Recombinant interferon drug targeting conserved HIV gp41 subunit membrane-proximal external region and use thereof

Abstract

Disclosed are a recombinant interferon drug targeting a conserved HIV gp41 subunit membrane-proximal external region (MPER) and a use thereof. Specifically, disclosed is a targeted interferon fusion protein ZP-IC, comprising a 10E8 antibody or an antigen-binding fragment thereof, and an integrated interferon α, wherein the 10E8 antibody or the antigen-binding fragment thereof is linked to the integrated interferon α by means of a linker. In the present invention, the 10E8 antibody capable of identifying a highly conserved gp41 subunit MPER of an HIV envelope glycoprotein is used as a targeting tool for the integrated interferon. Therefore, a target exposed by a conformational change of a viral envelope protein during the infection of a cell can be accurately identified, viral infection is prevented, and a neutralizing effect is exerted; and in addition, a carried interferon molecule can more effectively bind to an interferon receptor on a cell around the HIV virus and achieve an antiviral role. Experiments have demonstrated that the ZP-IC has excellent antiviral activity and the capability of inhibiting an overwhelming majority of HIV strains across a broad spectrum.

Description

Recombinant interferon drug targeting the conserved HIV gp41 subunit membrane-proximal extracellular domain and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a recombinant interferon drug targeting the conserved extracellular region of the HIV gp41 subunit proximal to the membrane and its application. Background Art

[0002] AIDS is a highly harmful infectious disease caused by infection with the Human Immunodeficiency Virus (HIV). HIV can damage the human immune system and destroy the CD4 + T lymphocytes are the main target of attack, and they are destroyed in large numbers, causing the body to lose its immune function. As a result, the body is susceptible to various diseases and may develop malignant tumors, with a high mortality rate. + When infecting T cells, the CD4 molecule on the cell surface is the primary receptor, and CCR5 or CXCR4 is a secondary receptor. In the first step of infection, the virus approaches the cell through its outer membrane protein gp120 binding to the receptor molecule CD4 on CD4+ T cells. Then gp120 further binds to the secondary receptor CCR5 or CXCR4 embedded in the host cell membrane, shortening the distance between the virus and the cell, triggering a conformational change in the viral outer membrane protein molecule gp120, exposing its other membrane protein subunit gp41. Subsequently, the HR1 and HR2 domains in the gp41 subunit fold to form a six-helix bundle (both subunits are trimers), ultimately bringing the virus and host cell infinitely closer. The outer membrane of the virus fuses with the host cell membrane, facilitating the invasion of viral RNA into CD4+ T host cells.

[0003] HIV antiviral drugs include five major categories: nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase strand transfer inhibitors, and binding and membrane fusion inhibitors. HIV-infected people need to take medication for life to control viral load. Long-term use of chemical drugs highlights the long-term toxicity and viral resistance of drugs, which will lead to reduced patient compliance. Therefore, it is necessary to find more effective and safer therapeutic drugs, such as antibody drugs or recombinant fusion protein drugs. They do not enter cells and usually act outside the cells, preventing viruses from invading cells by neutralizing the virus, thereby controlling the infection.

[0004] Therapeutic neutralizing antibodies for HIV target the HIV outer membrane glycoprotein. The only antigenic outer membrane glycoprotein on the surface of HIV virions is gp160, a trimer (gp160)3 that can be cleaved into (gp120 / gp41)3. Many dominant epitopes on the HIV outer membrane protein are highly variable, resulting in low binding potency of antibodies produced by infected individuals themselves. However, there are also several recognized, relatively conserved sites on the HIV outer membrane protein that serve as epitopes for broadly neutralizing antibodies (bNAbs). One of these is the CD4 binding site on the gp120 subunit, which is responsible for binding to the human T cell differentiation antigen CD4 receptor molecule; another is a domain on the gp41 subunit close to the membrane, commonly referred to as the "membrane-proximal external region" (MPER), whose function is related to membrane fusion. (Huang JH, et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature. 2012, 491 (7424): 406-412. doi: 10.1038 / nature11544.).

[0005] The HIV gp41 subunit has a molecular weight of approximately 41 kDa. Its major structural domains, starting from the N-terminus, are the fusion peptide (FP), the α-helical heptad repeat domain HR1 (or NHR), the α-helical heptad repeat domain HR2 (or CHR), the membrane-proximal extracellular region (MPER), and the transmembrane domain (TM). Once the outer gp120 subunit completes its task of locating target cells and receptors, it undergoes conformational changes to expose the hidden gp41 subunit. The N-terminal fusion peptide FP translocates and inserts into the host cell membrane, while the C-terminal transmembrane domain remains anchored to the viral membrane, resulting in a transient, extended conformation of the gp41 subunit known as the "pre-hairpin intermediate." In this state, the NHR, CHR, and MPER domains are all exposed, facilitating recognition by membrane fusion inhibitors or related antibodies.

[0006] Many polypeptide membrane fusion inhibitors are designed by mimicking the sequence structure of CHR. They competitively bind to NHR, thereby preventing the folded binding of NHR and CHR, destroying the membrane fusion link of the virus and preventing viral infection. This type of fusion peptide includes the HIV fusion inhibitor drug T20 (enfuvirtide) launched in 2003, upgraded peptide candidate drugs such as albuvirtide, T1249, and T2635 launched in 2018 (Eggink D, et al. Detailed Mechanistic Insights into HIV-1 Sensitivity to Three Generations of Fusion Inhibitors. J Virol. 2011, 85(20): 10785-97. doi: 10.1128 / JVI.05331-11.), and peptide-lipid candidate drugs such as LP51 and LP98 (Xue J, et al. Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide. Cell, 2022.185(1):131-144.e18.doi:10.1016 / j.cell.2021.11.032.).

[0007] Some antibodies, such as 2F5, 4E10, Z13e1, and 10E8, target the MPER domain of gp41. Their mechanism of action is to attack the pre-hairpin intermediate of gp41, preventing the NHR and CHR domains from folding into a six-helix bundle. Because the MPER domain of gp41 is always shielded by the gp120 subunit in native viral particles, these antibodies are characterized by very high affinity for the pre-fusion pre-hairpin intermediate of gp41, while exhibiting very low affinity for the trimeric HIV membrane protein (gp160, composed of gp120 and gp41). Among antibodies targeting the MPER domain, 10E8 is the most extensively studied. According to literature reports, 10E8 targets the MPER region of the HIV gp41 subunit, with key binding sites including Trp672, Phe673, and Trp676 in the MPER region, as well as Tyr681, Ile682, and Arg683 at its C-terminus. More detailed studies, using X-ray crystallographic analysis of the 10E8 Fab fragment and a substrate peptide, revealed that the gp41 MPER region consists of two helices, with 10E8 primarily recognizing the second helix starting at position 671. This helix encompasses not only the C-terminus of the MPER region but also extends to several amino acids at the N-terminus of the transmembrane domain (TMD). The peptide structure underlying antibody recognition must extend to at least position 690 of the TMD. Structural analysis shows that the tip of the third complementarity determining region (CDRH3) on the 10E8 antibody heavy chain can penetrate the lipid bilayer and bind to the N-terminal helical structure of the TMD, thereby locking the antibody's binding to the MPER helical structure (Rujas E, et al. Neutralization of HIV-1 through the molecular recognition of 10E8 helical epitope at the membrane interface. Sci Rep 6, 38177 (2016). https: / / doi.org / 10.1038 / srep38177.).In terms of affinity, according to literature, the affinity (dissociation equilibrium constant) of 10E8 to the pre-hairpin intermediate of gp41 (including TMD) is about 1.85nM, the affinity to the full-length MPER region peptide (656-683) is 17nM, and the binding force to gp140 where the MPER region is hidden is extremely weak (Chen J, et al. Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41. J Virol. 2014, 88(2): 1249-58. doi: 10.1128 / JVI.02664-13.) (Huang JH, et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody.Nature.2012,491(7424):406-412.doi:10.1038 / nature11544.).

[0008] 10E8 was used to neutralize pseudoviruses constructed based on HIV outer membrane proteins of different sequences. 50 The concentration of the monoclonal antibody is less than 50 μg / ml (about 330 nM), and it can neutralize 98% of the 181 pseudoviruses tested. 50The judgment standard is below 1μg / ml (about 6.6nM). This monoclonal antibody can neutralize 72% of the tested viruses, which is much higher than the 4E10 monoclonal antibody whose binding site is also in the MPER region (the latter's neutralization coverage is only 37%). In addition, although the binding site of 10E8 is very close to the viral envelope, its binding ability to the phospholipid bilayer is relatively weak, indicating that it has good safety (Huang JH, et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature. 2012, 491(7424): 406-412. doi: 10.1038 / nature11544.) (Chen J, et al. Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41. J Virol. 2014, 88(2): 1249-58. doi: 10.1128 / JVI.02664-13.). However, the two antibodies 2F5 and 4E10 react strongly with the lipid membrane of cells and are not suitable for subsequent development. Therefore, 10E8 is an antibody with broad neutralizing activity and is safer than other antibodies with the same target.

[0009] Interferons (IFNs) are a group of active proteins (primarily glycoproteins) with multiple functions. They possess broad-spectrum antiviral, antitumor, and immunomodulatory effects and are biologically classified as cytokines. Interferons can be divided into type I and type II interferons based on factors such as the cells that produce them, their receptors, and their biological activity. Type I interferons (IFN-α, IFN-β, and IFN-ω) inhibit viral replication, fight parasites, inhibit the proliferation of various cells, stimulate the cytotoxic activity of immune cells, participate in immunomodulation, and have anti-tumor effects. Type II interferons (only IFN-γ) primarily activate T cells and natural killer cells, exerting an immunomodulatory effect. All type I interferon molecules bind to the same receptor, a heterodimer composed of two subunits: IFNAR1 and IFNAR2. After binding to its receptor, interferon activates the downstream non-receptor tyrosine kinase family, Jak1 and Tyk2, which further recruit and activate the signal transduction and transcription activator proteins STAT1 and STAT2. It then forms a complex with interferon regulatory factor 9 (IRF9), ISGF3 (IFN-stimulated gene factor 3), thereby activating the expression of a series of interferon-stimulated genes (ISGs), inducing these downstream genes to exert antiviral, antitumor, and immunomodulatory functions. Interferon functions in two ways: first, by interacting with relevant receptors on surrounding uninfected cells, stimulating the synthesis of various effector proteins, such as the antiviral protein tetherin, APOBEC1, and protein kinase R, thereby exerting an antiviral effect; second, by enhancing the activity of lymphocytes, macrophages, and natural killer cells, it exerts immunomodulatory and anti-tumor effects.

[0010] Type I interferons play a crucial role in the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Several recombinant type I interferons are now on the market, including Roche's Pegasys, Merck's PegIntron, and domestically produced products like Yundesu, Cerikin, and Endafin. However, in the treatment of HIV infection, the therapeutic or adjuvant effects of type I interferons are less clear. Some scholars believe that type I interferon plays a dual role in the progression of HIV infection: in the acute phase of HIV infection, interferon plays an antiviral role by stimulating effector proteins and the immune system; but in the chronic infection phase, continuous exposure to interferon can lead to desensitization of the body and activation of harmful immune responses ([1] Scagnolari C, et al. Type I interferon and HIV: Subtle balance between antiviral activity, immunopathogenesis and the microbiome. Cytokine Growth Factor Rev. 2018, 40: 19-31. doi: 10.1016 / j.cytogfr.2018.03.003. [2] Zhang Liguo. The role of type I interferon in HIV-1 infection is two-sided. Progress in Biochemistry and Biophysics Biophysics.2018,45(9):966-970.doi:10.16476 / j.pibb.2018.0191.[3]Sugawara S,etal.People with HIV-1demonstrate type 1interferon refractoriness associated with upregulated USP18.J Virol.2021,95(10):e01777-20.doi:10.1128 / JVI.01777-20.). These views undoubtedly make the application prospects of interferon in the field of HIV treatment unclear, and it gradually disappears from the list of candidate drugs for the treatment of HIV infection.

[0011] Human type I interferon includes 12 IFN-α subtypes, 1 IFN-β and 1 IFN-ω, and the antiviral activity of each interferon varies greatly (Bekisz J, et al. Human Interferons Alpha, Beta and Omega. Growth Factors. 2004, 22(4): 243-251. doi: 10.1080 / 08977190400000833). In an HIV treatment research project with 20 clinical cases, it was shown that pegylated interferon alfa-2a monotherapy can inhibit the replication of HIV-1 virus and reduce the integration of HIV DNA in cells (Azzoni L, et al. Pegylated Interferon alfa-2a monotherapy results in suppression of HIV type 1 replication and decreased cell-associated HIV DNA integration. J Infect Dis. 2013, 207(2): 213-222. doi: 10.1093 / infdis / jis663.). In addition, different subtypes of type I interferon also have different anti-HIV functions. This is mainly due to the differences in the protein sequences of different subtypes of interferon, which will lead to a difference of tens or hundreds of times in their affinity for the shared receptors IFNAR1 and IFNAR2, and the resulting downstream signal transduction and antiviral ability are also different. Researchers compared the anti-HIV abilities of different molecules of type I α-subtype interferon and found that molecules such as IFNα8, IFNα6, and IFNα14 had higher activity, while the common IFNα1 and IFNα2 had weaker anti-HIV abilities ([1] Harper MS, et al. Interferon-αSubtypes in an Ex Vivo Model of Acute HIV-1 Infection: Expression, Potency and Effector Mechanisms. PLoS Pathog. 2015, 11(11): e1005254. doi: 10.1371 / journal.ppat.1005254. [2] Lavender KJ, et al. Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo. J Virol. 2016, 90(13): 6001-6013. doi: 10.1128 / JVI.00451-16.).Therefore, the research on the drugability of type I interferon in the field of HIV anti-infection is still in its early stages and requires continuous exploration and improvement.

[0012] Summary of the Invention

[0013] The purpose of the present invention is to provide a drug for treating HIV infection with better anti-HIV virus ability. The technical problems to be solved are not limited to the technical themes described herein, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0014] To achieve the above objectives, the present invention first provides a fusion protein named ZP-IC, wherein the fusion protein ZP-IC comprises the 10E8 antibody or its antigen-binding fragment, and integrated interferon α, wherein the 10E8 antibody or its antigen-binding fragment and the integrated interferon α are connected via a linker.

[0015] Furthermore, the integrated interferon α can be fused to the C-terminus of the 10E8 antibody or its antigen-binding fragment via the linker.

[0016] Furthermore, the amino acid sequence of the integrated interferon α may be positions 483-648 of SEQ ID No. 1.

[0017] Furthermore, the linker may be a flexible peptide linker, such as a peptide linker comprising glycine and / or serine residues.

[0018] Furthermore, the linker includes but is not limited to GSGSGS, GGGSGGGS, GGGSGGG, GGSSGG, GSGSGSG, GGGGS.

[0019] Furthermore, the linker is GSGSGS (positions 477-482 of SEQ ID No. 1).

[0020] Furthermore, the amino acid sequence of the heavy chain of the fusion protein may be SEQ ID No. 1, and the amino acid sequence of the light chain of the fusion protein may be SEQ ID No. 3.

[0021] Among them, positions 1-476 of SEQ ID No. 1 are the amino acid sequence of the heavy chain of the 10E8 antibody targeting HIV gp41 protein, positions 477-482 of SEQ ID No. 1 are the amino acid sequence of the linker, and positions 483-648 of SEQ ID No. 1 are the amino acid sequence of integrated interferon α.

[0022] SEQ ID No. 3 is also the amino acid sequence of the light chain of the 10E8 antibody targeting HIV gp41 protein.

[0023] The fusion protein ZP-IC is also a recombinant interferon targeting the conserved HIV gp41 subunit membrane-proximal external region.

[0024] The present invention also provides a nucleic acid molecule, which can encode any fusion protein described herein.

[0025] Furthermore, the nucleic acid molecule may include a nucleic acid molecule encoding the heavy chain of the fusion protein ZP-IC and / or a nucleic acid molecule encoding the light chain of the fusion protein ZP-IC.

[0026] Furthermore, the nucleic acid molecule may be a DNA molecule whose coding sequence is SEQ ID No. 2 and / or a DNA molecule whose coding sequence is SEQ ID No. 4.

[0027] Among them, positions 1-1428 of SEQ ID No. 2 are the nucleotide sequence of the heavy chain of the 10E8 antibody targeting HIV gp41 protein, positions 1429-1446 of SEQ ID No. 2 are the nucleotide sequence of the linker, and positions 1447-1950 of SEQ ID No. 2 are the nucleotide sequence of integrated interferon α.

[0028] SEQ ID No. 4 is also the nucleotide sequence of the light chain of the 10E8 antibody targeting HIV gp41 protein.

[0029] The nucleic acid molecule may also include a nucleic acid molecule obtained by modifying the codon preference based on the nucleotide sequence shown in SEQ ID No. 2 and / or SEQ ID No. 4.

[0030] The present invention also provides a biomaterial, which may be any of the following:

[0031] A1) an expression cassette containing the nucleic acid molecule;

[0032] A2) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in A1);

[0033] A3) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the expression cassette described in A1), or a recombinant microorganism containing the recombinant vector described in A2);

[0034] A4) a recombinant cell containing the nucleic acid molecule, or a recombinant cell containing the expression cassette of A1), or a recombinant cell containing the recombinant vector of A2).

[0035] Wherein, the recombinant microorganism described in A3) and the recombinant cell described in A4) can express the fusion protein ZP-IC.

[0036] The vector described herein refers to a vector capable of transporting exogenous DNA or a gene of interest into a host cell for amplification and expression. The vector may be a cloning vector or an expression vector, including but not limited to: a plasmid, a phage (such as lambda phage or M13 filamentous phage), a cosmid (i.e., a cosmid), a viral vector (such as a baculovirus vector, a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, or a herpes virus (such as herpes simplex virus)). In one or more embodiments of the present invention, the vector is a pcDNA3.1Neo(+) vector and / or a pcDNA3.1Zeo(+) vector.

[0037] The microorganisms described herein may be yeast, bacteria, algae, or fungi. The bacteria may be from, but are not limited to, Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., or Bacillus sp. In one or more embodiments of the present invention, the microorganism is Escherichia coli Trans10.

[0038] The cells (host cells) described herein refer to cells that can be used to introduce vectors, including but not limited to eukaryotic cells (such as yeast cells, Aspergillus cells), animal cells (such as mammalian cells, insect cells), plant cells, or prokaryotic cells (such as Escherichia coli or Bacillus subtilis). In one or more embodiments of the present invention, the cells are CHO cells (CHO-DG44 cells).

[0039] The recombinant vectors described herein include the ZP-IC light chain expression vector pcDNA3.1 Zeo(+) / ZP-IC LC and the ZP-IC heavy chain expression vector pcDNA3.1 Neo(+) / ZP-IC HC.

[0040] pcDNA3.1 Zeo(+) / ZP-IC LC is a recombinant expression vector expressing the fusion protein ZP-IC light chain, obtained by replacing the fragment between the EcoRI recognition site and the EcoRV recognition site of the pcDNA3.1Zeo(+) vector with the ZP-IC light chain gene having the nucleotide sequence of SEQ ID No. 4, while keeping the other nucleotide sequences of the pcDNA3.1Zeo(+) vector unchanged.

[0041] pcDNA3.1 Neo(+) / ZP-IC HC is a recombinant expression vector expressing the fusion protein ZP-IC heavy chain, obtained by replacing the fragment between the EcoRI recognition site and the EcoRV recognition site of the pcDNA3.1Neo(+) vector with the ZP-IC heavy chain gene having the nucleotide sequence of SEQ ID No. 2, while keeping the other nucleotide sequences of the pcDNA3.1Neo(+) vector unchanged.

[0042] The recombinant microorganisms described herein include recombinant bacteria obtained by introducing the heavy chain gene (SEQ ID No. 2) and / or the light chain gene (SEQ ID No. 4) of the fusion protein into Escherichia coli.

[0043] The recombinant cells described herein include recombinant cells obtained by expressing the heavy chain gene (SEQ ID No. 2) and the light chain gene (SEQ ID No. 4) of the fusion protein in mammalian cells (such as CHO cells).

[0044] The present invention also provides any of the following uses of any of the fusion proteins, nucleic acid molecules or biomaterials described herein:

[0045] B1) Use in the preparation of a product for preventing or treating HIV infection;

[0046] B2) Use in the preparation of products for preventing or treating AIDS;

[0047] B3) Use in the preparation of products for inhibiting HIV viral activity;

[0048] B4) Use in the preparation of a product for binding or detecting gp41 protein;

[0049] B5) Use in the preparation of a product for binding or detecting an MPER polypeptide.

[0050] The products include reagents, kits, drugs or pharmaceutical compositions.

[0051] The present invention also provides a pharmaceutical composition, which may comprise any of the fusion proteins described herein and one or more pharmaceutically acceptable carriers.

[0052] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant, but is not limited thereto.

[0053] The pharmaceutical composition has a neutralizing antiviral effect of inhibiting or neutralizing HIV activity and is used to improve, prevent or treat HIV infection or AIDS.

[0054] The present invention also provides a reagent or kit for binding or detecting gp41 protein or MPER polypeptide, wherein the reagent or kit comprises any one of the fusion proteins described herein.

[0055] The present invention also provides a method for preparing any of the fusion proteins described herein, which may include: constructing recombinant expression vectors containing the heavy chain and light chain genes of the fusion protein respectively; introducing the recombinant expression vectors into host cells to obtain recombinant cells expressing the fusion protein; culturing the recombinant cells, and obtaining the fusion protein through separation and purification.

[0056] Furthermore, the nucleotide sequence of the fusion protein heavy chain gene may be SEQ ID No. 2.

[0057] Furthermore, the nucleotide sequence of the fusion protein light chain gene may be SEQ ID No.4.

[0058] The fusion protein of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the fusion protein of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells. The transfected host cells are cultured under specific conditions to express the fusion protein of the present invention. Those skilled in the art can select conventional host cells, expression vectors, methods for introducing the expression vectors into host cells, and methods for isolating and purifying the fusion protein as needed.

[0059] The present invention provides a targeted interferon drug (i.e., the fusion protein ZP-IC of the present invention) targeting HIV (human immunodeficiency virus) outer membrane protein gp41 and its application. The drug is a recombinantly expressed protein in mammalian cells (such as CHO cells), and its structure is an antibody-cytokine fusion protein. The antibody structure portion of the fusion protein can target and recognize the gp41 subunit in the HIV outer membrane protein gp160, and is responsible for carrying the interferon molecule to the vicinity of HIV or HIV-infected cells, thereby guiding the interferon's directional antiviral effect. Specifically, the targeted interferon is a fusion molecule of an antibody molecule and an interferon, wherein the interferon molecule is located at the C-terminus of the heavy chain of the antibody molecule. The antibody molecule is composed of its heavy chain variable region (VH) and light chain variable region (VL) to form a functional region that recognizes the HIV gp41 subunit and is responsible for specific binding to the corresponding sequence of the gp41 subunit. The constant region of the antibody molecule structure is of IgG1 type. At the C-terminus of the constant region CH3, a molecule of type I interferon (α or β subtype) is connected. The targeted interferon drug is used to treat HIV infection, and achieves precise drug administration through targeted sites, thereby reducing drug dosage and side effects while improving the antiviral effect of the drug.

[0060] In this invention, the antibody 10E8, which recognizes the highly conserved MPER region of the gp41 subunit of the HIV outer membrane glycoprotein, was selected as the targeting tool for the integrated interferon. It performs two functions: First, it accurately identifies the target site exposed by the conformational changes of the outer membrane protein in infected cells, preventing viral infection and exerting a neutralizing effect. Simultaneously, the carried interferon molecule can more effectively bind to interferon receptors on cells surrounding HIV, activating the intracellular interferon signaling pathway and allowing the cells to exert their antiviral function.

[0061] Specifically, the present invention uses genetic engineering technology to fuse the antibody 10E8, which can recognize the highly conserved MPER region of the gp41 subunit of the HIV outer membrane glycoprotein gp160, with a highly active integrated interferon to form the antibody-cytokine fusion protein ZP-IC, which exerts the function of targeted interferon. The product ZP-IC of the present invention targets the highly conserved MPER domain of HIV gp41. The heavy chain variable region CDR region (complementarity determining region) and light chain variable region CDR region sequences of the antibody structure portion of ZP-IC are both derived from the corresponding partial sequences of the monoclonal antibody 10E8. The backbone structure of the rest of the antibody adopts an IgG1 antibody structure. A molecule of integrated interferon is connected to the C-terminus of the antibody constant region CH3. This targeted interferon molecule ZP-IC recognizes and binds to the HIV viral outer membrane protein gp41 through the 10E8 antibody portion, blocking the virus's membrane fusion invasion process; through its integrated interferon functional portion, it mobilizes and binds to interferon receptors on cells surrounding the virus, activates the intracellular interferon signaling pathway, produces antiviral effector proteins, and exerts its antiviral function. This invention utilizes an innovative design, enabling the antibody molecule to perform dual functions: identifying pathogens and neutralizing viruses; and guiding the localization and localized enrichment of interferon. Thus, the invention cleverly combines the neutralizing effect of antibodies with the antiviral effects of interferon, achieving a practical effect of "1 + 1 > 2." Cell experiments have demonstrated that this "antibody-interferon" fusion molecule, ZP-IC, inhibits viral replication far more effectively than either the interferon molecule itself or the antibody itself.

[0062] U.S. Patent No. US20160333076A1 documents the monoclonal antibody 10E8, targeting the MPER region of the HIV gp41 subunit, as well as its mutants and applications. International Patent No. WO 2017079479A1 and U.S. Patent No. US20190077849A1 document the commercialized mutational engineering of the monoclonal antibody 10E8, targeting the MPER region of the HIV gp41 subunit, including further enhancements to its virus neutralization ability, solubility, and immunogenicity. These patent applications demonstrate that the 10E8 antibody has attracted significant attention due to its broad neutralizing activity. However, to date, no patents or articles have reported the fusion of 10E8 with interferon to achieve its targeted antiviral effects, demonstrating the innovative and novel nature of the ZP-IC molecular drug design. Cellular experiments with ZP-IC demonstrate that this "antibody-interferon" fusion molecule exhibits significantly greater antiviral efficacy than either interferon or the antibody itself.

[0063] The essence of the present invention is a genetically recombinant targeted interferon biotechnology drug. The targeted interferon is used to treat HIV infection. Through targeted and precise drug delivery, it reduces the dosage of the drug and reduces the side effects while improving the antiviral effect of the drug. The recommended clinical dosage of this drug should be less than 0.5 mg / dose each time, and the treatment interval should be no less than 3 days. The recommended method of use of this drug is intravenous injection. The recommended method of treating HIV infection with this drug is combined use, that is, combined with conventional HIV treatment drug cocktails (such as tenofovir, lamivudine, efavirenz, etc.) to exert synergistic antiviral capabilities.

[0064] In summary, compared with the prior art, the advantages and features of the present invention are:

[0065] (1) Through high-affinity, broad-spectrum antibody fragments that bind to viral glycoproteins, an integrated interferon molecule is precisely delivered to the vicinity of HIV-infected cells, blocking viral infection and mobilizing the cells' antiviral ability through the interferon signaling pathway, truly playing the role of a "biological missile."

[0066] (2) Compared with interferon, "targeted interferon" has a more precise effect. It will form a microenvironment with a higher concentration of interferon near HIV-infected cells, which is more conducive to the antiviral effect of interferon and the clearance of diseased cells. It also reduces the concentration of free interferon in the blood, which helps to reduce the toxic side effects of interferon.

[0067] (3) Compared with other antibodies targeting HIV outer membrane proteins, antibodies targeting the MPER region of the gp41 subunit have a broader spectrum. Because the gp41 subunit is responsible for membrane fusion, all functional regions related to membrane fusion are very conserved, including FP, MPER, and the heptad repeat sequence NHR, which are all highly conserved. Therefore, the 10E8 antibody can neutralize the vast majority of HIV strains. Choosing it as an antibody targeting interferon can make the function of interferon more broad-spectrum and more effective, and minimize the immune escape of the virus.

[0068] (4) Compared with the antibody drugs with the same target, “targeted interferon” is more effective. We used the in vitro HIV virus replication inhibition cell model to evaluate its effectiveness. The experiment showed that ZP-IC has an IC of 100% for 8 different types of HIV epidemic virus strains tested. 50 The IC values ​​of the isotype antibody ZP-ab were between 0.01 and 0.2 μg / mL. 50 Each comparative data shows that the half-maximal inhibitory concentration IC of the drug is higher than that of the target interferon ZP-IC and its isotype antibody. 50 Both were reduced by more than 200 times, that is, the efficacy was improved by at least 2 orders of magnitude, indicating that the synergistic effect of "targeted blocking" + "interferon antiviral" makes the molecule have better antiviral activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 is a schematic diagram of the structural design of the fusion protein ZP-IC.

[0070] FIG2 compares the amino acid sequences of 12 subtypes of interferon-α. The most frequently occurring consensus sequence is the sequence of integrated interferon-α.

[0071] FIG3 shows the enzyme digestion identification of the recombinant eukaryotic expression vector of ZP-IC.

[0072] Figure 4 shows the affinity chromatography purification and reducing SDS-PAGE of ZP-IC. In Figure 4, the symbol ① represents the heavy chain of ZP-IC, and the symbol ② represents the light chain of ZP-IC.

[0073] Figure 5 is a Western blot of ZP-IC.

[0074] FIG6 shows the affinity of the fusion protein ZP-IC to HIV outer membrane protein gp41 detected by ELISA.

[0075] FIG7 shows the affinity of the fusion protein ZP-IC to the membrane-proximal MPER polypeptide of the HIV outer membrane protein gp41 subunit detected by ELISA.

[0076] FIG8 shows the binding of ZP-IC to IFNR2. DETAILED DESCRIPTION

[0077] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0078] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0079] The pcDNA3.1Neo(+) vector in the following examples is a product of Invitrogen, catalog number V79020.

[0080] The pcDNA3.1Zeo(+) vector in the following examples is a product of Invitrogen, catalog number V86020.

[0081] Example 1. Design and structure of fusion protein ZP-IC

[0082] To enhance the antiviral activity of HIV-targeted interferon, the present invention employed a fusion protein of recombinant interferon α with an antibody molecule (10E8 antibody) that recognizes and binds to HIV outer membrane glycoprotein gp41. This fusion protein, designated ZP-IC (also known as targeted interferon ZP-IC), has a schematic structure shown in Figure 1.

[0083] Integrated interferon α, also known as consensus interferon α (Interferon alfacon-1), is a non-natural type I interferon. It is a new type of interferon that is synthesized by comparing the amino acid sequences of 12 types of interferon-α and assigning the most frequently occurring amino acids to their respective corresponding positions (Figure 2). Comparison of the biological activity of integrated interferon α with that of natural type I IFN-α shows stronger antiviral activity. For example, the specific activity of integrated interferon α (defined as the antiviral activity unit per milligram of protein) has been reported to be 5 to 20 times higher than that of other subtypes of IFN-α. Generally, the specific activity of other subtypes of IFN-α ranges from 1×10 8 U / mg to 4×10 8 U / mg protein, while the specific activity of integrated interferon α was 2×10 9U / mg protein (Blatt LM, et al. The biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon. J Interferon Cytokine Res. 1996, 16(7): 489-499. doi: 10.1089 / jir. 1996.16.489.).

[0084] The light chain variable region sequence of the ZP-IC antibody was derived from the literature: [Huang JH, et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature, 2012, 491(7424): 406-12. doi: 10.1038 / nature11544.].

[0085] The light chain constant region of the ZP-IC antibody adopts the human immunoglobulin kappa light chain constant region (IGKC, https: / / www.uniprot.org / uniprot / P01834).

[0086] The heavy chain variable region of the ZP-IC antibody was derived from the literature: [Huang JH, et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature, 2012, 491(7424): 406-12. doi: 10.1038 / nature11544.].

[0087] The heavy chain constant region of the ZP-IC antibody portion uses a human immunoglobulin heavy constant gamma (IgG), including the heavy chain constant region of human IgG1 (IGHG1, https: / / www.uniprot.org / uniprot / P01857), or the heavy chain constant region of human IgG2 (IGHG2, https: / / www.uniprot.org / uniprot / P01859), or the heavy chain constant region of human IgG4 (IGHG4, https: / / www.uniprot.org / uniprot / P01861). In this example, the heavy chain constant region of human IgG1 is used, and the sequence is shown at positions 149-476 of SEQ ID No. 1.

[0088] At the C-terminus of the heavy chain constant region, an integrated interferon α or consensus interferon α with stronger antiviral activity is fused via a flexible protein linker (e.g., positions 477-482 of SEQ ID No. 1). The amino acid sequence of the integrated interferon α is shown in positions 483-648 of SEQ ID No. 1.

[0089] The fusion protein of the present invention comprises the 10E8 antibody and integrated interferon α, wherein the integrated interferon α is connected to the constant region of the C-terminus of the 10E8 antibody via a linker. The specific sequence is shown below:

[0090] The amino acid sequence of the heavy chain of the fusion protein ZP-IC is SEQ ID No. 1; the nucleotide sequence of the heavy chain of the fusion protein ZP-IC is SEQ ID No. 2.

[0091] The amino acid sequence of the light chain of the fusion protein ZP-IC is SEQ ID No. 3; the nucleotide sequence of the light chain of the fusion protein ZP-IC is SEQ ID No. 4.

[0092] Among them, positions 1-476 of SEQ ID No. 1 are the amino acid sequence of the heavy chain of the 10E8 antibody targeting HIV gp41 protein, positions 477-482 of SEQ ID No. 1 are the amino acid sequence of the linker, and positions 483-648 of SEQ ID No. 1 are the amino acid sequence of integrated interferon α.

[0093] Positions 1-1428 of SEQ ID No. 2 are the nucleotide sequence of the heavy chain of the 10E8 antibody targeting HIV gp41 protein, positions 1429-1446 of SEQ ID No. 2 are the nucleotide sequence of the linker, and positions 1447-1950 of SEQ ID No. 2 are the nucleotide sequence of integrated interferon α.

[0094] SEQ ID No. 3 is also the amino acid sequence of the light chain of the 10E8 antibody targeting HIV gp41 protein.

[0095] SEQ ID No. 4 is also the nucleotide sequence of the light chain of the 10E8 antibody targeting HIV gp41 protein.

[0096] Example 2. Preparation of fusion protein ZP-IC

[0097] 1. Construction of recombinant eukaryotic expression vector and transfection of CHO cells

[0098] Commercial eukaryotic expression vectors such as pCHO1.0 (Invitrogen), pcDNA TM 3.1 / Neo(+ / -) and pcDNA TM 3.1 / Zeo(+ / -) (Invitrogen) or other commercial eukaryotic expression vectors can be used to express ZP-IC recombinant protein. Alternatively, a self-developed eukaryotic expression vector (e.g., Shao Yong, Hu Xianwen, et al., "A High-Efficiency Animal Cell Expression Vector and Application Based on GS Screening System," Patent No. ZL201410037976.6) can be used to express ZP-IC recombinant protein.

[0099] Based on the light and heavy chain gene sequences of the fusion protein, the light and heavy chain genes were optimized for CHO cell expression. EcoRI enzyme recognition sites and Kozak sequences were added upstream, and EcoRV enzyme recognition sites were added downstream. The optimized light chain gene (SEQ ID No. 4) and heavy chain gene (SEQ ID No. 2) of the fusion protein ZP-IC were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The light and heavy chain genes, pcDNA3.1Neo(+) vector, and pcDNA3.1Zeo(+) vector (Invitrogen) were double-digested with EcoRI / EcoRV, respectively. The relevant genes and vectors were recovered, and the light chain gene and pcDNA3.1Zeo(+) vector were ligated with T4 ligase, while the heavy chain gene and pcDNA3.1Neo(+) vector were ligated with T4 ligase. The cells were then transformed into Escherichia coli Trans10 (Beijing Quanshijin Biotechnology Co., Ltd.).

[0100] Colonies harboring the pcDNA3.1Zeo(+) vector ligated with the light chain gene were selected as PCR templates. Colony PCR was performed using CMV-F / BGH-R as upstream and downstream primers. The PCR reaction conditions were as follows: initial denaturation at 94°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and chain extension at 68°C for 1 minute, for 30 cycles. Following each cycle, a single extension at 72°C for 5 minutes was performed. Positive clones were selected for gene sequencing using primers CMV-F and BGH-R, respectively. Plasmids were extracted from clones that were sequenced correctly, yielding the eukaryotic expression vector pcDNA3.1Zeo(+) / ZP-IC LC expressing the ZP-IC light chain. EcoRI / EcoRV double enzyme digestion of this vector is shown in lane 2 of Figure 3. In Figure 3, pcDNA3.1 / Zeocin represents the pcDNA3.1Zeo(+) vector gene, and zp-ic lc represents the ZP-IC light chain gene.

[0101] CMV-F:5'-TAATACGACTCACTATAGGG-3' (SEQ ID No. 7);

[0102] BGH-R: 5'-TAGAAGGCACAGTCGAGG-3' (SEQ ID No. 8).

[0103] The ZP-IC light chain expression vector pcDNA3.1 Zeo(+) / ZP-IC LC is a recombinant expression vector expressing the fusion protein ZP-IC light chain, obtained by replacing the fragment between the EcoRI recognition site and the EcoRV recognition site of the pcDNA3.1Zeo(+) vector with the ZP-IC light chain gene having the nucleotide sequence of SEQ ID No.4, while keeping the other nucleotide sequences of the pcDNA3.1Zeo(+) vector unchanged.

[0104] Colonies harboring the pcDNA3.1Neo(+) vector ligated with the heavy chain gene were selected as PCR templates. Colony PCR was performed using CMV-F / ZP-IC-HC-R as upstream and downstream primers. The PCR reaction conditions were as follows: initial denaturation at 94°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, and chain extension at 68°C for 60 seconds, for 30 cycles. Following each cycle, a single extension at 72°C for 5 minutes was performed. Positive clones were selected for gene sequencing. Plasmids were extracted from clones containing the correct sequence, yielding the eukaryotic expression vector pcDNA3.1 Neo(+) / ZP-IC HC expressing the ZP-IC heavy chain. EcoRI / EcoRV double enzyme digestion of this vector is shown in lane 1 of Figure 3. In Figure 3, pcDNA3.1 / Neomycin represents the pcDNA3.1 Neo(+) vector gene, and zp-ic hc represents the ZP-IC heavy chain gene.

[0105] CMV-F:5'-TAATACGACTCACTATAGGG-3' (SEQ ID No. 7);

[0106] ZPIC-HC-R: 5'-ACAACCCAGTGCGGCTGTGC-3' (SEQ ID No. 9).

[0107] The ZP-IC heavy chain expression vector pcDNA3.1 Neo(+) / ZP-IC HC is a recombinant expression vector expressing the fusion protein ZP-IC heavy chain, obtained by replacing the fragment between the EcoRI recognition site and the EcoRV recognition site of the pcDNA3.1Neo(+) vector with the ZP-IC heavy chain gene having the nucleotide sequence of SEQ ID No. 2, while keeping the other nucleotide sequences of the pcDNA3.1Neo(+) vector unchanged.

[0108] Co-transfection of pcDNA3.1Neo(+) / ZP-IC HC and pcDNA3.1 Zeo(+) / ZP-IC LC vectors into CHO-DG44 cells (Thermo Fisher Scientific) was performed using electrofection or lipofection according to the procedures described in the relevant operation manual. When electrofection is performed, the Nucleofector cell electroporator manufactured by Lonza and the described method can be used; when lipofection is performed, the transfection reagent Lipofectamine manufactured by Invitrogen can be used. TM 2000 or Lipofectamine TM 3000 and the method described above.

[0109] The transfected CHO-DG44 cells were inoculated into T-shaped square flasks and cultured in a serum-free medium with a clear chemical composition (such as CD FortiCHO produced by Gibco). TMAfter 24 hours of culture, the stably transfected cell lines were pressure-selected using serum-free medium containing 250-400 μg / mL bleomycin (ZeocinTM) and 500-700 μg / mL geneticin (G418 sulfate). The medium was changed every 3 days, and the cells were cultured continuously for 2-3 weeks using serum-free medium containing ZeocinTM and G418 to screen for stably transfected CHO cell colonies. Afterwards, these stable colonies were trypsinized and monoclonal cultured and screened in 96-well culture plates using the limiting dilution method. Monoclonal cell lines that highly expressed the target protein were selected by ELISA, passaged, and expanded. Stably transfected CHO engineered cell lines (recombinant cells) that highly expressed the ZP-IC recombinant protein were frozen using 10% DMSO cell freezing medium. The recombinant cells are cells expressing the fusion protein ZP-IC obtained by co-introducing the recombinant vectors pcDNA3.1 Neo(+) / ZP-IC HC and pcDNA3.1 Zeo(+) / ZP-IC LC into CHO-DG44 cells. The recombinant cells contain the ZP-IC heavy chain gene shown in SEQ ID No. 2 and the ZP-IC light chain gene shown in SEQ ID No. 4.

[0110] 2. Expression of fusion protein ZP-IC

[0111] Resuscitate the stably transfected CHO cell line that highly expresses the ZP-IC recombinant protein and subculture and expand it in a square flask using serum-free medium. The expanded CHO cell line was inoculated into a 2-liter shake flask, and the initial cell density was maintained at 0.2×10 6 ~0.6×10 6 cells / mL, the cell culture volume is 15% to 35% of the shake flask volume, and the cell culture flask is cultured in a shaker (speed 135 rpm, temperature 37°C, CO2 5%). Serum-free culture medium can be CD FortiCHO produced by Gibco TM Alternatively, use domestic serum-free culture media, such as Media C produced by Shanghai Duoning Biotechnology Co., Ltd. or OPM-CHO CD08 Medium produced by Shanghai Aopuma Biotechnology Co., Ltd. After 3-4 days of culture, subculture the cells at a ratio of 1 to 3 or 1 to 4. After 7 days of culture in a shake flask, collect the culture supernatant and purify the fusion protein ZP-IC.

[0112] 3. Purification of fusion protein ZP-IC

[0113] The antibody protein with the Fc domain was purified from the culture supernatant using a Protein A affinity chromatography column (GE Mabselect SURE). The culture supernatant was first clarified by filtration through a 0.45 μM filter membrane and then loaded onto a Protein A affinity chromatography column (GE Mabselect SURE). The specific procedure followed the recommended protocol for GE Protein A affinity chromatography medium. The purification instrument used was a Zhongke Senhui Protein Purification System PPS-HD-100. The loading pH was controlled at neutral (pH 7.0), and the elution pH was controlled at pH 3.0. The purified fusion protein ZP-IC was quantified using a Nanodrop instrument. The molecular weight and purity of the purified recombinant fusion protein were determined by polyacrylamide gel electrophoresis under reducing conditions. SDS-PAGE results showed that the heavy chain molecular weight of the recombinant protein was approximately 80 kDa, and the light chain molecular weight was approximately 25 kDa (Figure 4), both consistent with the designed target molecular weights.

[0114] Example 3: Reaction of ZP-IC with anti-type I interferon-specific antibodies

[0115] The affinity-purified fusion protein ZP-IC was first subjected to SDS-PAGE electrophoresis. A positive control (recombinant interferon IFN-Fc; for the sequence of IFN-Fc, see invention patent: ZL 202310085137.0, which carries the same interferon gene as ZP-IC, i.e., integrated interferon α) and a negative control (IgG, Shanxi Kangbao Biological Products Co., Ltd.) were set up. Two replicates of the same sample sequence were spotted. After electrophoresis, the PAGE gel was cut in half, one for stained and the other for Western blot. Semi-dry transfer was performed using a PVDF membrane (immobilon-P PVDF Membrane Catalog No.: IPVH00005, Millipore). After transfer, the membrane was washed three times with PBST solution and placed in a PBST solution containing 5% skim milk powder and blocked overnight at 4°C. Subsequently, the membrane was washed three times with PBST solution. Simultaneously, PBST containing 1% skim milk powder was prepared as the antibody incubation solution. Add 1:1000 diluted rabbit anti-interferon polyclonal antibody (Anti-Interferon alpha 2antibody, catalog number: ab193055, abcam) to the antibody incubation solution, and incubate with the membrane at 37°C for 1 hour with shaking. Afterwards, wash three times with PBST solution, each time for no less than 3 minutes. Add 1:1500 diluted goat anti-rabbit IgG (HRP labeled, Zhongshan Jinqiao, catalog number ZB2301) to the antibody incubation solution, and incubate with the membrane at 37°C for 1 hour with shaking. Afterwards, wash three times with PBST solution, each time for no less than 3 minutes. Using Enlight TMWestern blotting substrate (Cat. No. 29050, Engreen) was used for color development, and images were taken using a chemiluminescence analyzer (Tanon 5200multi). The Western blot results show that ZP-IC can be visualized at a specific molecular weight (80 kD), which corresponds exactly to the size of the heavy chain of the molecule (Figure 5). This indicates that the ZP-IC molecule carries the interferon molecule on its heavy chain.

[0116] Example 4: ELISA to detect the binding affinity between ZP-IC and HIV gp41 glycoprotein

[0117] Recombinant HIV gp41 protein (purchased from abcam, catalog number ab49070) was diluted to 1.0 μg / mL with coating buffer and coated onto an ELISA plate at 100 ng / well overnight at 4°C. Each well was then blocked for 1.5-2 hours with PBST containing 2% BSA. During the incubation period, the blocking buffer was diluted with PBST to a final BSA concentration of 0.5% BSA. This was used as the antibody diluent, and the antibody fusion protein ZP-IC was serially diluted from 10 μg / mL to 0.15 ng / mL. Human IgG (Shanxi Kangbao Biological Products Co., Ltd.) was used as control 1, and the antibody ZP-ab, which targets the same target (ZP-ab heavy chain amino acid sequence is SEQ ID No. 5, ZP-ab heavy chain nucleotide sequence is SEQ ID No. 6, ZP-ab light chain amino acid sequence is the same as that of the ZP-IC light chain amino acid sequence, and ZP-ab light chain nucleic acid sequence is the same as that of the ZP-IC light chain nucleic acid sequence) was used as control 2. After the ELISA plate is blocked, wash it thoroughly. Add serially diluted antibodies, 100 μL / well, incubate at 37°C for 1 hour, wash thoroughly, then add 100 μL of 1:2500 diluted goat anti-human IgG / horseradish enzyme label (Zhongshan Jinqiao, product number ZB2304), incubate at 37°C for 1 hour. After washing thoroughly, use TMB solution to develop color for 15 minutes, stop with 2M HCl, and read the absorbance at 450 nm. The measurement results were analyzed using GraphPad Prism Software 6.0, and nonlinear regression analysis was selected to calculate the half-maximal binding effect concentration (EC 50 ). The data analysis results showed that the half-maximal binding concentration EC of the fusion protein ZP-IC and the recombinant protein gp41 50 =74 ng / mL (about 0.37 nM), and ZP-ab (EC 50 =60.7 ng / mL) affinity remained consistent (Figure 6); the affinity EC of human immunoglobulin IgG to recombinant protein gp41 50 The concentration of ZP-IC is approximately 948 ng / mL, indicating that ZP-IC can specifically bind to the HIV viral outer membrane protein gp41 subunit.

[0118] Example 5: ELISA to detect the binding affinity between ZP-IC and MPER polypeptide

[0119] The MPER polypeptide is located in the gp41 membrane-proximal extracellular region. The MPER polypeptide sequence reference (Huang JH, et al. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature. 2012, 491(7424):406-412. doi:10.1038 / nature11544.) is: NEQELLELDKWASL WNWFDITNWLWYIR (positions 656-683, a total of 28 aa, synthesized by GenScript Biotech Co., Ltd.). Because the gp41 subunit is responsible for membrane fusion, all functional regions related to membrane fusion are highly conserved, including the FP, MPER, and heptad repeats (NHRs). Therefore, antibodies targeting the MPER region of the gp41 subunit have a broader spectrum of activity.

[0120] MPER peptide was dissolved in an alkaline solution and diluted to 1.5 μg / mL with PBS. 100 μL / well was added to the ELISA plate for antigen coating overnight at 4°C. Each well was then blocked for 1.5-2 hours with PBST containing 2% BSA. During this incubation period, the blocking solution was diluted with PBST to a final BSA concentration of 0.5% BSA. This was used as the antibody diluent, and serial dilutions of the antibody fusion protein ZP-IC were made, starting at 500 μg / mL and ending at less than 0.25 μg / mL. Human IgG (Shanxi Kangbao Biological Products Co., Ltd.) was used as a control. After blocking, the plate was washed thoroughly. 100 μL / well of the serially diluted antibody solution was added and incubated at 37°C for 1 hour. After thorough washing, 100 μL of a 1:2500 dilution of goat anti-human IgG / horseradish enzyme conjugate (Zhongshan Jinqiao, Cat. No. ZB2304) was added and incubated at 37°C for 1 hour. After thorough washing, the cells were developed with TMB solution for 15 min, terminated with 2 M HCl, and the absorbance was read at 450 nm. The results were analyzed using GraphPad Prism Software 6.0, and nonlinear regression analysis was used to calculate the half-maximal binding effect concentration (EC 50(See Figure 7). Software calculations showed that the half-maximal binding concentration of ZP-IC was 7.2 μg / mL, approximately 40 nM; in contrast, the half-maximal binding concentration of the control IgG antibody was >500 μg / mL (i.e., >3.3 μM). This indicates that ZP-IC specifically binds to the MPER peptide on HIV gp41, exhibiting broad-spectrum inhibition against the vast majority of HIV strains and minimizing viral immune escape.

[0121] Example 6: ELISA to detect the binding affinity between ZP-IC and interferon receptor (IFNR2)

[0122] Recombinant interferon receptor 2 (Human IFN-alpha / beta R2 Protein, His Tag, Catalog No.: IF2-H5224, purchased from ACROBiosystems) was diluted to 1.0 ug / mL with coating solution and coated onto an ELISA plate at 100 ng / well at 4°C overnight. A PBST solution containing 2% BSA was then added to each well for blocking for 1.5-2 hours. During the incubation period, the blocking solution was diluted with PBST to a final BSA concentration of 0.5% BSA. This was used as the antibody diluent, and the antibody fusion protein ZP-IC was serially diluted 3-fold, starting from 90 μg / mL to 1.5 ng / mL. Human IgG (Shanxi Kangbao Biological Products Co., Ltd.) was used as a control. After the ELISA plate was blocked, it was washed thoroughly. Add the diluted sample to be tested, 100 μL / well, incubate at 37°C for 1 hour, wash thoroughly, and then add 100 μL of 1:2500 diluted goat anti-human IgG / horseradish enzyme label (Zhongshan Jinqiao, product number ZB2304) and incubate at 37°C for 1 hour. After thorough washing, use TMB solution to develop color for 15 minutes, 2M HCl to stop, and read the absorbance value at 450nm. The measurement results were analyzed using GraphPad Prism Software 6.0 software, and nonlinear regression analysis was selected to calculate the half-maximal binding effect concentration (EC 50 ). The data analysis results showed that the half-maximal binding concentration of the fusion protein ZP-IC and the recombinant human interferon receptor 2 was EC 50 =140 ng / mL (about 0.7 nM) ( FIG8 ), indicating that ZP-IC can specifically bind to the interferon receptor with high affinity (<1 nM), thereby activating the interferon signaling pathway.

[0123] Example 7: Antiviral activity test of ZP-IC

[0124] Test cell line: TZM-bl cells, derived from the HeLa cell line JC.53, stably express high levels of CD4 and CCR5, endogenously express CXCR4, and have been engineered to incorporate luciferase and Escherichia coli β-galactosidase under the control of the HIV-1 promoter. The TZM-bl cell line is highly sensitive to infection with different HIV-1 strains and is suitable for quantifying neutralizing antibody potency. This cell line was gifted by Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme through the AIDS Reagent Program of the National Institutes of Health and is currently maintained at the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences.

[0125] The test HIV virus strain was obtained from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. The information is shown in Table 1:

[0126] Table 1. Tested HIV strains

[0127] The antiviral activity test steps are as follows: the antibody fusion protein ZP-IC to be tested (or the control antibody drug ZP-ab, HIV-DNS) is diluted 3 times in a 96-well cell culture plate, 50 μL / well, with 3 replicates and 9 gradients; 50 μL of recombinant virus (100 TCID 50 ) (see the test virus strains listed in Table 1) were added to the culture wells and placed at 37°C for 60 min; then 1×10 4 TZM-bl cells. After adding DEAE-dextran at a final concentration of 15 μg / mL, incubate in a cell culture incubator at 37°C and 5% CO2 for about 48 hours. After carefully discarding the culture medium, use 30 μL of cell lysis solution to lyse the cells at room temperature for 15 minutes per well, then add 100 μL / well Luciferaese Assay reagent, mix thoroughly, and transfer 100 μL to a 96-well white plate. Use a chemiluminescence detector to measure RLU (relative luminescence unit), and use GraphPad Prism Software to calculate the IC of the drug using the S-shaped dose-response formula in nonlinear regression analysis. 50 Calculation method: half effective inhibitory concentration (IC 50 ) refers to the dose of the drug that can cause 50% of the maximum effect. 50 When all drug concentrations were converted to log 10(concentration value), the sample fluorescence inhibition rate was calculated for each sample well at each concentration, and the calculation formula was: inhibition rate = [1-(RLU value of the drug-added well-RLU value of the negative control well) / (RLU mean of the positive control well-RLU mean of the negative control well)] × 100%. Data analysis was performed using GraphPad Prism Software using the sigmoidal dose-response formula in nonlinear regression analysis to analyze the IC of each drug. 50 The half effective inhibitory concentration (IC50) of drug ZP-IC on HIV virus strain TRO11 and other cells infected with the virus was 100%. 50 ) The measurement results (μg / mL) are shown in Table 2. The half effective inhibitory concentration (IC 50 ) The measurement results (μg / mL) are shown in Table 3.

[0128] Table 2. ZP-IC half-effective inhibitory concentration (IC50) of HIV virus infection in cells 50 )

[0129] Table 3. The half effective inhibitory concentration (IC) of ZP-IC on HIV virus infection in cells 50 )

[0130] From the above test data, it can be seen that the 8 different types of HIV epidemic virus strains tested all maintain good sensitivity to the candidate drug ZP-IC. 50 Except for 0.2μg / mL, the half inhibition concentration of the other tested strains was at the level of 0.01-0.06μg / mL. The antibody drug ZP-ab with the same target has a half inhibition concentration IC of 0.01-0.06μg / mL for HIV strain X1632. 50 Above 33 μg / mL, the half-maximal inhibitory concentration IC of the other three test virus strains (CNE8, CE70301, BJOX002000) 50 The dosage range is also between 3 and 16 μg / mL, and its antiviral activity is more than 200-fold lower than that of the fusion protein ZP-IC. This indicates that ZP-ab, through its targeted blocking effect alone, can only achieve antiviral effects at the microgram level. However, through the design of the fusion protein of the present invention, namely targeted interferon, ZP-IC has significantly improved its antiviral ability compared to the ZP-ab antibody, with an efficacy increase of at least two orders of magnitude. Therefore, in actual dosage, the dosage of ZP-IC can be reduced by two orders of magnitude compared to the antibody ZP-ab with the same target, while still achieving the same antiviral effect.

[0131] HIV-DNS is a bispecific antibody targeting gp120 (see Chinese patent: ZL 202210196757.7, "Multispecific antibodies targeting HIV gp120 protein and human CD3 molecule and their applications"). In this test, the ability of HIV-DNS to inhibit different HIV strains was tested in parallel. From several sets of data, the half-maximal inhibitory concentration IC of HIV strain X1632 was 2.37, which was 1.37. 50 The IC of ZP-IC was 0.166 μg / mL, which was similar to that of ZP-IC. 50 The half-maximal inhibitory concentration IC of the other three test virus strains (CNE8, CE70301, BJOX002000) is close. 50 The inhibitory activity was between 0.3-0.5 μg / mL, which was not as high as that of ZP-IC (IC 50 The ZP-IC concentration ranged from 0.02 to 0.06 μg / mL. This indicates that, by targeting interferon, ZP-IC exhibits nearly an order of magnitude higher in vitro anti-HIV activity than HIV-DNS. This also demonstrates that the fusion protein ZP-IC has a very strong antiviral effect.

[0132] In this cellular antiviral experiment, we also submitted recombinant long-acting interferon (IFN-Fc, see invention patent: ZL 202310085137.0 for sequence) for activity testing. Unfortunately, in several preliminary experiments, the test party did not detect the in vitro antiviral activity of IFN-Fc. Therefore, in the formal experiment, the antiviral test of IFN-Fc was not performed. This shows that interferon alone cannot play a role in directional guidance and activation of cellular antiviral effects.

[0133] We use the following data to establish a data connection between the antiviral activity of the fusion protein ZP-IC and IFN-Fc at the cellular level. In the invention patent: ZL 202310085137.0, the antiviral activity of HIV-DNS and recombinant long-acting interferon (IFN-Fc) were compared in parallel. Multiple sets of data showed that the in vitro antiviral activity of IFN-Fc was one order of magnitude lower than that of HIV-DNS (Supplementary data: IFN-Fc and HIV-DNS against HIV strain HIV-1 ⅢB Inhibitory capacity of IC for replication in TZM-bl cells 50 The inhibitory effects of IFN-Fc and HIV-DNS on HIV-1 were 52.17±15.37nM and 4.55±4.10nM, respectively. IIIB Inhibitory effect of IC on replication in C8166 cells 50The results were 25.02±8.15nM and 1.63±0.71nM, respectively. These data indicate that the in vitro antiviral activity of ZP-IC > HIV-DNS > IFN-Fc. This indicates that the in vitro antiviral activity of ZP-IC, which targets interferon molecules, is much higher than that of IFN-Fc in cells.

[0134] The above data and analysis demonstrate that the design of targeted interferon selectively enriches interferon molecules around the virus, enhancing the antiviral signal and achieving a superior antiviral effect. Its antiviral effect is at least 1-2 orders of magnitude stronger than the monoclonal antibody ZP-ab and the long-acting interferon molecule IFN-Fc with the same target. Therefore, the targeted interferon molecule (ZP-IC) has extremely high anti-HIV activity.

[0135] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A fusion protein, characterized in that The fusion protein is formed by connecting the 10E8 antibody or its antigen-binding fragment with integrated interferon α via a linker; The amino acid sequence of the heavy chain of the fusion protein is SEQ ID No.1, and the amino acid sequence of the light chain of the fusion protein is SEQ ID No.

3.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecules are a DNA molecule whose coding sequence is SEQ ID No.2 and a DNA molecule whose coding sequence is SEQ ID No.

4.

4. Biomaterial, characterized in that The biological material is any of the following: A1) an expression cassette containing the nucleic acid molecule according to claim 2; A2) a recombinant vector containing the nucleic acid molecule according to claim 2; A3) a recombinant yeast or Escherichia coli containing the nucleic acid molecule of claim 2; A4) A recombinant animal cell containing the nucleic acid molecule of claim 2.

5. Any of the following uses of the fusion protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the biomaterial according to claim 4: B1) Use in the preparation of products for preventing or treating HIV infection; B2) Use in the preparation of products for the prevention or treatment of AIDS; B3) Use in the preparation of products for inhibiting HIV virus activity; B4) Use in the preparation of a product for binding or detecting HIV gp41 protein; B5) Use in the preparation of a product for binding or detecting HIV MPER polypeptide.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fusion protein of claim 1 and one or more pharmaceutically acceptable carriers.

7. A reagent or kit for binding or detecting HIV gp41 protein or HIV MPER polypeptide, characterized in that: The reagent or kit comprises the fusion protein according to claim 1.

8. The method for preparing the fusion protein according to claim 1, characterized in that: The method comprises: constructing recombinant expression vectors containing the heavy chain and light chain genes of the fusion protein respectively; introducing the recombinant expression vectors into host cells to obtain recombinant cells expressing the fusion protein; culturing the recombinant cells and obtaining the fusion protein through separation and purification.

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