A PEGylated multivalent anti-West Nile virus single-chain antibody

Abstract

The present invention provides a PEGylated multivalent anti-West Nile virus single-chain antibody, relating to the field of biomedicine. The present invention provides a method for improving the antigen-binding ability of a ScFv single-chain antibody. Using phage library screening technology, an anti-West Nile virus ScFv single-chain antibody with antigen-binding and in vitro neutralizing activity was obtained. The multivalent anti-West Nile virus ScFv single-chain antibody obtained through PEGylation has a lower risk of ADE and higher antigen-binding ability, providing new research directions and tools for studying the mechanisms of West Nile virus-related diseases and developing therapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a pegylated multivalent anti-West Nile virus single-chain antibody. Background Art

[0002] It is generally believed that the ADE effect is related to the binding of the Fc segment of the antibody to the Fc receptor expressed on the surface of the target cell. That is, the antibody not only cannot neutralize the virus, but instead forms an infectious immune complex with the virus, and binds to the Fc receptor on the surface of the target cell, such as macrophages and monocytes, through its Fc segment, thereby promoting the target cell's uptake of the virus-antibody complex, and ultimately enhancing the virus's infection of the target cell and the virus's replication in the cell.

[0003] ScFv (single-chain antibody fragment) is composed of a light chain variable region (VL) and a heavy chain variable region (VH) connected by a flexible linker sequence. It lacks an Fc region, significantly reducing the risk of inducing ADE. Compared to full-length antibodies, ScFv single-chain antibodies have a smaller molecular weight, superior tissue penetration, and greater modification flexibility; however, they have weaker affinity, a short half-life, and are relatively unstable.

[0004] In view of this, the present invention provides an anti-West Nile virus ScFv single-chain antibody and a method for improving the antigen binding ability of the ScFv single-chain antibody. Summary of the Invention

[0005] To address the above issues, the present invention provides an anti-West Nile virus ScFv single-chain antibody and a method for improving the antigen-binding ability of ScFv single-chain antibodies. Through phage library screening, an anti-West Nile virus ScFv single-chain antibody with antigen-binding and in vitro neutralizing activity was obtained. Polymerization with polyethylene glycol yielded a multivalent anti-West Nile virus ScFv single-chain antibody, enhancing its antigen-binding ability.

[0006] To achieve the above objectives, the specific technical solutions provided by the present invention are as follows:

[0007] In a first aspect, the present invention provides a single-chain antibody against West Nile virus, which consists of a heavy chain variable region, a flexible linker sequence, and a light chain variable region, wherein the heavy chain variable region includes HCDR1-HCDR3 shown in SEQ ID NOs: 1-3, and the light chain variable region includes LCDR1-LCDR3 shown in SEQ ID NOs: 4-6.

[0008] West Nile virus (WNV) is an enveloped, positive-stranded RNA virus belonging to the genus Flavivirus in the family Flaviviridae. It infects humans, birds, horses, poultry, and other animals through mosquito bites. Its major structural and nonstructural proteins include the capsid protein (C); pre-membrane / membrane proteins (prM / M); envelope protein (E), which includes three key domains (DI–DIII); and nonstructural proteins (NS), including NS1, NS3, and NS5.

[0009] In the context of the present invention, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (e.g., diabodies) formed from at least two intact antibodies, so long as they exhibit the desired biological activity.

[0010] In the present invention, the antibody sequences obtained by modification also fall within the scope of protection of the present invention. The term "modification" refers to any form of modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to the reduction of one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to a change in the amino acid sequence that results in the addition of one or more amino acids compared to the original amino acid sequence. In the present invention, the modification preferably occurs in an area outside the variable region, such as the constant region or framework region of the antibody, and the modified antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen binding properties.

[0011] In the present invention, the terms "heavy chain variable region" and "light chain variable region" refer to the structure of an antibody, from N-terminus to C-terminus. Each antibody heavy chain has a heavy chain variable region, also known as a heavy chain variable domain, followed by three heavy chain constant domains: CH1, CH2, and CH3. Similarly, from N-terminus to C-terminus, each antibody light chain has a light chain variable region, also known as a light chain variable domain, followed by a light chain constant domain. The heavy and light chain variable regions can be further divided into hypervariable regions (i.e., CDRs) and relatively conserved regions intervening between the hypervariable regions (i.e., framework regions, FRs).

[0012] In the present invention, the term "CDR" refers to the "hypervariable region" or "complementarity determining region" of an antibody. Both the heavy and light chain variable regions have three CDRs, which together form the antibody's antigen-binding site and spatially complement the antigenic determinant. The position of the CDR within the variable region is denoted differently according to different numbering schemes, including IMGT, Chothia, Kabat, and others. In specific embodiments of the present invention, the numbering scheme is Kabat.

[0013] Furthermore, the connection order of the single-chain antibody is light chain variable region-flexible connection sequence-heavy chain variable region.

[0014] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; and the flexible linker sequence is a (GGGGS) 3 linker.

[0015] A second aspect of the present invention provides a biomaterial, comprising any one of the following:

[0016] A1) A nucleic acid molecule encoding the single-chain antibody according to the first aspect of the present invention.

[0017] A2) An expression vector comprising the nucleic acid molecule described in A1).

[0018] A3) A recombinant cell, comprising the nucleic acid molecule described in A1) and / or the expression vector described in A2).

[0019] In the present invention, the term "nucleic acid molecule" or "nucleic acid" refers to a polymeric form comprising nucleotides of any length, including deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, and also includes DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases. Therefore, the nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc. Once the coding sequence of the single-chain antibody of the present invention or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto is isolated, the single-chain antibody can be obtained in large quantities using recombinant technology.

[0020] In the present invention, the term "expression vector" or "vector" refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, herpes virus vectors and other viral vectors. Other viral vectors may include phage vectors, baculovirus vectors, animal virus vectors, plant virus vectors, and may include papovavirus vectors, herpes virus vectors, poxvirus vectors, RNA virus vectors, and may include bovine papillomavirus vectors and Epstein-Barr virus vectors, retrovirus vectors, etc.

[0021] In the present invention, the term "recombinant cell" or "recombinant host cell" refers to any cell type suitable for transformation, transfection, transduction, etc. with an expression vector comprising a nucleic acid molecule provided by the present invention. Recombinant cells include any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. The recombinant cells include prokaryotic cells and eukaryotic cells; the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasmas, chlamydia, and rickettsia; the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes a T cell, a NK cell, an iNKT cell, a B cell, a CTL cell, a monocyte, a myeloid cell, a dendritic cell, a macrophage, or any combination thereof.

[0022] The third aspect of the present invention provides a derivative of a single-chain antibody, wherein the single-chain antibody is the single-chain antibody according to the first aspect of the present invention, and the derivative includes any one of the following:

[0023] B1) The derivatives are monomers and polymers of single-chain antibodies, or multimers.

[0024] B2) The derivative is a fusion protein comprising a single-chain antibody.

[0025] B3) The derivative is a single-chain antibody and a solid phase carrier coupled thereto.

[0026] B4) The derivative is a single-chain antibody, the monomer and multimer described in B1), the multimer described in B1), the fusion protein described in B2), or the solid phase carrier described in B3), and a detectable label coupled thereto.

[0027] In the present invention, a multimer refers to a complex formed by two or more antibody molecules bound together by covalent or non-covalent bonds. Multimers include homomultimers, heteromultimers, and antibody fragment multimers. In a specific embodiment of the present invention, the multimer is a homomultimer.

[0028] Furthermore, the polymer is formed by PEGylation.

[0029] In some embodiments, the PEG molecules used for PEGylation include amine-reactive PEGylation reagents such as MS(PEG) 4, SAT(PEG) 4, TMS(PEG) 4; sulfhydryl-reactive PEGylation reagents such as MM(PEG) 12, PEG-4-arm-Mal; PEGylated amino acids and amine compounds such as CA(PEG) 4, CA(PEG) 8; PEGylated carboxyl and thiol compounds such as CT(PEG) 12, MT(PEG) 4, ML(PEG) 4.

[0030] The PEG molecule used in the PEGylation is PEG-four-arm-maleimide (PEG-4-arm-Mal).

[0031] Furthermore, the PEGylation step includes: mildly reducing the single-chain antibody to expose the Cys site at the C-terminus of the single-chain antibody, and adding PEG-four-arm-maleimide.

[0032] The mild reduction is achieved by using TCEP.

[0033] The molar ratio of the PEG-four-arm-maleimide to the single-chain antibody is 1:1.

[0034] Furthermore, the form of the fusion protein includes at least one of a multivalent antibody, a fusion protein with an effector molecule, and a fusion protein with a functional protein.

[0035] In some embodiments, the effector molecules include immunotoxins, such as Pseudomonas aeruginosa exotoxin and ricin toxin; cytokines, such as IL-2, TNF, IFN, etc.; enzymes, such as alkaline phosphatase, urokinase, etc., which can be used to design "antibody-guided enzyme-prodrug" therapy; targeting molecules, such as cell-permeable polypeptides; receptors or ligands and other biologically active effector molecules.

[0036] In some embodiments, the fusion protein includes a fusion protein formed by fusing a single-chain antibody with an immunoglobulin Fc segment, serum albumin (HSA), a molecular chaperone (GST, TRX) or other functional proteins.

[0037] The solid phase carrier includes at least one of a natural polymer carrier, a synthetic polymer carrier, an inorganic carrier, and a composite carrier.

[0038] In some embodiments, the natural polymer carriers are such as agarose, cellulose, chitosan, etc., the synthetic polymer carriers are such as polystyrene, polymethyl methacrylate, polylactic acid-glycolic acid copolymer, etc., the inorganic carriers are such as magnetic nanoparticles, silica, gold nanoparticles, etc., and the solid phase carriers also include composite carriers of the above carriers, metal organic frameworks, hydrogels and other new carriers.

[0039] The detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme.

[0040] The fourth aspect of the present invention provides uses of the single-chain antibody according to the first aspect of the present invention, the biomaterial according to the second aspect of the present invention, or the derivative according to the third aspect of the present invention, wherein the uses include any of the following:

[0041] C1) Use in the preparation of products for the diagnosis, prevention or treatment of diseases related to West Nile virus infection.

[0042] C2) Use in the preparation of products for detecting West Nile virus and / or West Nile virus EDIII protein.

[0043] C3) Application in the study of the disease mechanisms associated with West Nile virus infection.

[0044] A fifth aspect of the present invention provides a method, comprising any one of the following:

[0045] D1) A method for detecting West Nile virus and / or West Nile virus EDIII protein in a test sample for non-therapeutic purposes, the method comprising: contacting the test sample with the derivative described in B4) of the third aspect of the present invention, and detecting the complex formed between West Nile virus and / or West Nile virus EDIII protein and the derivative using a detectable marker.

[0046] D2) A method for producing the single-chain antibody of the first aspect of the present invention, the method comprising: artificially synthesizing the single-chain antibody; or culturing the recombinant cell of the third aspect of the present invention, and isolating and purifying the single-chain antibody of the first aspect of the present invention from the culture product.

[0047] In certain embodiments, the single-chain antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, single-chain antibodies of the present invention are obtained by direct amino acid synthesis. In certain embodiments, single-chain antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0048] D3) A method for preparing the recombinant cell described in the third aspect of the present invention, the method comprising: introducing the nucleic acid molecule or expression vector described in the third aspect of the present invention into the cell, wherein the introduction method includes calcium phosphate transfection, DEAE, dextrose-mediated transfection, electroporation, and phage infection.

[0049] Advantages and beneficial effects of the present invention: The present invention provides an anti-West Nile virus ScFv single-chain antibody and a method for improving the antigen-binding ability of the ScFv single-chain antibody. The multivalent anti-West Nile virus ScFv single-chain antibody obtained by polyethylene glycol polymerization has a lower risk of producing an ADE effect and has a higher antigen-binding ability, providing new research directions and tools for the study of the mechanism of West Nile virus infection-related diseases and the development of therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the SDS-PAGE electrophoresis diagram of ScFvC single-chain antibody.

[0051] Figure 2 This is the SDS-PAGE electrophoresis diagram of PEGylated ScFvC single-chain antibody.

[0052] Figure 3 Figure 3 is the SDS-PAGE electrophoresis diagram of each component of ScFvC-PEGylation, where ① is the PEGylated ScFvC monomer; ② is the PEGylated ScFvC dimer; ③ is the PEGylated ScFvC polymer mixture (including dimer, trimer and tetramer).

[0053] Figure 4 This is a graph showing the antigen binding activity of each component of ScFvC-PEGylated. DETAILED DESCRIPTION

[0054] The various reagents involved in the technical solutions and experimental procedures described in this invention are all commonly used or commercially available reagents that are well known and readily available to those skilled in the art based on their professional knowledge and routine practice. The description of the reagents in this invention is intended to clearly illustrate the material basis involved in the technical solutions, and those skilled in the art, based on their professional qualities and industry common sense, will be able to successfully obtain and correctly use these reagents to achieve the technical objectives of this invention.

[0055] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0056] Example

[0057] 1. Experimental Methods

[0058] 1. Expression plasmid construction: Based on the ScFv sequence information, the light chain (VL) and heavy chain (VH) variable region sequences of the ScFv antibody were linked via a (GGGGS)3 linker in the order of light chain variable region-(GGGGS)3 linker-heavy chain variable region. A cysteine ​​sequence (C) was introduced at the C-terminus. The sequence was then sent to Shanghai Bioengineering for full sequence synthesis. The synthesized gene sequence was ligated into the expression vector pCDNA3.1 to construct the recombinant expression plasmid pCDNA3.1-ScFvC. pCDNA3.1-ScFvC was confirmed by sequencing.

[0059] 2. Protein Expression and Purification: pCDNA3.1-ScFvC was transfected into mammalian ExpiCHO cells using the ExpiCHO transfection kit according to the kit instructions. Culture the cells in a shaker at 125 rpm, 37°C, and 5% CO2. After 8-10 days, the cell culture supernatant was collected and purified using a GE Protein L column. Elution was performed with pH 3.0 citrate buffer. The flow-through was collected and immediately neutralized with 1 mol / L TRIS-HCl buffer, pH 8.5. The column was dialyzed against 0.01 mol / L PBS, pH 7.2, for 72 hours and sterilized by filtration through a 0.22 μm filter. The purified antibody concentration was determined by BCA assay, and protein purity was assessed by SDS-PAGE electrophoresis.

[0060] 3. PEG polymerization: 5 mg of ScFvC single-chain antibody solution (2 mg / mL, PBS, pH 7.4) was added with TCEP (final concentration 0.5 mM). After mixing by inversion at room temperature for 2 h, PEG-quadruple-maleimide was added at a molar ratio of 1:1. Mixing by inversion at room temperature for another 2 h was continued. The PEGylation efficiency was analyzed by SDS-PAGE electrophoresis.

[0061] 4. ELISA: Dilute WNV DⅢ-his to 1 μg / ml in coating solution, add 100 μl per well to the enzyme-linked plate and incubate at 4°C overnight; wash three times with PBST, block with 5% milk, and incubate at 37°C for 1 h; discard the blocking solution, add the antibody serially diluted in blocking solution to a starting concentration of 1 μg / ml, and incubate at 37°C for 1 h; wash three times with PBST, add goat anti-human-HRP secondary antibody and react at room temperature for 45 min; wash three times with PBST, develop color with TMB, terminate with 1 mol / L sulfuric acid, and measure the absorbance at 450 nm (A450).

[0062] 2. Experimental Results

[0063] 1. Expression and purification of ScFvC single-chain antibody: The single-chain antibody expression plasmid pCDNA3.1-ScFvC targeting West Nile virus E protein DIII was transfected into ExpiCHO-S cells. After 8-10 days, the cell culture supernatant was collected and purified using Protein L affinity purification technology. The molecular weight was determined by SDS-PAGE. The electrophoresis results are as follows: Figure 1 As shown, the purity of the single-chain antibody is higher than 90%. Under reducing conditions, it appears as a protein band with a relative molecular mass (Mr) of approximately 30×10 3 .

[0064] 2. Polyethylene glycol polymerization of ScFvC single-chain antibody: First, the single-chain antibody protein was mildly reduced with TCEP to expose the Cys site at the C-terminus. Then, maleimide-activated polyethylene glycol derivative PEG-quadruple-maleimide (10 kD) was added at a protein:PEG (molar ratio of 1:1) to perform site-specific polyethylene glycol polymerization of the unpaired cysteine ​​at the C-terminus of the ScFvC single-chain antibody. The results are shown in Figure 2. Figure 2 As shown, the PEGylated ScFvC single-chain antibody fragment constitutes the main part of the protein band, most of which are PEGylated ScFvC monomers and dimers with observed molecular weights of approximately 50 and 90 kD, respectively. A small part is PEGylated ScFvC multimers, including trimers and tetramers.

[0065] 3. Purification and separation of PEGylated ScFvC single-chain antibody: Protein L column was used to separate the components of PEGylated ScFvC single-chain antibody fragments. The separation results are shown in the figure. Figure 3 As shown, the PEGylated conjugate is mainly separated into three parts: ① PEGylated ScFvC monomer; ② PEGylated ScFvC dimer and ③ PEGylated ScFvC multimer mixture (including dimer, trimer and tetramer).

[0066] 4. Binding of ScFvC-PEGylated components to target antigens: In order to detect the effect of PEGylation on the antigen recognition ability of ScFvC single-chain antibody, ELISA was further used to detect the binding of purified and separated ScFvC-PEGylated components to the target antigen WNVEDIII. Figure 4 As shown in Figure 3, PEGylation does not affect the WNV EDIII binding properties of the antibody fragments. Specifically, the antigen-binding activity of the PEGylated ScFvC monomer is comparable to that of the parental ScFvC single-chain antibody, while the PEGylated ScFvC multimer enhances the binding to the target antigen through an avidity effect.

[0067] Table 1. Sequence Listing

[0068]

[0069]

[0070] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A single-chain antibody against West Nile virus, characterized in that The single-chain antibody consists of a heavy chain variable region, a flexible linker sequence, and a light chain variable region, wherein the heavy chain variable region includes HCDR1-HCDR3 shown in SEQ ID NOs: 1-3, and the light chain variable region includes LCDR1-LCDR3 shown in SEQ ID NOs: 4-6.

2. The single-chain antibody according to claim 1, characterized in that The connection sequence of the single-chain antibody is light chain variable region-flexible connection sequence-heavy chain variable region.

3. The single-chain antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; and the flexible linker sequence is a (GGGGS) 3 linker.

4. A biomaterial, characterized in that The biological material includes any one of the following: A1) a nucleic acid molecule encoding the single-chain antibody according to any one of claims 1 to 3; A2) an expression vector comprising the nucleic acid molecule described in A1); A3) A recombinant cell, comprising the nucleic acid molecule described in A1) and / or the expression vector described in A2).

5. A derivative of a single-chain antibody, wherein the single-chain antibody is the single-chain antibody according to any one of claims 1 to 3, characterized in that: The derivatives include any of the following: B1) The derivative is a monomer or multimer of a single-chain antibody, or a multimer; B2) the derivative is a fusion protein comprising a single-chain antibody; B3) the derivative is a single-chain antibody and a solid phase carrier coupled thereto; B4) The derivative is a single-chain antibody, the monomer and multimer described in B1), the multimer described in B1), the fusion protein described in B2), or the solid phase carrier described in B3), and a detectable label coupled thereto.

6. The derivative according to claim 5, characterized in that The polymer is formed by PEGylation; The PEG molecule used in the PEGylation is PEG-four-arm-maleimide.

7. The derivative according to claim 6, characterized in that The PEGylation step includes: mildly reducing the single-chain antibody to expose the Cys site at the C-terminus of the single-chain antibody, and adding PEG-four-arm-maleimide; The mild reduction is achieved by using TCEP; The molar ratio of the PEG-four-arm-maleimide to the single-chain antibody is 1:

1.

8. The derivative according to claim 5, characterized in that The form of the fusion protein includes at least one of a multivalent antibody, a fusion protein with an effector molecule, and a fusion protein with a functional protein; The solid phase carrier includes at least one of a natural polymer carrier, a synthetic polymer carrier, an inorganic carrier, and a composite carrier; The detectable label comprises at least one of a radioisotope, a metal nanomaterial, a fluorescein, a biotin, avidin, a biotin / avidin complex, a biotin / avidin complex, a chromophore, an electron-dense substance, and an enzyme.

9. Use of the single-chain antibody according to any one of claims 1 to 3, the biomaterial according to claim 4, or the derivative according to any one of claims 5 to 8, characterized in that: The application includes any of the following: C1) Use in the preparation of products for diagnosing, preventing or treating diseases related to West Nile virus infection; C2) Use in the preparation of products for detecting West Nile virus and / or West Nile virus EDIII protein; C3) Application in the study of the disease mechanisms associated with West Nile virus infection.

10. A method, characterized in that The method includes any of the following: D1) A method for detecting West Nile virus and / or West Nile virus EDIII protein in a sample for non-therapeutic purposes, the method comprising: contacting the sample with the derivative described in B4) of claim 5, and detecting a complex formed between the West Nile virus and / or West Nile virus EDIII protein and the derivative using a detectable marker; D2) A method for producing the single-chain antibody according to any one of claims 1 to 3, the method comprising: artificially synthesizing the single-chain antibody; or culturing the recombinant cell according to claim 4, and isolating and purifying the single-chain antibody according to any one of claims 1 to 3 from the culture product; D3) A method for preparing the recombinant cell of claim 4, comprising: introducing the nucleic acid molecule or expression vector of claim 4 into the cell, wherein the introduction method comprises calcium phosphate transfection, DEAE, dextrose-mediated transfection, electroporation, or phage infection.

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