Humanized antibody aiming at vaccinia virus membrane protein A33R and application thereof

Abstract

The invention discloses a humanized antibody aiming at vaccinia virus membrane protein A33R 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 in an in-vivo protection experiment, mice can be completely protected from death caused by vaccinia virus infection by injecting the humanized antibody in advance, and the humanized antibody has great potential in treatment and prevention of vaccinia virus infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibodies, and particularly relates to a humanized antibody against a membrane protein A33R of vaccinia virus and application thereof. BACKGROUND

[0002] Vaccinia virus (VACV) belongs to Orthopoxvirus of Poxviridae, is a complex double-stranded DNA virus, and has a brick or oval shape with a size of about 200x250nm. The virus core contains a complete genome and an enzyme system involved in virus replication, and the outer layer is wrapped by a lipid protein envelope, and the envelope is inlaid with various viral proteins, which play a key role in the process of virus invasion into host cells. Vaccinia virus has strong independent replication ability and does not need to rely on many replication mechanisms of host cells, and can complete its own replication cycle in the cytoplasm of host cells. VACV infectious virus particles have mature virus form (MV) and envelope virus form (EV).

[0003] Antibody, also known as immunoglobulin, is a kind of glycoprotein with specific binding ability to antigen, which is produced by plasma cells formed by B lymphocytes under the stimulation of antigen. The molecular structure of antibody is Y-shaped, which is composed of two identical heavy chains (H chain) and two identical light chains (L chain) connected by disulfide bond. The structure can be divided into variable region (V region) and constant region (C region). The variable region is the specific binding site of antibody and antigen, and the amino acid sequences of variable regions of different antibodies are different, thereby determining the specificity of antibody binding to antigen. The constant region has various biological functions, such as activating complement system, binding to cell surface Fc receptor, etc., and mediates the effector function of antibody.

[0004] In 1975, Kohler and Milstein invented the hybridoma technology, successfully prepared monoclonal antibodies, and accelerated the wide application of antibodies in the field of biological medicine. At this stage, the prepared monoclonal antibodies are mainly mouse-derived antibodies, which have the advantages of high specificity and large-scale preparation, and have been widely used in disease diagnosis and basic research. However, mouse-derived antibodies have strong immunogenicity in the human body, and when they are used for human treatment, they can easily induce human anti-mouse antibody (HAMA) reaction. HAMA reaction not only shortens the half-life of mouse-derived antibodies in the human body, reduces their therapeutic effect, but also can cause allergic reactions, serum sickness and other adverse reactions, which seriously limit the application of mouse-derived antibodies in human disease treatment. On this basis, people began to use DNA recombination technology to humanize mouse-derived antibodies, so that their amino acid sequences are as close as possible to the amino acid sequences of human antibodies, thereby reducing their immunogenicity in the human body and improving their safety and effectiveness in the human body. Humanized antibodies not only retain the ability of parent antibodies (such as mouse-derived antibodies) to specifically bind to antigens, but also have lower immunogenicity, can better exert their biological effects in the human body, reduce adverse reactions caused by immunogenicity, such as allergic reactions, antibody-dependent cellular cytotoxicity, etc., and to some extent, improve the drug possibility of antibodies.

[0005] The vaccinia virus A33R protein is a homodimer type II transmembrane glycoprotein present on the EEV envelope, which can regulate virion movement and actin tail nucleation, and is responsible for correctly transporting B5 to EEV-specific membranes and correctly forming infectious EEV. A33R is essential for the efficient formation of EEV and the long-distance transmission of viruses in the host body, so A33R is an important target for neutralizing antibody response to EEV. Previous literature has reported the preparation of high-affinity antibodies against A33R, such as A27D7 antibodies that can bind to A33R dimers. The literature reports the specific binding site of the antibody and its protective effect on vaccinia in mice. However, the A27D7 antibody is a mouse-derived antibody with strong immunogenicity in the human body, which greatly reduces the drug possibility of the antibody, and a humanized antibody needs to be developed, which can maintain the antiviral activity of the antibody and increase the humanization degree of the antibody to increase its drug possibility. SUMMARY

[0006] The technical problem to be solved by the present application is how to prevent or / and treat poxviruses. Without solving the technical problem, the present application provides the following technical solutions: The present application provides an antibody or antigen-binding fragment thereof that binds to a poxvirus membrane protein A33R, the antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being as set forth in SEQ ID NO: 1; and / or the amino acid sequence of the light chain variable region being as set forth in SEQ ID NO: 2.

[0007] In the present application, the heavy chain variable region comprises three heavy chain variable region complementarity determining regions designated as HCDR1, HCDR2 and HCDR3, respectively, and four heavy chain variable region framework regions designated as HFR1, HFR2, HFR3 and HFR4, respectively. The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are as set forth in SEQ ID NO: 1 at positions 26-33, 51-58 and 97-108, respectively.

[0008] In the present application, the light chain variable region comprises three light chain variable region complementarity determining regions designated as LCDR1, LCDR2 and LCDR3, respectively, and four light chain variable region framework regions designated as LFR1, LFR2, LFR3 and LFR4, respectively. The amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 2, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region are as set forth in SEQ ID NO: 2 at positions 27-39, 56-58 and 95-102, respectively.

[0009] In the present application, the complementarity determining regions (CDRs) are defined based on the IMGT numbering scheme.

[0010] The antibody or antigen-binding fragment thereof can be humanized.

[0011] The term "antibody" in the present application is a heterotetrasaccharide protein of about 150,000 daltons having the same structural features, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain. In the antibody or antigen-binding fragment thereof, the binding can be specific binding.

[0012] The specific binding refers to the affinity of one molecule binding to another molecule being significantly higher than the affinity of binding to any cross-reacting antigen or off-target antigen (collectively, non-target antigen). The affinity is determined using experimental techniques such as surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, Kinetic Exclusion Assay (KinExA), isothermal titration calorimetry (ITC), radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective reaction will be at least 1.5 times the non-target signal or non-target binding noise.

[0013] In the present application, the antibody or antigen-binding fragment thereof can further include a constant region.

[0014] In the present application, the constant region of the heavy chain is IgG1 or IgG4.

[0015] In the present application, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 3.

[0016] In the present application, the constant region of the light chain is kappa or lambda.

[0017] In the present application, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 4.

[0018] The present application also provides a nucleic acid molecule that can encode the above-mentioned antibody or antigen-binding fragment thereof.

[0019] In the above-mentioned nucleic acid molecule, the coding gene of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as follows A1) or A2): A1) a DNA molecule having a coding sequence as shown in SEQ ID NO: 5; A2) a DNA molecule having more than 70% identity with the DNA molecule shown in A1) and encoding the heavy chain variable region.

[0020] In the above-mentioned nucleic acid molecule, the coding gene of the light chain variable region of the antibody or antigen-binding fragment thereof is as follows B1) or B2): B1) a DNA molecule having a coding sequence as shown in SEQ ID NO: 6; B2) a DNA molecule having more than 70% identity with the DNA molecule shown in B1) and encoding the light chain variable region.

[0021] In the above-mentioned nucleic acid molecule, the coding gene of the heavy chain constant region of the antibody or antigen-binding fragment thereof is as follows C1) or C2): C1) a DNA molecule having a coding sequence as shown in SEQ ID NO: 7; C2) a DNA molecule having 70% or more identity to the DNA molecule of C1), and encoding the heavy chain constant region.

[0022] Among the above nucleic acid molecules, the coding gene for the light chain constant region of the antibody or antigen-binding fragment thereof is as follows: D1) a DNA molecule having a coding sequence as set forth in SEQ ID NO: 6; D2) a DNA molecule having 70% or more identity to the DNA molecule of D1), and encoding the light chain constant region.

[0023] The present application also provides an expression cassette containing the above nucleic acid molecule.

[0024] The present application also provides a recombinant vector containing the above nucleic acid molecule or containing the above expression cassette.

[0025] The present application also provides a recombinant cell containing the above nucleic acid molecule, containing the above expression cassette, or containing the above recombinant vector.

[0026] In the present application, the cell can be a prokaryotic cell or a eukaryotic cell.

[0027] In the present application, the cell can be a cell from an animal, a plant, or a microorganism.

[0028] In the present application, the cell from an animal can be an ex vivo animal cell.

[0029] In the present application, the ex vivo animal cell includes, but is not limited to, ExpiCHO-S™ cells, Chinese hamster ovary cells (CHO), 293F cells, 293E cells, 293-6E cells, and the like.

[0030] In one specific embodiment of the present application, the ex vivo animal cell is a 293F cell.

[0031] In the present application, the microorganism can be a bacterium (such as Escherichia coli), a yeast, an algae, or a fungus. In the present application, the bacterium can be Escherichia coli.

[0032] The present application also provides a method for preparing the above antibody or antigen-binding fragment thereof, the method comprising the step of expressing the coding gene of the antibody or antigen-binding fragment thereof in a mammalian cell to obtain the antibody or antigen-binding fragment thereof. In the above method, the cell can be an ex vivo cell or a non-human animal body cell.

[0033] The present application also provides a medicament or a pharmaceutical composition comprising the above antibody or antigen-binding fragment thereof.

[0034] In the present application, the medicament or pharmaceutical composition can also comprise a pharmaceutically acceptable excipient or carrier.

[0035] The present application also provides the use of at least one of the following E1 ) the use of the above-mentioned nucleic acid molecule for the preparation of the above-mentioned antibody; E2) the use of the above-mentioned expression cassette for the preparation of the above-mentioned antibody; E3) the use of the above-mentioned recombinant vector for the preparation of the above-mentioned antibody; E4) the use of the above-mentioned recombinant cell for the preparation of the above- mentioned antibody; E5) the use of the above-mentioned antibody for the preparation of a product for the prevention or treatment of a disease caused by a poxvirus infection, for the preparation of a product for detecting the level of a poxvirus and / or the level of a poxvirus A33R antigen, for the preparation of a product for the diagnosis or the aid diagnosis of a disease caused by a poxvirus infection or / and for the preparation of a product for the inhibition or neutralization of the activity of a poxvirus; E6) the use of the above-mentioned nucleic acid molecule for the preparation of a product for the prevention or treatment of a disease caused by a poxvirus infection, for the preparation of a product for detecting the level of a poxvirus and / or the level of a poxvirus A33R antigen, for the preparation of a product for the diagnosis or the aid diagnosis of a disease caused by a poxvirus infection or / and for the preparation of a product for the inhibition or neutralization of the activity of a poxvirus; E7) the use of the above-mentioned expression cassette for the preparation of a product for the prevention or treatment of a disease caused by a poxvirus infection, for the preparation of a product for detecting the level of a poxvirus and / or the level of a poxvirus A33R antigen, for the preparation of a product for the diagnosis or the aid diagnosis of a disease caused by a poxvirus infection or / and for the preparation of a product for the inhibition or neutralization of the activity of a poxvirus; E8) the use of the above-mentioned recombinant vector for the preparation of a product for the prevention or treatment of a disease caused by a poxvirus infection, for the preparation of a product for detecting the level of a poxvirus and / or the level of a poxvirus A33R antigen, for the preparation of a product for the diagnosis or the aid diagnosis of a disease caused by a poxvirus infection or / and for the preparation of a product for the inhibition or neutralization of the activity of a poxvirus; E9) the use of the above-mentioned recombinant cell for the preparation of a product for the prevention or treatment of a disease caused by a poxvirus infection, for the preparation of a product for detecting the level of a poxvirus and / or the level of a poxvirus A33R antigen, for the preparation of a product for the diagnosis or the aid diagnosis of a disease caused by a poxvirus infection or / and for the preparation of a product for the inhibition or neutralization of the activity of a poxvirus.

[0036] The product can be a medicament or a pharmaceutical composition.

[0037] The product can also be a reagent or a kit.

[0038] The above-mentioned antibody or antigen-binding fragment thereof or a composition comprising the above-mentioned antibody or antigen-binding fragment thereof for use as a medicament also falls within the scope of the present application.

[0039] In some embodiments of the present application, the medicament can be a medicament for preventing or treating poxvirus or poxvirus infection.

[0040] In some embodiments of the present application, the poxvirus is Vaccine virus strain WR.

[0041] The present application also provides use of the above-mentioned antibody or antigen binding fragment thereof, medicament or pharmaceutical composition for preventing and / or treating poxvirus or poxvirus infection.

[0042] The present application also provides a method for treating or / and preventing poxvirus or poxvirus infection, which comprises the step of administering an effective dose of the above-mentioned antibody or antigen binding fragment thereof, medicament or pharmaceutical composition to a subject to treat or / and prevent poxvirus or poxvirus infection.

[0043] In the present application, the subject can be a poxvirus susceptible population or a poxvirus infected person.

[0044] In some embodiments of the present application, the poxvirus is vaccinia virus. More particularly, the vaccinia virus is Vaccine virus strain WR. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 SDS-PAGE figure for antibody IPB-POX2.1 purification.

[0046] Figure 2 SPR detection of binding kinetics activity of antibody IPB-POX2.1.

[0047] Figure 3 Poxvirus infected mouse model detection of protective effect of antibody IPB-POX2.1.

[0048] Figure 4 Heavy / light chain humanization analysis of A27D7 and IPB-POX2.1.

[0049] Figure 5 Sequence in the present application. DETAILED DESCRIPTION

[0050] I. Terms in the present application: Examples of resources that describe many of the terms related to molecular biology used herein can be found in Alberts et al., Molecular Biology of The Cell, 5th Ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Ed., Springer- Verlag: New York, 1991 ; King et al., A Dictionary of Genetics, 6th Ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0051] Any reference cited herein, including, for example, all patents, published patent applications and non-patent publications, is incorporated by reference in its entirety.

[0052] For the purposes of understanding the present application, several terms and abbreviations used herein are defined as follows: In the present application, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence (or a nucleotide sequence) can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI home page website. For example, the identity of a pair of amino acid sequences can be calculated by searching 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 using the Advanced BLAST 2.1, and then the value of the identity (%) can be obtained.

[0053] In particular, the greater than 70% identity can be greater than 75% identity. In particular, the greater than 75% identity can be greater than 80% identity. In particular, the greater than 80% identity can be greater than 85% identity. In particular, the greater than 85% identity can be greater than 90% identity. In particular, the greater than 90% identity can be greater than 91% identity, greater than 92% identity, greater than 93% identity, greater than 94% identity, greater than 95% identity, greater than 96% identity, greater than 97% identity, greater than 98% identity, or greater than 99% identity. More particularly, the greater than 70% identity can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0054] The term "and / or" when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e. A alone, B alone, or A and B in combination. The expression "A, B, and / or C" is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0055] The term "comprising", is intended to mean inclusive, and not exclusive. It is intended to mean that the compositions, methods, and systems include the listed elements, but not excluding additional elements. The term "comprising" encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably in this document.

[0056] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to polymers of amino acids linked via peptide (amide) bonds. These terms refer to a protein, peptide, or polypeptide of any size, structure or function. Typically, a protein, peptide, or polypeptide is at least 3 amino acids in length. A protein, peptide, or polypeptide can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, e.g., by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnyl group, an isofarnyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide can also be a single molecule or can be a multimeric complex. A protein, peptide, or polypeptide can be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. Any protein provided herein can be produced by any method known in the art. For example, a protein provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising peptide linkers.

[0057] Complementarity determining regions (CDRs) are the antigen binding sites in antibodies. CDRs can be defined using various terms: (i) Complementarity determining regions (CDRs) are based on sequence variability. (ii) "Hypervariable regions," "HVRs," or "HV" refer to the regions of an antibody variable domain that are highly variable in structure relative to other variable domain sequences, as defined by Chothia and Lesk. The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definitions for antigen binding sites. As used herein, the terms "CDR," "CDR1," "CDR2," "CDR3" include CDRs defined by any of the methods described above, Kabat, Chothia, or IMGT, unless otherwise explicitly stated in the specification. Framework regions (FW) are adjacent to and located between the CDRs.

[0058] As used herein, the term "fragment" refers to a polypeptide that substantially retains the same biological function or activity of an antibody of the application. A polypeptide fragment of the application can be (i) a polypeptide having one or more conservative or non-conservative amino acid residue substitutions (preferably conservative amino acid residue substitutions), where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusing a mature polypeptide to another compound (such as a compound that prolongs the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence or a protein for purifying the polypeptide or a proprotein sequence, or a fusion protein with a 6xHis tag).

[0059] The term "antigen binding fragment" refers to a fragment of an antibody of the present application, which generally includes at least a portion of an antigen binding or variable region (e.g., one or more CDRs) of a parental antibody. Such an antigen binding fragment retains at least certain binding specificities of the parental antibody. Generally, such an antigen binding fragment retains at least 10% of the binding activity of the parental antibody, when activity is expressed on a molar basis. Specifically, such an antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parental antibody to the target.

[0060] In the present application, the antibody or antigen binding fragment thereof includes, but is not limited to, a recombinant antibody, a bivalent antibody, a multivalent antibody, a multispecific antibody, a single heavy chain antibody, a Fab, a Fab' fragment, a F(ab')2 fragment, a minimal recognition unit (MRU), a Fv antibody, a single chain antibody, or a heavy chain antibody.

[0061] The term "bivalent antibody" or "multivalent antibody" refers to a fusion polypeptide formed by linking two or more of the antibodies. The antibodies in the "bivalent antibody" or "multivalent antibody" are formed by covalent or non-covalent linkage via a linker molecule, or are formed by non-covalent linkage via mixing with a multimer.

[0062] The term "multispecific antibody" refers to a fusion polypeptide having multispecificity formed by linking the antibody with an antigen binding fragment specific for another antigen.

[0063] The term "multispecific" refers to an antigen binding protein (the antibody or antigen binding fragment thereof) having two or more different antigen binding specificities.

[0064] The term "Fab" is a fragment of antigen binding (Fab), which is composed of one complete antibody light chain and the VH (variable region of heavy chain) and CH1 (constant region 1 of heavy chain) domains of a heavy chain. That is, a heterodimer formed by the combination of the Fd of the heavy chain and the complete light chain through a disulfide bond.

[0065] The term "Fab' fragment" contains one complete antibody light chain and a portion of one antibody heavy chain containing the VH domain and CH1 domain and the region between CH1 and CH2 domains. Thus, an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0066] The term "F(ab')2 fragment" is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0067] The term "minimal recognition unit (MRU)" refers to a single CDR structure in the variable region only, with a molecular mass of only about 1% of the intact antibody, which can bind to the corresponding antigen.

[0068] The term "Fv antibody" is a protein consisting of the variable region of the heavy chain and the variable region of the light chain of an antibody. The variable region of the heavy chain and the variable region of the light chain are connected by non-covalent bonds.

[0069] The term "single chain antibody" (ScFv) is a protein in which the variable region of the heavy chain and the variable region of the light chain of an antibody are connected by a short peptide.

[0070] The term "specific binding", "specific recognition" or "specific for" as used herein refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.

[0071] The term "specificity" refers to the selective recognition of an antibody for a specific epitope of an antigen.

[0072] The specific binding refers to the affinity of one molecule to bind to another molecule significantly higher than the affinity to bind to any cross-reacting antigen or off-target antigen (collectively referred to as non-target antigen). The affinity is determined using experimental techniques, such as surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS) analysis, Kinetic Exclusion Assay (KinExA), isothermal titration calorimetry (ITC), radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA).

[0073] Unless otherwise indicated, the terms "nucleic acid", "nucleotide" and "polynucleotide" encompass both DNA and RNA.

[0074] The host cell can be a prokaryotic cell, such as a bacterial cell, more specifically, such as an E. coli cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0075] The nucleic acid sequence encoding the antibody or antigen-binding fragment thereof disclosed herein can be introduced into a cell by "transfection", "transformation" or "transduction". As used herein, "transfection", "transformation" or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.

[0076] The term "transformation" means the introduction of one or more exogenous polynucleotides into a bacterial cell that has the ability to be transformed, for example, by using dimethyl sulfoxide, divalent cations (such as calcium) or polyethylene glycol. Many transformation techniques are known in the art and include heat shock and electroporation.

[0077] The terms "express" and "expression" mean permitting or causing the information in a gene or DNA sequence to be produced. For example, expression can take the form of production of a protein by activating cellular functions involved in transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. An expression product itself, for example, a resulting protein, can also be said to be "expressed" by a cell. An expression product can be characterized as intracellular, extracellular, or transmembrane.

[0078] The term "biological material" refers to any material that carries genetic information and is capable of self-replication or capable of being replicated in a biological system, such as genes, plasmids, microorganisms, animals, and plants, etc.

[0079] The terms "treat" and "treatment" refer to therapeutic treatment, wherein the object is to slow or stop the undesired physiological change or disease, or to provide beneficial or desired clinical outcomes during the course of treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and / or remission (whether partial or total), whether or not detectable. "Treatment" can also mean prolonging survival as compared to expected survival of a subject if the subject were not receiving treatment. Those in need of treatment include those already with the undesired physiological change or disease as well as those prone to have a physiological change or disease. Treatment can involve a therapeutic agent, also referred to herein as a "medicament" or "medication," which can be intended to help achieve the beneficial or desired clinical outcome of interest by its action. The therapeutic agent or medicament can be administered to a subject by a number of routes, including at least intravenous and oral routes. The term "intravenous" in connection with administration of a therapeutic agent or medicament means that the therapeutic agent or medicament is administered intravenously. The term "oral" in connection with administration of a therapeutic agent or medicament means that the therapeutic agent or medicament is administered via the oral passage such as the mouth.

[0080] In the present application, a "subject" includes a human being who is a patient being treated for a disease or to prevent a disease. The methods described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, mammals of the order Rodentia, such as mice and rats, and mammals of the order Logomorpha, such as rabbits. Mammals can be of the order Carnivora, including the family Felidae (cats) and the family Canidae (dogs). Mammals can be of the order Artiodactyla, including the family Bovidae (cattle) and the family Suidae (pigs), or of the order Perssodactyla, including the family Equidae (horses). Mammals can be of the order Primate, Ceboid, or Simoid (monkeys) or hominids.

[0081] The term "effective," as applied to dose or amount, refers to the amount of a compound or pharmaceutical composition that, when administered to a subject in need, is sufficient to effect the desired activity. Note that when a combination of active ingredients is administered, the effective amount of the combination can, and typically does, include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0082] II. Examples The application is further described in detail by reference to specific embodiments given only by way of illustration. The examples provided below serve only as a guide to one of ordinary skill in the art for further improving the present technology, and do not in any way constitute a limitation on the scope of the present application.

[0083] The experimental methods in the following examples are routine methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the instructions of the products, unless otherwise specified. The materials, reagents, and the like used in the following examples are commercially available, unless otherwise specified. The quantitative tests in the following examples are all set up in triplicate, and the results are averaged.

[0084] 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." The public can obtain this biological material from the application in accordance with the relevant regulations of national biosafety, and the obtained biological material can only be used for experimental verification of the application and cannot be used for other purposes.

[0085] The A27D7 antibody in the following examples is described in the literature "Matho MH, Schlossman A, Meng X, et al. Structural and Functional Characterization of Anti-A33 Antibodies Reveals a Potent Cross-Species Orthopoxviruses Neutralizer. PLoS Pathog. 2015;11(9):e1005148."

[0086] Example 1, Expression and purification of antibody IPB-POX2.1 The humanized antibody IPB-POX2.1 is a monoclonal antibody (hereinafter referred to as IPB-POX2.1 or POX2.1), which is composed of two heavy chains and two light chains.

[0087] The heavy chain is divided into a heavy chain variable region and a heavy chain constant region. The heavy chain variable region includes three heavy chain variable region complementarity determining regions named HCDR1, HCDR2 and HCDR3, and four heavy chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1. The amino acid sequences of HCDR1, HCDR2 and HCDR3 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.

[0088] The coding sequence of the coding gene of the heavy chain variable region is shown in SEQ ID NO: 5. The coding sequence of the coding gene of the heavy chain constant region is shown in SEQ ID NO:.

[0089] The light chain is divided into a light chain variable region and a light chain constant region. The light chain variable region includes three light chain variable region complementarity determining regions named LCDR1, LCDR2 and LCDR3, and four light chain variable region framework regions named LFR1, LFR2, LFR3 and LFR4. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 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.

[0090] The coding sequence of the coding gene of the light chain variable region is shown in SEQ ID NO: 7. The coding sequence of the coding gene of the light chain constant region is shown in SEQ ID NO: 8.

[0091] The specific preparation method of the antibody IPB-POX2.1 is as follows: I. Construction of a recombinant expression vector The DNA fragment between the EcoRI and BglII enzyme cutting sites of the vector pAb-hIgG1-CH containing the coding sequence of the human heavy chain constant region (IgG1) is replaced with a DNA molecule (humanized heavy chain variable region coding gene) having the nucleotide sequence shown in SEQ ID NO: 5, and the other sequences of the pAb-hIgG1-CH vector remain unchanged, to obtain recombinant plasmid A.

[0092] The vector pAb-hIgG1-CH containing the human heavy chain constant region is obtained by replacing the DNA fragment between the BglII and NheI enzyme cutting sites of the Pfuse-hIgG1-Fc2 vector (invivoGen, pfuse-hglfc2) with a DNA molecule (human heavy chain constant region (IgG1) coding sequence) having the nucleotide sequence shown in SEQ ID NO: 7, and the other sequences of the Pfuse-hIgG1-Fc2 vector remain unchanged.

[0093] The DNA fragment between the EcoRI and BglII enzyme cutting sites of the vector pAb-hIgG1-CL containing the human light chain (κ subtype) constant region sequence is replaced with a DNA molecule having the nucleotide sequence shown in SEQ ID NO: 6 (humanized light chain variable region coding gene), and the other sequences of the vector pAb-hIgG1-CL remain unchanged, to obtain recombinant plasmid B.

[0094] Among them, the vector pAb-hIgG1-CL containing the human light chain (κ subtype) constant region sequence is obtained by replacing the DNA fragment between the BglII and NheI restriction sites of the Pfuse-hIgG1-Fc2 vector (invivoGen, pfuse-hglfc2) with a DNA molecule (human light chain constant region coding sequence) with the nucleotide sequence shown in SEQ ID NO:8, while keeping other sequences of the Pfuse-hIgG1-Fc2 vector unchanged.

[0095] II. Expression of IPB-POX2.1 using a eukaryotic cell expression system. The day before transfection, Expi293F TM Cells (Thermo Fisher, A39250) were processed at a rate of 1 × 10⁻⁶. 6 After densely stocking the flasks, allow all reagents to stand at room temperature for 10 minutes before transfection. The following procedure uses 100 mL of cells as an example: Prepare two clean centrifuge tubes. Dilute 50 μg of plasmid DNA (25 μg each of recombinant plasmid A and recombinant plasmid B) into 10 mL of serum-free Expi 293 medium (Gibco), and pipette 3-4 times. Add 75 μL of transfection reagent FectoPRO to the other tube. ® Reagent (polyplus, 101000007). Pour the diluted plasmid DNA all at once into the FectroPro Reagent transfection reagent and mix gently 3-4 times immediately; let stand at room temperature for 10 minutes. Evenly drop the transfection mixture into the cell culture flask, gently shake to disperse the transfection complex, and add 50 μL of Booster (polyplus) within 0-4 hours to enhance protein expression efficiency. Place the cell culture flask in an 8% CO2, 37°C constant temperature shaker. After 5 days, collect the cell culture medium and analyze protein expression levels using SDS-PAGE.

[0096] III. Purification of IPB-POX2.1 The purification medium used in this embodiment was Protein A magnetic beads (Nanjing Genscript Biotech Co., Ltd.). Antibody purification was performed according to the instructions for these commercially available magnetic beads. The protein was replaced using a 15kDa ultrafiltration tube (Millipore), and the final antibody protein was dissolved in PBS. The concentration was measured, and purity was determined by SDS-PAGE and Coomassie Brilliant Blue staining. The purified antibody was then aliquoted and stored at -80℃ for later use. Figure 1 Coomassie brilliant blue staining results showed that the purity of the purified IPB-POX2.1 antibody protein was greater than 90%.

[0097] Example 2: Determination of the affinity between IPB-POX2.1 and A33R protein The affinity of the antibody was determined using a BIAcore T200 biomolecular interaction analyzer (GE Life Sciences), which is a multifunctional and high-sensitivity surface plasmon resonance (SPR) system. When determining the interaction between A33R protein and IPB-POX2.1 humanized antibody prepared in Example 1, the A33R recombinant protein (also referred to as VACV-A33R, with an amino acid sequence as shown in SEQ ID NO: 9) was first coated on the sensor chip, and then the IPB-POX2.1 humanized antibody was used as the flow phase to determine the association constant, dissociation constant and affinity constant. The specific steps are as follows: 1. Coupling A33R protein to CM5 chip The protein coupling temperature was 25 degrees Celsius, and the buffer was PBS-P (PBS, 0.05% P20 (BR100054 (cytiva)), pH 7.4). The program template Immuobilization was selected, channel 2 of the CM5 chip was selected for amino coupling, the ligand was 10 μg / mL A33R protein, the protein buffer system was pH 4.0 sodium acetate, 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.

[0098] 2. Affinity and kinetic determination of A33R, murine antibody A27D7 and humanized antibody IPB-POX2.1 The multi-cycle kinetic template was selected, the determination temperature was 25 degrees Celsius, the buffer was PBS-P, the sample flow path was 2-1, 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 (murine antibody A27D7 or humanized antibody IPB-POX2.1) concentration was serially diluted (0.8 nM, 0.4 nM, 0.2 nM, 0.1 nM, 0.05 nM). Finally, the data obtained was analyzed by Biacore Evaluation Software to calculate the association constant (ka), dissociation constant (kd) and affinity constant (KD). The chip, reagent and buffer used in the above Biacore analysis were all products of GE Life Sciences.

[0099] The results are shown in Table 1 and Figure 2 The results show that the affinity of the humanized antibody IPB-POX2.1 is slightly higher than that of the murine antibody A27D7.

[0100] Table 1 BIAcore T200 results of IPB-POX2.1 and A33R protein affinity determination

[0101] Example 3, Evaluation of the preventive protective effect of IPB-POX2.1 in a mouse infection model To understand the antiviral activity of IPB-POX2.1 under in vivo conditions, a 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, named IPB-POX2.1 experimental group, A27D7 experimental group and PBS control group, respectively. There were 8-10 mice in each group. 24 hours before infection, IPB-POX2.1 antibody, A27D7 antibody or equal volume of PBS was injected intraperitoneally, with an antibody injection dose of 5 mg / kg per mouse. After 24 hours (recorded as Day 0), each mouse was inoculated with 0.5 x 10 5 PFU of vaccinia virus Vaccine virus strain WR. The mice were returned to the cage and the body weight of the mice was recorded daily, and the state of the mice was observed. According to the requirements of the animal experiment ethics review, when the body weight of the mice was less than 20% of the original body weight or the mice could not eat, etc., it was determined as death, and the death of the mice was recorded. A total of 14 days of observation, the body weight change curve and the survival rate curve of the mice were drawn.

[0102] The body weight change and survival curve of the mice in the IPB-POX2.1 experimental group, the A27D7 experimental group and the PBS control group are shown in Figure 3 The body weight change curve is shown in B, and the survival curve is shown in A. From the figure, it can be seen that the mice in the PBS control group began to rapidly lose weight on the 4th day after infection with vaccinia virus and eventually all died on the 7th day, but the mice injected with IPB-POX2.1 antibody did not show significant changes in body weight during the experimental period, and no mice died until the end of the experiment. The experimental results prove that the injection of IPB-POX2.1 antibody in advance shows excellent protective effect in the mouse model infected with vaccinia virus.

[0103] Example 4, Heavy / light chain humanization analysis of A27D7 and IPB-POX2.1 The AbNatiV of Chemistry of Health-softwae website (website: https: / / www-cohsoftware.ch.cam.ac.uk / ) was used to analyze the degree of humanization of the heavy / light chain sequences of antibodies A27D7 and IPB-POX2.1.

[0104] Figure 4Figure A and Figure B in the above table represent the degree of humanization of the heavy chain and light chain variable region of A27D7 and IPB-POX2.1, respectively. From the results, it can be seen that the degree of humanization of the sequence of A27D7 of mouse origin is low, but the degree of humanization of the heavy chain and light chain of IPB-POX2.1 is greatly improved (close to 1.00). This indicates that the modified IPB-POX2.1 has a high degree of humanization, which greatly makes up for the regret that A27D7 cannot be further applied due to its mouse origin.

[0105] The sequences in the present application are as shown in Figure 5

[0106] The present application has been described in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without unnecessary experiments. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application is intended to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application.​

Claims

1. An antibody or its antigen-binding fragment that binds to the vaccinia virus membrane protein A33R, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that: The antibody or its antigen-binding fragment further includes a constant region; The constant region of the heavy chain is IgG1 or IgG4, and the constant region of the light chain is κ or λ.

3. A nucleic acid molecule, characterized by: The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: the coding gene for the heavy chain variable region encoding the antibody or its antigen-binding fragment is as shown in A1) or A2) below: A1) A DNA molecule with a coding sequence as described in SEQ ID NO:5; A2) has more than 70% identity with the DNA molecule shown in A1) and is the DNA molecule encoding the heavy chain variable region; The gene encoding the light chain variable region of the antibody or its antigen-binding fragment is shown in B1) or B2) below: B1) A DNA molecule with a coding sequence as described in SEQ ID NO:6; B2) has more than 70% identity with the DNA molecule shown in B1) and is a DNA molecule encoding the light chain variable region; The gene encoding the heavy chain constant region of the antibody or its antigen-binding fragment is shown in C1) or C2) below: C1) DNA molecules with coding sequences as described in SEQ ID NO:7; The DNA molecule shown in C2) has more than 70% identity with the DNA molecule shown in C1), and is the DNA molecule encoding the heavy chain constant region; The gene encoding the light chain constant region of the antibody or its antigen-binding fragment is shown in D1) or D2) below: D1) A DNA molecule with a coding sequence as described in SEQ ID NO:6; The DNA molecules shown in D2) have more than 70% similarity to those shown in D1) and encode the DNA molecule of the light chain constant region.

5. An expression box, characterized in that: The expression cassette contains the nucleic acid molecule as described in claim 3 or 4.

6. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule as described in claim 3 or 4, or the expression cassette as described in claim 5.

7. Recombinant cells, characterized by: The recombinant cells contain the nucleic acid molecules of claim 3 or 4, the expression cassette of claim 5, or the recombinant vector of claim 6.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the antibody or its antigen-binding fragment as described in claim 1 or 2.

9. Application, characterized in that: The application is shown in at least one of the following: E1) The use of the nucleic acid molecule of claim 3 or 4 in the preparation of the antibody of claim 1 or 2; E2) Use of the expression cassette of claim 5 in the preparation of the antibody of claim 1 or 2; E3) The use of the recombinant vector according to claim 6 in the preparation of the antibody according to claim 1 or 2; E4) Use of the recombinant cells of claim 7 in the preparation of the antibody of claim 1 or 2; E5) The use of the antibody of claim 1 or 2 in the preparation of products for the prevention or treatment of diseases caused by poxvirus infection, in the preparation of products for the detection of poxvirus levels and / or poxvirus A33R antigen levels, in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection, and / or in the preparation of products for the inhibition or neutralization of poxvirus activity. E6) The use of the nucleic acid molecule of claim 3 or 4 in the preparation of products for the prevention or treatment of diseases caused by poxvirus infection, in the preparation of products for the detection of poxvirus levels and / or poxvirus A33R antigen levels, in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection, and / or in the preparation of products for the inhibition or neutralization of poxvirus activity. E7) The expression cassette of claim 5 is used in the preparation of products for the prevention or treatment of diseases caused by poxvirus infection, in the preparation of products for the detection of poxvirus levels and / or poxvirus A33R antigen levels, in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection, and / or in the preparation of products for the inhibition or neutralization of poxvirus activity. E8) The use of the recombinant vector of claim 6 in the preparation of products for the prevention or treatment of diseases caused by poxvirus infection, in the preparation of products for the detection of poxvirus levels and / or poxvirus A33R antigen levels, in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection, and / or in the preparation of products for the inhibition or neutralization of poxvirus activity. E9) The use of the recombinant cells of claim 7 in the preparation of products for the prevention or treatment of diseases caused by poxvirus infection, in the preparation of products for the detection of poxvirus levels and / or poxvirus A33R antigen levels, in the preparation of products for the diagnosis or auxiliary diagnosis of diseases caused by poxvirus infection, and / or in the preparation of products for the inhibition or neutralization of poxvirus activity.

10. A method for preparing the antibody or antigen-binding fragment of claim 1 or 2, the method comprising the step of expressing the encoding gene of the antibody or antigen-binding fragment in mammalian cells to obtain the antibody or antigen-binding fragment.

Images