ability against HIV infection caused by the occurrence of HIV escape mutations, thereby achieving a long-term and even permanent therapeutic effect against HIV infection with a single injection.

[0058] The antibody-like molecule, consisting of multiple single-chain antibody variable region fragments (scFv) based on the above expression framework, shows effective blood drug concentrations after being delivered into mice via lentivirus and adeno-associated virus vectors, has in vitro cytological HIV-1 virus neutralizing activity, and simultaneously has broad-spectrum neutralizing ability against two types of CXCR4 and CCR5-tropic viruses, providing a highly potential technological route for the development of a broad-spectrum neutralizing antibody gene therapy drug for the anti-AIDS virus.

[0059] The scope of application of the present invention includes various forms of anti-AIDS virus gene therapy based on genetic expression of broadly neutralizing antibodies. [Brief description of the drawings]

[0060] The drawings of the present invention will now be described in detail. [Figure 1] FIG. 1 is a schematic diagram of the mature molecular structure of a predicted triphilic anti-HIV neutralizing antibody-like molecule. [Diagram 2] FIG. 1 is a schematic diagram showing the structure of the gene sequence of a Miphil anti-HIV neutralizing antibody-like molecule. [Diagram 3] FIG. 1 is a schematic diagram of the mature molecular structure of a predicted biphilic anti-HIV neutralizing antibody-like molecule. [Figure 4] FIG. 1 is a schematic diagram showing the structure of the gene sequence of a biphilic anti-HIV neutralizing antibody-like molecule. [Diagram 5] FIG. 1 is a schematic diagram of a gene construct in which the gene sequence of a biphilic (KL-BsHIV01) or triphilic (KL-BsHIV01-003) anti-HIV neutralizing antibody-like molecule was cloned into a lentivirus vector. [Figure 6] 1 is a graph of the latest generation lentiviral vector pKL-kan-lenti-EF1α-WPRE applied to the present invention. [Figure 7] 1 is a graph of the latest generation adeno-associated virus vector pAAV-MCS-CMV-EGFP(trans) applied to the present invention. [Figure 8]FIG. 1 is a schematic diagram of a gene construct in which the gene sequence of a biphilic (KL-BsHIV01) or triphilic (KL-BsHIV01-003) anti-HIV neutralizing antibody-like molecule was cloned into an adeno-associated virus vector. [Figure 9] The expression of anti-HIV neutralizing antibody-like molecules in 293T cells was detected by Western blot analysis. After transfection of 293T cells with a plasmid of a gene construct in which the gene sequence of the anti-HIV neutralizing antibody-like molecule was cloned into an adeno-associated virus vector, the same volume of 293T cell culture supernatant was used as a sample for Western blot analysis under non-reducing conditions, and the protein of the anti-HIV neutralizing antibody-like molecule was detected with a goat anti-human IgG1 Fc fragment antibody. M, pre-stained protein size marker; 1, blank plasmid transfection control; 2, KL-BsHIV01; 3, KL-BsHIV01-003; 4, KL-BsHIV01-C34; 5, KL-BsHIV01-T20. The high molecular weight bands (much larger than 180 kDa) in the Western blot are the anti-HIV neutralizing antibody-like molecules (under non-reducing conditions) that are expected to exist in the form of dimers. [Figure 10]The expression of anti-HIV neutralizing antibody-like molecules in C2C12 myotube cells was detected by Western blot analysis. The gene sequence of the anti-HIV neutralizing antibody-like molecule was cloned into an adeno-associated virus vector, and the recombinant adeno-associated virus was transfected into 293T cells using the plasmid of the gene construct and other helper plasmids, and then packaged and purified. C2C12 cells were infected and transduced with the same infection coefficient, and the same volume of the C2C12 cell culture supernatant was used as a sample for Western blot analysis under non-reducing conditions, and the protein of the anti-HIV neutralizing antibody-like molecule was detected with a goat anti-human IgG1 Fc fragment antibody. M, pre-stained protein size marker; 1, KL-BsHIV01; 2, KL-BsHIV01-003; 3, KL-BsHIV01-C34; 4, KL-BsHIV01-T20. The high molecular weight bands (much larger than 180 kDa) in the Western blot are anti-HIV neutralizing antibody-like molecules (under non-reducing conditions) that are expected to exist in the form of dimers. The band at the position corresponding to 180 kDa is a non-specific band. [Figure 11] HIV neutralizing antibody. Neutralizing activity against HIV virus of culture supernatant of 293T cells transduced with lentiviral gene therapy vector. VCN, copy number of lentiviral vector after transduction of 293T cells. [Figure 12] HIV neutralizing antibody: Virus neutralizing activity of the culture supernatant of C2C12 cells transduced with an adeno-associated virus gene therapy vector. [Figure 13] Anti-HIV neutralizing antibody expression in BALB / c mice after intramuscular injection of adeno-associated virus gene therapy vector. The levels of anti-HIV neutralizing antibody secreted in mouse serum were measured by ELISA. [Figure 14] Neutralizing activity against HIV virus strains of biphilic and triphilic HIV neutralizing antibody-like molecules secreted into mouse serum at different concentrations. Data in the figure are the mean ± SD of three parallel determinations for each sample. This experiment was repeated at least twice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Broadly neutralizing antibodies (bNAbs) with HIV-1 neutralizing activity have been produced in a small proportion of HIV-1 infected individuals over the course of several years. They can efficiently bind to a broad range of HIV viral surface glycoproteins, neutralize the infectious activity of HIV to CD4+ T cells, and represent a promising method to prevent or combat HIV-1 infection. However, the specific mechanism of their production is unclear, and most HIV-1 infected individuals can only produce non-neutralizing antibodies. Currently, there have been no successful studies in inducing the production of broadly neutralizing antibodies in healthy subjects by standard immunization methods.

[0062] Without wishing to be bound by theory, in some embodiments, it is believed that recombinant-derived broadly neutralizing antibodies against HIV-1 virus can effectively reduce the viral load in the patient's body, and even if they cannot completely eliminate HIV in the body, they can contribute to controlling the development of HIV infection to AIDS. Compared with small molecule anti-AIDS virus drugs, recombinant-derived neutralizing antibodies can also greatly reduce toxic side effects and increase patient administration compliance. In clinical practice, recombinant broadly neutralizing antibodies can not only be used in vaccine replacement products in certain situations to prevent the occurrence of AIDS virus infection, but can also be used as antiviral drugs for different disease steps.

[0063] However, like highly efficient small molecule anti-AIDS virus drugs, specific broadly neutralizing antibodies are only directed against a single epitope on a single viral protein (HIV-gp160), and it is still difficult to avoid the escape phenomenon caused by viral mutation. In addition, the costs of developing, producing, and using broadly neutralizing antibodies are all much higher than those of small molecule anti-AIDS virus drugs, and there is no advantage to combination drugs. Therefore, the large number of broadly neutralizing antibody drugs for the anti-AIDS virus that are under research have been difficult to apply to clinical practice so far.

[0064] In order to address the above challenges, research and development of broadly neutralizing antibody drugs against the AIDS virus needs to make breakthroughs in the following two areas: 1. Research and develop biphilic or polyphilic neutralizing antibodies or antibody-like polymer drugs to cover multiple targets and avoid escape due to viral mutation. 2. Genetization of broadly neutralizing protein antibodies or antibody-like polymer drugs. Anti-AIDS virus infection gene therapy drugs delivered by recombinant viruses or non-viral vectors can stably express neutralizing antibodies or antibody-like polymers in vivo for a long period of time through genome integration or non-integration, so that a single treatment can be effective for a long period of time, even for life, and greatly reduce the production and use costs of polymer antibody drugs.

[0065] The present invention employs a polyphilic antibody molecular structure, i.e., two or more single-chain variable fragments (scFv) of monoclonal broadly neutralizing antibodies are connected in series by a linker polypeptide to form an antigen-binding region.

[0066] A specific embodiment includes constructing a series of antibody molecule gene constructs containing biphilic / triphilic antibody single chain variable fragments (scFv) and cloning them into recombinant adeno-associated virus and recombinant lentivirus vectors. Then, packaging the corresponding adeno-associated virus and lentivirus in 293T cells, and infecting 293T cells and differentiated and undifferentiated muscle cell lines with a certain biological titer of virus. The antibody molecules produced in the cell supernatant are quantitatively and qualitatively detected, and the neutralizing activity against HIV-1 wild type virus strains is detected in the infection activity assay of HIV-1 wild type virus strains and virus-sensitive reporter gene cell line TZM-bl.

[0067] The structure of the anti-HIV neutralizing antibody of the present invention, the composition of the gene sequence construct, and related terminology will be described in more detail below in combination with examples.

[0068] Antibody: may be an immunoglobulin, an antigen-binding fragment, or a protein molecule derived therefrom that can specifically recognize and bind to an antigen (e.g., HIV-1 gp41 antigen). In the present invention, "antibody" is broadly defined and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, polyphilic antibodies (e.g., biphilic antibodies, triphilic antibodies), and antibody fragments, so long as they have specific antigen-binding activity. Specific examples of antibodies include complete immunoglobulins, as well as antibody variants and fragments that retain antigen-binding affinity. Examples of 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 generated after hydrolysis by proteases), single-chain antibody molecules (e.g., scFv), diabodies, nanobodies, and polyphilic antibodies formed from a combination of antibody fragments. Antibody fragments include antigen-binding fragments generated by modification from complete antibodies, or antigen-binding fragments synthesized de novo using recombinant DNA technology.

[0069] A single-chain antibody (scFv) is a molecule obtained by genetic engineering, which contains one or more antibody light chain variable regions (VL) and heavy chain variable regions (VH), each fragment being connected in series with a suitable linker polypeptide to form a single fused single-chain molecule. The tandem order of VL and VH in a single-chain antibody molecule usually does not affect its antigen-binding function, so any single-chain antibody consisting of two tandem arrangements (VL-VH or VH-VL) can be used.

[0070] An antibody may have one or more antigen-binding sites. When an antibody has one or more antigen-binding sites, the binding sites may be the same or different. For example, a naturally occurring immunoglobulin has two identical antigen-binding sites, whereas a Fab fragment produced from an immunoglobulin by hydrolysis of papain has only one antigen-binding site, and a biphilic single-chain antibody (scFv) has two different antigen-binding sites.

[0071] Normally, a naturally produced immunoglobulin consists of a light chain and a heavy chain, which are connected by disulfide bonds. Immunoglobulin genes include gamma, alpha, delta, epsilon, mu, lambda, and kappa constant region genes, as well as numerous immunoglobulin variable region genes. There are two types of light chains, gamma and kappa. There are five main types of heavy chains (gamma, alpha, delta, epsilon, mu), which determine the functional typing of antibody molecules, and are IgG, IgA, IgD, IgE, and IgM, respectively.

[0072] Each heavy and light chain comprises one constant region and one variable region. VH represents the variable region of the heavy chain of an antibody, including the heavy chain variable region of an antigen-binding fragment Fv, scFv, or Fab. VL represents the variable region of the light chain of an antibody, including the light chain variable region of an antigen-binding fragment Fv, scFv, or Fab. In the following examples, VH and VL together play a role in specifically recognizing and binding to an antigen.

[0073] VH and VL are composed of three separate hypervariable regions (also called complementarity determining regions (CDRs)) and a frame region. The sequences of the frame regions of different light and heavy chains are relatively conservative within the same genus. The frame region of an antibody determines the position of the complementarity determining regions in the three-dimensional structure. The complementarity determining regions are mainly responsible for binding to the antigenic determinant cluster of an antigen. The three complementarity determining regions in the light chain are designated LCDR1, LCDR2, and LCDR3, respectively, from the N-terminus to the C-terminus. The three complementarity determining regions in the heavy chain are designated HCDR1, HCDR2, and HCDR3, respectively, from the N-terminus to the C-terminus.

[0074] The VH and VL protein sequences of the present invention include, in addition to the sequences disclosed in the Examples below, any other sequence carrying a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity thereto.

[0075] Antibody constant region fragment: A region in which the amino acid sequence of an immunoglobulin is relatively stable other than the variable region, which has a large variation in the amino acid sequence near the N-terminus, and includes the constant domain in the antigen-binding fragment (located at the N-terminus of the hinge region, including the light chain constant domain and the heavy chain constant domain) and the constant domain of the crystallizable fragment of the heavy chain (called the Fc fragment, located at the C-terminus of the hinge region). The Fc fragment usually 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 include a part or all of the hinge region sequence located at its N-terminus.

[0076] Anti-HIV-1 neutralizing antibodies or antigen-binding fragments: The neutralizing antibodies or antigen-binding fragments specifically bind to HIV-1 envelope proteins (e.g., bind to gp41) and thereby inhibit biological functions associated with the HIV-1 envelope (e.g., the ability to bind to target receptors). In the following examples, the anti-HIV-1 neutralizing antibodies or antigen-binding fragments reduce the infectivity of HIV-1 virus strains of different tropisms to cells.

[0077] Biphilic or polyphilic antibodies: These antibodies are recombinant molecules consisting of two or more different antigen binding domains and therefore can bind to two or more different antigenic determinant clusters. Biphilic or polyphilic antibodies include molecules in which two or more different antigen binding domains are connected by chemical synthesis or genetic engineering methods. The antigen binding domains can be connected via a linker polypeptide. The antigen binding domains can be monoclonal antibodies, antigen binding fragments (e.g., scFv or Fab), or a combination of antigen binding domains of different origin.

[0078] Linker polypeptide: for connecting two protein molecules or fragments into one continuous single fusion molecule, for example, in the following examples, it connects two or more antibody molecules or antigen-binding fragments (e.g., scFv) to form a polyphilic antibody molecule having two or more antigen-binding sites, or connects the light chain variable region (VL) and heavy chain variable region (VH) of one antibody to form one single-chain antigen-binding sequence, or connects an antibody molecule or antigen-binding fragment to another effector molecule, for example, connects an antigen-binding fragment scFv to an HIV-1 membrane fusion inhibitor polypeptide to form one fusion protein. The linker polypeptide is usually rich in glycine (Gly or G) to improve the flexibility of the linker, and contains serine (Ser or S) or threonine (Thr or T) to improve solubility, for example, (GGGGS) of different lengths used in some of the following examples. n linker, where n may be 1 or more than 1. However, the linker polypeptide sequences used in the examples are not limited thereto and also include other alternative linker polypeptide sequences.

[0079] Antigenic determinant cluster: A specific chemical group or polypeptide sequence on a molecule that has antigenicity, i.e., is capable of eliciting a specific immune response in a host. An antibody specifically binds to a specific antigenic determinant cluster on a polypeptide, for example, in the following example, an antibody that can specifically bind to an antigenic determinant cluster on gp41.

[0080] HIV-1 envelope protein: HIV-1 envelope protein is first synthesized in the form of a precursor protein with a size of 845-870 amino acid residues, called HIV gp160. gp160 forms a homotrimer in the host cell, is glycosylated, sheared to remove the signal peptide, and is cleaved at 511 / 512 amino acid residues by a single intracellular protease to generate gp120 and gp41 polypeptide chains. gp120 and gp41 are associated in homotrimers as gp120 / gp41 protomers. Mature gp120 consists of 31-511 amino acid residues of HIV-1 envelope protein, is a highly N-glycosylated protein, and constitutes the majority of the domain exposed on the envelope surface of the HIV-1 envelope protein trimer. gp120 is responsible for binding to the human CD4 cell receptor and co-receptors (e.g., chemotactic factor receptors CCR5 or CXCR4). gp41 is composed of 512-860 amino acid residues of the HIV-1 envelope protein, and includes an intraenvelope domain, a transmembrane domain, and an extraenvelope domain. The extraenvelope domain of gp41 contains 512-644 amino acid residues and binds to gp120 to form a protomer, which together constitutes the HIV-1 envelope protein homotrimer. The domain protruding outside the envelope of the HIV-1 envelope protein homotrimer undergoes several structural rearrangements, from a blocking structure before fusion with the host cell membrane that can avoid antibody recognition, and an intermediate structure that binds to the human CD4 cell receptor and co-receptor, to a structure after membrane fusion.

[0081] HIV membrane fusion inhibitor polypeptide: Membrane fusion between the virus and the host cell is an important step in the infection of cells by HIV. After the glycoprotein gp120 / gp41 identical trimer on the HIV envelope binds to the human CD4 cell receptor and co-receptor, it undergoes several steps of conformational changes, and finally the gp41 trimer incorporated on the HIV envelope is inserted into the host cell membrane, completing the fusion of the virus and the host cell membrane, and the genetic material of HIV enters the cell. The HIV membrane fusion inhibitor polypeptide binds to the viral envelope protein to prevent the occurrence of the conformational changes required for the fusion of the virus with the host CD4 cell membrane, thereby inhibiting HIV infection of CD4 cells. The HIV membrane fusion inhibitor polypeptide used in the present invention includes the P52, C34, and T20 sequences disclosed in the following examples, or homologous sequences having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0082] Genetic sequence construct: A vector consisting of a recombinant polynucleotide sequence, in which an expression control sequence and a nucleic acid sequence to be expressed are connected. An expression vector contains sufficient cis-acting elements, and other expression elements are provided by the host cell. Expression vectors include all vectors in the present invention, including, for example, plasmids and viruses (e.g., recombinant lentiviruses and recombinant adeno-associated viruses) into which a recombinant polynucleotide sequence has been incorporated. A vector contains a nucleic acid sequence (DNA or RNA) that allows 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. A viral vector is a recombinant nucleic acid vector, and contains at least a portion of a nucleic acid sequence derived from one or more viruses. In some examples below, the viral vector contains one or more nucleic acid sequences encoding an antibody or antigen-binding fragment capable of specifically binding to the disclosed HIV-1 gp160 and neutralizing HIV-1. In some examples, the viral vector is a recombinant adeno-associated virus (AAV) vector or a recombinant lentivirus vector. These viral vectors are replication-defective vectors and require other helper plasmids or vectors carrying gene functions or components necessary for viral replication to amplify and package in cells to produce viral particles. The purified viral vectors or viral particles cannot be replicated in host cells during the course of treating patients.

[0083] Treatment of HIV infection: HIV is a virus that attacks the human immune system. It mainly attacks CD4 T lymphocytes, the most important cell in the human immune system, destroying them in large numbers, causing the body to lose immune function. Many patients in the acute HIV infection stage have flu-like symptoms 2-4 weeks after infection, which may last for several days to several weeks. There is a large amount of HIV in the blood of patients in the acute infection stage, and they are highly infectious. Then, they enter the asymptomatic chronic infection stage, also called the HIV latency stage. However, HIV in the patient's body is still active, continues to grow, and transmits HIV. The average latency period of HIV in the human body is 8-9 years, and during the HIV virus latency period, HIV-infected people can live and work for many years without any symptoms. However, if HIV-infected people do not undergo anti-HIV infection treatment, the HIV will progress through the latency stage to the most serious HIV infection stage, that is, the AIDS stage. AIDS patients have a high viral load, are easily able to transmit HIV to others, and have severely damaged immune systems, making the human body more susceptible to various diseases, leading to the development of malignant tumors and a high mortality rate.

[0084] At present, there is still no effective drug that can cure HIV infection in the whole world. The treatment goal at the current stage is to reduce the viral load in a sustained manner to the maximum extent, obtain the restructuring and maintenance of immune function, improve the quality of life, and reduce the HIV-related morbidity and mortality. The currently commonly used method of anti-HIV infection is antiretroviral combination therapy (i.e., cocktail therapy), which has significantly improved the therapeutic effect of anti-HIV and significantly improved the quality of life and prognosis of patients. For example, treatment is started after HIV infection is confirmed during the acute phase of HIV infection, i.e., the first few months after infection. However, the side effects and limitations of cocktail therapy, such as the need to maintain long-term and continuous compliance with medication, and the gradually emerging drug-resistant strains of HIV, still cause long-term economic and social burdens, significant decline in quality of life, and significant suffering to a vast number of AIDS patients.

[0085] Broadly neutralizing antibodies with HIV-1 neutralizing activity identified and isolated in a small proportion of HIV-infected individuals can efficiently bind to the glycoproteins on the surface of the HIV virus over a broad range, neutralizing the infectious activity of the HIV virus to human CD4+ T cells, and representing a promising method to prevent or fight HIV-1 infection. Compared with small molecule anti-HIV drugs, recombinantly derived neutralizing antibodies can significantly reduce toxic side effects and increase patient compliance. At present, the only anti-HIV neutralizing antibody in clinical use is Ibalizumab, which targets the CD4 receptor on the surface of human T cells and blocks the binding of the glycoproteins on the surface of the HIV virus to the CD4 receptor by binding to the CD4 receptor, thereby neutralizing the infectious activity of the virus to CD4+ T cells, and showing excellent therapeutic effects on multi-resistant patients infected with the AIDS virus.

[0086] However, a specific broadly neutralizing antibody is only directed against a single epitope of a single viral protein (HIV-gp160), so it is still difficult to avoid the escape phenomenon caused by viral mutation, and the development, production and use costs of broadly neutralizing antibodies are all much higher than those of small molecule anti-AIDS virus drugs, and there is no advantage in combination drugs. The present invention researches and develops biphilic and polyphilic neutralizing antibodies or antibody-like polymer drugs, and covers multiple targets to avoid escape caused by viral mutation, and gene-izes protein broadly neutralizing antibodies or antibody-like polymer drugs to make a breakthrough in the research and development of anti-HIV broadly neutralizing antibody drugs. Gene therapy drugs for anti-HIV infection delivered by recombinant viruses or non-viral vectors can stably express neutralizing antibodies or antibody-like polymers in vivo for a long period of time through genome integration or non-integration, and a single treatment can be effective for a long period of time (for example, the drug effect lasts for one or several years) and even for life, greatly reducing the production and use costs of polymeric antibody drugs.

[0087] The following examples are illustrative of the present invention, but the present invention is not limited to the following examples. EXAMPLES

[0088] 1. Design of a structure expressing HIV neutralizing antibodies The structure of the anti-HIV neutralizing antibody and antibody-like molecule applied to the present invention is as follows: triphilic anti-HIV neutralizing antibody-like molecule: the protein molecule is structured from the N-terminus to the C-terminus as follows: -signal peptide-single chain variable region fragment of anti-HIV-gp41 broadly neutralizing antibody (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-inhibiting short peptide. The gene coding sequence shown is expressed in cells and translated into protein, which then forms a dimer and is secreted outside the cells. The mature molecular structure of the expected anti-HIV neutralizing antibody-like molecule is shown in Figure 1, and the gene structure is shown in Figure 2.

[0089] II. Gene sequence expressing HIV neutralizing antibodies Here, the anti-HIV-gp41 broadly neutralizing antibody single-chain variable region fragment (scFv) sequences are signal peptide (protein sequence is SEQ ID NO:1), anti-HIV-1-gp41-MHER monoclonal antibody (10E8v4-5R+100cF)-scFv (protein sequence is SEQ ID NO:2), anti-human CD4 monoclonal antibody (Ibalizumab) single-chain variable region fragment scFv (protein sequence is SEQ ID NO:3), human IgG1 Fc fragment (protein sequence is SEQ ID NO:4), HIV membrane fusion inhibitor short peptide P52 (protein sequence is SEQ ID NO:5), HIV membrane fusion inhibitor short peptide C34 (protein sequence is SEQ ID NO:6), and HIV membrane fusion inhibitor short peptide T20 (protein sequence is SEQ ID NO:7).

[0090] Third, four anti-HIV neutralizing antibody gene expression frameworks were constructed, which are as follows: 1. Anti-HIV biphilic neutralizing antibody-like molecule KL-BsHIV01 (protein sequence see SEQ ID NO:8) (DNA sequence see SEQ ID NO:9) comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF-scFv (see Figures 3 and 4), 2. Anti-HIV triphilic neutralizing antibody-like molecule KL-BsHIV01-003 (protein sequence see SEQ ID NO: 10) (DNA sequence see SEQ ID NO: 11) comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF-scFv (see Figures 1 and 2); 3. Anti-HIV triphilic neutralizing antibody-like molecule KL-BsHIV01-C34 (protein sequence see SEQ ID NO: 12) (DNA sequence see SEQ ID NO: 13) containing anti-HIV neutralizing antibody 10E8v4-5R+100cF-scFv (see Figures 1 and 2); 4. Anti-HIV triphilic neutralizing antibody-like molecule KL-BsHIV01-T20 (protein sequence see SEQ ID NO:14) (DNA sequence see SEQ ID NO:15) comprising anti-HIV neutralizing antibody 10E8v4-5R+100cF-scFv (see Figures 1 and 2).

[0091] IV. Examples of HIV neutralizing antibody gene therapy vector constructs As shown in Figure 5, the monoclonal antibody molecule gene expression framework was cloned into the latest generation lentiviral vector pKL-kan-lenti-EF1α-WPRE (Figure 6) (DNA sequence see SEQ ID NO: 16) currently used in the manner of multi-fragment recombination ligation. The lentiviral vector includes: 5'LTR, in which the promoter region of the LTR is replaced with the CMV promoter; ψ packaging signal; retroviral output element RRE; cPPT; promoter CBH; polynucleotide encoding the polypeptide of HIV neutralizing antibody fragment; post-transcriptional regulatory element WPRE; PPT; ΔU3 3'LTR; and poly(A) signal. The neutralizing antibody gene expression framework designed in this embodiment (FIG. 4) was synthesized by GenScript (Nanjing) Co. Ltd., and then cloned between the CBH promoter (sequence see SEQ ID NO: 17) and the multiple cloning site EcoRI / EcoRV on the lentiviral vector frame pKL-kan-lenti-EF1α-WPRE by a method of homologous recombination well known in the art (FIG. 6). After cloning was completed, the sequence information was confirmed by sequencing, and the plasmid was named pKL-Kan-lenti-CBH-KL-BsHIV01 (sequence see SEQ ID NO: 18) (FIG. 5). The coding sequence of HIV fusion inhibitor short peptide P52 or C34 or T20 is cloned between the multiple cloning sites EcoRV on the lentiviral vector frame pKL-kan-lenti-EF1α-WPRE by the method of homologous recombination well known in the art, and after the cloning is completed, the sequence information is confirmed by sequencing, and the plasmid is named pKL-Kan-lenti-CBH-KL-BsHIV01-003 respectively (see SEQ ID NO: 19 for the sequence) (Figure 5).

[0092] As shown in Figure 5, the HIV neutralizing antibody gene expression frameworks CBH-KL-BsHIV01, CBH-KL-BsHIV01-003, CBH-KL-BsHIV01-C34, and CBH-KL-BsHIV01-T20 present in pKL-Kan-lenti-CBH-KL-BsHIV01 were cloned between the multi-cloning site MluI / SalI of the latest generation adeno-associated virus vector pAAV-MCS-CMV-EGFP(trans) (sequence see SEQ ID NO:20) currently applied in the manner of WPRE and multi-fragment recombination connection (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. The plasmids are designated pAAV-CBH-KL-BsHIV01-WPRE (see SEQ ID NO:21 for sequence), pAAV-CBH-KL-BsHIV01-003-WPRE (see SEQ ID NO:22 for sequence), pAAV-CBH-KL-BsHIV01-C34-WPRE (see SEQ ID NO:23 for sequence), and pAAV-CBH-KL-BsHIV01-T20-WPRE (see SEQ ID NO:24 for sequence) (Figure 8).

[0093] 5. Packaging and purification of viruses expressing HIV neutralizing antibodies The antibody gene therapy lentiviral vector was packaged in 293T cell line using lentiviral vectors (pKL-Kan-lenti-CBH-KL-BsHIV01, pKL-Kan-lenti-CBH-KL-BsHIV01-003). The antibody gene lentiviral vectors constructed in the examples (pKL-Kan-lenti-CBH-KL-BsHIV01, pKL-Kan-lenti-CBH-KL-BsHIV01-003, envelope plasmid (pKL-Kan-Vsvg, the nucleotide sequence of which is shown in SEQ ID NO: 25) and packaging plasmids (pKL-Kan-Rev, the nucleotide sequence of which is shown in SEQ ID NO: 26; pKL-Kan-GagPol, the nucleotide sequence of which is shown in SEQ ID NO: 27) were used. After mixing the 293T cells with the HIV neutralizing antibody gene therapy lentivirus (purchased from American Type Culture Collection (ATCC), with the ATCC deposit number CRL-3216), 293T cells were transfected at the same time to package the HIV neutralizing antibody gene therapy lentivirus into the 293T cell line. The transfection method was PEI cationic polymer-mediated eukaryotic cell transient transfection, the PEI cationic polymer was PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), the transfection procedure was performed with reference to the standardized procedure recommended by the manufacturer, and the transfection scale was a 15 cm cell culture dish. 48 hours after the transfection was completed, the lentivirus vector (transfected cell culture supernatant) was harvested and first centrifuged at 4000 rpm for 5 minutes at room temperature in a tabletop bucket centrifuge to remove cell fragments, and then further centrifuged at 4°C. The virus particles were precipitated by centrifugation at 10,000 g for 4 hours, and the centrifugation supernatant was removed. 1 mL of DMEM complete medium was then added to the virus particle precipitate, and the virus particles were resuspended using a microinjector. The prepared virus resuspension was aliquoted and frozen and stored at -80°C.

[0094] The antibody gene therapy AAV vector was packaged in 293T cell line using AAV expression vectors (pAAV-CBH-KL-BsHIV01-WPRE, pAAV-CBH-KL-BsHIV01-003-WPRE, pAAV-CBH-KL-BsHIV01-C34-WPRE, pAAV-CBH-KL-BsHIV01-T20-WPRE). The antibody gene AAV vector constructed in the examples, capsid plasmid (AAV2 / 8, the nucleotide sequence of which is shown in SEQ ID NO: 28) and packaging plasmid (pHelper, the nucleotide sequence of which is shown in SEQ ID NO: 29) were mixed and then transfected simultaneously into 293T cells, and the HIV neutralizing antibody gene therapy vector AAV was packaged in the 293T cell line. The transfection method was PEI cationic polymer-mediated transient transfection of eukaryotic cells, the PEI cationic polymer was PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), the transfection operation was performed with reference to the standardized operation recommended by the manufacturer, and the transfection scale was 15 cm cell culture dish. 7 h after the transfection was completed, the supernatant was aspirated and discarded, and replaced with 25 ml of toxic production medium. 120 hours after the transfection was completed, the supernatant and cells were collected and centrifuged at 4200 rpm for 10 min, and after the centrifugation was completed, the supernatant and cells were separated, and the lysate and ribozyme were added to the cells, lysed and digested for 1 h, and centrifuged at 10000 g for 10 min to obtain the lysate supernatant. The lysate supernatant and medium supernatant were purified by affinity chromatography and then frozen and stored at -80 ° C.

[0095] 6. Functional verification of HIV neutralizing antibodies expressed in the supernatant of cells transduced with lentiviral gene therapy vectors The packaged lentiviral vectors pKL-Kan-lenti-CBH-KL-BsHIV01 and pKL-Kan-lenti-CBH-BsHIV01-003 were infected into 293T cells at different MOIs. After 48 hours, the supernatants and aliquot cells were collected. The lentiviral vector infection copy number was detected by the probe method. 1. Collect 293T cells infected with lentiviral vectors, wash the cells with PBS, and then collect the cells by centrifugation at 4200 rpm for 5 minutes. Resuspend the cells in 20 μl of high-speed extraction solution (QE DNA Extraction Solution). Run the following program in a PCR machine to lyse the cells and extract total DNA.

[0096] [Table A]

[0097] The lentiviral copy number (VCN) of 293T cell infection is calculated by quantitative PCR using methods well known in the art.

[0098] 2. TZM-bl cells were plated at 2E4 cells / well. 50μL of cell antibody expression supernatant with VCN of 0.05 and 0.25 was mixed with 50μL of HIV virus pAD-8 or pNL4-3, respectively, and then incubated at 37℃ for 30min, and added to TZM-bl cells. The wells with only pAD-8 or pNL4-3 were negative controls, and the wells without HIV virus were blank. At 24 hours, the supernatant was discarded, 100μL of cell lysis solution was added, and the lysed cells were collected after 10min and centrifuged at 8000rpm for 5min. 100μL of firefly luciferase detection reagent was added to 50μL of lysed cells. RLU (relative light unit) was detected and measured using a multi-function microplate reader with chemiluminescence function. As shown in the results (Figure 11), in vitro cell tests showed that at the same infectious copy number (VCN), the culture supernatant of 293T cells transduced with the HIV neutralizing antibody KL-BsHIV01-003 lentiviral gene therapy vector contained antibodies that thoroughly neutralized the HIV virus, and the neutralizing effect was significantly superior to that of the HIV bispecific neutralizing antibody KL-BsHIV01 lentiviral gene therapy vector.

[0099] 7. Functional verification of HIV neutralizing antibodies expressed in the supernatant of cells transduced with AAV gene therapy vectors In a 24-well cell culture plate of C2C12 mouse myoblasts, each well had 1E5 cells. They were differentiated into myotubes in 2% horse serum medium. Packaged adeno-associated viruses pAAV-CBH-KL-BsHIV01, pAAV-CBH-KL-BsHIV0-003, pAAV-CBH-KL-BsHIV01-C34, and pAAV-CBH-KL-BsHIV01-T20 were infected into differentiated C2C12 cells according to different MOIs. Supernatants were collected after 96 hours.

[0100] 1. The synthesized HIV MHER polypeptide was coated on an ELISA PLATE (Corning, Catalog No.: 42592), and the expression supernatant and purified quantitative KL-BsHIV01 standard were used as the primary antibody, HRP-labeled goat anti-human IgG Fc (KPL, Catalog No.: 04-10-20) was used as the secondary antibody, coloring was performed with TMB, and the OD value at 450 nm was detected with a microplate reader. The ELISA results (Table 1) show that the anti-HIV neutralizing antibody adeno-associated virus vector effectively expressed HIV neutralizing antibody after transducing cells in vitro and secreted mature HIV neutralizing antibody protein into the cell culture supernatant. At the same MOI, the expression level in the supernatant of KL-BsHIV01 was the highest, and the expression level decreased after adding HIV fusion inhibitor short peptide (see also Figure 9 and Figure 10).

[0101] [Table 1]

[0102] 2.TZM-bl cells were plated at 2E4 cells / well. The cell expression supernatants containing the same amount of antibody were mixed with 50μL of HIV virus pAD-8 or pNL4-3, then incubated at 37℃ for 30min, and added to TZM-bl cells. The wells with only pAD-8 or pNL4-3 were negative controls, and the wells without HIV virus were blank. At 24 hours, the supernatant was discarded, 100μL of cell lysis solution was added, and the lysed cells were collected after 10min and centrifuged at 8000rpm for 5min. 100μL of firefly luciferase detection reagent was added to 50μL of lysed cells. RLU (relative light unit) was detected and measured using a multi-function microplate reader with chemiluminescence function. As a result (Figure 12), in an in vitro cell test, when the same amount of HIV neutralizing antibody was added, the neutralizing effect of the trispecific HIV neutralizing antibody KL-BsHIV01 plus an HIV fusion inhibitor short peptide was not weaker than that of the original antibody, and the neutralizing effect of KL-BsHIV01-003 against pAD-8 and pNL-3 was significantly superior to that of the HIV bispecific neutralizing antibody KL-BsHIV01.

[0103] 8. Expression in BALB / c mice after intramuscular injection of adeno-associated virus gene therapy vectors AAV gene therapy vectors (pAAV-CBH-KL-BsHIV01-WPRE, pAAV-CBH-KL-BsHIV01-003-WPRE) were injected intramuscularly into the hind thigh muscles of mice. Blood was collected every week, serum was separated, and the antibody concentration expressed in the serum was detected by ELISA.

[0104] 1. The synthesized HIV MHER polypeptide was coated on an ELISA PLATE (Corning, Catalog No.: 42592), and the expression supernatant and purified quantification KL-BsHIV01 standard were used as the primary antibody, HRP-labeled goat anti-human IgG Fc (KPL, Catalog No.: 04-10-20) was used as the secondary antibody, coloring was performed with TMB, and the OD value at 450 nm was detected with a microplate reader. The ELISA results (Figure 13) show that the anti-HIV neutralizing antibody adeno-associated virus vector can persistently and effectively express HIV neutralizing antibodies in mice. 2.TZM-bl cells were plated at 2E4 cells / well. The serum dilutions containing the same amount of antibody were mixed with 50μL of HIV virus pAD-8 or pNL4-3, then incubated at 37℃ for 30min, and added to TZM-bl cells. The wells with only pAD-8 or pNL4-3 were negative controls, and the wells without HIV virus were blank. After overnight incubation, the supernatant was discarded, 100μL of cell lysis solution was added, and the lysed cells were collected after 10min and centrifuged at 8000rpm for 5min. 100μL of firefly luciferase detection reagent was added to 50μL of lysed cells. RLU (relative light unit) was detected and measured using a multi-function microplate reader with chemiluminescence function. As a result, in the cell test, both the anti-HIV biphilic (KL-BsHIV01) and triphilic (KL-BsHIV01-003) neutralizing antibodies had strong HIV neutralizing activity (Figure 14), with IC50s of 0.878ng / mL and 0.150ng / mL for neutralizing the HIV virus strain pAD-8, respectively, and IC50s of 0.117ng / mL and 0.017ng / mL for neutralizing the HIV virus strain pNL4-3, respectively. Obviously, after adding the same amount of HIV neutralizing antibody, the neutralizing effect of the triphilic HIV neutralizing antibody KL-BsHIV01-003 against pAD-8 and pNL4-3 is superior to that of the biphilic HIV neutralizing antibody KL-BsHIV01 (Figure 14). These two HIV neutralizing antibodies of the present invention have stronger neutralizing activity against HIV than the previously reported broadly neutralizing antibodies against HIV.

[0105] SEQ ID NO: 1 MARPLCTLLLLMATLAGALA SEQ ID NO: 2 SELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSS SEQ ID NO: 3 DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS SEQ ID NO: 4 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 5 WEQKIEELLKKAEEQQKKNEEELKKLEK SEQ ID NO: 6 YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF SEQ ID NO: 7 LLEQENKEQQNQSEEILSHILSTYNNIERDWEMW

[0106] SEQ ID NO: 8 MARPLCTLLLLMATLAGALASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0107] SEQ ID NO: 9

[0108] SEQ ID NO: 10 MARPLCTLLLLMATLAGALASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSWEQKIEELLKKAEEQQKKNEEELKKLEK

[0109] SEQ ID NO: 11

[0110] SEQ ID NO: 12 MARPLCTLLLLMATLAGALASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

[0111] SEQ ID NO: 13

[0112] SEQ ID NO: 14 MARPLCTLLLLMATLAGALASELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS LLEQENKEQQNQSEEILSHILSTYNNIERDWEMW

[0113] SEQ ID NO: 15

[0114] SEQ ID NO: 16 TGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0115] SEQ ID NO: 17 CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGT

[0116] SEQ ID NO: 18 TGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTACCGGTCGCCACCATGGCGAGACCCCTGTGCACATTACTTCTGTTGATGGCTACCCTGGCAGGCGCCCTCGCCAGCGAGCTGACACAGGACCCTGCCGTGTCCGTGGCCCTGAAGCAGACCGTGACAATCACCTGCAGAGGCGATTCCCTGAGATCCCACTACGCCTCCTGGTACCAGAAGAAGCCTGGCCAGGCCCCCGTGCTGCTGTTTTACGGCAAGAATAACCGCCCCAGCGGCATCCCCGATAGATTTTCCGGCAGCGCCTCCGGCAACAGAGCCAGCCTGACAATCACCGGCGCCCAGGCCGAGGACGAGGCTGATTACTACTGCAGCTCCAGAGATAAGAGCGGCAGCAGACTGTCCGTGTTTGGCGGCGGCACCAAGCTGACCGTGCTCGGAGGAGGAGGAAGCGGAGGAGGAGGCTCAGGCGGCGGCGGCTCTGAGGTGAGGCTGAGAGAGTCCGGCGGCGGCCTGGTGAAGCCCGGAGGATCTCTGAGGCTGTCCTGCTCCGCCTCCGGCTTCGATTTTGACAATGCCTGGATGACCTGGGTGAGACAGCCCCCTGGCAAGGGCCTG

[0117] SEQ ID NO: 19 TGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCGCTAGCGTCGACCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0118] SEQ ID NO: 20 TTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGAATTCGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0119] SEQ ID NO: 21 CGAATTCGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0120] SEQ ID NO: 22 GAATTCGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0121] SEQ ID NO: 23 CGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGAATTCGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0122] SEQ ID NO: 24 TGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGAATTCGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGTTTAAACAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0123] SEQ ID NO: 25 CCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCCTTGCTTAGATACCACCTTCCCCTGAAG TGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTAGAGGTCTACTTGAAGAAAAAACAGGGGCATGGTTTGACTGTC CTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0124] SEQ ID NO: 26 GTCAGGAGCTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGATCTGAGCCTGGGAGATCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCAGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTGTCGACCATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0125] SEQ ID NO: 27

[0126] SEQ ID NO: 28

[0127] SEQ ID NO: 29 GTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCG TAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGATGGATCC

Claims

1. A gene sequence construct for gene therapy of AIDS virus (HIV) infection, comprising: the gene coding sequence for one or more antibody molecules capable of inhibiting HIV infection and the gene coding sequence for one or more polypeptides (consisting of 2 to 50 amino acid residues) capable of inhibiting HIV infection are included, so as to express a fusion protein molecule containing an anti-HIV antibody molecule and a polypeptide, the fusion protein being encoded by a single gene; The fusion protein molecule has two or more targets of action. The gene sequence construct.

2. the antibody molecule comprises a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and / or the light chain constant region comprises a kappa or lambda light chain light chain constant region. The gene sequence construct of claim 1.

3. (a) The gene coding sequence of the antibody molecule having the ability to inhibit HIV infection and the gene coding sequence of the polypeptide having the ability to inhibit HIV infection are directly or indirectly connected in series via the coding sequence of a linker polypeptide. (b) the gene sequence construct comprises two or more gene coding sequences for antibody molecules capable of inhibiting HIV infection, the gene coding sequences for the antibody molecules being directly or indirectly connected in series via a coding sequence for a linker polypeptide; and / or (c) the gene sequence construct comprises two or more gene coding sequences for polypeptides having the ability to inhibit HIV infection, and the gene coding sequences for the polypeptides are directly or indirectly connected in series between each other via a coding sequence for a linker polypeptide; A gene sequence construct according to claim 1 or 2.

4. (i) The gene coding sequence of one or more antibody molecules capable of suppressing HIV infection includes a gene coding sequence of an antibody molecule of anti-HIV-1-gp160 (or its cleavage products gp120 and gp41), (ii) the gene coding sequence for one or more antibody molecules capable of inhibiting HIV infection comprises a gene coding sequence for an antibody molecule that binds to the human CD4 receptor site; and / or (iii) the gene coding sequence for one or more polypeptides capable of suppressing HIV infection includes a gene coding sequence for a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane; The gene sequence construct of claim 1.

5. The antibody comprises two or more gene coding sequences for antibody molecules having the ability to inhibit HIV infection, and one or more gene coding sequences for polypeptides having the ability to inhibit HIV infection, the gene coding sequences for two or more antibody molecules capable of inhibiting HIV infection include a gene coding sequence for an antibody molecule of anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) and a gene coding sequence for an antibody molecule that binds to the human CD4 receptor site; The gene sequence construct according to claim 1, wherein the gene coding sequence for one or more polypeptides capable of inhibiting HIV infection includes a gene coding sequence for a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane.

6. (A) (i) Gene coding sequences for the light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) of an anti-HIV-1-gp160 (or its cleavage products gp120 and gp41) monoclonal antibody; (ii) gene coding sequences for the light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) and heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) of a monoclonal antibody that binds to the human CD4 receptor site; (iii) a gene coding sequence for a human IgG Fc fragment, and (iv) a gene coding sequence for a polypeptide that inhibits fusion of HIV with the CD4+ T cell membrane; The antibody coding sequences are directly or indirectly connected in tandem via the linker polypeptide coding sequence, regardless of the order. and / or (B) (i) Gene coding sequences for the light chain variable region (VL) and heavy chain variable region (VH) of an anti-HIV-1-gp160 (including its cleavage products gp120 and gp41) monoclonal antibody; (ii) gene coding sequences for the light chain variable region (VL) and heavy chain variable region (VH) of a monoclonal antibody that binds to the human CD4 receptor site; (iii) a gene coding sequence for a human IgG Fc fragment, and (iv) a gene coding sequence for a polypeptide that inhibits fusion of HIV with the CD4+ T cell membrane; The antibody coding sequences are directly or indirectly connected in tandem via the linker polypeptide coding sequence, regardless of the order. The gene sequence construct of claim 1.

7. (a) further comprising a promoter located upstream of a gene coding sequence for an antibody molecule having the ability to inhibit HIV infection and a gene coding sequence for a polypeptide having the ability to inhibit HIV infection; and / or (b) further comprising a secretory signal peptide coding sequence located upstream of the gene coding sequence for an antibody molecule having the ability to inhibit HIV infection and the gene coding sequence for a polypeptide having the ability to inhibit HIV infection; The gene sequence construct of claim 1.

8. a gene coding sequence for a first antibody molecule having the ability to suppress HIV infection, a gene coding sequence for a second antibody molecule having the ability to suppress HIV infection, and a gene coding sequence for a polypeptide having the ability to suppress HIV infection; VL2-linker-VH2-linker-VL1-linker-VH1-linker-CH2-CH3-linker-peptide inhibitor; VL2 and VH2 are variable region fragments of the light chain and heavy chain of a first antibody molecule, respectively; VL1 and VH1 are variable region fragments of the light chain and heavy chain of a second antibody molecule, respectively; CH2-CH3 are Fc fragments of the constant region of human IgG; linker is a linker polypeptide; and peptide inhibitor is a polypeptide that suppresses HIV infection (e.g., a polypeptide that inhibits the fusion of HIV with the CD4+ T cell membrane). The gene sequence construct of claim 1. (a) the protein sequences of VL2 and VH2 comprise SEQ ID NO:2, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; (b) the protein sequence of VL1 and VH1 comprises SEQ ID NO: 3, or a functional fragment thereof, or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; (c) the linker sequence is selected from GGGGS, (GGGGS) 2 , (GGGGS) 3 , (GGGGS) 4 , (GGGGS) 5 , (GGGGS) 6 , and (GGGGS) 7 , or other alternative linker polypeptide sequences; and / or (d) The peptide inhibitor that inhibits fusion of HIV with the CD4+ T cell membrane may be selected from membrane fusion inhibitor polypeptides such as P52, C34, and T20; Optionally, The sequence of the membrane fusion-inhibiting polypeptide P52 comprises SEQ ID NO: 5 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; The polypeptide sequence of C34 comprises SEQ ID NO: 6 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; The polypeptide sequence of T20 comprises SEQ ID NO: 7 or a homologous sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; The gene sequence construct of claim 8.

10. A viral vector genome comprising the construct of claim 1.

11. A viral vector system comprising the genome of claim 10, optionally a lentiviral vector system or an adeno-associated viral vector system.

12. A viral particle comprising the genome of the construct of claim 1.

13. A method for producing a virus comprising administering to a subject a virus vector system according to claim 11 or a virus particle according to claim 12, and a pharmaceutically acceptable carrier or diluent, optionally comprising cells that are muscle cells, liver cells, or CD4+ T cells; Pharmaceutical compositions.

14. Use of cells transduced by the viral vector system of claim 11 or the viral particles of claim 12 in the manufacture of a medicament for inhibiting HIV infection.

15. A pharmaceutical composition for use in suppressing HIV infection in a subject, comprising cells transduced by the viral vector system of claim 11 or the viral particles of claim 12.

16. Use of a cell transduced by the viral vector system of claim 11 or the viral particle of claim 12 in the manufacture of a medicament for treating HIV infection in a subject, Optionally, the subject comprises an HIV-infected individual with early stage HIV infection, or an HIV-infected individual who has already received cocktail drug therapy, or an HIV-infected individual who is resistant to cocktail drug therapy. The above use.

17. A pharmaceutical composition for use in treating HIV infection in a subject, comprising cells transduced by the viral vector system of claim 11 or the viral particle of claim 12, Optionally, the subject comprises an HIV-infected individual with early stage HIV infection, or an HIV-infected individual who has already received cocktail drug therapy, or an HIV-infected individual who is resistant to cocktail drug therapy. The pharmaceutical composition.

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