Humanized antibody aiming at poxvirus membrane associated protein L1R and application thereof

Abstract

The invention discloses a humanized antibody aiming at poxvirus membrane associated protein L1R and application of the humanized antibody. The amino acid sequence of a heavy chain variable region of the humanized antibody is as shown in SEQ ID No.1, and the amino acid sequence of a light chain variable region of the humanized antibody is as shown in SEQ ID No.2. Experiments prove that the humanized antibody provided by the invention can efficiently neutralize vaccinia virus and monkey pox virus in an in-vitro experiment, and in an in-vivo protection experiment, mice can be completely protected from death caused by vaccinia virus infection by injecting the antibody in advance. In addition, the humanized antibody provided by the invention is a monoclonal antibody which is best in poxvirus non-complement-dependent neutralizing antibodies reported at present and has great potential in the aspect of treating and preventing poxvirus infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a humanized antibody targeting poxvirus membrane-associated protein L1R and an application thereof. Background Art

[0002] Monkeypox is a viral disease caused by Monkeypox virus (MPXV). Monkeypox virus is classified as Orthopoxvirus in the Poxviridae family. It is one of the four Orthopoxviruses that are pathogenic to humans. The other three are smallpox virus, vaccinia virus and cowpox virus. Under the electron microscope, monkeypox virus particles are brick-shaped or oval, with a size of 200nm×250nm, and have an envelope. The virus particles contain structural proteins and DNA-dependent RNA polymerase. The genome is double-stranded DNA with a length of about 197kb. Orthopoxvirus has a large and complex proteome and is one of the most structurally complex viruses in the world. During the infection process, poxvirus particles exist in two forms: mature virus form (MV) and lipid envelope form (EV).

[0003] Antibodies are immunoglobulins produced by plasma cells differentiated from B cells. When the immune system is stimulated, they will react specifically with the corresponding antigen. At the end of the last century (1890), the first successful use of serum injection to treat diseases opened up a new path for modern medicine. Later, people determined that its active ingredient was an antibody, and in 1975, the first monoclonal antibody was prepared using hybridoma technology. According to the source of antibody production, it is divided into normal antibodies (natural antibodies), such as anti-A and anti-B antibodies in blood type ABO, and immune antibodies such as anti-microbial antibodies. According to the source of the reactive antigen, it is divided into xenoantibodies, heterophilic antibodies, alloantibodies and autoantibodies. According to the agglutination state of the antigen reaction, it is divided into complete antibodies IgM and incomplete antibodies IgG. Antibodies are widely used in medical practice. For example, they have a certain role in the prevention, diagnosis and treatment of diseases. Clinically, immunoglobulin is used to prevent viral hepatitis, measles, rubella, etc., and anti-Rh immunoglobulin is used internationally to prevent hemolysis caused by Rh blood type incompatibility. In diagnosis, rheumatoid factor is used for rheumatoid arthritis, antinuclear antibodies (ANA) and anti-DNA antibodies are used for systemic lupus erythematosus, and antisperm antibodies are used for the diagnosis of primary infertility; in treatment, antitoxin treatment is used for poisoning and immunodeficiency diseases.

[0004] Since the first mouse monoclonal antibody drug was introduced in 1986, there are now nearly 100 monoclonal antibody drugs on the market worldwide. The preparation technology of monoclonal antibody drugs has also gone through four stages of development: the first generation: mouse monoclonal antibody (momab): hybridoma monoclonal antibody technology; the second generation: human-mouse chimeric monoclonal antibody (ximab): chimeric antibody and humanized monoclonal antibody technology; the third generation: humanized monoclonal antibody (zumab): fully humanized monoclonal antibody technology; the fourth generation: fully humanized monoclonal antibody (mumab): natural fully humanized monoclonal antibody technology.

[0005] More than a hundred years ago, the revelation of the principles of antibody-antigen specific binding and antibody passive immune characteristics opened up a new way to diagnose diseases, and the advent of monoclonal antibody technology in 1975 accelerated the widespread application of this method. In the early days, most monoclonal antibodies used in clinical practice were mouse-derived. Due to the species specificity of humans and mice, the first generation of mouse-derived antibodies could be recognized by the human immune system, causing human anti-mouse antibody reactions, which weakened the efficacy of monoclonal antibody drugs and caused serious adverse reactions. Therefore, the clinical application of the first generation of monoclonal antibody drugs was greatly limited. Due to the various limitations of mouse-derived antibodies in clinical applications, people use DNA recombination technology to humanize mouse antibodies to humanize antibodies. Humanized monoclonal antibodies and fully humanized monoclonal antibodies can overcome human anti-mouse antibody reactions, prevent monoclonal antibody molecules from being quickly cleared by the immune system as heterologous proteins, and improve the efficacy of monoclonal antibody drugs. Antibody humanization is an important part of the recombinant antibody production and preparation experiment.

[0006] The M1R protein of monkeypox virus is homologous to the L1R protein of vaccinia virus and is a transmembrane protein found on the surface of mature IMV particles. Encoded by the L1R ORF, it is highly conserved, participates in the assembly of virions, and plays an important role in the entry and maturation of the virus. L1R interacts with EFC and acts synergistically with other known entry proteins. Monkeypox virus infection poses a major threat to public health. There is currently no effective antiviral treatment. Previous literature reports have shown that neutralizing antibodies with L1R as a binding target can exert non-complement-dependent neutralizing activity. However, there are two main problems with current antibodies: on the one hand, multiple antibodies are required to work together to exert strong antiviral activity; on the other hand, the source of the antibody, such as the 7D11 antibody, which is a potent neutralizing antibody derived from mice. Therefore, it is urgent to develop a humanized antibody that can neutralize multiple poxviruses. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a highly humanized antibody capable of neutralizing a variety of poxviruses and its application. The technical problem to be solved is not limited to the technical subject matter as described, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0008] In order to solve the above technical problems, the present invention first provides an antibody, which is a humanized neutralizing antibody against vaccinia virus membrane-associated protein L1R, and is named IPB-POX1.1 in the present invention. The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown in positions 26-33, 51-58 and 97-108 of SEQ ID No.1, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are shown in positions 27-39, 56-58 and 95-102 of SEQ ID No.2, respectively.

[0009] Among them, CDR1, CDR2 and CDR3 are three complementarity determining regions in the heavy chain variable region or the light chain variable region, and the sequences of the complementarity determining regions are defined according to IMGT.

[0010] Furthermore, the heavy chain variable region and the light chain variable region also include a framework region.

[0011] The structure of the heavy chain variable region is: framework region FR1-complementarity determining region CDR1-framework region FR2-complementarity determining region CDR2-framework region FR3-complementarity determining region CDR3-framework region FR4. The amino acid sequence of the framework region FR1 is shown in positions 1-25 of SEQ ID No.1; the amino acid sequence of the framework region FR2 is shown in positions 34-50 of SEQ ID No.1; the amino acid sequence of the framework region FR3 is shown in positions 59-96 of SEQ ID No.1; and the amino acid sequence of the framework region FR4 is shown in positions 109-119 of SEQ ID No.1.

[0012] The structure of the light chain variable region is: framework region FR1-complementarity determining region CDR1-framework region FR2-complementarity determining region CDR2-framework region FR3-complementarity determining region CDR3-framework region FR4. The amino acid sequence of the framework region FR1 is shown in positions 1-26 of SEQ ID No.2; the amino acid sequence of the framework region FR2 is shown in positions 40-55 of SEQ ID No.2; the amino acid sequence of the framework region FR3 is shown in positions 56-94 of SEQ ID No.2; and the amino acid sequence of the framework region FR4 is shown in positions 103-112 of SEQ ID No.2.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region is any one of the following A1)-A4): A1) The amino acid sequence is the protein shown in SEQ ID No. 1; A2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1; A3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.1; A4) A protein having 90% or more identity with the amino acid sequence shown in SEQ ID No. 1 and having the same function.

[0014] The amino acid sequence of the light chain variable region is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID No. 2; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 2; B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; B4) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID No. 2 and having the same function.

[0015] In the protein described in A2) or B2) above, the tag refers to a polypeptide or protein that is fused and expressed together with the target antibody using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target antibody. The tag includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0016] In the protein described in A3) or B2) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues. In certain embodiments, substitution and / or deletion and / or addition of one or more amino acid residues may occur in one or more complementary determining regions and / or framework regions of the antibodies described in the present invention, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as is well known to those skilled in the art, conservative substitutions of amino acids do not change the properties and functions of the protein) may be made to the complementary determining regions and / or framework regions, which do not substantially reduce the binding affinity.

[0017] In the protein described in A4) or B4), the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be calculated. The identity includes amino acid sequences having 80% or more, or 85% or more, or 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 of the present invention.

[0018] The protein described in A1) or A2) or A3) or A4) or B1) or B2) or B3) or B4) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0019] Furthermore, the antibody also includes a heavy chain constant region and a light chain constant region.

[0020] The heavy chain constant region is a heavy chain constant region of human IgG1. Specifically, the amino acid sequence of the heavy chain constant region is SEQ ID No.3.

[0021] The light chain constant region is a light chain constant region of human Kappa. Specifically, the amino acid sequence of the light chain constant region is SEQ ID No.4.

[0022] In order to solve the above technical problems, the present invention further provides any of the following biomaterials: C1) An active fragment of an antibody, which is any of the following derived from the above antibodies: antigen-binding fragment, Fab fragment, Fab′ fragment, F(ab′) 2 fragment, single-chain antibody, single-domain antibody, bispecific antibody, minimal recognition unit; C2) a nucleic acid molecule encoding the above-mentioned antibody or an active fragment of the antibody described in C1); C3) An expression cassette, recombinant vector, recombinant cell or recombinant microorganism containing the nucleic acid molecule described in C2).

[0023] In the above C2), the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown in positions 76-99, 151-174 and 289-321 of SEQ ID No. 5, respectively.

[0024] The nucleotide sequences encoding CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are shown in SEQ ID No. 6 at positions 79-117, 166-174 and 283-306, respectively.

[0025] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the antibody is SEQ ID No. 5, or a sequence having at least 75% identity with SEQ ID No. 5.

[0026] The nucleotide sequence encoding the light chain variable region of the antibody is SEQ ID No. 6, or a sequence having at least 75% identity with SEQ ID No. 6.

[0027] Furthermore, the nucleotide sequence encoding the heavy chain constant region of the antibody is SEQ ID No.7, or a sequence having at least 75% identity with SEQ ID No.7.

[0028] The nucleotide sequence encoding the light chain constant region of the antibody is SEQ ID No. 8, or a sequence having at least 75% identity with SEQ ID No. 8.

[0029] Those skilled in the art can easily mutate the nucleotide sequence encoding the variable region or constant region of the antibody of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the variable region or constant region of the antibody, as long as they encode the variable region or constant region of the antibody and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention. If the degeneracy of the codons is taken into account, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; it is also possible to artificially synthesize and transform the gene according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody, that is, to obtain variants of the antibody by using methods known in the art, which are all included in the scope of the present invention. In addition, polynucleotides containing sequences that optimize antibody specificity or neutralizing activity by applying directed evolution to any nucleic acid sequence of the present invention are also within the scope of the present invention.

[0030] The identity refers to sequence similarity with natural nucleic acid sequences. "Identity" includes nucleotide sequences that have 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity with the nucleotide sequence of the present invention encoding the protein composed of the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4. The identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0031] In the above C3), the expression cassette refers to a DNA capable of expressing the antibody in a host cell, and the DNA may include not only a promoter for initiating transcription of the antibody encoding gene sequence, but also a terminator for terminating transcription of the antibody encoding gene sequence.

[0032] The vector includes, but is not limited to, plasmid, bacteriophage (such as lambda phage or M13 filamentous phage), cosmid (i.e., cosmid), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus or herpes virus (such as herpes simplex virus), etc. The recombinant vector refers to a recombinant DNA molecule constructed by connecting the above nucleic acid molecule or the above expression cassette with the vector in vitro. The recombinant vector can directly or indirectly introduce the above nucleic acid molecule or the above expression cassette into the host cell for gene expression by means of transformation, transfection or transduction. Specifically, the recombinant vector can be the recombinant plasmid A or recombinant plasmid B mentioned below.

[0033] The cells (host cells) include but are not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells) or prokaryotic cells. The recombinant cells refer to cells containing the above-mentioned recombinant vectors. Specifically, the recombinant cells are Freestyle 293F cells containing the above-mentioned recombinant vectors.

[0034] The microorganisms include but are not limited to yeast, bacteria, algae or fungi. Among them, the bacteria can be from the genus Escherichia The recombinant microorganism refers to a microorganism containing the above-mentioned recombinant vector.

[0035] In order to solve the above technical problems, the present invention also provides a pharmaceutical composition.

[0036] The pharmaceutical composition provided by the present invention contains the above-mentioned antibody and a pharmaceutically acceptable carrier.

[0037] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, agents, absorption enhancers, adsorption carriers, surfactants or lubricants but are not limited thereto.

[0038] Wherein, the pharmaceutical composition is used to prevent or treat diseases caused by poxvirus infection.

[0039] In order to solve the above technical problems, the present invention further provides any one of the following applications D1)-D6): D1) Use of the above biological materials in the preparation of the above antibodies; D2) Use of the above antibody, the above biological material or the above pharmaceutical composition in the preparation of a product for preventing or treating a disease caused by poxvirus infection; D3) Use of the above antibody, the above biological material or the above pharmaceutical composition in the preparation of a product for inhibiting poxvirus infection; D4) Use of the above antibody or the above biological material or the above pharmaceutical composition in the preparation of a product for detecting poxvirus levels and / or poxvirus L1R antigen; D5) Use of the above antibody, the above biological material or the above pharmaceutical composition in the preparation of a product for diagnosing or assisting in the diagnosis of a disease caused by poxvirus infection; D6) Use of the above antibody, the above biological material or the above pharmaceutical composition in the preparation of a product that inhibits or neutralizes the activity of poxvirus.

[0040] In the above application, the products for detecting poxvirus levels and / or poxvirus L1R antigens include but are not limited to products for detecting antigen-antibody binding using enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination assay or immunoturbidimetry.

[0041] In the above application, the product may be a reagent or a kit or a chip or a test paper. The reagent or the kit or the chip or the test paper contains the antibody. The kit includes but is not limited to a chemiluminescent immunoassay kit, an enzyme-linked immunosorbent assay kit, a colloidal gold immunoassay kit, or a fluorescent immunoassay kit.

[0042] In order to solve the above technical problems, the present invention also provides a kit; the kit has any of the following uses: E1) Prevention or treatment of diseases caused by poxvirus infection; E2) Inhibition of poxvirus infection; E3) Detection of poxvirus levels and / or poxvirus L1R antigen; E4) Diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection; E5) inhibits or neutralizes poxvirus activity.

[0043] The kit provided by the present invention contains the above-mentioned antibody or the above-mentioned biological material or the above-mentioned pharmaceutical composition.

[0044] Any of the poxviruses mentioned above includes, but is not limited to, monkeypox virus, cowpox virus, rabbitpox virus, mousepox virus, vaccinia virus, smallpox virus, and camelpox virus.

[0045] Diseases caused by any of the above-mentioned poxvirus infections include, but are not limited to, smallpox, monkeypox, cowpox, rabbitpox, mousepox, and camelpox.

[0046] In order to solve the above technical problems, the present invention finally provides a method for preparing the above antibody.

[0047] The method for preparing the above-mentioned antibody provided by the present invention comprises the following steps: expressing the coding gene of the above-mentioned antibody in a host cell to obtain the antibody.

[0048] Furthermore, the method for expressing the above-mentioned antibody encoding gene in the host cell is to introduce the above-mentioned antibody encoding gene into the host cell.

[0049] The introduction can be carried out by any known transfection method such as calcium phosphate co-precipitation method, liposome-mediated method, electroporation method or virus vector method to transform the host cell with the vector carrying the antibody encoding gene.

[0050] Furthermore, the cells (host cells) include but are not limited to eukaryotic cells (such as yeast cells, Aspergillus cells), animal cells (such as mammalian cells, insect cells) or prokaryotic cells.

[0051] Furthermore, the cells may be Freestyle 293F cells.

[0052] In some embodiments, the antibody encoding gene is introduced into the host cell via the recombinant plasmid A and recombinant plasmid B described below.

[0053] The term "antigen binding fragment" refers to an antigen binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antigen binding fragment retains at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parental antibody's binding activity to a target. Affinity.

[0054] The term "Fab fragment" refers to a heterodimer formed by the heavy chain Fd and the complete light chain bound by disulfide bonds, and contains only one antigen binding site. The coding genes of the heavy chain Fd and the complete light chain are connected and fused with the bacterial protein signal peptide gene, and the Fab antibody (Fab fragment) can be secreted and expressed in Escherichia coli, and has complete three-dimensional folding and intra-chain and inter-chain disulfide bonds. The heavy chain Fd refers to about 1 / 2 of the H chain part in Fab (containing about 225 amino acid residues, including VH, CH1 and part of the hinge region).

[0055] The term "Fab' fragment" comprises a light chain and a portion of a heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form F(ab') 2 molecular.

[0056] The term "F(ab') 2 A "fragment" contains two light chains and two heavy chains including a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F(ab') 2The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0057] The term "single-chain antibody (ScFv)" refers to the use of appropriate oligonucleotide linkers to connect the light chain and heavy chain variable region genes to express a single polypeptide chain, called a single-chain antibody (ScFv). The polypeptide chain can spontaneously fold into a natural conformation, maintaining the specificity and affinity of Fv.

[0058] The term "single-domain antibody (nanobody)" refers to an antibody containing only the VH fragment obtained by expressing the heavy chain V region of the antibody through genetic engineering methods. The ability of single-domain antibodies to bind to antigens and their stability are basically the same as those of complete antibodies.

[0059] The term "bispecific antibody" refers to the introduction of two sets of light chain and heavy chain genes into myeloma cells, and the selection of appropriate antibody constant regions and Ig types to obtain bispecific antibodies with high yield, uniformity and purity. In addition, bispecific antibodies can also be obtained by chemical cross-linking technology or hybridization-hybridoma technology.

[0060] The term “minimum recognition unit (MRU)” refers to a structure containing only a single CDR in the variable region, with a molecular mass of only About 1% of the complete antibody can bind to the corresponding antigen.

[0061] The present invention selects the binding mode of the 7D11 antibody as a template, and uses a computer-aided design method to undergo multiple rounds of iterative humanization transformation, and finally obtains the antibody IPB-POX1.1 with a high degree of humanization. Through humanization analysis, affinity, and in vitro and in vivo neutralization activity identification of the antibody IPB-POX1.1, it is found that the antibody IPB-POX1.1 has a significant improvement in both affinity and in vitro and in vivo neutralization activity compared to 7D11, and is at the leading level among the currently reported single neutralizing antibodies for poxviruses.

[0062] The beneficial effects of the present invention are as follows: The present invention provides a humanized antibody IPB-POX1.1 with high-efficiency vaccinia virus and monkeypox virus neutralizing activity, which is specific to vaccinia virus L1R (homologous to monkeypox virus core antigen M1R), can be expressed in large quantities using a mammalian cell expression system, and has good solubility, low cost and good biological activity. At the same time, the antibody IPB-POX1.1 has a high degree of humanization, which greatly improves its application potential. Since L1R is a highly conserved protein in poxvirus, and the embodiments prove that the antibody can efficiently neutralize vaccinia virus and monkeypox virus in in vitro experiments, in in vivo protection experiments, the advance injection of the antibody IPB-POX1.1 can completely protect mice from death caused by vaccinia virus infection. In addition, the humanized antibody provided by the present invention is the best performing monoclonal antibody among the currently reported non-complement-dependent neutralizing antibodies for poxviruses, and has great potential in the treatment and prevention of poxvirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the SDS-PAGE picture of the purification of antibody IPB-POX1.1.

[0064] Figure 2 The binding kinetic activity of antibody IPB-POX1.1 was detected by SPR.

[0065] Figure 3 To detect the neutralizing activity of antibody IPB-POX1.1 against vaccinia virus in vitro.

[0066] Figure 4 To detect the activity of antibody IPB-POX1.1 in neutralizing monkeypox virus in vitro.

[0067] Figure 5 The protective effect of antibody IPB-POX1.1 was tested in a mouse model of vaccinia virus infection. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0069] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0070] The vaccinia virus strain WR in the following example is described in the literature "Li M, Ren Z, Wang Y, et al. Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus. Emerg Microbes Infect . 2023;12(2):2223669.”

[0071] The monkeypox virus Mpox virus (lineage IIbc.1) in the following examples is recorded in the literature "Yu J, Zhang X, Liu J, et al. Phylogeny and molecular evolution of the first local monkeypox virus cluster in Guangdong Province, China. Nat Commun . 2023;14(1):8241. Published 2023 Dec 12.”

[0072] The 7D11 antibody in the following examples is described in the document “Wolffe EJ, Vijaya S, Moss B. Amyristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies. Virology. 1995 Aug 1; 211(1): 53-63.”

[0073] The antibody AI-1028 in the following example is described in the document “Yang X, Duan H, Liu X, Zhang X, Pan S, Zhang F, Gao P, Liu B, Yang J, Chi X, Yang W. Broad Sarbecovirus Neutralizing Antibodies Obtained by Computational Design and Synthetic Library Screening. J Virol. 2023 Jul 27;97(7):e0061023.”

[0074] Example 1. Expression and purification of antibody IPB-POX1.1 The heavy chain of the antibody IPB-POX1.1 of the present invention consists of a heavy chain variable region and a heavy chain constant region, and the light chain consists of a light chain variable region and a light chain constant region.

[0075] The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are shown in SEQ ID No. 1 at positions 26-33, 51-58 and 97-108, respectively. The amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 3.

[0076] The amino acid sequence of the light chain variable region is shown in SEQ ID No. 2, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown in SEQ ID No. 2, respectively, at positions 27-39, 56-58 and 95-102. The amino acid sequence of the light chain constant region is shown in SEQ ID No. 4.

[0077] The specific preparation method of antibody IPB-POX1.1 is as follows: 1. Construction of recombinant expression vector The DNA fragment between the EcoRI and BglII restriction sites of the vector pAb-hIgG1-CH containing the human heavy chain constant region sequence was replaced with the human heavy chain variable region sequence shown in SEQ ID No. 5, and the other sequences of the pAb-hIgG1-CH vector were kept unchanged to obtain recombinant plasmid A. Among them, the vector pAb-hIgG1-CH containing the human heavy chain constant region was obtained by replacing the DNA fragment between the BglII and NheI restriction sites of the Pfuse-hIgG1-Fc2 vector (invivoGen, pfuse-hg1fc2) with the human heavy chain constant region sequence shown in SEQ ID No. 7, and keeping the other sequences of the Pfuse-hIgG1-Fc2 vector unchanged.

[0078] The vector pAb-hIgG1-CL containing the human light chain (κ subtype) constant region sequence κ The DNA fragment between the restriction sites EcoRI and BglII was replaced with the human light chain variable region sequence shown in SEQ ID No.6, and the vector pAb-hIgG1-CL was maintained. κ The other sequences remain unchanged to obtain recombinant plasmid B. Among them, the vector pAb-hIgG1-CL containing the human light chain (κ subtype) constant region sequence κ The vector is obtained by replacing the DNA fragment between the BglII and NheI restriction sites of the Pfuse-hIgG1-Fc2 vector (invivoGen, pfuse-hg1fc2) with the human light chain constant region sequence shown in SEQ ID No.8, while keeping the other sequences of the Pfuse-hIgG1-Fc2 vector unchanged.

[0079] 2. Expression of IPB-POX1.1 using eukaryotic cell expression system One day before transfection, Expi293F™ cells (Thermo Fisher, A39250) were plated at 1×10 6 Density spread, place all reagents at room temperature for 10 minutes before transfection. The following operation takes 100mL cells as an example: prepare two clean centrifuge tubes, dilute 50μg plasmid DNA (25μg each of recombinant plasmid A and recombinant plasmid B) into 10mL serum-free expi293 medium (Gibco), pipette 3-4 times; add 75μL transfection reagent FectoPRO to another tube ®Reagent (polyplus, 101000007). Pour all the diluted plasmid DNA into the transfection reagent FectroPro Reagent at once, and gently mix 3-4 times immediately; let stand at room temperature for 10 minutes. Drop the transfection mixture evenly into the cell culture bottle, shake gently to disperse the transfection complex evenly, and add 50μL of booster (polyplus, 101000007) within 0-4 hours to enhance protein expression efficiency. Place the cell culture bottle in 8% CO 2 , in a constant temperature shaker at 37°C. After 5 days, the cell culture medium was collected and the protein expression level was detected by SDS-PAGE.

[0080] 3. Purification of IPB-POX1.1 The purification medium used in this example is Protein A magnetic beads (Nanjing GenScript Biotechnology Co., Ltd.). Antibody purification was performed according to the instructions of the commercial magnetic beads, and the protein system was replaced using a 15 kDa ultrafiltration tube (Millipore). Finally, the antibody protein was dissolved in a PBS system, the concentration was measured, and the purity was identified by SDS-PAGE and Coomassie Brilliant Blue staining, and then the antibody protein was aliquoted and stored at -80°C for use. Figure 1 The Coomassie Brilliant Blue staining results showed that the purity of the purified IPB-POX1.1 antibody protein was greater than 90%.

[0081] Example 2: Determination of affinity between IPB-POX1.1 and L1R protein The affinity of the antibody was determined using the BIAcore T200 biomolecular interaction instrument (GE Life Sciences). The BIAcore T200 is a versatile and highly sensitive surface plasmon resonance (SPR) system. When determining the interaction between the L1R protein and the IPB-POX1.1 humanized antibody prepared in Example 1, the L1R protein was first coated on a sensor chip, and then the IPB-POX1.1 humanized antibody was used as the mobile phase to determine the binding constant, dissociation constant, and affinity constant. The specific steps are as follows: 1. L1R protein coupled CM5 chip The RBD protein coupling temperature was 25 degrees Celsius, and the buffer was PBS-P (PBS, 0.05% P20, pH 7.4). The program template Immuobilization, CM5 chip channel 4 amino coupling was selected, the ligand was 10μg / mL L1R recombinant protein (Sino Biological, 40903-V07H), the protein buffer system was pH 5.5 sodium acetate, the target coupling amount was 300RU, and the eluent was 50mM NaOH. The chip activator was 50mmol / L N-hydroxysuccinimide (NHS) and 200mmol / L 3-(3-dimethylaminopropyl) carboxylic acid diimide hydrochloride (EDC) to activate the chip, and the blocking agent was 1mol / L ethanolamine hydrochloride.

[0082] 2. Affinity and kinetics determination of L1R with mouse antibody 7D11 and humanized antibody IPB-POX1.1 The multi-cycle kinetic template was selected, the measurement temperature was 25 degrees Celsius, the buffer was PBS-P, the sample flow path was 4-3, the sample binding time was 180s, the flow rate was 30μL / min, the dissociation time was 500s, the regeneration eluent was Glycine-HCL2.5, the regeneration liquid binding time was 30s, the flow rate was 30μL / min, the stabilization time was 0s, and the antibody (mouse antibody 7D11 or humanized antibody IPB-POX1.1) concentration was serially diluted. The data obtained were finally analyzed by BiacoreEvaluation Software to calculate the binding constant (ka), dissociation constant (kd) and affinity constant (KD). The chips, reagents and buffers used in the above Biacore analysis are all products of GE Life Sciences.

[0083] The results are shown in Table 1 and Figure 2 As shown. The results show that the affinity constant KD value of the humanized antibody obtained by the present invention and the L1R protein has approached the detection limit of the instrument, as low as 0.039nM. KD is the equilibrium dissociation constant between the antibody and its antigen, that is, the ratio of ka / kd. KD is inversely proportional to affinity. The KD value is related to the concentration of the antibody (the amount of antibody required for a specific experiment), so the lower the KD value (the lower the concentration), the higher the affinity of the antibody. Therefore, the humanized antibody IPB-POX1.1 obtained by the present invention has a relatively excellent affinity for the L1R protein.

[0084] Table 1

[0085] Example 3: In vitro vaccinia virus neutralization activity assay of IPB-POX1.1 Cell preparation: BS-C-1 cells (Wuhan Punosai, CL-0039) were plated in a 48-well plate the day before the neutralization experiment, so that the monolayer of cells covered the bottom of the well the next day. Experimental process First, the antibody + vaccinia virus mixture was prepared according to the following scheme: serum-free DMEM medium was used to dilute the antibody (antibody IPB-POX1.1 prepared in Example 1) at a 10-fold dilution ratio, with a starting concentration of 10 μg / mL, and a total of 7 concentration gradients were set, and then the antibody was mixed with 100 PFU vaccinia virus Vaccine virus strain WR, the volume ratio of antibody to virus was 1:1, and the infection volume per well was 100 μL (i.e., 50 μL each of antibody and virus), 3 duplicate wells were set for each gradient, and a PBS group (antibody concentration was 0) and an empty cell control group were set on each plate. After the mixture was placed in a 37°C, 5% carbon dioxide incubator for 1 hour, the cell culture medium was discarded, and the mixture was added to the BS-C-1 monolayer cells at 100 μL / well. The plate was placed in the incubator for infection for 2 hours, and the plate was gently shaken every half an hour or so. The infected material was then discarded, and 0.4 mL of MEM culture medium containing 1% methylcellulose and 2.5% serum was added to each well. After culturing in a 37°C incubator for 2-3 days, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then 0.1% crystal violet solution was added for staining and the plaques were counted.

[0086] The results of the vaccinia virus neutralization test are shown in Table 2 and Figure 3 As shown, the results showed that the neutralizing activity of the humanized antibody IPB-POX1.1 was improved compared with the prototype 7D11.

[0087] Table 2

[0088] Example 4: In vitro monkeypox virus neutralization activity assay of IPB-POX1.1 Cell preparation: Vero-E6 cells (ATCC, CRL-1586) were plated in a 24-well plate the day before the neutralization experiment, so that the monolayer of cells covered the bottom of the well the next day. Experimental process First, the antibody + monkeypox virus mixture was prepared according to the following scheme: serum-free DMEM medium was used to dilute the antibody (antibody IPB-POX1.1 prepared in Example 1) at a 10-fold dilution ratio, with a starting concentration of 100 μg / mL, and a total of 7 concentration gradients were set, and then the antibody was mixed with 50-100 PFU monkeypox virus Mpox virus (lineage IIbc.1), the volume ratio of antibody to virus was 1:1, and the infection volume per well was 200 μL (i.e., 100 μL of antibody and virus each), and 3 duplicate wells were set for each gradient, and at the same time, a PBS group (antibody concentration was 0) and an empty cell control group were set for each plate. After the mixture was incubated in a 37°C, 5% carbon dioxide incubator for 1 hour, the cell culture medium was discarded, and the mixture was added to the Vero-E6 monolayer cells at 200 μL / well. The plate was placed in the incubator for infection for 2 hours, and the plate was gently shaken every half an hour or so. The infection was then discarded, and 1 mL of DMEM medium containing 1% methylcellulose and 2.5% serum was added to each well. After culturing in a 37°C incubator for 2-3 days, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then 0.1% crystal violet solution was added for staining and the plaques were counted. All the above operations were performed in a BSL-3 laboratory.

[0089] The results of the neutralization activity test of monkeypox virus are shown in Table 3 and Figure 4 As shown, the results showed that the activity of IPB-POX1.1 was significantly improved compared with 7D11.

[0090] Table 3

[0091] Example 5: Evaluation of the preventive and protective effect of IPB-POX1.1 in a mouse infection model In order to understand the antiviral activity of IPB-POX1.1 under in vivo conditions, the mouse model infected with VACV-WR was used for evaluation. The experimental procedure is as follows: SPF female Balb / c mice were randomly divided into three groups, with 8-10 mice in each group. 24 hours before virus infection, IPB-POX1.1 antibody and control antibody (neutralizing antibody AI-1028 of a coronavirus, denoted as Control Ab) were injected intraperitoneally, the injection dose of IPB-POX1.1 antibody was 10 mg / kg (denoted as IPB-POX1.1-10 mg / kg experimental group) or 5 mg / kg (denoted as IPB-POX1.1-5 mg / kg experimental group), and the injection dose of Control Ab was 10 mg / kg (denoted as Control Ab control group). 24 hours later (denoted as Day 0), each mouse was inoculated with 0.5×10 5 PFU of vaccinia virus strain WR. The mice were returned to the cages and their weight was recorded daily. The mice were observed. According to the ethical review requirements for animal experiments, mice were considered dead when their weight was less than 20% of their original weight or they were unable to eat. The death of the mice was recorded. The observation lasted for 14 days, and the weight change curve and survival rate curve of the mice were statistically drawn.

[0092] The weight changes and survival curves of mice in the IPB-POX1.1-10mg / kg experimental group, IPB-POX1.1-5mg / kg experimental group, and Control Ab control group are shown in Figure 5 As shown, the left figure is the weight change curve, and the right figure is the survival curve. As can be seen from the figure, the mice injected with the control antibody began to lose weight sharply on the 4th day after infection with the vaccinia virus and eventually all died on the 7th day. However, the mice injected with the IPB-POX1.1 antibody did not show a significant change in weight during the experimental period, regardless of whether the dose was 10 mg / kg or 5 mg / kg, and no mice died until the end of the experiment. The experimental results show that the early injection of IPB-POX1.1 antibody shows excellent protective effects in the mouse model infected with vaccinia virus.

[0093] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. An antibody, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown in SEQ ID No. 1 at positions 26-33, 51-58 and 97-108, respectively; The amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are shown in SEQ ID No. 2 at positions 27-39, 56-58 and 95-102, respectively.

2. The antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is any one of the following A1)-A4): A1) The amino acid sequence is the protein shown in SEQ ID No. 1; A2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1; A3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.1; A4) a protein having 90% or more identity with the amino acid sequence shown in SEQ ID No.1 and having the same function; And / or, the amino acid sequence of the light chain variable region is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID No. 2; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 2; B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; B4) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID No. 2 and having the same function.

3. The antibody according to claim 1 or 2, characterized in that: The antibody further comprises a heavy chain constant region and a light chain constant region; And / or, the heavy chain constant region is the heavy chain constant region of human IgG1; And / or, the light chain constant region is a human Kappa light chain constant region.

4. Any of the following biological materials: C1) An active fragment of an antibody, which is any of the following derived from the antibody of any one of claims 1 to 3: an antigen-binding fragment, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a single-chain antibody, a single-domain antibody, a bispecific antibody, and a minimum recognition unit; C2) a nucleic acid molecule encoding the antibody according to any one of claims 1 to 3 or an active fragment of the antibody according to C1); C3) An expression cassette, a recombinant vector, a recombinant cell or a recombinant microorganism containing the nucleic acid molecule described in C2).

5. The biomaterial according to claim 4, characterized in that: In the nucleic acid molecule described in C2), the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown in positions 76-99, 151-174 and 289-321 of SEQ ID No. 5, respectively; And / or, in the nucleic acid molecule described in C2), the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are shown in positions 79-117, 166-174 and 283-306 of SEQ ID No.6, respectively.

6. The biomaterial according to claim 4 or 5, characterized in that: In C2), the nucleotide sequence encoding the heavy chain variable region of the antibody is SEQ ID No. 5, or a sequence having at least 75% identity with SEQ ID No. 5; And / or, in the nucleic acid molecule of C2), the nucleotide sequence encoding the light chain variable region of the antibody is SEQ ID No. 6, or a sequence having at least 75% identity with SEQ ID No.

6.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the antibody according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

8. Any application of D1) to D6) below: D1) Use of the biological material according to any one of claims 4 to 6 in the preparation of the antibody according to any one of claims 1 to 3; D2) Use of the antibody according to any one of claims 1 to 3, the biological material according to any one of claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a product for preventing or treating a disease caused by poxvirus infection; D3) Use of the antibody according to any one of claims 1 to 3, the biological material according to any one of claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a product for inhibiting poxvirus infection; D4) Use of the antibody according to any one of claims 1 to 3, the biological material according to any one of claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a product for detecting poxvirus levels and / or poxvirus L1R antigen; D5) Use of the antibody according to any one of claims 1 to 3, the biological material according to any one of claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a product for diagnosing or assisting in the diagnosis of a disease caused by poxvirus infection; D6) Use of the antibody according to any one of claims 1 to 3, the biological material according to any one of claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of a product that inhibits or neutralizes the activity of poxvirus.

9. A kit, characterized in that The kit contains the antibody according to any one of claims 1 to 3 or the biological material according to any one of claims 4 to 6 or the pharmaceutical composition according to claim 7, and the kit has any of the following uses: E1) Prevention or treatment of diseases caused by poxvirus infection; E2) Inhibition of poxvirus infection; E3) Detection of poxvirus levels and / or poxvirus L1R antigen; E4) Diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection; E5) Inhibit or neutralize poxvirus activity.

10. A method for preparing the antibody according to any one of claims 1 to 3, comprising the following steps: expressing the coding gene of the antibody according to any one of claims 1 to 3 in a host cell to obtain the antibody according to any one of claims 1 to 3.

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