APC Conjugates for Treating and / or Preventing HIV-Related Diseases, Their Preparation Methods and Applications

Abstract

The present invention provides an APC conjugate for treating and / or preventing HIV-related diseases, its preparation method and uses. Specifically, the present invention provides an antibody-polypeptide conjugate of the structure shown in Formula I or a pharmaceutically acceptable salt thereof. On the basis of retaining the antiviral activity of each single molecule, the APC conjugate of the present invention achieves a dual-drug antiviral spectrum; and the APC conjugate does not affect the pharmacokinetics of the anti-HIV antibody while prolonging the half-life of ABT.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to an APC conjugate for treating and / or preventing HIV-related diseases, its preparation method and application. Background Art

[0002] AIDS, the full name is "Acquired Immunodeficiency Syndrome" (AIDS), is a highly lethal malignant infectious disease caused by the HIV virus, that is, the human immunodeficiency virus. The HIV virus invades the human body, can destroy the human immune system, making the infected gradually lose the resistance to various diseases, and finally leading to death. At present, there is no vaccine to prevent it, nor an effective drug or method to cure this disease.

[0003] Alberix (ABT) is a polypeptide developed by Frontier Biopharma for the treatment or prevention of HIV infection. It belongs to an HIV-1 fusion inhibitor, acts on the HIV-1 envelope glycoprotein gp41, and prevents the virus from entering cells.

[0004] Human immunodeficiency virus neutralizing antibodies are a class of antibodies isolated from the bodies of HIV-infected individuals through biotechnology. Such antibodies can recognize the envelope glycoprotein gp120 of the HIV virus, can directly neutralize the HIV virus to reduce the level of HIV virus in the body, and at the same time, the binding to gp120 can prevent the virus from entering cells, thereby reducing the replication of the virus in the body. This antibody can also kill the virus by stimulating other immune components in the body.

[0005] If an anti-HIV drug with more precise targeting and a broader antiviral spectrum can be developed, it will surely bring new breakthroughs for the treatment or prevention of HIV infection. Summary of the Invention

[0006] The object of the present invention is to provide an APC conjugate for treating and / or preventing HIV infection, which is formed by conjugating ABT to an antibody molecule that specifically recognizes HIV antigens, and can rely on the targeting of the antibody to precisely kill free HIV viruses or T cells infected with the HIV virus; this APC conjugate can not only retain the antiviral activities of Alberix (ABT) and anti-HIV antibodies respectively, but also extend the half-life of ABT, realize the long-term antiviral activity of the drug while achieving the dual-drug antiviral spectrum, and increase patient compliance.

[0007] In the first aspect of the present invention, there is provided an antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof. The antibody-polypeptide conjugate includes an anti-HIV antibody and a polypeptide linked thereto. The polypeptide is Alberix (ABT), and the structure of the antibody-polypeptide conjugate is shown in Formula I:

[0008] Ab-(J-U)n (Ⅰ)

[0009] In the formula,

[0010] Ab is an anti-HIV antibody;

[0011] Each U is independently a polypeptide chain of ABT, which has the amino acid sequence shown in SEQ ID NO:3;

[0012] J is a linker in ABT: AEEA-MPA;

[0013] n is 0 or a positive integer;

[0014] "-" is a chemical bond.

[0015] In another preferred example, the anti-HIV antibody includes a monospecific antibody, a bispecific antibody, a multispecific antibody (such as a trispecific antibody).

[0016] In another preferred example, the anti-HIV antibody includes: a monoclonal antibody, a single-chain antibody (scFv), a nanobody.

[0017] In another preferred example, the anti-HIV antibody includes a monovalent, divalent or multivalent antibody.

[0018] In another preferred example, the anti-HIV antibody includes an antibody in a multimeric form.

[0019] In another preferred example, the anti-HIV antibody specifically binds to HIV.

[0020] In another preferred example, the anti-HIV antibody includes a HIV monovalent antibody, a divalent antibody and / or a multivalent antibody.

[0021] In another preferred example, the HIV is human HIV or HIV of a non-human mammal (such as mouse HIV).

[0022] In another preferred example, the anti-HIV antibody is a human or non-human mammalian antibody.

[0023] In another preferred example, the non-human mammal is selected from the group consisting of: camel, alpaca, mouse, cynomolgus monkey.

[0024] In another preferred example, the anti-HIV antibody is an anti-HIV antibody or a derivative antibody thereof.

[0025] In another preferred example, the derivative antibody is a modification of the anti-HIV antibody, including but not limited to linking the HIV antibody to an Fc fragment, human serum albumin, polyethylene glycol PEG, forming a divalent antibody and / or a multivalent antibody.

[0026] In another preferred embodiment, the anti-HIV antibody includes a humanized antibody, a camel-derived antibody, and a chimeric antibody.

[0027] In another preferred embodiment, the anti-HIV antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2; or

[0028] the anti-HIV antibody comprises a heavy chain as shown in SEQ ID NO: 4 and a light chain as shown in SEQ ID NO: 2; or a mutant antibody thereof.

[0029] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified, and / or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to bind to HIV.

[0030] In another preferred embodiment, the anti-HIV antibody sequence comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0031] In another preferred embodiment, the mutant antibody is 10-1074 or VRC01.

[0032] In another preferred embodiment, the ABT is linked to the terminal amino group or side chain amino group of the heavy chain constant region or the heavy chain variable domain of the anti-HIV antibody.

[0033] In another preferred embodiment, the ABT is linked to the thiol group of the anti-HIV antibody.

[0034] In another preferred embodiment, the ABT is linked to the anti-HIV antibody at a specific site and / or randomly (i.e., in formula I, the U is linked to Ab at a specific site and / or randomly).

[0035] In another preferred embodiment, the U is linked to Ab at a specific site.

[0036] In another preferred embodiment, the chemical bond couples the anti-HIV antibody and the ABT through the 13th Lys of the polypeptide chain shown in SEQ ID NO: 3.

[0037] In another preferred embodiment, the degree of polymerization of the chemical bond is a positive integer greater than or equal to 1.

[0038] In a second aspect of the present invention, there is provided a pharmaceutical composition for treating and / or preventing HIV-related diseases, the pharmaceutical composition comprising:

[0039] (a) An antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention;

[0040] (b) A pharmaceutically acceptable carrier.

[0041] In another preferred embodiment, the pharmaceutical composition further comprises:

[0042] (c) Other bioactive drugs, such as drugs for treating HIV-related diseases.

[0043] In another preferred embodiment, the pharmaceutical composition further includes other therapeutic agents for treating and / or preventing AIDS or HIV-related diseases.

[0044] In another preferred embodiment, the pharmaceutical composition is a single-agent drug, a combination drug, or a synergistic drug.

[0045] In another preferred embodiment, the administration mode of the pharmaceutical composition is selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or nasal and oral spray and aerosol inhalation.

[0046] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: liquid, solid, or gel state.

[0047] In another preferred embodiment, the pharmaceutical composition is used for treating and / or preventing HIV-related diseases.

[0048] In another preferred embodiment, the HIV is of the HIV-1 serotype.

[0049] In another preferred embodiment, the HIV is selected from the group consisting of: AC10.0.29, CH110, CNE15, CAAN5342, or a combination thereof, preferably AC10.0.29.

[0050] The third aspect of the present invention provides a method for preparing an antibody-polypeptide conjugate as described in the first aspect of the present invention, the method comprising the steps of:

[0051] (1) Reacting an anti-HIV antibody with a reducing reagent in a buffer to obtain a reduced anti-HIV antibody;

[0052] (2) Then adding ABT to the reduced anti-HIV antibody for a cross-linking reaction to obtain the antibody-polypeptide conjugate.

[0053] In another preferred embodiment, the antibody in step (1) is reduced by a reducing reagent, so that the inter-chain disulfide bonds of the antibody are reduced to generate thiol groups.

[0054] In another preferred embodiment, the reducing agent in step (1) is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or dithiothreitol (DTT).

[0055] In a fourth aspect of the present invention, there is provided an application of an antibody-polypeptide conjugate as described in the first aspect of the present invention, or a pharmaceutical composition as described in the second aspect of the present invention, in the preparation of a drug for treating and / or preventing HIV-related diseases.

[0056] In another preferred embodiment, the HIV is of the HIV-1 serotype.

[0057] In another preferred embodiment, the HIV is selected from the group consisting of AC10.0.29, CH110, CNE15, CAAN5342, or a combination thereof, preferably AC10.0.29.

[0058] In a fifth aspect of the present invention, there is provided a kit, which comprises:

[0059] (1) a first container, and an anti-HIV antibody located within the first container, and a pharmaceutically acceptable carrier;

[0060] (2) a second container, and ABT located within the second container, and a pharmaceutically acceptable carrier;

[0061] and (3) an optional instruction manual.

[0062] In a sixth aspect of the present invention, there is provided a method for treating and / or preventing HIV-related diseases, comprising: administering to a subject in need an antibody-polypeptide conjugate drug as described in the first aspect of the present invention or a pharmaceutical composition as described in the second aspect of the present invention.

[0063] In another preferred embodiment, the subject in need includes humans and non-human mammals.

[0064] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Shows the HIC detection chromatogram of APC conjugate 1 in Example 1 of the present invention.

[0066] Figure 2 Shows the HIC detection chromatogram of APC conjugate 2 in Example 1 of the present invention.

[0067] Figure 3Shows the inhibitory activity (nM) of APC conjugate 1 against recombinant virus AC10.0.29 in Example 2 of the present invention.

[0068] Figure 4 Shows the inhibitory activity (nM) of APC conjugate 1 against recombinant virus CH110 in Example 2 of the present invention.

[0069] Figure 5 Shows the inhibitory activity (nM) of APC conjugate 1 against recombinant virus CAAN5342 in Example 2 of the present invention.

[0070] Figure 6 Shows the inhibitory activity (nM) of APC conjugate 1 against recombinant virus CNE15 in Example 2 of the present invention.

[0071] Figure 7 Shows the anti-HIV antibody 1 and ABT plasma concentration-time curves after intravenous injection of APC conjugate 1 in SD rats in Example 3 of the present invention.

[0072] Figure 8 Shows the plasma concentration-time curve of anti-HIV antibody 1 after injection of APC conjugate 1 and anti-HIV antibody 1 in SD rats in Example 3 of the present invention.

[0073] Figure 9 Shows the ABT plasma concentration-time curve after injection of APC conjugate 1 and ABT in SD rats in Example 3 of the present invention. Detailed implementation mode

[0074] The inventors have, through extensive and in-depth research, for the first time screened and identified an anti-HIV antibody and developed an anti-HIV antibody and ABT polypeptide conjugate. Specifically, the dual-target antibody conjugate of the present invention has excellent anti-HIV virus activity. The HIV antibody and ABT polypeptide conjugate developed by the present invention exhibits outstanding anti-HIV effects and a novel mechanism of action, and has clinical development and application value. The present invention has been completed on this basis.

[0075] Terms

[0076] Antibody of the present invention

[0077] As used herein, the terms "antibody of the present invention", "HIV-targeting antibody of the present invention", and "anti-HIV antibody of the present invention" are used interchangeably and all refer to antibodies that specifically recognize and bind to HIV (including human or murine HIV). Particularly preferred is an antibody (anti-HIV antibody 1) whose amino acid sequence of the heavy chain is as shown in SEQ ID NO: 1 and the light chain sequence is as shown in SEQ ID NO: 2.

[0078] As used herein, the terms "antibody-drug conjugate of the present invention", "dual-targeting antibody-drug conjugate of the present invention", and "HIV antibody and ABT polypeptide conjugate of the present invention" are used interchangeably and all refer to a novel drug molecule formed by conjugating an antibody that specifically recognizes and binds to HIV (including human or murine HIV) and its derivative proteins with an ABT polypeptide. The antibody in the antibody-drug conjugate is particularly preferably an antibody (anti-HIV antibody 1) whose amino acid sequence of the heavy chain is as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain is as shown in SEQ ID NO: 2.

[0079] It should be understood that the HIV antibodies of the present invention are other antibodies with similar functions, such as antibody 10-1074, VRC01, etc. (see Cristina Possas, et al. HIV cure: global overview of bNAbs' patents and related scientific publications. Expert Opin Ther Pat. 2018 Jul; 28(7): 551-560.).

[0080] In another preferred embodiment of the present invention, the antibody is a mutant antibody obtained by mutating on the basis of anti-HIV antibody 1, hereinafter referred to as "molecule J" or "anti-HIV antibody 2", whose amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4 and the amino acid sequence of the light chain is as shown in SEQ ID NO: 2.

[0081] The "anti-HIV antibodies" in the present invention include "anti-HIV antibody 1" and "anti-HIV antibody 2" as described above and are prepared by a series of biotechnologies. Specifically, single B lymphocytes of patients are sorted by flow cytometry to obtain antibody sequences, the mRNA of monoclonal antibodies is obtained from single B lymphocytes, reverse transcribed into cDNA by PCR technology, and then the DNA sequences of the heavy and light chains of monoclonal antibodies are obtained by sequencing technology. The heavy and light chain DNAs are synthesized by gene synthesis technology, and then the heavy and light chain DNAs are cloned into expression vectors respectively by restriction enzyme digestion and ligation technology to prepare expression plasmids. The expression plasmids are transfected into CHO stable cell lines, and the heavy and light chains are recombined into the CHO cell genome by MSX pressure screening to construct a stable expression cell line. The master cell bank and working cell bank are constructed by cell passage culture technology. Upstream and downstream process development are carried out successively to optimize the upstream cell culture conditions and downstream purification conditions for antibody expression. After a series of method validations and microbial validations, aseptic preparation filling is carried out.

[0082] As used herein, the terms "antibody" or "immunoglobulin" refer to a heterotetrameric glycoprotein of approximately 150,000 daltons having the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a plurality of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0083] As used herein, the term "variable" indicates that certain portions of the variable regions in an antibody differ in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable regions are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally in a β-sheet conformation and are connected by three CDRs that form connecting loops and can form a partial β-sheet structure in some cases. The CDRs in each chain are brought closely together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell cytotoxicity.

[0084] As is known to those skilled in the art, immunoconjugates and fusion expression products include: conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention.

[0085] As used herein, the terms "antibody-drug conjugate" and "antibody-drug conjugate" are used interchangeably. As is known to those skilled in the art, an antibody conjugate is a special form of antibody-drug conjugate, which is a drug molecular form formed by conjugating an antibody or a derivative protein with a drug, toxin, cytokine, radionuclide, enzyme, and other diagnostic or therapeutic molecules, and can be used for antiviral therapy, tumor therapy, drug delivery, and in vivo imaging, etc., and has broad clinical application value.

[0086] As used herein, the term "heavy chain variable region" and "V H " are used interchangeably.

[0087] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0088] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0089] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and a heavy chain constant region.

[0090] Furthermore, in the present invention, the Fc segment of the antibody can be wild-type or mutant, for example, containing mutations such as LS, so as to extend the half-life and / or increase the ADCC / CDC activity.

[0091] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to HIV, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0092] The present invention also provides other proteins or fusion expression products having the antibody of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is the same as or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present invention.

[0093] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called variable regions (CDR), which divide this segment into four framework regions (FR). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures, and the β-sheets formed by the FRs in between are close to each other in the spatial structure. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

[0094] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least some of them are involved in antigen binding. Therefore, the present invention includes those molecules having an antibody heavy chain variable region with CDRs, as long as the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0095] The present invention includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of the said antibodies.

[0096] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0097] The antibodies of the present invention refer to polypeptides having HIV-binding activity and including the above-mentioned CDR regions. This term also includes variant forms of polypeptides containing the above-mentioned CDR regions and having the same function as the antibodies of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or comparable properties, the function of the protein is usually not changed. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0098] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the DNA encoding the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using the antiserum against the antibodies of the present invention.

[0099] The present invention also provides other polypeptides, such as fusion proteins comprising an antibody or a fragment thereof. In addition to the almost full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Generally, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0100] In the present invention, the antibodies of the present invention also contain conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or close properties compared with the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0101] Table 1

[0102]

[0103]

[0104] The present invention also provides polynucleotide molecules encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0105] The polynucleotides encoding the antibodies of the present invention include: coding sequences encoding only the antibodies of the present invention; coding sequences of the antibodies of the present invention and various additional coding sequences; coding sequences of the antibodies of the present invention (and optional additional coding sequences) and non-coding sequences.

[0106] The term "polynucleotide encoding a polypeptide" can be a polynucleotide including the polynucleotide encoding this polypeptide, or can also be a polynucleotide further including additional coding and / or non-coding sequences.

[0107] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0108] The full-length nucleotide sequence or a fragment thereof of the antibody of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Generally, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0109] Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0110] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0111] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0112] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0113] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well-known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well-known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0114] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell. After the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0115] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0116] The antibodies of the present invention can be used alone or in combination or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or any combination of the above substances.

[0117] Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0118] Therapeutic agents that can be bound or conjugated to the antibodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Drug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. Chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0119] Linker

[0120] According to the mechanism of drug release in cells, the "linker" or "linker of antibody-drug conjugate" can be divided into two categories: non-cleavable linkers and cleavable linkers.

[0121] For antibody-drug conjugates containing non-cleavable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed into the cell, the antibody is enzymatically degraded in the lysosome, releasing an active molecule composed of a small molecule drug, a linker, and an antibody amino acid residue. The resulting change in the drug molecular structure does not weaken its cytotoxicity, but since the active molecule is charged (amino acid residue), it cannot penetrate into neighboring cells. Therefore, such active drugs cannot kill tumor cells (bystander effect) that do not express the target antigen (antigen-negative cells) in the vicinity.

[0122] A cleavable linker, as the name implies, can be cleaved within the target cell to release the active drug (the small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically labile linkers and enzymatically labile linkers. Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, etc. pH-sensitive linkers are usually also called acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of the blood (pH 7.3 - 7.5), but will be hydrolyzed within weakly acidic endosomes (pH 5.0 - 6.5) and lysosomes (pH 4.5 - 5.0). Most of the first-generation antibody-drug conjugates used such linkers, such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on such linkers usually have a short half-life (2 - 3 days). This short half-life has limited the application of pH-sensitive linkers in new-generation antibody-drug conjugates to a certain extent.

[0123] For glutathione-sensitive linkers, also known as disulfide bond linkers. Drug release is caused by the difference in the high concentration of glutathione (in the millimolar range) within the cell and the relatively low concentration of glutathione (in the micromolar range) in the blood. This is especially true for tumor cells, whose low oxygen content leads to enhanced activity of reductases, thus resulting in a higher glutathione concentration. Disulfide bonds have thermodynamic stability and therefore have good stability in plasma.

[0124] Enzyme-labile linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as Cathepsin B or plasmin (the content of such enzymes increases in some tumor tissues). This peptide linkage is considered to be very stable in plasma circulation because the inappropriate extracellular pH value and serum protease inhibitors render proteases usually inactive. Given the high plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme-labile linkers include Val-Cit (VC), Phe-Lys, etc.

[0125] Self-immolative linkers are generally embedded between the cleavable linker and the active drug, or are themselves part of the cleavable linker. The mechanism of action of self-immolative linkers is that when the cleavable linker breaks under appropriate conditions, the self-immolative linker can spontaneously rearrange its structure and then release the active drug linked thereto. Common self-immolative linkers include p-aminobenzyl alcohol (PAB) and β-glucuronide, etc.

[0126] The present invention provides a linker or coupling reagent comprising a diarylthio maleimide unit and a coupling group. The diarylthio maleimide unit is used to crosslink the thiol groups (after reduction) between antibody chains, while the coupling group is used to couple with a drug or a drug-linker unit. Due to the bidentate binding of the diarylthio maleimide unit to the two sulfur atoms of the open cysteine-cysteine disulfide bond in the antibody, these ADCs are homogeneous and have stronger stability than ADCs containing monodentate linkers. Therefore, they will have an increased in vivo half-life, reduce the amount of cytotoxins released systemically, and have more favorable drug properties than ADCs with monodentate linkers.

[0127] In another aspect, the resulting drug-linker unit is conjugated to the antibody through the linker to generate a partially interchain-crosslinked conjugate. Compared with traditional antibody-drug conjugates, the drug / antibody ratio (DAR) distribution of the antibody-drug conjugates prepared by the method of the present invention is narrower, thereby greatly improving the product homogeneity and the homogeneity of pharmacological properties. The antibody-drug conjugate can be used for targeted delivery of drugs to target cell populations, such as tumor cells. The antibody-drug conjugate can specifically bind to cell surface proteins, and the resulting conjugate is then endocytosed by the cell. Inside the cell, the drug is released in the form of an active drug to produce an effect. Antibodies include chimeric antibodies, humanized antibodies, human antibodies; antibody fragments capable of binding to antigens; or antibody Fc fusion proteins; or proteins. "Drug" is a highly active drug (see the definition section), and in some cases, the drug can be polyethylene glycol.

[0128] The antibody-polypeptide conjugate (APC) of the present invention

[0129] As used herein, the terms "conjugate of the present invention", "APC conjugate of the present invention", and "antibody-polypeptide conjugate of the present invention" are used interchangeably and all refer to the conjugate formed by an anti-HIV antibody and albiglutide (ABT). In a preferred embodiment of the present invention, the anti-HIV antibody is anti-HIV antibody 1, and the conjugate formed by it and ABT is APC conjugate 1. In another preferred embodiment of the present invention, the anti-HIV antibody is molecule J, i.e., a mutant antibody of anti-HIV antibody 1 (anti-HIV antibody 2), and the conjugate formed by it and ABT is molecule J-ABT (APC conjugate 2).

[0130] The present invention provides an antibody-polypeptide conjugate obtained by conjugating a monoclonal antibody (such as anti-HIV antibody 1) with albiglutide (ABT) through a chemical linker formed by a free sulfhydryl group after partial reduction of the antibody part and a maleimide group on a lysine side chain of ABT.

[0131] In the present invention, anti-HIV antibody 1 has the following heavy and light chains of the antibody:

[0132] Heavy chain of anti-HIV antibody 1 (anti-HIV antibody 1HC):

[0133] QVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVWGSGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1)

[0134] Light chain of anti-HIV antibody 1 (anti-HIV antibody 1 LC):

[0135] DIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2)

[0136] In the present invention, the anti-HIV antibody 1 mutant antibody, namely molecule J (anti-HIV antibody 2), has the following heavy and light chains of the antibody:

[0137] Heavy chain of molecule J, which has two amino acid mutations, M434L and N440S, relative to the heavy chain of anti-HIV antibody 1 (SEQ ID NO.1):

[0138] QVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVWGSGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV L HEALH S HYTQKSLSLSPGK (SEQ ID NO:4)

[0139] Light chain of molecule J:

[0140] DIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:2)

[0141] Aibovirtide (ABT) is a polypeptide used for treating HIV-related diseases and belongs to an HIV-1 fusion inhibitor. It acts on the outer membrane glycoprotein gp41 of HIV-1 to prevent the virus from entering cells. The ABT includes a polypeptide chain, and the polypeptide chain is a polypeptide having the following sequence:

[0142] WEEWDREINNYTKLIHELIEESQNQQEKNEQELL(SEQ ID NO:3);

[0143] Aibovirtide (ABT) is a polypeptide in which Lys at the 13th position of the polypeptide chain is attached with an AEEA-MPA linker, and its chemical structural formula is shown as Formula A:

[0144]

[0145] Typically, the antibody-drug conjugate includes the antibody and an effector molecule. The antibody is conjugated with the effector molecule, and preferably, it is chemically conjugated. Among them, the effector molecule is preferably a drug with therapeutic activity or a drug with immune function promoting activity.

[0146] The antibody of the present invention and the effector molecule can be conjugated through a coupling agent. Examples of the coupling agent can be any one or several of a non-selective coupling agent, a coupling agent using carboxyl groups, a peptide chain, and a coupling agent using disulfide bonds. The non-selective coupling agent is a compound that forms a covalent bond connection between the effector molecule and the antibody, such as glutaraldehyde, etc. The coupling agent using carboxyl groups can be any one or several of cis-aconitic anhydride coupling agents (such as cis-aconitic anhydride) and acylhydrazone coupling agents (the coupling site is acylhydrazone).

[0147] Certain residues on the antibody (such as Cys or Lys, etc.) are used to be linked to a variety of functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly linked to the antibody through a linker.

[0148] The antibody can be conjugated with a drug to form an antibody-drug conjugate (ADCs). Typically, an ADC contains a linker located between the drug and the antibody. The linker can be a degradable or non-degradable linker. The degradable linker typically degrades easily in the intracellular environment, for example, the linker degrades at the target site, so that the drug is released from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by proteases.

[0149] Before being linked to the antibody, the linker has reactive functional groups capable of reacting with certain amino acid residues, and the linkage is achieved through the reactive functional groups. Thiol-specific reactive functional groups are preferred and include, for example, maleimide compounds, haloamides (such as iodine, bromine, or chlorine-substituted), haloesters (such as iodine, bromine, or chlorine-substituted), halomethyl ketones (such as iodine, bromine, or chlorine-substituted), benzyl halides (such as iodine, bromine, or chlorine-substituted), vinyl sulfones, pyridyl disulfides, mercury derivatives such as 3,6-di-(mercurimethyl) dioxane, and the counterions are acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonates. The linker can include, for example, maleimide linked to the antibody through succinimide.

[0150] It should be understood that the drug can generally be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In the present invention, the drug is a drug that activates or promotes an immune response, for example, activating the innate immune response to assist the activation of the adaptive immune response. In a specific embodiment, the drug is a TLR receptor agonist.

[0151] In an embodiment, the linker connects an antibody and a drug, and the drug has a functional group capable of bonding to the linker. For example, the drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a keto group capable of bonding to the linker. When the drug is directly linked to the linker, the drug has a reactive functional group before being linked to the antibody.

[0152] In the present invention, the drug-linker can be used to form an ADC in a single simple step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step method. For example, a cysteine residue reacts with a reactive moiety of the linker in the first step, and in a subsequent step, a functional group on the linker reacts with the drug to form an ADC.

[0153] Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive functional group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkyne group on the drug moiety. The drug is covalently bound to the linker through a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other conjugation strategies, such as those described in Bioconjugate Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for either the linker or the drug.

[0154] The present invention also provides a method for preparing an APC conjugate, which may further include: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (APC).

[0155] In certain embodiments, the method of the present invention includes: combining an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further includes: combining the antibody-linker conjugate with a polypeptide moiety under conditions sufficient to covalently link the polypeptide moiety to the antibody through the linker.

[0156] In some embodiments, the antibody-polypeptide conjugate APC includes an anti-HIV antibody and a polypeptide linked thereto, the polypeptide being Albiglutide (ABT), and the structural formula of the antibody-polypeptide conjugate is as shown in the following molecular formula:

[0157] Ab-(J-U)n (Ⅰ)

[0158] In the formula,

[0159] Ab is an anti-HIV antibody;

[0160] Each U independently is a polypeptide chain of ABT;

[0161] J is a linker in ABT: AEEA-MPA;

[0162] n is 0 or a positive integer;

[0163] "-" is a chemical bond.

[0164] In the present invention, the chemical bond preferably couples the anti-HIV antibody with ABT through Lys at the 13th position of the polypeptide chain shown in SEQ ID NO:3.

[0165] In the present invention, a preferred antibody is anti-HIV antibody 1.

[0166] Application

[0167] The present invention also provides the use of the antibody of the present invention and the APC conjugate containing the antibody of the present invention, for example, for preparing a diagnostic agent, or for preparing a drug for preventing and / or treating HIV-related diseases.

[0168] Pharmaceutical composition

[0169] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier or excipient, and optionally other bioactive substances. Usually, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0170] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the antibody (or its conjugate) of the present invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. In addition, the APC conjugate of the present invention can also be used together with other therapeutic agents.

[0171] In another embodiment of the present invention, the APC conjugate of the present invention can be co-administered with other HIV therapeutic drugs to prevent the replication of HIV (including HIV-1, HIV-2, and all other serotypes thereof) or SIV virus particles in the patient's body.

[0172] In another embodiment of the present invention, the APC conjugate of the present invention is co-administered with one or more additional compounds for the treatment and / or prevention of HIV or HIV-induced diseases. These other drugs that can be co-administered with the APC conjugate include, but are not limited to, trimetrexate glucuronate (for the treatment of Pneumocystis carinii pneumonia); ganciclovir (for the treatment of cytomegalovirus retinitis); aerosolized pentamidine (for the treatment of Pneumocystis carinii pneumonia); erythropoietin (for the treatment of zidovudine-related anemia); atovaquone (for the treatment of Pneumocystis carinii pneumonia); rifabutin (for the treatment of Mycobacterium avium); V1STIDE (for the treatment of recurrent cytomegalovirus retinitis); and SEROSTIM (for the treatment of AIDS-related wasting).

[0173] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight - about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are within the scope of the skills of a skilled physician.

[0174] Preparation method of the antibody-polypeptide conjugate

[0175] The present invention also provides a method for preparing the antibody-polypeptide conjugate described in the first aspect of the present invention: the inter-chain disulfide bonds of the antibody are reduced to generate 2n (such as 4) thiol groups; the substituted maleimide linker-polypeptide conjugate (i.e., ABT) of the present invention is cross-linked with the reduced thiol groups of the antibody to generate the corresponding antibody-polypeptide conjugate.

[0176] Specifically, the method includes the steps:

[0177] (1) React an anti-HIV antibody with a reducing reagent in a buffer solution to obtain a reduced anti-HIV antibody;

[0178] (2) Then add ABT to the reduced anti-HIV antibody for cross-linking reaction to obtain the antibody-polypeptide conjugate.

[0179] In another preferred example, the antibody in step (1) is reduced by a reducing reagent, so that the inter-chain disulfide bonds of the antibody are reduced to generate thiol groups.

[0180] In another preferred example, the reducing reagent in step (1) is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or dithiothreitol (DTT).

[0181] The main advantages of the present invention include:

[0182] (a) Since Aplevirate and the anti-HIV antibody act on gp41 and gp120 of the viral glycoprotein at the stage of HIV virus entering human T cells respectively, gp41 and gp120 form gp160. First, the gp120 part of HIV binds to human CD4, triggering binding to the co-receptor in the human body, resulting in conformational changes, dissociation of gp120 from gp41, full exposure of gp41 and formation of a trimeric transition state, and then fusion to form a hexameric virus, which successfully enters human T cells;

[0183] The present invention forms a targeted antibody-polypeptide conjugate by conjugating Aplevirate acting on gp41 with an anti-HIV antibody acting on gp120, which is beneficial to improving the defect of insufficient virus coverage of the antibody or ABT. And experiments show that the conjugate has a single-molecule (bimolecular splicing) dual mechanism, retains their respective antiviral activities, realizes a dual-drug antiviral spectrum, and expands the broad-spectrum nature of drugs for treating and / or preventing HIV-related diseases;

[0184] (b) And experiments show that the APC of the present invention does not affect the pharmacokinetics of the anti-HIV antibody while prolonging the half-life of ABT, realizing the long-acting nature of drugs for treating and / or preventing HIV-related diseases.

[0185] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0186] Example 1 Preparation of the APC conjugate of the present invention

[0187] 1. Sample preparation

[0188] 1.1 Conjugation of anti-HIV antibody 1 with ABT (APC conjugate 1)

[0189] 1.1.1 Conjugation steps

[0190] a) Take an appropriate amount of anti-HIV antibody 1 from 2 - 8°C, dilute it 1-fold with D-PBS. After dilution, add the reducing agent TCEP for reduction reaction;

[0191] b) Add 3-fold molar equivalent ratio of the reducing agent TCEP and react at 37°C for 1.5 h;

[0192] c) After the reduction reaction is completed, add an ABT solution with a molar ratio of 1:10 to the reduction reaction system and react at room temperature for 2 h;

[0193] d) Take a sample for HIC detection and analyze the DAR value.

[0194] 1.1.2 Purification steps

[0195] Use a 30Kda ultrafiltration tube to ultrafilter 12 ml of the sample each time. After all the samples are ultrafiltered, use D-PBS as the buffer and ultrafilter 12 times to analyze the DAR, purity, and ABT residue of the conjugate product.

[0196] 1.2 Conjugation of molecule J (anti-HIV antibody 2) with ABT (APC conjugate 2)

[0197] 1.2.1 Conjugation steps

[0198] Operate according to the same method as in step 1.1.1 above, except that anti-HIV antibody 1 is replaced with anti-HIV antibody 2.

[0199] 1.2.2 Purification steps

[0200] Purify according to the same method as in step 1.1.2 above.

[0201] 2. HIC Detection

[0202] The prepared APC conjugate was detected by hydrophobic interaction chromatography.

[0203] The HIC detection chromatogram of APC conjugate 1 is as shown in Figure 1 and that of APC conjugate 2 is as shown in Figure 2 shown. The number of conjugated drugs (DAR value) is 0, 2, 4, 6, 8 respectively, denoted as D0, D2, D4, D6, D8.

[0204] Example 2 Verification of the in vitro activity of the APC conjugate of the present invention

[0205] The APC conjugate 1 and APC conjugate 2 prepared in Example 1 were used for in vitro antiviral activity determination.

[0206] 2.1 Anti-HIV-1 virus AC10.0.29 and CH110 activities of APC conjugate 1

[0207] Brief introduction of the antiviral test procedure is as follows: The drug was serially diluted 3-fold in a 96-well cell culture plate, 50 μL / well, with 3 replicates and 9 gradients set; 50 μL of recombinant virus (100 TCID50) was added to the culture wells and placed at 37 °C for 60 min; then 100 μL of 1×10 4 TZM-bl cells were added to each well. After adding DEAE dextran at a final concentration of 15 μg / ml, the cells were incubated in a 37 °C, 5% CO2 cell culture incubator for about 48 h. After carefully discarding the culture medium, each well was lysed with 30 μl of cell lysis solution at room temperature for 15 min, and then 100 μL / well of Luciferaese Assay reagent was added. After thorough mixing, 100 μl was taken and transferred to a 96-well white plate. The RLU was measured using a chemiluminescence detector, and the IC50, IC90 values of the drug and the drug inhibition curve were calculated using the sigmoidal dose-response formula in non-linear regression analysis with GraphPad Prism Software.

[0208] Using APC conjugate 1 for in vitro antiviral experiments on recombinant HIV-1 viruses AC10.0.29 and CH110 according to the above method, the IC 50 values were determined, and at the same time, parallel experimental groups using anti-HIV antibody 1, ABT-HSA and ABT were used as controls. A total of 3 repeated experiments were carried out, and the results are shown in Table 2 - Table 4. The summary of the results of the 3 experiments is shown in Table 5, as well as Figure 3 and Figure 4 shown.

[0209] Table 2 Inhibitory Activity IC of Samples against Recombinant HIV-1 Virus 50 - nM (Results of the First Test)

[0210]

[0211] Table 3 Inhibitory Activity IC of Samples against Recombinant HIV-1 Virus 50 - nM (Results of the Second Test)

[0212]

[0213] Table 4 Inhibitory Activity IC of Samples against Recombinant HIV-1 Virus 50 - nM (Results of the Third Test)

[0214]

[0215] Table 5 Inhibitory Activity IC50 of Samples against Recombinant HIV-1 Virus nM (Summary of Results of Three Tests)

[0216]

[0217] In addition, the IC of the APC conjugate 1 against the recombinant HIV-1 viruses AC10.0.29 and CH110 in vitro was also determined 90 values. A total of 3 replicate experiments were carried out, and parallel experimental groups using anti-HIV antibody 1, ABT-HSA, and ABT were used as controls at the same time. The results are shown in Tables 6 - 8. The summary of the results of the 3 experiments is shown in Table 9

[0218] Table 6 Inhibitory Activity IC of Samples against Recombinant HIV-1 Virus 90 - nM (Results of the First Test)

[0219]

[0220] Table 7 Inhibitory Activity IC of Samples against Recombinant HIV-1 Virus 90 -- nM (Results of the Second Test)

[0221]

[0222] Table 8 Inhibitory Activity IC of Samples against Recombinant HIV-1 Virus 90 - nM (Results of the Third Test)

[0223]

[0224] Table 9 Inhibitory activity IC of the sample against the HIM-1 recombinant virus 90 - nM (Summary of the results of 3 tests)

[0225]

[0226] The above results show that for the strain CH110 which is sensitive to both ABT and anti-HIV antibody 1, APC conjugate 1 shows the inhibitory activity of the higher one of the two;

[0227] For the strain AC10.0.29 which is not sensitive to anti-HIV antibody 1 but sensitive to ABT, APC conjugate 1 shows the activity of ABT;

[0228] The virus inhibitory activity of APC conjugate 1 is overall superior to that of ABT-HSA (the in vivo active form of ABT).

[0229] 2.2 Anti-HIV-1 virus CNE15 and CAAN5342 activities of APC conjugate 1

[0230] In vitro antiviral experiments were carried out on the recombinant HIV-1 viruses CNE15 and CAAN5342 using APC conjugate 1 to determine the IC 50 value, and at the same time, parallel experimental groups using anti-HIV antibody 1, ABT-HSA and ABT were used as controls. A total of 3 repeated experiments were carried out, and the results are shown in Table 10 - Table 12. The summary of the results of the 3 experiments is shown in Table 13, and Figure 5 , Figure 6 as shown.

[0231] Table 10 Inhibitory activity IC50 - nM of the sample against the HIV-1 recombinant virus (Results of the 1st test)

[0232]

[0233] Table 11 Inhibitory activity IC50 - nM of the sample against the HIV-1 recombinant virus (Results of the 2nd test)

[0234]

[0235] Table 12 Inhibitory activity IC50 - nM of the sample against the HIV-1 recombinant virus (Results of the 3rd test)

[0236]

[0237] Table 13 Inhibitory activity IC50 - nM of the sample against the HIV-1 recombinant virus (Summary of the results of 3 tests)

[0238]

[0239] The above results indicate that for the strain CAAN5342 that is insensitive to ABT but sensitive to anti-HIV antibody 1, APC conjugate 1 shows the activity of anti-HIV antibody 1;

[0240] For the strain CNE15 that is insensitive to anti-HIV antibody 1 but sensitive to ABT, APC conjugate 1 shows the activity of ABT;

[0241] The virus inhibitory activity of APC conjugate 1 is overall superior to that of ABT-HSA (the in vivo active form of ABT).

[0242] 2.3 Activity of molecule J-ABT against HIV-1 viruses CNE15 and CAAN5342

[0243] An in vitro antiviral experiment was conducted on recombinant HIV-1 viruses CNE15 and CAAN5342 using APC conjugate 2 (molecule J-ABT) to determine the IC 50 and IC 90 values, and the parallel experimental groups using anti-HIV antibody 1, molecule J, ABT, and APC conjugate 1 were used as controls. The experiment was repeated three times, and the results of the three experiments were summarized. The results are shown in Tables 14 and 15.

[0244] Table 14 Inhibitory activity IC 50 (nM)

[0245]

[0246] Table 15 Inhibitory activity IC 90 (nM)

[0247]

[0248] As can be seen from the above Tables 14 - 15, for the strain CAAN5342 that is insensitive to ABT but sensitive to anti-HIV antibody 1, molecule J-ABT shows the anti-HIV virus activity of anti-HIV antibody 1; for the strain CNE15 that is insensitive to anti-HIV antibody 1 but sensitive to ABT, molecule J-ABT shows the anti-HIV virus activity of ABT.

[0249] Example 3 Pharmacokinetic study of APC conjugate 1 of the present invention in rats

[0250] Test article: 12 mg / mL APC conjugate 1;

[0251] Route of administration: Slowly inject intravenously after sterilization through a 0.22 μm filter membrane, and the administration time is at least 1 minute;

[0252] Frequency of administration: Single administration;

[0253] Administration volume: 5 mL / kg;

[0254] Administration site: caudal vein;

[0255] Sampling time: before administration, 5 m (±1 minute), 4 h (±5 minutes), 8 h (±5 minutes), 24 h (±30 minutes), 48 h, 72 h, 7 d (168 h), 14 d (336 h), 21 d (504 h), 28 d (672 h), 35 d (840 h), 42 d (1008 h), 49 d (1176 h), 56 d (1344 h) after administration;

[0256] Blood sample treatment: Whole blood samples are placed in an ice box before centrifugation, centrifuged at 2 - 8°C and a centrifugal force of 1800×g for 10 minutes to separate 2 tubes of plasma. One tube contains 50 μL, and the remaining plasma is placed in the other tube. After sample aliquoting, it is stored at -66°C or below for testing.

[0257] The dosing regimen is shown in the following table:

[0258] Table 16 Dose Design

[0259]

[0260] Bioanalysis: There is no method to detect the anti-HIV antibody 1-ABT conjugate. Therefore, the ELISA method is used to detect the anti-HIV antibody 1 and ABT drug concentrations in plasma samples respectively, indirectly reflecting the drug content of the APC conjugate 1.

[0261] The results are shown in Tables 17 - 21, and Figure 7 as follows.

[0262] Table 17 and Figure 7 are the summary results of the anti-HIV antibody 1 and ABT drug concentrations (μM) in plasma samples after intravenous injection of 60 mg / kg of APC conjugate 1 to 4 SD rats. Tables 18 and 19 show the specific data of the ABT drug concentration in plasma samples of 4 rats (2 males and 2 females) after intravenous injection of APC conjugate 1, presented in μM and μg / mL respectively; Tables 20 and 21 show the specific data of the anti-HIV antibody 1 drug concentration in plasma samples of 4 rats (2 males and 2 females) after intravenous injection of APC conjugate 1, presented in μM and μg / mL respectively.

[0263] Table 17 Anti-HIV antibody 1 and ABT drug concentrations in SD rats after injection of APC conjugate 1 ( μM )

[0264]

[0265] Table 18 ABT Plasma Drug Concentrations in sD Rats after Intravenous Injection of 60 mg / kg APC Conjugate 1 μM )

[0266]

[0267] Note: In the table, 1M01, 1M02, 1F01, and 1F02 represent the experimental rat groups, M = Male, F = Female, and 01 and 02 represent the rat numbers; BLQ: Below the limit of quantification.

[0268] Table 19 ABT Plasma Drug Concentrations (μg / mL) in SD Rats after Intravenous Injection of 60 mg / kg APC Conjugate 1

[0269]

[0270] Note: In the table, 1M01, 1M02, 1F01, and 1F02 represent the experimental rat groups, M = Male, F = Female, and 01 and 02 represent the rat numbers; BLQ: Below the limit of quantification.

[0271] Table 20 Anti-HIV Antibody 1 Plasma Drug Concentrations in SD Rats after Intravenous Injection of 60 mg / kg APC Conjugate 1 μM )

[0272]

[0273] Note: In the table, 1M01, 1M02, 1F01, and 1F02 represent the experimental rat groups, M = Male, F = Female, and 01 and 02 represent the rat numbers; BLQ: Below the limit of quantification.

[0274] Table 21 Anti-HIV Antibody 1 Plasma Drug Concentrations (μg / mL) in SD Rats after Intravenous Injection of 60 mg / kg APC Conjugate 1

[0275]

[0276] Note: In the table, 1M01, 1M02, 1F01, and 1F02 represent the experimental rat groups, M = Male, F = Female, and 01 and 02 represent the rat numbers; BLQ: Below the limit of quantification.

[0277] From the above data, it can be seen that after intravenous injection of 60 mg / kg of APC conjugate 1 in SD rats, the mean half-lives of anti-HIV antibody 1 and ABT were 161.52 h and 191.86 h, respectively.

[0278] Meanwhile, the blood drug concentrations of anti-HIV antibody 1 or ABT in blood samples were also detected after separately administering anti-HIV antibody 1 or ABT, and the results are shown in Tables 22 - 24, as well as Figures 8 - 9 as follows. Among them, Tables 22 - 23 and Figure 8 show the data of the drug concentration of anti-HIV antibody 1 in plasma samples after intravenous injection of anti-HIV antibody 1 in SD rats, presented in μM and μg / mL respectively; Tables 24 and Figure 9 show the data of the drug concentration of ABT in blood samples after intravenous injection of ABT in SD rats.

[0279] Table 22 Blood drug concentration of anti-HIV antibody 1 (μM) after intravenous injection of anti-HIV antibody 1 in SD rats - Toxicokinetics experiment

[0280]

[0281] Table 23 Blood drug concentration of anti-HIV antibody 1 (μg / ml) after intravenous injection of anti-HIV antibody 1 in SD rats - Toxicokinetics experiment

[0282]

[0283] Table 24 Blood drug concentration of ABT after intravenous injection of 9 mg / kg ABT in DD rats ( μM ) - Toxicokinetics Study

[0284]

[0285] Based on the above data, the results show that: After intravenous injection of APC conjugate 1 in SD rats, plasma anti-HIV antibody 1 and ABT can be detected by the previous ELISA method. The pharmacokinetic characteristics of anti-HIV antibody 1 in APC conjugate 1 are basically the same as those of the separate anti-HIV antibody 1. After ABT binds to anti-HIV antibody 1 (i.e., APC conjugate 1), the half-life is significantly extended from 28.4 hours to 191.86 hours.

[0286] According to the method described above, a pharmacokinetic experiment was conducted on APC conjugate 2 (molecule J - ABT) of the present invention, and similar results (data not shown) were obtained as those of APC conjugate 1.

[0287] All the documents mentioned in the present invention are cited in this application as references, just as if each document was separately cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. Sequence Listing <110> Frontier Biopharma Co., Ltd. (Nanjing) <120> APC Conjugates for Treating and / or Preventing HIV-Related Diseases, Their Preparation Methods and Applications <130> P2022-1306 <150> CN2021107216365 <151> 2021-06-28 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Heavy chain of anti-HIV antibody 1 <400> 1 Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp Tyr 20 25 30 Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val 35 40 45 Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln Phe 50 55 60 Gln Gly Arg Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp Thr 65 70 75 80 Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr Ala 85 90 95 Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp Val 100 105 110 Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 2 <211> 206 <212> PRT <213> Artificial sequence <220> <223> Anti - HIV antibody light chain <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp Tyr 20 25 30 Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly Ser 35 40 45 Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly Arg Arg Trp Gly 50 55 60 Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile Ala 65 70 75 80 Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val Pro Gly Thr Arg Leu 85 90 95 Asp Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 100 105 110 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 115 120 125 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 130 135 140 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 145 150 155 160 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 165 170 175 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 180 185 190 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 195 200 205 <210> 3 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Polypeptide chain of ABT <400> 3 Trp Glu Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Lys Leu Ile His 1 5 10 15 Glu Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30 Leu Leu <210> 4 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Anti-HIV antibody 2 heavy chain <400> 4 Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp Tyr 20 25 30 Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val 35 40 45 Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln Phe 50 55 60 Gln Gly Arg Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp Thr 65 70 75 80 Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr Ala 85 90 95 Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp Val 100 105 110 Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450

Claims

1. An antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-polypeptide conjugate comprises an anti-HIV antibody and a polypeptide linked thereto, the polypeptide being Aibofovir ABT, and the structure of the antibody-polypeptide conjugate is shown in Formula I: Ab-(J-U)n (I) In the formula: Ab is an anti-HIV antibody; U is the polypeptide chain of ABT, and its amino acid sequence is shown in SEQ ID NO: 3; J is the linker in ABT: AEEA-MPA; n is a positive integer; "-" is a chemical bond; Among them, The anti-HIV antibody comprises a heavy chain shown in SEQ ID NO: 1 and a light chain shown in SEQ ID NO: 2; or The anti-HIV antibody comprises a heavy chain shown in SEQ ID NO: 4 and a light chain shown in SEQ ID NO:

2.

2. The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein ABT is linked to the sulfhydryl group of the anti-HIV antibody, wherein the antibody is reduced by a reducing agent, so that the interchain disulfide bond of the antibody is reduced to produce sulfhydryl groups.

3. A pharmaceutical composition for treating and / or preventing HIV, comprising: (a) The antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2; and (b) A pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, The HIV is of the HIV-1 serotype.

5. The pharmaceutical composition according to claim 3, wherein The HIV is selected from the group consisting of AC10.0.29, CH110, CNE15, CAAN5342, or a combination thereof.

6. The pharmaceutical composition according to claim 3, wherein, The HIV is AC10.0.

29.

7. The method for preparing the antibody-polypeptide conjugate according to claim 1 or 2, characterized in that, The method comprises the steps of: (1) Reacting an anti-HIV antibody with a reducing agent in a buffer solution to obtain a reduced anti-HIV antibody; (2) Then adding ABT to the reduced anti-HIV antibody for a cross-linking reaction to obtain the antibody-polypeptide conjugate.

8. The method according to claim 7, wherein In step (1), the antibody is reduced by a reducing agent, so that the interchain disulfide bond of the antibody is reduced to produce sulfhydryl groups.

9. The method according to claim 7, wherein In step (1), the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride, beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or dithiothreitol.

10. Use of the antibody-polypeptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3 in the preparation of a drug for treating and / or preventing HIV.

11. The use according to claim 10, characterized in that, The HIV is of the HIV-1 serotype.

12. The use according to claim 10, wherein The HIV is selected from the group consisting of AC10.0.29, CH110, CNE15, CAAN5342, or a combination thereof.

13. The use according to claim 10, characterized in that, The HIV is AC10.0.29.

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