Humanized antibodies against poxvirus membrane-associated protein l1r and uses thereof

Abstract

The application discloses a humanized antibody for poxvirus membrane-associated protein L1R and application thereof. An amino acid sequence of a heavy chain variable region of the humanized antibody is shown as SEQ ID No. 1, and an amino acid sequence of a light chain variable region is shown as SEQ ID No. 2. Experiments prove that the humanized antibody provided by the application can efficiently neutralize vaccinia virus and monkeypox virus in an in-vitro experiment, and in an in-vivo protection experiment, the antibody injected in advance can completely protect mice from death caused by vaccinia virus infection. In addition, the humanized antibody provided by the application is the best monoclonal antibody among currently reported poxvirus non-complement-dependent neutralizing antibodies, and has great potential in treatment and prevention of poxvirus infection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a humanized antibody against the vaccinia virus membrane-associated protein L1R and its application. Background Technology

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

[0003] Antibodies are immunoglobulins produced by plasma cells differentiated from B cells. When the immune system is stimulated, they specifically bind to corresponding antigens. The successful first use of serum injection to treat disease in the late 19th century (1890) opened a new path for modern medicine. Later, the effective component was identified as antibodies, and in 1975, the first monoclonal antibody was prepared using hybridoma technology. Antibodies are classified according to their origin: normal antibodies (natural antibodies), such as anti-A and anti-B antibodies in the ABO blood group classification, and immune antibodies, such as antimicrobial antibodies. They are also classified according to the source of the antigen they react with: heterologous antibodies, heterophilic antibodies, alloantibodies, and autoantibodies. Furthermore, they are classified according to the agglutination state of the antigen reaction: complete antibodies (IgM) and incomplete antibodies (IgG), etc. Antibodies have a wide range of applications in medical practice. They play a role in disease prevention, diagnosis, and treatment. Clinically, gamma globulin is used to prevent viral hepatitis, measles, rubella, etc., and internationally, anti-Rh immunoglobulin is used to prevent hemolytic disease 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 therapy is used for poisoning and for the treatment of immunodeficiency diseases.

[0004] Since the first murine monoclonal antibody drug was introduced in 1986, there are nearly 100 monoclonal antibody drugs on the market worldwide. The preparation technology of monoclonal antibody drugs has experienced four development stages: the first generation: murine 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 human monoclonal antibody technology; the fourth generation: fully humanized monoclonal antibody (mumab): natural fully human monoclonal antibody technology.

[0005] More than a hundred years ago, the discovery of the principle of specific binding of antibodies to antigens and passive immunization of antibodies opened up a new way for disease diagnosis. The advent of monoclonal antibody technology in 1975 accelerated the widespread application of this method. In the early stage, the monoclonal antibodies used in clinical practice were mostly of murine origin. Due to the species specificity between humans and mice, the first generation of murine antibodies can be recognized by the human immune system, causing human anti-mouse antibody (HAMA) reaction, reducing the efficacy of monoclonal antibody drugs, and causing serious adverse reactions. Therefore, the clinical application of the first generation of monoclonal antibody drugs is greatly limited. Due to the limitations of murine antibodies in clinical application, DNA recombination technology is used to humanize murine antibodies, making antibodies humanized. Humanized antibodies and fully humanized antibodies can overcome the HAMA reaction, avoid the rapid clearance of antibody molecules by the immune system as foreign proteins, and improve the efficacy of monoclonal antibody drugs. Antibody humanization is an important part of recombinant antibody production and preparation experiments.

[0006] The monkeypox virus M1R protein is homologous to the vaccinia virus L1R protein, which is a transmembrane protein found on the surface of mature IMV particles. It is encoded by the L1R ORF and is highly conserved, involved in virion assembly, and plays an important role in the process of virus entry and maturation. L1R interacts with EFC and cooperates with other known entry proteins. Monkeypox virus infection poses a significant threat to public health, and there is currently no effective antiviral treatment. Previous literature reports that neutralizing antibodies targeting L1R can exhibit non-complement-dependent neutralizing activity. However, the current antibodies have two main problems: first, multiple antibodies are needed to work together to exert strong antiviral activity; second, the source of the antibodies, such as the 7D11 antibody, is a potent neutralizing antibody derived from mice. Therefore, there is an urgent need to develop a humanized antibody that can neutralize multiple poxviruses. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a highly humanized antibody that can neutralize multiple poxviruses and its application. The technical problem to be solved is not limited to the technical subject as described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0008] To solve the above technical problems, the present application first provides an antibody, which is a humanized neutralizing antibody against the membrane-associated protein L1R of vaccinia virus, named IPB-POX1.1 in the present application, 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 as follows: 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 as follows: 27-39, 56-58 and 95-102, respectively.

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

[0010] Further, the heavy chain variable region and the light chain variable region further comprise 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 SEQ ID No. 1 as follows: 1-25; the amino acid sequence of the framework region FR2 is shown in SEQ ID No. 1 as follows: 34-50; the amino acid sequence of the framework region FR3 is shown in SEQ ID No. 1 as follows: 59-96; and the amino acid sequence of the framework region FR4 is shown in SEQ ID No. 1 as follows: 109-119.

[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 SEQ ID No. 2 as follows: 1-26; the amino acid sequence of the framework region FR2 is shown in SEQ ID No. 2 as follows: 40-55; the amino acid sequence of the framework region FR3 is shown in SEQ ID No. 2 as follows: 56-94; and the amino acid sequence of the framework region FR4 is shown in SEQ ID No. 2 as follows: 103-112.

[0013] Further, the amino acid sequence of the heavy chain variable region is any one of the following A1)-A4):

[0014] A1) the amino acid sequence is the protein shown in SEQ ID No. 1;

[0015] A2) a fusion protein having the same function as the protein shown in SEQ ID No. 1, obtained by connecting a tag at the N terminus and / or C terminus of the amino acid sequence shown in SEQ ID No. 1;

[0016] A3) a protein having the same function as the protein shown in SEQ ID No. 1, obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID No. 1;

[0017] A4) a protein having 90% or more identity to the amino acid sequence shown in SEQ ID No. 1 and having the same function.

[0018] The amino acid sequence of the light chain variable region is any one of the following B1) - B4):

[0019] B1) the amino acid sequence is the protein shown in SEQ ID No. 2;

[0020] B2) a fusion protein having the same function as the protein shown in SEQ ID No. 2, obtained by connecting a tag at the N terminus and / or C terminus of the amino acid sequence shown in SEQ ID No. 2;

[0021] B3) a protein having the same function as the protein shown in SEQ ID No. 2, obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID No. 2;

[0022] B4) a protein having 80% or more identity to the amino acid sequence shown in SEQ ID No. 2 and having the same function.

[0023] In the protein of A2) or B2) above, the tag refers to a polypeptide or protein that is expressed in fusion with the antibody of interest by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and / or purification of the antibody of interest. The tag includes but is not limited to: a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA tag protein, a Myc tag protein, a GFP (green fluorescent protein), a CFP (cyan fluorescent protein), a YFP (yellow-green fluorescent protein), a mCherry (monomeric red fluorescent protein) or an AviTag tag protein.

[0024] 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 not more than 10 amino acid residues. In certain embodiments, the substitution and / or deletion and / or addition of one or several amino acid residues can occur within one or more complementarity determining regions and / or framework regions of the antibody described in the present application, provided that such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions of amino acids, which are well known to those skilled in the art, substitution of amino acids that are conservative in nature do not change the properties and functions of the protein) can be made to the complementarity determining regions and / or framework regions, which do not substantially reduce the binding affinity.

[0025] In the protein described in A4) or B4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using the homology search site on the Internet, such as the BLAST page of the NCBI homepage website. For example, the identity of a pair of amino acid sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and performing a search in Advanced BLAST 2.1, and then the value of the identity (%) can be obtained. The identity includes an amino acid sequence having 80% or more, or having 85% or more, or having 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 to the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2.

[0026] The protein described in A1) or A2) or A3) or A4) or B1) or B2) or B3) or B4) above can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically to obtain.

[0027] Further, the antibody further comprises a heavy chain constant region and a light chain constant region.

[0028] The heavy chain constant region is the heavy chain constant region of human IgG1. Specifically, the amino acid sequence of the heavy chain constant region is SEQ ID No. 3.

[0029] The light chain constant region is the light chain constant region of human Kappa. Specifically, the amino acid sequence of the light chain constant region is SEQ ID No. 4.

[0030] To solve the above technical problems, the present application further provides any one of the following biological materials:

[0031] C1) an active fragment of the antibody, which is any one of the following derived from the above antibody: 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, a minimal recognition unit;

[0032] C2) a nucleic acid molecule encoding the above antibody or the active fragment of the antibody of C1);

[0033] C3) an expression cassette, a recombinant vector, a recombinant cell or a recombinant microorganism containing the nucleic acid molecule of C2).

[0034] In the above C2), the nucleotide sequences encoding CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown in SEQ ID No. 5, positions 76-99, 151-174 and 289-321, respectively.

[0035] The nucleotide sequences encoding CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are shown in SEQ ID No. 6, positions 79-117, 166-174 and 283-306, respectively.

[0036] Further, 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.

[0037] 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.

[0038] Further, 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.

[0039] 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.

[0040] The nucleotide sequence encoding the antibody variable region or the constant region of the present application can be easily mutated by those skilled in the art using known methods, such as methods of directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence encoding the antibody variable region or the constant region of the present application, as long as they encode the antibody variable region or the constant region and have the same function, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application. The gene sequence encoding the above antibody can be modified in its coding region without changing the amino acid sequence, as considering the degeneracy of codons, to obtain a gene encoding the same antibody; or the modified gene can be artificially synthesized according to the codon bias of the host expressing the antibody to improve the expression efficiency of the antibody, i.e. the variants of the antibody can be obtained by using methods known in the art, all of which are included in the scope of the present application. In addition, polynucleotides containing sequences optimized for antibody specificity or neutralizing activity by applying directed evolution methods to any nucleic acid sequence of the present application are also within the scope of the present application.

[0041] The identity refers to the sequence similarity with the natural nucleic acid sequence. The identity includes nucleotide sequences having 75% or more, having 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting 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 of the present application. The identity can be evaluated by naked eyes or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%) which can be used to evaluate the identity between related sequences.

[0042] In the above C3), the expression cassette refers to DNA capable of expressing the antibody in a host cell, which can include not only a promoter initiating the transcription of the antibody coding gene sequence, but also a terminator terminating the transcription of the antibody coding gene sequence.

[0043] The vector includes, but is not limited to, plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e. cosmids), viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpes viruses (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 a host cell for gene expression by transformation, transfection or transduction, etc. Specifically, the recombinant vector can be specifically recombinant plasmid A or recombinant plasmid B as follows.

[0044] 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 aforementioned recombinant vector. Specifically, the recombinant cells are Freestyle 293F cells containing the aforementioned recombinant vector.

[0045] The microorganisms mentioned include, but are not limited to, yeast, bacteria, algae, or fungi. Among them, bacteria may be derived from the genus *Escherichia*.

[0046] The recombinant microorganisms include *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*; the yeast may be *Pichia pastoris*. The recombinant microorganisms refer to microorganisms containing the above-mentioned recombinant vectors.

[0047] To address the aforementioned technical problems, the present invention also provides a pharmaceutical composition.

[0048] The pharmaceutical composition provided by this invention contains the above-mentioned antibody and a pharmaceutically acceptable carrier.

[0049] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, humectant, or disintegrant.

[0050] Agents, absorption promoters, adsorbent carriers, surfactants, or lubricants, but not limited to these.

[0051] The pharmaceutical composition is used to prevent or treat diseases caused by poxvirus infection.

[0052] To address the aforementioned technical problems, the present invention also provides any one of the following applications (D1)-D6):

[0053] D1) The application of the above-mentioned biomaterials in the preparation of the above-mentioned antibodies;

[0054] D2) The use of the above-mentioned antibody, biological material, or pharmaceutical composition in the preparation of products for the prevention or treatment of diseases caused by poxvirus infection;

[0055] D3) The use of the above-mentioned antibody, biological material, or pharmaceutical composition in the preparation of a product for inhibiting poxvirus infection;

[0056] D4) Use of the above-mentioned antibody, biological material, or pharmaceutical composition in the preparation of products for detecting poxvirus levels and / or poxvirus L1R antigen;

[0057] D5) Use of the antibody or the biological material or the pharmaceutical composition in the preparation of a product for detecting or aiding in the detection of a disease caused by a poxvirus infection.

[0058] D6) Use of the antibody or the biological material or the pharmaceutical composition in the preparation of a product for inhibiting or neutralizing the activity of a poxvirus.

[0059] In the above use, the product for detecting the level of a poxvirus and / or a poxvirus L1R antigen includes but is not limited to a product for detecting the binding of an antigen and an antibody by using enzyme-linked immunosorbent assay, immunofluorescence detection, radioimmunoassay, luminescence immunoassay, colloidal gold immunochromatography, agglutination, or immunoturbidimetry.

[0060] In the above use, the product can 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 chemiluminescence immunoassay kit, an enzyme-linked immunosorbent assay kit, a colloidal gold immunoassay kit, or a fluorescence immunoassay kit.

[0061] To solve the above technical problems, the present application further provides a kit; the kit has any one of the following uses:

[0062] E1) preventing or treating a disease caused by a poxvirus infection;

[0063] E2) inhibiting a poxvirus infection;

[0064] E3) detecting the level of a poxvirus and / or a poxvirus L1R antigen;

[0065] E4) diagnosing or aiding in the diagnosis of a disease caused by a poxvirus infection;

[0066] E5) inhibiting or neutralizing the activity of a poxvirus.

[0067] The kit provided by the present application contains the above-mentioned antibody or the above-mentioned biological material or the above-mentioned pharmaceutical composition.

[0068] Any of the above-mentioned poxviruses includes but is not limited to a monkeypox virus, a cowpox virus, a rabbitpox virus, a mousepox virus, a vaccinia virus, a smallpox virus, and a camel pox virus.

[0069] Any of the above-mentioned diseases caused by a poxvirus infection includes but is not limited to smallpox, monkeypox, cowpox, rabbitpox, mousepox, and camel pox.

[0070] To solve the above technical problems, the present application finally provides a preparation method of the above-mentioned antibody.

[0071] The preparation method of the above-mentioned antibody provided by the present application includes the following steps: expressing a coding gene of the above-mentioned antibody in a host cell to obtain the antibody.

[0072] Further, the method for expressing the coding gene of the antibody in the host cell is to introduce the coding gene of the antibody into the host cell.

[0073] The introduction can be by any known transfection method such as calcium phosphate coprecipitation, liposome-mediated, electroporation or viral vector method, etc. to transform the host cell with the vector carrying the coding gene of the antibody.

[0074] Still further, the cell (host cell) includes but is not limited to eukaryotic cell (such as yeast cell, aspergillus), animal cell (such as mammalian cell, insect cell) or prokaryotic cell.

[0075] Still further, the cell can be Freestyle 293F cell.

[0076] In some embodiments, the coding gene of the antibody is introduced into the host cell by recombinant plasmid A and recombinant plasmid B below.

[0077] The term "antigen-binding fragment" refers to an antigen-binding fragment and antibody analog of an antibody, which generally includes at least a part of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody. The antigen-binding fragment retains at least some of the binding specificity of the parental antibody. Generally, the antigen-binding fragment retains at least 10% of the binding activity of the parental antibody when activity is expressed on a molar basis. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding activity of the parental antibody to the target.

[0078] The term "antigen-binding fragment" refers to an antigen-binding fragment and antibody analog of an antibody, which generally includes at least a part of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody. The antigen-binding fragment retains at least some of the binding specificity of the parental antibody. Generally, the antigen-binding fragment retains at least 10% of the binding activity of the parental antibody when activity is expressed on a molar basis. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding activity of the parental antibody to the target.

[0079] The term "Fab fragment" is a heterodimer consisting of a heavy chain Fd and a complete light chain bound by disulfide bonds, containing only one antigen-combining site. The coding gene of the heavy chain Fd and the complete light chain is ligated and fused with the bacterial protein signal peptide gene, and then the Fab antibody (Fab fragment) can be expressed in E. coli with complete stereo folding and intra-chain and inter-chain disulfide bonds. The heavy chain Fd refers to about 1 / 2 of the H chain portion in the Fab (about 225 amino acid residues, including VH, CH1 and part of the hinge region).

[0080] The term "Fab' fragment" contains a light chain and a part of a heavy chain comprising a VH domain and a CH1 domain and a region between CH1 and CH2 domains, whereby an inter-chain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0081] The term "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0082] The term "single-chain antibody (ScFv)" refers to the light chain and heavy chain variable region genes connected by a suitable oligonucleotide linker, so that a single polypeptide chain is expressed, which is called a single-chain antibody (ScFv). The polypeptide chain can spontaneously fold into a native conformation, maintaining the specificity and affinity of the Fv.

[0083] The term "single-domain antibody (nanobody)" refers to the antibody heavy chain V region expressed by genetic engineering methods, obtaining an antibody containing only the VH fragment. The ability of single-domain antibodies to bind to antigens and their stability are basically consistent with complete antibodies.

[0084] 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, which can obtain bispecific antibodies with large yield, high uniformity and purity. In addition, bispecific antibodies can also be obtained by chemical cross-linking technology or hybrid-hybridoma technology.

[0085] The term "minimal recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only

[0086] about 1% of the complete antibody, which can bind to the corresponding antigen.

[0087] The present application selects the binding mode of the 7D11 antibody as a template, and uses the method of computer-aided design to carry out humanization modification through multiple rounds of iteration, and finally obtains the antibody IPB-POX1.1 with high degree of humanization. Through the 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 greater optimization and improvement in affinity and in vitro and in vivo neutralization activity compared with 7D11, and is at the leading level among the currently reported single neutralizing antibodies of poxviruses.

[0088] The beneficial effects of the present application are as follows: the present application provides a humanized antibody IPB-POX1.1 with high-efficiency vaccinia virus and monkeypox virus neutralization activity, which is specific to vaccinia virus L1R (homologous to monkeypox virus core antigen M1R), can be expressed in large quantities by using a mammalian cell expression system, 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 poxviruses, and the examples demonstrate that the antibody can efficiently neutralize vaccinia virus and monkeypox virus in in vitro experiments, and in in vivo protection experiments, pre-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 application is the best monoclonal antibody among the currently reported poxvirus complement-independent neutralizing antibodies, and has great potential in the treatment and prevention of poxvirus infection. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 SDS-PAGE figure for purification of the antibody IPB-POX1.1.

[0090] Figure 2 SPR detection of the binding kinetics activity of the antibody IPB-POX1.1.

[0091] Figure 3 In vitro detection of the activity of the antibody IPB-POX1.1 in neutralizing vaccinia virus.

[0092] Figure 4 In vitro detection of the activity of the antibody IPB-POX1.1 in neutralizing monkeypox virus.

[0093] Figure 5 Detection of the protection effect of the antibody IPB-POX1.1 in a vaccinia virus-infected mouse model. DETAILED DESCRIPTION

[0094] The present application will be further described in detail below in conjunction with specific embodiments, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0095] The experimental methods in the following examples are all routine methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified. In the quantitative tests in the following examples, three repeated experiments were set up, and the results were averaged.

[0096] The vaccine virus strain WR in the following examples 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."

[0097] The Mpox virus (lineage IIbc.1) in the following examples is described 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."

[0098] The 7D11 antibody in the following examples is described in the literature "Wolffe EJ, Vijaya S, Moss B. A myristylated 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."

[0099] The antibody AI-1028 in the following examples is described in the literature "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."

[0100] Example 1, Expression and purification of antibody IPB-POX1.1

[0101] The heavy chain of the antibody IPB-POX1.1 of the present application is composed of a heavy chain variable region and a heavy chain constant region, and the light chain is composed of a light chain variable region and a light chain constant region.

[0102] 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.

[0103] 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 at positions 27-39, 56-58 and 95-102, respectively. The amino acid sequence of the light chain constant region is shown in SEQ ID No. 4.

[0104] The specific preparation method of the antibody IPB-POX1.1 is as follows:

[0105] I. Construction of recombinant expression vector

[0106] The DNA fragment between EcoRI and BglII enzyme cutting sites of the vector pAb-hIgG1-CH containing human heavy chain constant region sequence was replaced by 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 human heavy chain constant region sequence is obtained by replacing the DNA fragment between BglII and NheI enzyme cutting 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.

[0107] The DNA fragment between EcoRI and BglII enzyme cutting sites of the vector pAb-hIgG1-CH containing human heavy chain constant region sequence was replaced by 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 human heavy chain constant region sequence is obtained by replacing the DNA fragment between BglII and NheI enzyme cutting 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. κ κ The DNA fragment between EcoRI and BglII enzyme cutting sites of the vector pAb-hIgG1-CH containing human heavy chain constant region sequence was replaced by 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 human heavy chain constant region sequence is obtained by replacing the DNA fragment between BglII and NheI enzyme cutting 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. κ The DNA fragment between EcoRI and BglII enzyme cutting sites of the vector pAb-hIgG1-CH containing human heavy chain constant region sequence was replaced by 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 human heavy chain constant region sequence is obtained by replacing the DNA fragment between BglII and NheI enzyme cutting 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.

[0108] II. Expression of IPB-POX1.1 using eukaryotic cell expression system

[0109] Expi293F™ cells (Thermo Fisher, A39250) were plated at a density of 1×10 6 All reagents were placed at room temperature for 10 minutes before transfection. The following operations are taken as an example of 100 mL cells: prepare two clean centrifuge tubes, dilute 50 μg of plasmid DNA (25 μg of recombinant plasmid A and 25 μg of recombinant plasmid B) into 10 mL serum-free expi293 medium (Gibco) with a pipette gun blowing and sucking 3-4 times; another tube is added with 75 μL of transfection reagent FectoPRO ® ​Reagent (polyplus, 101000007). Diluted plasmid DNA was poured into the transfection reagent FectroPro Reagent all at once, and immediately mixed gently for 3-4 times; room temperature for 10 minutes. The transfection mixture was evenly dropped into the cell culture bottle, and the transfection complex was evenly dispersed by gently shaking. 50 μL of booster (polyplus, 101000007) was added within 0-4 hours to enhance the protein expression efficiency. The cell culture bottle was placed in an 8% CO2, 37°C constant temperature incubator, and the cell culture solution was collected after 5 days. The protein expression amount was detected by SDS-PAGE.

[0110] III. Purification of IPB-POX1.1

[0111] The purification medium used in this example is Protein A magnetic beads (Nanjing Kingsrui Biological Technology Co., Ltd.). According to the instructions of the commercial magnetic beads, the antibody purification was carried out. The protein was replaced by a 15 kDa ultrafiltration tube (Millipore). The final antibody protein was dissolved in PBS system. The concentration was determined and the purity was identified by SDS-PAGE and Coomassie blue staining. Then it was stored at -80°C for use. Figure 1 The Coomassie blue staining results of IPB-POX1.1 antibody protein showed that the purity of the purified IPB-POX1.1 antibody protein was greater than 90%.

[0112] Example 2, IPB-POX1.1 and L1R protein affinity determination

[0113] The BIAcore T200 biomolecular interaction instrument (GE Life Sciences Co.) was used for affinity determination of the antibody. BIAcore T200 is a multifunctional and high sensitivity surface plasmon resonance (SPR) system. In the determination of the interaction between L1R protein and IPB-POX1.1 humanized antibody prepared in Example 1, L1R protein was first coated on the sensor chip, and then IPB-POX1.1 humanized antibody was used as the mobile phase to determine the association constant, dissociation constant and affinity constant. The specific steps are as follows:

[0114] 1. L1R protein coupling CM5 chip

[0115] The RBD protein was coupled at a temperature of 25 degrees Celsius and in a PBS-P (PBS, 0.05% P20, pH 7.4) buffer. The program template was Immuobilization, and channel 4 of the CM5 chip was selected for amino coupling. The ligand was 10 μg / mL L1R recombinant protein (Yiqiao God, 40903-V07H), and the protein buffer system was sodium acetate at pH 5.5. The target coupling amount was 300 RU, and the eluent was 50 mM NaOH. The chip activator was 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the chip, and the blocking agent was 1 mol / L ethanolamine hydrochloride.

[0116] 2. Affinity and kinetic determination of L1R and mouse antibody 7D11 and humanized antibody IPB-POX1.1

[0117] The multi-cycle kinetic template was selected, the determination temperature was 25 degrees Celsius, the buffer was PBS-P, the sample flow path was 4-3, the sample binding time was 180 s, the flow rate was 30 μL / min, the dissociation time was 500 s, the regeneration eluent was glycine-HCL 2.5, the regeneration liquid binding time was 30 s, the flow rate was 30 μL / min, the stabilization time was 0 s, and the antibody (mouse antibody 7D11 or humanized antibody IPB-POX1.1) was diluted in series. The final data was analyzed by Biacore Evaluation Software, and the association constant (ka), dissociation constant (kd), and affinity constant (KD) were calculated. The chip, reagent, and buffer used in the above Biacore analysis were all products of GE Life Sciences.

[0118] The results are shown in Table 1 and Figure 2 The results show that the affinity constant KD value of the humanized antibody obtained by the present application and the L1R protein has approached the lower limit of detection of the instrument, as low as 0.039 nM. 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 application has a relatively excellent affinity for the L1R protein.

[0119] Table 1

[0120]

[0121] Example 3, determination of the in vitro vaccinia virus neutralization activity of IPB-POX1.1

[0122] Cell preparation: One day before the neutralization experiment, BS-C-1 cells (Wuhan Ponsay, CL-0039) were plated in 48-well plates so that the monolayer cells would cover the bottom of the wells the next day. First, the antibody + vaccinia virus mixture was prepared according to the following scheme: the antibody (IPB-POX1.1 prepared in Example 1) was diluted in serum-free DMEM medium at a dilution ratio of 10 times, with an initial concentration of 10 μg / mL, and a total of 7 concentration gradients were set up. Then the antibody was mixed with 100 PFU of vaccinia virus Vaccine virus strain WR, with a volume ratio of antibody to virus of 1:1, and the infection volume per well was 100 μL (i.e. 50 μL of antibody and 50 μL of virus), 3 replicate wells were set up for each gradient, and a PBS group (antibody concentration of 0) and a blank cell control group were set up for each plate. The above mixture was incubated in a 37°C, 5% carbon dioxide incubator for 1 h, then the cell culture medium was discarded, and the above mixture was added to the BS-C-1 monolayer cells at 100 μL / well, and the plate was placed in the incubator for 2 h, during which the plate was gently shaken every half hour or so. Then the infection was discarded, 0.4 mL of MEM medium containing 1% methyl cellulose and 2.5% serum was added to each well, and after 2-3 days of culture in a 37°C incubator, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, then 0.1% crystal violet solution was added for staining, and the plaques were counted.

[0123] The results of the vaccinia virus neutralization test are shown in Table 2 and Figure 3 The results show that the neutralization activity of the humanized antibody IPB-POX1.1 is improved compared to the prototype 7D11.

[0124] Table 2

[0125]

[0126] Example 4, In vitro monkeypox virus neutralization activity determination of IPB-POX1.1

[0127] Cell preparation: One day before the neutralization experiment, Vero-E6 cells (ATCC, CRL-1586) were plated in 24-well plates so that the monolayer cells would cover the bottom of the wells the next day. First, the antibody + monkeypox virus mixture was prepared according to the following scheme: the antibody (IPB-POX1.1 prepared in Example 1) was diluted in serum-free DMEM medium at a dilution ratio of 10 times, with an initial concentration of 100 μg / mL, a total of 7 concentration gradients were set, and then the antibody was mixed with 50-100 PFU of monkeypox virus Mpox virus (lineage IIbc.1), the volume ratio of antibody to virus was 1:1, the infection volume of each well was 200 μL (i.e. 100 μL of antibody and virus), 3 replicate wells were set for each gradient, and PBS groups (antibody concentration of 0) and empty cell control groups were also set for each plate. The above mixture was incubated in a 37°C, 5% CO2 incubator for 1 h, then the cell culture medium was discarded, and the above mixture was added to the Vero-E6 monolayer cells at 200 μL / well, and the plate was placed in the incubator for 2 h, during which the well plate was gently shaken every half hour or so. Then the infection was discarded, 1 mL of DMEM medium containing 1% methyl cellulose and 2.5% serum was added to each well, and after 2-3 days of culture in a 37°C incubator, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, then 0.1% crystal violet solution was added for staining and plaque counting. The above operations were all carried out in a BSL-3 laboratory.

[0128] The results of the monkeypox virus neutralization activity detection are shown in Table 3 and Figure 4 The results show that the activity of IPB-POX1.1 is greatly improved compared with 7D11.

[0129] Table 3

[0130]

[0131] Example 5, Evaluation of the preventive protection effect of IPB-POX1.1 in a mouse infection model

[0132] To understand the antiviral activity of IPB-POX1.1 under in vivo conditions, a mouse model infected with VCV-WR was used for evaluation. The experimental procedure was as follows: SPF-grade female Balb / c mice were randomly divided into three groups, with 8-10 mice in each group. Twenty-four hours before viral infection, IPB-POX1.1 antibody and a control antibody (a neutralizing antibody against a coronavirus, AI-1028, denoted as Control Ab) were administered intraperitoneally. The IPB-POX1.1 antibody dose was 10 mg / kg (IPB-POX1.1-10 mg / kg experimental group) or 5 mg / kg (IPB-POX1.1-5 mg / kg experimental group), and the Control Ab dose was 10 mg / kg (Control Ab control group). Twenty-four hours later (Day 0), each mouse was inoculated intranasally with 0.5 × 10⁻⁶ mmol / L of the antiviral antibody. 5 PFU-based Vaccine Virus Strain WR. Mice were returned to their cages, and their weight and condition were recorded daily. In accordance with animal experiment ethics requirements, mice were considered dead when their weight fell below 20% of their original weight or when they refused to eat. Mortality was recorded. The observation period was 14 days, and the mouse weight change curve and survival rate curve were plotted.

[0133] The weight changes and survival curves of mice in the IPB-POX 1.1-10 mg / kg experimental group, IPB-POX 1.1-5 mg / kg experimental group, and Control Ab control group are shown in the figure below. Figure 5 As shown in the figures, the left graph represents the weight change curve, and the right graph represents the survival curve. The figures reveal that mice injected with the control antibody experienced a sharp decline in weight starting on day 4 after vaccinia virus infection, ultimately leading to death by day 7. However, mice injected with IPB-POX1.1 antibody, regardless of the dose (10 mg / kg or 5 mg / kg), did not show significant weight changes throughout the experimental period, and no mice died by the experimental endpoint. These results demonstrate that pre-injection of IPB-POX1.1 antibody provides excellent protection in a mouse model of vaccinia virus infection.

[0134] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An antibody against vaccinia virus membrane-associated protein L1R, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are shown as 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 variable region of the light chain of the antibody are shown as positions 27-39, 56-58 and 95-102 of SEQ ID No. 2, 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 that of the protein shown in SEQ ID No. 1; A2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 1; A3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1; A4) Proteins that have 90% or more of the same amino acid sequence as shown in SEQ ID No. 1 and have 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 that of the protein shown in SEQ ID No. 2; B2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 2; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; B4) is a protein that has 80% or more of the same amino acid sequence as the one shown in SEQ ID No. 2 and has the same function.

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

4. Any of the following biological materials: C1) The active fragment of the antibody is any of the following derived from any of the antibodies described in any of claims 1-3: antigen-binding fragment, bispecific antibody; C2) A nucleic acid molecule, said nucleic acid molecule encoding the antibody according to any one of claims 1-3 or the active fragment of the antibody according to C1); C3) Expression cassettes, recombinant vectors, recombinant cells or recombinant microorganisms containing the nucleic acid molecules described in C2).

5. The biomaterial according to claim 4, characterized in that: In the nucleic acid molecule described in C2), the nucleotide sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region encoding 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 of CDR1, CDR2 and CDR3 in the light chain variable region encoding 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 the nucleic acid molecule described 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 described in 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 as described in any one of claims 1-3, and a pharmaceutically acceptable carrier.

8. Any one of the following applications (D1)-D6): D1) The use of the biomaterials of any one of claims 4-6 in the preparation of the antibodies of any one of claims 1-3; D2) Use of the antibody of any one of claims 1-3, the biomaterial of any one of claims 4-6, or the pharmaceutical composition of claim 7 in the preparation of a product for the prevention or treatment of diseases caused by poxvirus infection; D3) Use of the antibody of any one of claims 1-3, the biomaterial of any one of claims 4-6, or the pharmaceutical composition of claim 7 in the preparation of a product for inhibiting vaccinia virus infection; D4) Use of any of the antibodies of claims 1-3, any of the biological materials of claims 4-6, or the pharmaceutical composition of claim 7 in the preparation of products for detecting poxvirus levels and / or poxvirus L1R antigen; D5) Use of the antibody of any one of claims 1-3, the biomaterial of any one of claims 4-6, or the pharmaceutical composition of claim 7 in the preparation of a product for the diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection; D6) Use of the antibody of any one of claims 1-3, the biomaterial of any one of claims 4-6, or the pharmaceutical composition of claim 7 in the preparation of a product that inhibits or neutralizes poxvirus activity; The poxvirus mentioned is either vaccinia virus or monkeypox virus.

9. A reagent kit, characterized in that, The kit contains the antibody of any one of claims 1-3, the biological material of any one of claims 4-6, or the pharmaceutical composition of claim 7, and the kit has any of the following uses: E1) Prevention or treatment of diseases caused by poxvirus infection; E2) Inhibits 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; The poxvirus mentioned is either vaccinia virus or monkeypox virus.

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

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