CN117430716B - Recombinant interferon drug targeting conserved HIV gp41 subunit near-membrane-end outer region and application thereof - Google Patents

Abstract

The invention discloses a recombinant interferon drug targeting a conserved HIV gp41 subunit near-membrane-end outer region and application thereof. Specifically disclosed is a targeting interferon fusion protein ZP-IC comprising a 10E8 antibody or antigen binding fragment thereof, and an integrated interferon alpha, wherein the 10E8 antibody or antigen binding fragment thereof is linked to the integrated interferon alpha through a linker. The invention uses the antibody 10E8 which can identify the gp41 subunit MPER region with high conservation of HIV outer membrane glycoprotein as a directional tool of integrated interferon, can accurately identify the target spot exposed by the outer membrane protein allosteric in infected cells of virus, prevent virus infection, play a neutralizing role, and simultaneously, the carried interferon molecule can be more effectively combined with interferon receptors on cells around the HIV virus to play an antiviral function. Experiments prove that ZP-IC has excellent antiviral activity and has the capability of broad-spectrum inhibition of most HIV strains.

Description

Recombinant interferon drug targeting conserved HIV gp41 subunit near-membrane-end outer region and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a recombinant interferon drug targeting a conserved HIV gp41 subunit near-membrane-end outer region and application thereof.

Background

AIDS is a very dangerous infectious disease caused by infection with human immunodeficiency virus (Human Immunodeficiency Virus, HIV). HIV can destroy the immune system of human body, and uses important CD4 ⁺ T lymphocyte in the immune system of human body as main target for attack to destroy the cell in great amount, so that the immune function of human body is lost. Therefore, the human body is easy to infect various diseases, malignant tumors can occur, and the death rate is high. When the human immunodeficiency virus invades CD4 ⁺ T cells, the CD4 molecules on the cell surface are used as main receptors, and CCR5 or CXCR4 is used as auxiliary receptors. In the first step of infection, the virus approaches the cell by combining the outer membrane protein gp120 with the receptor molecule CD4 on the CD4+ T cell, further combining the gp120 with the auxiliary receptor CCR5 or CXCR4 embedded on the host cell membrane, shortening the distance between the virus and the cell, triggering the allosteric of the outer membrane protein molecule gp120 of the virus, exposing the other membrane protein subunit gp41, and then folding the HR1 and HR2 domains in the gp41 subunits to form a six-helix bundle (subunits are all trimers), finally infinitely pulling the virus and the host cell, fusing the outer membrane of the virus and the host cell membrane, and facilitating the invasion of virus RNA into the CD4+ T host cell.

The HIV antiviral drugs include nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase chain transfer inhibitors, binding and membrane fusion inhibitors 5 major classes. HIV-infected individuals need to take drugs for life to control viral load. The long-term toxicity and virus resistance of the drug which is obvious from the long-term administration of the chemical drug can lead to reduced patient compliance. There is a need to find more effective and safer therapeutic agents, such as antibodies or recombinant fusion protein drugs. They do not enter the cell, typically act extracellularly, and control infection by neutralizing the virus, etc., to prevent it from invading the cell.

Therapeutic neutralizing antibodies to HIV target HIV outer membrane glycoproteins. The only antigen outer membrane glycoprotein on the surface of HIV virus particles is gp160, the trimer (gp 160) ₃, which can be cleaved into (gp 120/gp 41) ₃. Many dominant epitopes on HIV outer membrane proteins are highly variable, resulting in poor binding of antibodies produced by the infected person themselves. However, there are also several recognized relatively conserved sites on HIV outer membrane proteins that become recognition epitopes for broadly neutralizing antibodies (broadly neutralizing antibodies, bNAb). One of them is the CD4 binding site on gp120 subunit responsible for binding to the human T cell differentiation antigen CD4 receptor molecule; also a domain on the gp41 subunit near the membrane region, commonly referred to as the "membrane proximal outer region" (MPER), which is functionally associated with membrane fusion .(Huang JH,et al.Broad and potent neutralization of HIV-1by a gp41-specific human antibody.Nature.2012,491(7424):406–412.doi:10.1038/nature11544.).

The HIV gp41 subunit has a molecular weight of about 41kDa, and its main domain is, in order from the N-terminus, a Fusion Peptide (FP), an α -helical heptad repeat domain HR1 (or NHR), an α -helical heptad repeat domain HR2 (or CHR), a near-membrane end outer region (MPER), a transmembrane region (TM). When the task of searching for target cells and receptors is completed, the outer gp120 subunit will be allosterically exposed to the hidden gp41 subunit. The N-terminal fusion peptide FP of the gp41 subunit translocates and intercalates into the host cell membrane, while the C-terminal transmembrane region remains anchored to the viral membrane, rendering the gp41 subunit in a transiently extended conformation, known as the "pre-HAIRPIN INTERMEDIATE intermediate". In this state, either the NHR, CHR or MPER domains are exposed, facilitating recognition of membrane fusion inhibitors or related antibodies.

Many polypeptide membrane fusion inhibitors are designed by simulating the sequence structure of CHR, and are competitively combined on NHR, so that the folding combination of NHR and CHR is prevented, the membrane fusion link of viruses is destroyed, and the infection of viruses is prevented. Such fusion polypeptides include HIV fusion inhibitor drug T20 (enfuvirtide) marketed in 2003, ai Kening (albuvirtide), T1249, T2635 and other upgrading and upgrading polypeptide candidate drugs (Eggink D,et al.Detailed Mechanistic Insights into HIV-1Sensitivity to Three Generations of Fusion Inhibitors.J Virol.2011,85(20):10785-97.doi:10.1128/JVI.05331-11.), and LP51, LP98 and other polypeptide-lipid candidate drugs marketed in 2018 (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.).

Some antibodies target the MPER domains of gp41, such as 2F5,4E10, Z13E1, 10E8, etc., by attacking the "pro-hairpin intermediates" of gp41, preventing the folding of the NHR and CHR domains to form a six-helix bundle. Since the mpr domain of gp41 is always masked by the gp120 subunit of the naturally occurring viral particle, this class of antibodies is characterized by a very high affinity for the "pre-hairpin intermediate" gp41, which is present before fusion, while exhibiting very low affinity for the trimeric HIV membrane proteins (gp 160, which is composed of gp120 and gp 41). Of the antibodies to the MPER domain, 10E8 was most widely studied. The literature reports that 10E8 targets the MPER region on HIV gp41 subunit, with key binding sites including the MPER regions Trp672, phe673, trp676, and Tyr681, ile682, arg683 at its C-terminus. More detailed studies have employed X-ray crystal structure analysis of the 10E8 Fab fragment with the substrate peptide, indicating that the MPER region of gp41 consists of two helices, whereas 10E8 recognizes primarily the second helix starting at position 671, which comprises not only the C-terminus of the MPER region, but also several amino acids extending the N-terminus of the transmembrane region (TMD). The peptide structural basis providing antibody recognition extends at least to position 690 of TMD. Structural analysis showed that the tip of the 3 rd complementarity determining region (CDRH 3) on the heavy chain of the 10E8 antibody could be immersed in the lipid bilayer to bind to the N-terminal helix of TMD, thus locking the binding (Rujas E,et al.Neutralization of HIV-1through the molecular recognition of 10E8 helical epitope at the membrane interface.Sci Rep 6,38177(2016).https://doi.org/10.1038/srep38177.). of the antibody to the MPER helix, as determined in literature, the affinity (dissociation equilibrium constant) of 10E8 to the pro-hairpin intermediate of gp41 (TMD-containing) was about 1.85nM, the affinity to the full-length peptide of the MPER region (656-683) was 17nM, and the binding to gp140 with the hidden MPER region was very 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-1by a gp41-specific human antibody.Nature.2012,491(7424):406–412.doi:10.1038/nature11544.).

10E8 is used for neutralizing pseudoviruses constructed based on HIV outer membrane proteins with different sequences, and if IC ₅₀ is lower than 50 mug/ml (about 330 nM) is used as a judgment standard, the monoclonal antibody can neutralize 98% of 181 test pseudoviruses; if IC ₅₀ is used as a criterion for less than 1 μg/ml (about 6.6 nM), the mab neutralizes 72% of these test viruses, much more than 4E10 mab whose binding site is also the MPER region (the latter has a neutralization coverage of only 37%). In addition, although the binding site of 10E8 is very close to the viral envelope, the binding capacity of the binding site to the phospholipid bilayer is weak, which indicates that the binding site has good safety (Huang JH,et al.Broad and potent neutralization of HIV-1by a gp41-specific human antibody.Nature.2012,491(7424):406–412.doi:10.1038/nature11 544.)(Chen J,et al.Mechanism of HIV-1neutralization by antibodies targeting a membrane-proximal region of gp41.J Virol.2014,88(2):1249-58.doi:10.1128/JVI.02664-13.)., and the two antibodies of 2F5 and 4E10 have strong reaction with the lipid membrane of cells, so that the binding site is not suitable for subsequent development. Thus, 10E8 is an antibody with broadly neutralizing activity and is safer than other antibodies with co-targets.

Interferon (IFN) is a group of active proteins (mainly glycoproteins) with multiple functions, which have broad-spectrum antiviral, antitumor and immunoregulatory effects, and are biologically a cytokine. Interferon can be classified into type I and type II interferons according to factors such as its production cell, receptor, and biological activity, etc. as a standard. The type I interferon (IFN-alpha, IFN-beta and IFN-omega) has the effects of inhibiting virus replication, resisting parasites, inhibiting proliferation of various cells, stimulating the killing activity of immune cells, participating in immune regulation, resisting tumors and the like; the main biological activity of type II (IFN-gamma only) is activation T cells, NK cells and the like, and plays an immunoregulatory role. All type I interferon molecules bind to the same receptor, i.e. a heterodimer consisting of two subunits IFNAR1 and IFNAR 2. After binding to its receptor, interferon activates downstream non-receptor tyrosine kinase families Jak1 and Tyk2, further recruits and activates signaling and transcriptional activator proteins STAT1 and STAT2, and forms a complex with interferon regulatory factor 9 (IRF 9) ISGF3 (IFN-stimulated gene factor 3), thereby activating expression of a series of interferon-stimulated genes (ISGs) that induce these downstream genes to exert antiviral, antitumor and immunomodulatory functions. The function of interferon includes the following two aspects: firstly, through the action of related receptors on surrounding uninfected cells, the cells are stimulated to synthesize various effector proteins, such as antiviral protein tetherin, APOBEC1 and protein kinase R, so as to play an antiviral role; secondly, the immunoregulation and anti-tumor effects are exerted by enhancing the activity of lymphocytes, macrophages and natural killer cells.

Type I interferon plays an important role in the treatment of infection with hepatitis b virus (HEPATITIS B VIRUS), hepatitis c virus (HEPATITIS C virus, HCV), and there are many types of recombinant type I recombinant interferons marketed, such as perhexn of rochan, pelargonic of moxadong, domestic transferrin, sirojin, andefen, etc. However, in the field of anti-HIV infection, the therapeutic or adjuvant therapeutic effect of type I interferon is not very obvious. The learner believes that type I interferon plays a dual role in the progression of HIV infection: in the acute phase of HIV infection, interferons exert antiviral effects by stimulating effector proteins and stimulating the immune system; however, during the chronic infection phase, continued exposure of interferon leads to activation of the body's desensitization and adverse immune response ([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 twofold. The ideas of biochemistry and biophysical progression Progress in Biochemistry and Biophysics.2018,45(9):966-970.doi:10.16476/j.pibb.2018.0191.[3]Sugawara S,et al.People with HIV-1demonstrate type 1interferon refractoriness associated with upregulated USP18.J Virol.2021,95(10):e01777-20.doi:10.1128/JVI.01777-20.). undoubtedly make the application prospect of interferon in the field of HIV treatment unclear and gradually vanish from the list of drug candidates for HIV infection treatment.

Human type I interferon comprises 12 IFN-alpha subtypes, 1 IFN-beta and 1 IFN-omega, antiviral activity of each interferon is greatly different (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 study item with 20 clinical cases, and polyethylene glycol modified interferon alpha-2 a single drug treatment can inhibit replication of HIV-1 virus and reduce integration (Azzoni L,et al.Pegylated Interferon alfa-2a monotherapy results in suppression of HIV type 1replication and decreased cell-associated HIV DNA integration.J Infect Dis.2013,207(2):213-222.doi:10.1093/infdis/jis663.). of HIV DNA in cells. This is due mainly to the fact that the different interferon protein subtypes differ in sequence, which results in tens or hundreds of times different affinities for the consensus receptors IFNAR1 and IFNAR2, and thus the magnitude of the downstream signaling and antiviral capacity that is triggered varies. Researchers compare the anti-HIV capability of different molecules of alpha interferon in type I, find that the activities of the molecules such as IFN alpha 8, IFN alpha 6, IFN alpha 14 are higher, and the anti-HIV capability of common IFN alpha 1, IFN alpha 2 is weaker ([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-1Infection In Vivo.J Virol.2016,90(13):6001-6013.doi:10.1128/JVI.00451-16.)., so that the drug-forming research of the type I interferon in the HIV anti-infection field is still in the primary stage, and continuous exploration and improvement are required.

Disclosure of Invention

The invention aims to provide an HIV infection treatment drug with better anti-HIV virus capability. The technical problems to be solved are not limited to the technical subject matter described herein, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To achieve the above object, the present invention provides, first, a fusion protein named ZP-IC comprising a 10E8 antibody or antigen-binding fragment thereof, and an interferon- α, to which the 10E8 antibody or antigen-binding fragment thereof is linked through a linker.

Further, the integrated interferon alpha may be fused to the C-terminus of the 10E8 antibody or antigen binding fragment thereof via the linker.

Further, the amino acid sequence of the integrated interferon alpha may be positions 483-648 of SEQ ID No. 1.

Further, the linker (linker) may be a flexible peptide linker, such as a peptide linker comprising glycine and/or serine residues.

Further, the joint includes, but is not limited to GSGSGS, GGGSGGGS, GGGSGGG, GGSSGG, GSGSGSG, GSGSG, GGGGS.

Further, the linker is GSGSGS (positions 477-482 of SEQ ID No. 1).

Further, 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.

Wherein, the 1 st-476 th site of SEQ ID No.1 is the amino acid sequence of the heavy chain of the 10E8 antibody targeting HIV gp41 protein, the 477 th-482 th site of SEQ ID No.1 is the amino acid sequence of a linker (linker), and the 483 rd-648 th site of SEQ ID No.1 is the amino acid sequence of an integrated interferon alpha.

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

The fusion protein ZP-IC is also a recombinant interferon targeting the conserved HIV gp41 subunit near-membrane-end outer region.

The invention also provides nucleic acid molecules that encode any of the fusion proteins described herein.

Further, the nucleic acid molecule may comprise 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.

Further, the nucleic acid molecule may be a DNA molecule having the coding sequence SEQ ID No.2 and/or a DNA molecule having the coding sequence SEQ ID No. 4.

Wherein, the 1 st to 1428 th positions of SEQ ID No.2 are nucleotide sequences of heavy chains of 10E8 antibodies targeting HIV gp41 proteins, the 1429 th to 1446 th positions of SEQ ID No.2 are nucleotide sequences of a linker (linker), and the 1447 th to 1950 th positions of SEQ ID No.2 are nucleotide sequences of an integrated interferon alpha.

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

The nucleic acid molecules may also include nucleic acid molecules which have been modified by codon preference on the basis of the nucleotide sequences indicated in SEQ ID No.2 and/or SEQ ID No. 4.

The present invention also provides a biomaterial which may be any one of the following:

a1 An expression cassette comprising said nucleic acid molecule;

a2 A recombinant vector comprising said nucleic acid molecule, or a recombinant vector comprising said expression cassette of A1);

a3 A recombinant microorganism comprising said nucleic acid molecule, or a recombinant microorganism comprising said expression cassette of A1), or a recombinant microorganism comprising said recombinant vector of A2);

A4 A recombinant cell comprising said nucleic acid molecule, or a recombinant cell comprising said expression cassette of A1), or a recombinant cell comprising said recombinant vector of A2).

Wherein the recombinant microorganism of A3) and the recombinant cell of A4) express the fusion protein ZP-IC.

The vectors described herein refer to vectors capable of carrying exogenous DNA or genes of interest into host cells for amplification and expression, and may be cloning vectors or expression vectors, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), viral vectors (e.g., baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (e.g., herpes simplex viruses), etc. In one or more embodiments of the invention, the vector is a pcdna3.1neo (+) vector and/or a pcdna3.1zeo (+) vector.

The microorganism described herein may be a yeast, bacterium, algae or fungus. Among them, bacteria may be derived from Escherichia (Escherichia sp.), erwinia (Erwinia sp.), agrobacterium (Agrobacterium sp.), flavobacterium (Flavobacterium sp.), alcaligenes (Alcaligenes sp.), pseudomonas (Pseudomonas sp.), bacillus (Bacillus sp.), etc., but are not limited thereto. In one or more embodiments of the invention, the microorganism is E.coli Trans10.

The cells (host cells) described herein refer to cells that can be used to introduce vectors, including but not limited to: eukaryotic cells (e.g., yeast cells, aspergillus), animal cells (e.g., mammalian cells, insect cells), plant cells, or prokaryotic cells (e.g., escherichia coli or bacillus subtilis). In one or more embodiments of the invention, the cell is a CHO cell (CHO-DG 44 cell).

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.

The pcDNA3.1Zeo (+)/ZP-IC LC is a recombinant expression vector which replaces a fragment between an EcoRI recognition site and an EcoRV recognition site of the pcDNA3.1Zeo (+) vector with a ZP-IC light chain gene with a nucleotide sequence of SEQ ID No.4, and keeps other nucleotide sequences of the pcDNA3.1Zeo (+) vector unchanged, thus obtaining the expression fusion protein ZP-IC light chain.

The pcDNA3.1Neo (+)/ZP-IC HC is a recombinant expression vector which replaces a fragment between an EcoRI recognition site and an EcoRV recognition site of the pcDNA3.1Neo (+) vector with a ZP-IC heavy chain gene with a nucleotide sequence of SEQ ID No.2, and keeps other nucleotide sequences of the pcDNA3.1Neo (+) vector unchanged, thus obtaining the expression fusion protein ZP-IC heavy chain.

The recombinant microorganism described herein includes a recombinant bacterium obtained by introducing a heavy chain gene (SEQ ID No. 2) and/or a light chain gene (SEQ ID No. 4) of the fusion protein into E.coli.

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.

The invention also provides any one of the following uses of the fusion protein, the nucleic acid molecule or the biological material described herein:

b1 Use of a composition for the preparation of a product for the prevention or treatment of HIV infection;

B2 The use of said composition for the preparation of a product for the prevention or treatment of AIDS;

B3 Use of a composition for inhibiting HIV viral activity;

B4 Use in the preparation of a product for binding or detecting gp41 protein;

B5 For the preparation of a product for binding or detecting an MPER polypeptide.

The product comprises an agent, a kit, a medicament or a pharmaceutical composition.

The invention also provides pharmaceutical compositions that may comprise any of the fusion proteins described herein together with one or more pharmaceutically acceptable carriers.

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

Wherein the pharmaceutical composition has a neutralizing antiviral effect that inhibits or neutralizes HIV activity. The pharmaceutical composition is used for improving, preventing or treating HIV infection or AIDS.

The invention also provides a reagent or kit for binding or detecting a gp41 protein or an MPER polypeptide, the reagent or kit comprising any of the fusion proteins described herein.

The invention also provides a method of preparing a fusion protein as described herein, which may comprise: respectively constructing recombinant expression vectors containing heavy chain genes and light chain genes of the fusion protein; introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the fusion protein; culturing the recombinant cells, and separating and purifying to obtain the fusion protein.

Further, the nucleotide sequence of the fusion protein heavy chain gene may be SEQ ID No.2.

Further, the nucleotide sequence of the fusion protein light chain gene may be SEQ ID No.4.

The fusion proteins of the invention may be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the fusion proteins of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors, then host cells are transfected, the transfected host cells are cultured under specific conditions, and the fusion proteins of the invention are expressed. The person skilled in the art can select host cells, expression vectors, methods for introducing the expression vectors into host cells and methods for isolating and purifying fusion proteins, which are conventional in the art, as required.

The invention provides a targeting interferon drug (namely the fusion protein ZP-IC) for targeting HIV (human immunodeficiency virus) outer membrane protein gp41 and application thereof. The medicine is a recombinant expression protein of mammalian cells (such as CHO cells), and the structure of the medicine is an antibody-cytokine fusion protein. The antibody structural part of the fusion protein can target and identify gp41 subunit in HIV outer membrane protein gp160, and is responsible for carrying interferon molecules to the vicinity of HIV or HIV-infected cells, so as to guide the directional antiviral effect of the interferon. Specifically, the targeting interferon is a fusion molecule of an antibody molecule and an interferon, wherein the interferon molecule is located at the C-terminal end of the heavy chain of the antibody molecule. The antibody molecule forms a functional region for identifying HIV gp41 subunit together with a heavy chain variable region (VH) and a light chain variable region (VL) and is responsible for specific binding with a corresponding sequence of gp41 subunit. The constant region of the antibody molecule structure is of the IgG1 type. At the C-terminal end of the constant region CH3, one molecule of type I interferon (subtype α or β) is attached. The targeted interferon medicament is used for treating HIV infection, realizes accurate administration through a targeted site, reduces medicament dosage, reduces side effects and improves the antiviral effect of the medicament.

In the invention, the antibody 10E8 which can identify the gp41 subunit MPER region highly conserved by HIV outer membrane glycoprotein is selected as a targeting tool of the integrated interferon, thereby playing two roles. Firstly, the target spot exposed by the outer membrane protein allosteric in the infected cell of the virus can be accurately identified, the virus infection is prevented, and the neutralization effect is exerted; meanwhile, the carried interferon molecules can be more effectively combined with interferon receptors on cells around HIV viruses, so that intracellular interferon signal paths are activated, and the cells can play an antiviral function.

Specifically, the invention combines the antibody 10E8 which can recognize the MPER region with highly conserved gp41 subunit in HIV outer membrane glycoprotein gp160 with high-activity integrated interferon to form an antibody-cytokine fusion protein ZP-IC by genetic engineering technology, thereby playing the function of targeting interferon. The ZP-IC of the present invention targets the highly conserved MPER domain of HIV gp 41. The heavy chain variable region CDR region (complementarity determining region) and light chain variable region CDR region sequences of the antibody structural portions of ZP-IC are both derived from the corresponding partial sequences of mab 10E 8. The framework structure of the rest part of the antibody adopts an IgG1 type antibody structure. At the C-terminal end of the antibody constant region CH3, one molecule of integrated interferon is linked. The targeting interferon molecule ZP-IC recognizes and combines with HIV virus outer membrane protein gp41 through 10E8 antibody part to block the membrane fusion invasion process of the virus; through its integrated interferon functional portion, it mobilizes and binds to interferon receptor on the cells around the virus, activates intracellular interferon signaling pathway, produces antiviral effector protein, and exerts antiviral function. The invention adopts innovative design to make antibody molecules play a dual role: namely, identifying pathogens, neutralizing viruses; directing the localization of the interferon and locally enriching the interferon. Therefore, the invention skillfully combines the neutralization effect of the antibody and the antiviral effect of the interferon, and plays a practical role of 1+1> 2. In cell experiments, it was demonstrated that such "antibody-interferon" fusion molecules of ZP-IC have a much stronger inhibitory effect on viral replication than the interferon molecule itself, as well as the antibody itself.

U.S. patent No. 20160333076A1 reports monoclonal antibody 10E8 targeting the MPER region of HIV gp41 subunit and mutants and uses thereof. International patent WO 2017079479A1, U.S. patent No. 20190077849A1, reports commercial mutational engineering of monoclonal antibody 10E8 targeting the MPER region of HIV gp41 subunit, including further enhancement of virus neutralization capacity, increased solubility, and immunogenicity engineering. These patent applications demonstrate that the 10E8 strain is valued by the developer for its broad neutralizing activity. However, up to now, no patent and article for fusion expression of 10E8 and interferon and targeting antiviral effect have been reported, and it is demonstrated that ZP-IC molecular drug design is innovative and novel. The cell experiment result of the ZP-IC proves that the antibody-interferon fusion molecule of the ZP-IC has much stronger antiviral effect than the interferon molecule and the antibody.

The essence of the invention is a gene recombination targeted interferon biotechnological drug. The targeting interferon is used for treating HIV infection, and the targeting interferon is used for targeting and accurate targeting administration, so that the antiviral effect of the medicine is improved while the dosage and side effects of the medicine are reduced. The recommended clinical dosage of the medicine should be lower than 0.5mg/dose each time, and the treatment interval time is not less than 3 days. The recommended method of use of the medicament is intravenous injection. The recommended mode of treating HIV infection by the medicine is combined, namely, the medicine is combined with a common HIV treatment medicine cocktail (such as tenofovir, lamivudine, efavirenz and the like) to play a role in synergistic antiviral.

In summary, compared with the prior art, the invention has the advantages and characteristics that:

(1) Through high affinity and broad spectrum combination of antibody fragments of virus glycoprotein, an integrated interferon molecule is accurately delivered to the vicinity of HIV virus infected cells, thereby blocking virus infection, and through interferon signal path way, the antiviral ability of the cells is mobilized, thus truly playing the role of biological missile.

(2) Compared with interferon, the effect of the targeting interferon is more accurate, a microenvironment with higher concentration of interferon can be formed near HIV infection cells, the antiviral effect of the interferon and the effect of eliminating pathological cells are better exerted, the concentration of free interferon in blood is reduced, and the toxic and side effects of the interferon are reduced.

(3) Antibodies targeting the MPER region of its gp41 subunit are more broad-spectrum than other antibodies targeting HIV outer membrane proteins. Because the gp41 subunit is responsible for membrane fusion, all functional regions associated with membrane fusion are highly conserved, including FP, MPER and heptad repeat NHR. Thus, the 10E8 antibody is able to neutralize the vast majority of HIV strains. The target antibody is selected as the target antibody of the interferon, so that the function of the interferon can be wider and more effective, and the immune escape of viruses can be avoided to the greatest extent.

(4) "Targeting interferons" are more effective than co-target antibody class drugs. The effectiveness of the HIV virus is evaluated by using an in-vitro HIV virus replication inhibition cell model, and experiments show that the IC ₅₀ of the ZP-IC pair test 8 different types of HIV epidemic strains is between 0.01 and 0.2 mug/mL, and the IC ₅₀ of the homotype antibody ZP-ab is above 5 mug/mL. Each comparison data shows that compared with the homotype antibody of the targeting interferon ZP-IC, the half maximum inhibitory concentration IC ₅₀ of the drug is reduced by more than 200 times, namely the drug effect is improved by at least 2 orders of magnitude, which indicates that the synergistic effect of the targeting interferon ZP-IC on 'targeting closure' + 'interferon antiviral' is achieved, so that the molecule has better antiviral activity.

Drawings

FIG. 1 is a schematic diagram showing the structural design of a fusion protein ZP-IC.

FIG. 2 is a diagram showing the comparison of the amino acid sequences of 12 subtypes of interferon-alpha, and the consensus sequence with the highest occurrence frequency is the sequence of integrated interferon-alpha.

FIG. 3 shows the identification of the cleavage of recombinant eukaryotic expression vectors for ZP-IC.

FIG. 4 shows affinity chromatography purification and reduction SDS-PAGE of ZP-IC. In FIG. 4, reference ① denotes the heavy chain of ZP-IC and reference ② denotes the light chain of ZP-IC.

FIG. 5 shows Western blot of ZP-IC.

FIG. 6 shows ELISA method for detecting the affinity of fusion protein ZP-IC with HIV outer membrane protein gp 41.

FIG. 7 shows ELISA method for detecting the affinity of fusion protein ZP-IC with HIV outer membrane protein gp41 subunit near membrane end MPER polypeptide.

FIG. 8 shows the binding of ZP-IC to IFNR 2.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The pcDNA3.1neo (+) vector in the examples described below is a product of Invitrogen company under the designation V79020.

The pcDNA3.1Zeo (+) vector in the examples described below is the product of Invitrogen company under the designation V86020.

Example 1 design and Structure of fusion protein ZP-IC

In order to improve the antiviral activity of the targeted HIV interferon, the invention selects the fusion expression of recombinant integrated interferon alpha and an antibody molecule (10E 8 antibody) capable of recognizing and combining HIV virus outer membrane glycoprotein gp 41. The fusion protein designed by the invention is named ZP-IC (also called targeting interferon ZP-IC), and the structural schematic diagram is shown in figure 1.

The integrated interferon alpha, also called consensus interferon alpha (Consensus Interferon-alpha, interferon alfacon-1), is a non-natural type I interferon, which is a novel interferon formed by comparing the amino acid sequences of 12 types of interferon-alpha and mainly distributing the amino acid with the highest occurrence frequency to the corresponding positions of each interferon (figure 2). The biological activity of integrated interferon alpha compared to the biological activity of native type I IFN-alpha shows a stronger antiviral activity, e.g. the specific activity of integrated interferon alpha (defined as units of antiviral activity per mg protein) has been reported 5-to 20-fold higher than other subtypes of IFN-alpha. In general, the specific activity of other subtypes of IFN- α ranges from 1X 10 ⁸ U/mg to 4X 10 ⁸ U/mg protein, while the specific activity of the integrated interferon- α is 2X 10 ⁹ U/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.).

The light chain variable region sequences of the ZP-IC antibody portion are from 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.].

The ZP-IC antibody portion light chain constant region uses a human immunoglobulin Kappa light chain constant region (human Immunoglobulin KAPPA CHAIN constant region, IGKC, https:// www.uniprot.org/uniprot/P01834).

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

The heavy chain constant region of the ZP-IC antibody portion employs a human immunoglobulin gamma heavy chain constant region (Human immunoglobulin heavy constant gamma, igG), including the heavy chain constant region of human IgG1 (IGHG 1, https:// www.uniprot.org/uniprot/P01857), or the heavy chain constant region of human IgG2 (IGHG 2, https:// www.uniprot.org/uniprot/P01859), or the heavy chain constant region of human IgG4 (IGHG 4, https:// www.uniprot.org/uniprot/P01861). The heavy chain constant region of human IgG1 is adopted in this example, and the sequence is shown in positions 149-476 of SEQ ID No. 1.

The C end of the heavy chain constant region is fused with integrated interferon alpha or consensus sequence interferon alpha (Consensus Interferon-alpha) with stronger antiviral activity through a flexible protein linker (as shown at 477-482 of SEQ ID No. 1). The amino acid sequence of the integrated interferon alpha is shown in 483-648 of SEQ ID No. 1.

The fusion protein comprises a 10E8 antibody and an integrated interferon alpha, wherein the integrated interferon alpha is connected with a constant region at the C end of the 10E8 antibody through a linker. The specific sequence is as follows:

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.

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.

Wherein, the 1 st-476 th site of SEQ ID No.1 is the amino acid sequence of the heavy chain of the 10E8 antibody targeting HIV gp41 protein, the 477 th-482 th site of SEQ ID No.1 is the amino acid sequence of a linker (linker), and the 483 rd-648 th site of SEQ ID No.1 is the amino acid sequence of an integrated interferon alpha.

Positions 1-1428 of SEQ ID No.2 are nucleotide sequences of heavy chains of 10E8 antibodies targeting HIV gp41 protein, positions 1429-1446 of SEQ ID No.2 are nucleotide sequences of a linker (linker), and positions 1447-1950 of SEQ ID No.2 are nucleotide sequences of an interferon alpha.

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

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

Example 2 preparation of fusion protein ZP-IC

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

ZP-IC recombinant proteins may be expressed using commercially available eukaryotic expression vectors such as pCHO1.0 (Invitrogen), pcDNA ^TM 3.1.1/Neo (+/-) and pcDNA ^TM 3.1.1/Zeo (+/-) (Invitrogen) or other commercially available eukaryotic expression vectors, or may be expressed using self-developed eukaryotic expression vectors (e.g., shao Yong, hu Xianwen, etc. an animal cell efficient expression vector based on the GS screening system and applications, patent number: ZL 201410037976.6).

Optimizing light chain and heavy chain genes which are preferentially expressed by CHO cells according to the light chain gene sequence and the heavy chain gene sequence of the fusion protein, adding an EcoRI enzyme recognition site and a Kozak sequence at the upstream of the genes, adding an EcoRV enzyme recognition site at the downstream of the genes, and entrusting the light chain gene (SEQ ID No. 4) and the heavy chain gene (SEQ ID No. 2) of the optimized fusion protein ZP-IC by Beijing qing biological science and technology Co., ltd. The related gene and vector were recovered by double-digestion of the light chain gene, heavy chain gene, pcDNA3.1neo (+) vector and pcDNA3.1Zeo (+) vector (Invitrogen) with EcoRI/EcoRV, ligation of the light chain gene and pcDNA3.1Zeo (+) vector with T4 ligase, ligation of the heavy chain gene and pcDNA3.1neo (+) vector with T4 ligase, and transformation of E.coli Trans10 (Beijing full gold Biotechnology Co.).

Taking a colony of a pcDNA3.1Zeo (+) vector connected with a light chain gene as a PCR template, and carrying out colony PCR identification by using CMV-F/BGH-R as an upstream primer and a downstream primer, wherein the PCR reaction conditions are as follows: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s; annealing temperature 60 ℃ for 30s; the chain extension temperature is 68 ℃ for 1min, and 30 cycles are total; after the circulation is completed, the extension is carried out once at 72 ℃ for 5 min. The positive clone is selected, the primer CMV-F and the primer BGH-R are used for gene sequencing, the correct sequence is adopted to clone and extract plasmid, and a eukaryotic expression vector pcDNA3.1Zeo (+)/ZP-IC LC for expressing the ZP-IC light chain is obtained, the EcoRI/EcoRV double enzyme digestion identification of the vector is shown as a 2 nd lane of FIG. 3, pcDNA3.1/Zeocin in FIG. 3 represents pcDNA3.1Zeo (+) vector gene, and ZP-IC LC represents the light chain gene of ZP-IC.

CMV-F:5’-TAATACGACTCACTATAGGG-3’(SEQ ID No.7)；

BGH-R:5’-TAGAAGGCACAGTCGAGG-3’(SEQ ID No.8)。

The ZP-IC light chain expression vector pcDNA3.1Zeo (+)/ZP-IC LC is a recombinant expression vector which replaces a fragment between an EcoRI recognition site and an EcoRV recognition site of the pcDNA3.1Zeo (+) vector with a ZP-IC light chain gene with a nucleotide sequence of SEQ ID No.4, and keeps other nucleotide sequences of the pcDNA3.1Zeo (+) vector unchanged, thus obtaining the expression fusion protein ZP-IC light chain.

Colony of pcDNA3.1neo (+) vector connected with heavy chain gene is selected as a PCR template, CMV-F/ZPIC-HC-R is used as an upstream primer and a downstream primer, colony PCR is carried out, and the PCR reaction conditions are as follows: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s; annealing temperature 60 ℃ for 30s; the chain extension temperature is 68 ℃ for 60s, and 30 cycles are total; after the circulation is completed, the extension is carried out once at 72 ℃ for 5 min. The positive clones are selected to be respectively subjected to gene sequencing, and plasmids are extracted from clones after the correct sequencing, so that a eukaryotic expression vector pcDNA3.1Neo (+)/ZP-IC HC for expressing the ZP-IC heavy chain is obtained, the EcoRI/EcoRV double enzyme digestion identification of the vector is shown as a lane 1 in FIG. 3, pcDNA3.1/Neomycin in FIG. 3 represents pcDNA3.1Neo (+) vector genes, and ZP-IC HC represents heavy chain genes of the ZP-IC.

CMV-F:5’-TAATACGACTCACTATAGGG-3’(SEQ ID No.7)；

ZPIC-HC-R:5’-ACAACCCAGTGCGGCTGTGC-3’(SEQ ID No.9)。

The ZP-IC heavy chain expression vector pcDNA3.1Neo (+)/ZP-IC HC is a recombinant expression vector which replaces a fragment between an EcoRI recognition site and an EcoRV recognition site of the pcDNA3.1Neo (+) vector with a ZP-IC heavy chain gene with a nucleotide sequence of SEQ ID No.2, and keeps other nucleotide sequences of the pcDNA3.1Neo (+) vector unchanged, thereby obtaining the expression fusion protein ZP-IC heavy chain.

Using electrotransfection or lipofection methods, pcDNA3.1neo (+)/ZP-IC HC and pcDNA3.1Zeo (+)/ZP-IC LC vectors were co-transfected into CHO-DG44 cells (Siemens Fedder) according to the procedure described in the relevant operating manual. When the step adopts an electrotransfection mode, a cell electrotransducer Nucleofector produced by the company Lonza and the method can be adopted for carrying out; when the liposome transfection method is used, the step may be performed using a transfection reagent Lipofectamine ^TM or Lipofectamine ^TM 3000 manufactured by Invitrogen corporation, and the method.

The transfected CHO-DG44 cells are inoculated into T-shaped square bottles for culture, and serum-free culture medium with definite chemical composition (such as CD FortiCHO ^TM serum-free culture medium produced by Gibco company) is adopted for culture. After 24 hours of incubation, stably transfected cell lines were pressure-screened with serum-free medium containing 250-400. Mu.g/mL bleomycin (Zeocin ^TM) and 500-700. Mu.g/mL geneticin (G418 sulfate). The medium was changed every 3 days, cultured continuously for 2-3 weeks in serum-free medium containing Zeocin ^TM and G418, and stably transfected CHO cell colonies were obtained by screening. After digestion of these stable colonies with pancreatin, monoclonal cultures and screens were performed in 96-well plates by limiting dilution. ELISA method selects monoclonal cell strain with high expression target protein, carries out passage and amplification, uses 10% DMSO cell freezing solution to freeze and stably transfected CHO engineering cell strain (recombinant cell) with high expression ZP-IC recombinant protein. The recombinant cell is a cell which expresses the fusion protein ZP-IC and is obtained after the recombinant vector pcDNA3.1 Neo (+)/ZP-IC HC and the recombinant vector pcDNA3.1 Zeo (+)/ZP-IC LC are jointly introduced into CHO-DG44 cells. The recombinant cell contains a heavy chain gene of ZP-IC shown in SEQ ID No.2 and a light chain gene of ZP-IC shown in SEQ ID No. 4.

2. Expression of fusion protein ZP-IC

Resuscitate the stably transfected CHO engineering cell strain expressing ZP-IC recombinant protein and adopt serum-free culture medium to culture and amplify in square bottle. The amplified CHO engineering cell strain is inoculated into 2 liter shake flasks, the initial cell density is maintained at 0.2X10 ⁶～0.6×10⁶ cells/mL, the cell culture volume is 15% -35% of the shake flask volume, and the cell culture flasks are placed in a shaker (rotation speed 135rpm, temperature 37 ℃, CO ₂%) for culture. As the serum-free Medium, CD FortiCHO ^TM produced by Gibco may be used, or as the Medium, such as Media C produced by Shanghai Duoning biosciences, inc., OPM-CHO CD08 Medium produced by Shanghai ao Pu Mai biosciences, inc., may be used. After 3-4 days of culture, subculture can be carried out according to the proportion of 1 to 3 or 1 to 4, culture supernatant is collected after 7 days of culture in a shake flask, and fusion protein ZP-IC in the culture supernatant is purified.

3. Purification of fusion protein ZP-IC

Antibody proteins with Fc domains were purified from the culture supernatants using Protein A affinity chromatography columns (Mabselect SURE, GE). The culture supernatant was first clarified by filtration through a 0.45. Mu.M filter and then applied to a Protein A affinity column (GE Mabselect SURE). Specific procedures reference is made to the recommended procedure for GE company affinity chromatography packing Protein A. The purification instrument adopts Zhongkesen Hui Protein purification SYSTEM PPS-HD-100. The pH of the sample was controlled to be neutral (pH 7.0), and the pH of the eluate was controlled to be pH3.0. Purified fusion protein ZP-IC was quantified by Nanodrop instrument. The purified recombinant fusion protein was checked for molecular weight and purity under reducing conditions using polyacrylamide gel electrophoresis. SDS-PAGE results show that the heavy chain molecular weight of the recombinant protein is about 80kDa, and the light chain molecular weight is about 25kDa (figure 4), which are consistent with the designed target molecular weight.

Example 3 reaction of ZP-IC with anti-type I Interferon-specific antibodies

The fusion protein ZP-IC purified by affinity chromatography is subjected to SDS-PAGE electrophoresis, and positive control (recombinant interferon IFN-Fc, IFN-Fc sequence see the patent ZL 202310085137.0, which carries an interferon gene consistent with ZP-IC, i.e. integrated interferon alpha) and negative control (IgG, shanxikang Biotechnology Co., ltd.) are set. The same samples were spotted in duplicate. After electrophoresis, the PAGE gel is cut into two parts, one part is stained, and the other part is subjected to Western blot. Semi-dry transfer was performed using PVDF Membrane (immobilon-P PVDF Membrane cat# IPVH00005, millipore). After transfer, the cells were washed three times with PBST solution, placed in PBST solution containing 5% skimmed milk powder, and blocked overnight at 4 ℃. Thereafter, the washing was performed three times with PBST solution. Meanwhile, PBST containing 1% skimmed milk powder was formulated as an antibody incubation liquid. Adding 1to the antibody incubation liquid: a1000 dilution of rabbit Anti-interferon polyclonal antibody (Anti-Interferon alpha Anti, cat# ab193055, abcam) was incubated with the membrane with shaking at 37℃for 1 hour. Thereafter, the washing was performed three times with the PBST solution for not less than 3 minutes each time. Adding 1to the antibody incubation liquid: goat anti-rabbit IgG (HRP-labeled, sequoyitol bridge, cat No. ZB 2301) diluted 1500 was incubated with the membrane with shaking for 1 hour at 37 ℃. Thereafter, the washing was performed three times with the PBST solution for not less than 3 minutes each time. Color development was performed using Enlight ^TM western blotting substrate (cat# 29050, engreen) and photographed using a chemiluminescent instrument (Tanon 5200 multi). As can be seen from the Western blot results, ZP-IC was able to develop a color at a specific molecular weight (80 kD), exactly corresponding to the size of the heavy chain of the molecule (FIG. 5). Indicating that ZP-IC molecules carry interferon molecules on their heavy chains.

Example 4 ELISA method for detecting binding force of ZP-IC to HIV gp41 glycoprotein

Recombinant HIV gp41 protein (purchased from abcam, cat# ab 49070) was diluted to 1.0ug/mL with coating solution and coated onto the ELISA plate at 100 ng/well overnight at 4 ℃. Thereafter each well was blocked by adding 2% BSA in PBST for 1.5-2 hours. During incubation, blocking solution was diluted with PBST to a final BSA concentration of 0.5% BSA as antibody diluent, and antibody fusion protein ZP-IC was subjected to a double dilution starting from 10. Mu.g/mL to 0.15ng/mL. Human IgG (Shanxikang biological products Co., ltd.) was used as control 1, and an antibody ZP-ab (the amino acid sequence of the ZP-ab heavy chain is SEQ ID No.5, the nucleotide sequence of the ZP-ab heavy chain is SEQ ID No.6, the amino acid sequence of the ZP-ab light chain is identical to that of the ZP-IC light chain, and the nucleic acid sequence of the ZP-ab light chain is identical to that of the ZP-IC light chain) of the same target was used as control 2. And after the ELISA plate is closed, washing fully. Each antibody diluted in a double ratio was added, incubated at 37℃for 1 hour at 100. Mu.L/well, and after extensive washing, 1 was added: goat anti-human IgG/horseradish enzyme label (sequoyins, cat No. ZB 2304) at 2500 dilution was 100 μl and incubated for 1 hour at 37 ℃. After extensive washing, color development was performed with TMB solution for 15min,2M HCl was stopped and the light absorbance was read at 450 nm. The assay results were analyzed using GRAPHPAD PRISM Software 6.0, and nonlinear regression analysis was chosen to calculate the half maximal binding effect concentration (EC ₅₀). The results of the data analysis showed that the half maximum binding concentration EC ₅₀ = 74ng/mL (about 0.37 nM) of the fusion protein ZP-IC to the recombinant protein gp41 was consistent with ZP-ab (EC ₅₀ = 60.7 ng/mL) (fig. 6); the affinity EC ₅₀ of human immunoglobulin IgG for recombinant protein gp41 is approximately 948ng/mL. It is demonstrated that ZP-IC is capable of specifically binding to gp41 subunit of HIV outer membrane protein.

Example 5 ELISA method for detecting binding force of ZP-IC to MPER polypeptide

The MPER polypeptide is located in the membrane proximal outer region of gp 41. The MPER polypeptide sequence reference (Huang JH,et al.Broad and potent neutralization of HIV-1by a gp41-specific human antibody.Nature.2012,491(7424):406–412.doi:10.1038/nature11544.) sequence is: NEQELLELDKWASLWNWFDITNWLWYIR (positions 656-683, 28 aa, from Kirschner Biotech Co., ltd.). Because the gp41 subunit is responsible for membrane fusion, all functional regions associated with membrane fusion are highly conserved, including FP, MPER and heptad repeat NHR. Thus, antibodies targeting the gp41 subunit MPER region are more broad-spectrum.

The MPER polypeptide was dissolved in an alkaline solution, diluted to 1.5. Mu.g/mL with PBS, and antigen coated by adding 100. Mu.L/well to an ELISA plate at 4℃overnight. Thereafter each well was blocked by adding 2% BSA in PBST for 1.5-2 hours. During incubation, blocking solution was diluted with PBST to a final BSA concentration of 0.5% BSA, and antibody dilution was performed as a double dilution of the antibody fusion protein ZP-IC starting from 500. Mu.g/mL to below 0.25. Mu.g/mL. Human IgG (shanxi kangbao biologicals inc.) was used as a control. And after the ELISA plate is closed, washing fully. Adding antibody solution diluted by multiple ratio, 100 mu L/hole, incubating for 1 hour at 37 ℃, and adding 1: goat anti-human IgG/horseradish enzyme label (sequoyins, cat No. ZB 2304) at 2500 dilution was 100 μl and incubated for 1 hour at 37 ℃. After extensive washing, color development was performed with TMB solution for 15min,2M HCl was stopped and the light absorbance was read at 450 nm. The assay results were analyzed using GRAPHPAD PRISM Software 6.0, and nonlinear regression analysis was selected to calculate half maximal binding effect concentration (EC ₅₀) (see fig. 7). The half maximal binding concentration of ZP-IC was calculated by software to be 7.2 μg/mL, about 40nM; whereas the half maximal binding concentration of control antibody IgG was >500 μg/mL (i.e., >3.3 μm). The ZP-IC can specifically bind to MPER polypeptide on HIV gp41, has the capability of broad-spectrum inhibition of most HIV strains, and can avoid the immune escape of the virus to the greatest extent.

Example 6 detection of binding of ZP-IC to Interferon receptor (IFNR 2) by ELISA

Recombinant interferon receptor 2 (Human IFN-alpha/beta R2 Protein, his Tag, cat# IF2-H5224, available from Beepstein ACROBiosystems) was diluted to 1.0ug/mL with coating solution and coated onto an ELISA plate at 100 ng/well overnight at 4 ℃. Thereafter each well was blocked by adding 2% BSA in PBST for 1.5-2 hours. During incubation, blocking solution was diluted with PBST to a final BSA concentration of 0.5% BSA, and antibody fusion protein ZP-IC was serially diluted 3-fold as antibody dilution, starting from 90. Mu.g/mL to 1.5ng/mL. Human IgG (shanxi kangbao biologicals inc.) was used as a control. And after the ELISA plate is closed, washing fully. Adding diluted sample to be tested, incubating for 1 hour at 37 ℃ at 100 mu L/hole, and adding 1: goat anti-human IgG/horseradish enzyme label (sequoyins, cat No. ZB 2304) at 2500 dilution was 100 μl and incubated for 1 hour at 37 ℃. After extensive washing, color development was performed with TMB solution for 15min,2M HCl was stopped and the light absorbance was read at 450 nm. The assay results were analyzed using GRAPHPAD PRISM Software 6.0, and nonlinear regression analysis was chosen to calculate the half maximal binding effect concentration (EC ₅₀). The results of the data analysis showed that the half maximal binding concentration of fusion protein ZP-IC to recombinant human interferon receptor 2 EC ₅₀ = 140ng/mL (about 0.7 nM) (fig. 8). It was demonstrated that ZP-IC was able to specifically bind to interferon receptors with higher affinity (< 1 nM), thereby activating the interferon signaling pathway.

Example 7 antiviral Activity test of ZP-IC

Testing cell lines: TZM-bl cells, derived from a Hela cell line JC.53, stably express a large amount of CD4 and CCR5, endogenously express CXCR4, and the genome of which is engineered to integrate luciferase under the control of HIV-1 promoter and beta-galactosidase of E.coli. TZM-bl cell lines are highly sensitive to infection by different HIV-1 strains and are suitable for quantitative detection of neutralizing antibody efficacy. The cell line is presented by John C.kappa.doctor, wu Xiaoyun doctor and Tranzyme company through the American national institutes of health AIDS reagent program, and is currently saved by the institute of pathogenic biology of the national academy of medical science.

The test HIV strains are derived from the institute of pathogenic biology of the national academy of medical science, and the information is shown in Table 1:

TABLE 1 test HIV strains

Test number	Strain name	Subtype type
			TRO11	TRO.11	B
CNE55	CNE55	A/E
			CH119	CH119.10	B/C
25710	HIV_25710-2.43	C
			X1632	X1632-S2-B10	G
CNE8	CNE8	A/E
			CE70301	CE703010217_B6	C
BJOX002000	BJOX002000.03.2	B/C

The antiviral activity test steps were as follows: 3 times of dilution is carried out on the antibody fusion protein ZP-IC to be detected (or the control antibody drug ZP-ab, HIV-DNS) in a 96-well cell culture plate, 50 mu L/well is provided with 3 compound wells and 9 gradients; a volume of 50. Mu.L of recombinant virus (100 TCID ₅₀) (see test strains listed in Table 1) was added to the culture wells and left at 37℃for 60min; a volume of 100. Mu.L of 1X 10 ⁴ TZM-bl cells was then added to each well. After adding DEAE-dextran at a final concentration of 15. Mu.g/mL, the mixture was incubated at 37℃in a 5% CO ₂ cell incubator for about 48 hours. After careful removal of the culture medium, cells were lysed with 30. Mu.L of cell lysate per well at room temperature for 15min, after which 100. Mu.L/well Luciferaese Assay of reagent was added, and after thorough mixing 100. Mu.L was transferred to a 96 well white plate. RLU (relative luminescence units) was measured using a chemiluminescent detector and the IC ₅₀ values and drug inhibition curves of the drug were calculated using the sigmoidal dose response equation in a nonlinear regression analysis using GRAPHPAD PRISM Software. Calculation method half-effective inhibitory concentration (IC ₅₀) refers to the dose at which the drug is capable of causing 50% of the maximum effect. When IC ₅₀ of the drug to HIV-1 virus is calculated, all drug concentrations are converted into log ₁₀ (concentration value), and each sample hole of each concentration is calculated to obtain the sample fluorescence inhibition rate, wherein the calculation formula is as follows: inhibition ratio = [1- (RLU value for dosing well-RLU value for negative control well)/(RLU mean for positive control well-RLU mean for negative control well) ]x100%. Data analysis IC ₅₀ values and drug inhibition curves for each drug were analyzed using the sigmoidal dose response equation in nonlinear regression analysis using GRAPHPAD PRISM Software. The measurement results (mug/mL) of half-effective inhibitory concentration (IC ₅₀) of the drug ZP-IC on infected cells of HIV strain TRO11 and the like are shown in Table 2, and the measurement results (mug/mL) of half-effective inhibitory concentration (IC ₅₀) of the drug ZP-IC and the like on infected cells of HIV strain X1632 and the like are shown in Table 3.

TABLE 2 half-effective inhibitory concentration of ZP-IC on HIV strain-infected cells (IC ₅₀)

Table 3, half-effective inhibitory concentrations (IC ₅₀) of ZP-IC and the like on HIV strain-infected cells

From the above test data, it can be seen that the tested 8 different types of HIV epidemic strains all maintain good sensitivity to the candidate drug ZP-IC. The half-inhibitory concentration of ZP-IC was at a level of 0.01-0.06. Mu.g/mL for the remaining test strains except for half-inhibitory concentration of IC ₅₀ for the X1632 strain. The half inhibition concentration IC ₅₀ of the antibody drug ZP-ab with the same target point on the HIV virus strain X1632 is more than 33 mug/mL, the half inhibition concentration IC ₅₀ on the other three test virus strains (CNE 8, CE70301 and BJOX 002000) is also between 3 and 16 mug/mL, the activity of inhibiting the virus is reduced by more than 200 times compared with that of the fusion protein ZP-IC, and the effect of resisting the virus, which can be achieved by the ZP-ab, is required to be in microgram grade only through the targeted blocking effect; by the design mode of the fusion protein, namely the targeting interferon, the inhibiting capability of the ZP-IC to viruses is greatly improved compared with that of ZP-ab antibodies, and the drug effect is improved by at least 2 orders of magnitude. Therefore, in the practical dosage, the dosage of ZP-IC can be reduced by two orders of magnitude compared with the dosage of the antibody ZP-ab of the same target point, and the same antiviral effect can be achieved.

HIV-DNS is a bispecific antibody targeting gp120 (see Chinese patent ZL 202210196757.7, a multispecific antibody targeting HIV gp120 protein and human CD3 molecules and application thereof), and in this test, the inhibition ability of HIV-DNS to different HIV strains is tested in parallel. From several groups of data, the half inhibitory concentration IC ₅₀ for HIV strain X1632 was 0.166 μg/mL, which is close to the IC ₅₀ value of ZP-IC. The half inhibitory concentration IC ₅₀ for the other three test strains (CNE 8, CE70301, BJOX 002000) was between 0.3-0.5 μg/mL, which is inferior to the inhibitory activity of ZP-IC (IC ₅₀ for ZP-IC was between 0.02-0.06 μg/mL). It is demonstrated that ZP-IC has approximately 1 order of magnitude higher anti-HIV viral activity in vitro than HIV-DNS by targeting this design of interferon. This also shows that the fusion protein ZP-IC has very good antiviral effect.

In the cell level antiviral experiment, recombinant long-acting interferon (IFN-Fc, see the invention patent ZL 202310085137.0 for sequence) is also submitted for activity detection. Unfortunately, none of the detection parties detected the in vitro antiviral activity of IFN-Fc in several pre-experiments. Thus, in the official experiments, no antiviral detection of IFN-Fc was performed. The interferon alone can not play a role in directional guidance and activating cell antiviral effect.

We established a data link to the magnitude of antiviral activity at the cellular level of the fusion proteins ZP-IC and IFN-Fc by the following data. In the invention patent: in ZL 202310085137.0, the antiviral activity of HIV-DNS and recombinant long-acting interferon (IFN-Fc) are compared in parallel, and multiple groups of data show that the in vitro antiviral activity of IFN-Fc is an order of magnitude lower than that of HIV-DNS (supplementary data: IFN-Fc and HIV-DNS have an inhibitory potency IC ₅₀ of 52.17 + -15.37 nM and 4.55+ -4.10 nM, respectively, for replication of HIV-strain HIV-1 _ⅢB in TZM-bl cells, and an inhibitory potency IC ₅₀ of IFN-Fc and HIV-DNS for replication of HIV-1 _IIIB in C8166 cells, respectively, of 25.02+ -8.15 nM and 1.63+ -0.71 nM). From the above data, it was found that in vitro antiviral activity ZP-IC > HIV-DNS > IFN-Fc. I.e. the antiviral activity of the in vitro cell level of the targeting interferon molecule ZP-IC is far higher than that of IFN-Fc.

From the data and analysis, the design of the targeting interferon can selectively enrich interferon molecules around viruses, enhance antiviral signals and achieve better antiviral effects. The antiviral effect is at least 1-2 orders of magnitude stronger than that of the monoclonal antibody ZP-ab with the same target spot and the homotypic long-acting interferon molecule IFN-Fc. Thus, the targeting interferon molecule (ZP-IC) has extremely high anti-HIV virus capacity.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (8)

1. The fusion protein is characterized by being formed by connecting a 10E8 antibody or an antigen binding fragment thereof and an integrated interferon alpha through a connector;

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 encoding the fusion protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is a DNA molecule having a coding sequence of SEQ ID No.2 and a DNA molecule having a coding sequence of SEQ ID No. 4.

4. A biomaterial characterized in that the biomaterial is any one of the following:

a1 An expression cassette comprising the nucleic acid molecule of claim 2;

a2 A recombinant vector comprising the nucleic acid molecule of claim 2;

a3 A recombinant yeast or E.coli comprising the nucleic acid molecule of claim 2;

A4 A recombinant animal cell comprising the nucleic acid molecule of claim 2.

5. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 2 or 3 or the biological material of claim 4 for any of the following:

B1 Use of a composition for the preparation of a product for the prevention or treatment of HIV infection;

B2 The use of said composition for the preparation of a product for the prevention or treatment of AIDS;

B3 Use of a composition for inhibiting HIV viral activity;

b4 Use in the preparation of a product for binding or detecting HIV gp41 protein;

B5 For the preparation of a product for binding or detecting HIV MPER polypeptides.

6. A pharmaceutical composition comprising 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, comprising the fusion protein of claim 1.

8. The method of preparing a fusion protein of claim 1, comprising: respectively constructing recombinant expression vectors containing heavy chain genes and light chain genes of the fusion protein; introducing the recombinant expression vector into a host cell to obtain a recombinant cell expressing the fusion protein; culturing the recombinant cells, and separating and purifying to obtain the fusion protein.