WO2010040281A1 - Anticorps humanisés se liant à l'hémagglutinine du sous-type h5 du virus de la grippe aviaire et leur utilisation - Google Patents

Anticorps humanisés se liant à l'hémagglutinine du sous-type h5 du virus de la grippe aviaire et leur utilisation Download PDF

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WO2010040281A1
WO2010040281A1 PCT/CN2009/001129 CN2009001129W WO2010040281A1 WO 2010040281 A1 WO2010040281 A1 WO 2010040281A1 CN 2009001129 W CN2009001129 W CN 2009001129W WO 2010040281 A1 WO2010040281 A1 WO 2010040281A1
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seq
antibody
amino acid
variable region
acid sequence
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PCT/CN2009/001129
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Chinese (zh)
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罗文新
陈毅歆
郑振华
李国强
陈鸿霖
张军
夏宁邵
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厦门大学
养生堂有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to a humanized antibody which specifically binds to the H5 subtype avian influenza virus hemagglutinin (HA) protein, and a conservative variant or active fragment thereof, and the use of the humanized antibody or fragment for the same Methods and uses for prevention and treatment.
  • Background technique H5 subtype avian influenza virus hemagglutinin (HA) protein, and a conservative variant or active fragment thereof, and the use of the humanized antibody or fragment for the same Methods and uses for prevention and treatment.
  • H5-type highly pathogenic avian influenza also broke out in more than a dozen provinces in China, and found H5 avian flu infected chickens, ducks, herons, tigers, cats and other animals in Hong Kong, Thailand, and the Netherlands. event. What is even more worrying is that there have been incidents of suspected human transmission in Thailand, and there are many reports in Malaysia.
  • Romania, Russia, Turkey and other European countries have successively discovered the death of avian influenza H5 avian influenza virus. Experts believe that this is the result of migratory bird migration, which makes the control of the highly pathogenic H5 avian influenza virus further spread. Communication has become more difficult.
  • the H5 avian influenza virus may be further spread to African countries with poor sanitation through the Eurasian and Asian-African continental bridges, allowing the H5 avian influenza virus to be fully reorganized with other human influenza viruses.
  • the opportunity and time will be likely to form a new flu virus that is highly deadly to humans, and the damage it will cause to humans will be difficult to estimate.
  • WHO statistics as of January 19, 2006, the world was infected by humans infected with the H5N1 virus. The number of cases has reached 80, which has brought great challenges to global public health security.
  • influenza virus treatments There are two main types of influenza virus treatments that have been determined to be effective over a long period of time: M2 ion channel inhibitors amantadine and rimantadine, and the ceramidase inhibitor oseltamivir (oseltamivir) And zanamivir (Monto, AS Vaccine, 2003. 21 (16): 1796-800).
  • the former two exert antiviral effects by inhibiting viral ion channel protein M2; the latter can selectively inhibit the activity of neuraminidase on the surface of the virus, prevent the replication and release of progeny virus particles in the host cell, and effectively prevent colds and It relieves symptoms and makes the currently effective anti-influenza virus drugs, but it has been continuously found that new H5N1 virus mutants are resistant to them. If these drugs are taken within the first 2 days of the onset, they will shorten the course of the disease and even save the patient's life.
  • ion channel inhibitors are useful for some subtypes of influenza viruses (Dolin, R. et al., N Engl J Med, 1982.
  • H5N1-specific humanized murine monoclonal antibodies, fully human monoclonal antibodies, and F(ab, )2 fragments have been shown to be effective in the prevention and treatment of H5N1 (Lu, J. et al., Respir Res., 2006, 7: 43; Simmons, CP et al., PLOS Medicine, 2007, 4 (5): 928-936; Hanson, BJ Et a l. , Respir Res.
  • the laboratory uses a plurality of avian influenza virus H5N1 representative strains isolated from different time, different locations and different hosts to immunize mice, and continuously prepares monoclonal antibodies, and has prepared more than 400 strains with hemagglutination inhibition (HI).
  • H5 monoclonal antibody library of active H5-specific monoclonal antibodies is provided.
  • H5N1 virus pool Based on phylogenetic analysis of nearly a thousand H5N1 viral genome sequences around the world, we screened 34 H5N1 representative strains from different regions, different evolutionary branches, and different host sources, plus the latest 7 strains isolated in 2006. 41 strains constitute a representative H5N1 virus pool. The 41 H5N1 viruses were assayed for HI activity of each monoclonal antibody, and the 400 monoclonal antibodies were divided into 10 types according to the difference in HI activity, and the 41 representative viruses were divided into eight HI active branches. . The antigenic diversity of H5N1 strains since 2002 was initially selected from 17 monoclonal antibodies.
  • H5N1 subtypes can be divided into four antigenic groups.
  • 13D4, 16F13 and other monoclonal antibodies have high neutralizing activity for most strains, laying a foundation for therapeutic antibody research (Wu, WL, Chen, Y., Wang, P., Song, W. , Lau, SY, Rayner, JM, Smi th, GJ, Webs ter, RG, Pe iris, JS, L in, T., Xia, N., Guan, Y. and Chen, H. 2008, J Vi ro l 82 (4), 1798-1807).
  • the underlying object of the present invention is to obtain a humanized antibody of 13D4 therapeutic monoclonal antibody, and a conservative variant or active fragment thereof, for use in the development of a therapeutic drug for H5N1 virus and related research. Summary of the invention
  • the present invention provides a humanized antibody that specifically binds to the H5 subtype avian influenza virus hemagglutinin (HA) protein, which can be used for the prevention and/or treatment of avian influenza viruses, particularly H5 subfamily. Diseases caused by avian influenza viruses.
  • the invention also provides related nucleic acid molecules, as well as pharmaceutical compositions containing the same.
  • Figure 1 shows the humanized template of 13D4 murine monoclonal antibody and the partial amino acid substitution of the FR region of the humanized antibody.
  • the underlined italic is the CDR region; the two amino acids in parentheses indicate that the site has two amino acid substitutions.
  • Figure 2 shows the PCR amplification strategy for the 13D4 humanized single-chain antibody library fragment.
  • Figure 3 shows the results of PCR amplification of the 13D4 humanized single-chain antibody library DM fragment.
  • Figure 4 shows the phagemid vector pCANTAB 5E with a 13D4 humanized single chain antibody.
  • Figure 5 shows the results of ELISA detection of phage antibodies on HA and YU22 antigens.
  • Figure 6 shows the murine antibody amino acid positions retained in four 13D4 humanized antibodies VH, VL.
  • Figure 7 shows the amino acid sites of the murine antibody retained in eight strains of 13D4 humanized antibody VH, VL.
  • Figure 8 shows the results of ELISA of 8 strains of 13D4 humanized phage single-chain antibody with HA1 and YU22 viruses.
  • M13 phage is a negative control.
  • the secondary antibody is HRP-ant i- M13 antibody.
  • Figure 9 shows the expression vector containing the human antibody CH and CK fragments
  • Figure 10 shows the eukaryotic expression vector of 13D4 humanized antibody
  • Figure 11 shows that hygromycin resistance screens for stable cell lines expressing humanized antibodies.
  • Figure 12 shows the results of SDS-PAGE of humanized antibodies after purification by Protein A.
  • Figure 13 shows the results of ELISA for the reaction of 13D4 humanized antibody with HA1.
  • hemagglutinin in the present invention refers to an envelope glycoprotein of avian influenza virus. Hemagglutinin proteins mediate the adsorption and entry of influenza viruses against host cells.
  • the hemagglutinin protein of avian influenza virus has 16 serological subtypes, HA1 - HA16, corresponding to 16 viral subtypes, namely Hl - H16.
  • antibody refers to any immunoglobulin, including monoclonal antibodies, polyclonal antibodies, bispecific or multispecific antibodies that bind to specific antigens.
  • a complete antibody contains two heavy chains and two light chains. Each heavy chain contains a variable region and first, second, and third constant regions; each light chain comprises a variable region and a constant region.
  • the antibody is of the "Y” type, and the neck of the "Y” type structure contains two heavy and second constant regions of the heavy chain which are formed by disulfide bonding. Each arm of the "Y” type structure contains a first constant region and a variable region (VH) of one of the heavy chains, and a variable region (VL) and a constant region of one light chain.
  • variable regions of the light and heavy chains determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called the complementarity determining regions (CDRs) (the CDRs of the light chain (L) comprise LCDRU LCDR2, LCDR3,
  • CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3 (which is named by Kabat et al., see Sequences of Proteins of Immunologica Inters t, Fif th Edi t ion (1991), Vol. 1-3, NIH Publ icat ion 91-3242, Bethesda Md) 0 wherein the three CDRs are separated by a framework region (FR).
  • FR framework region
  • the framework regions are more conserved than the CDR regions and form a shelf-like structure to support the hypervariable regions.
  • the constant regions and antigens of the heavy and light chains The binding is irrelevant, but has multiple effector functions.
  • the antibody can be divided into several categories according to the amino acid sequence of the heavy chain constant region, mainly: IgA, IgD, IgE, IgG and IgM, some of which are further divided into subclasses, such as IgGl, IgG2. , IgG3, IgG4, IgAl or IgA2, and the like.
  • antibody in the present invention, except the specific immunoglobulin
  • fragments of immunoglobulins eg, at least one immunologically active segment of an immunoglobulin molecule
  • immunoglobulins eg, at least one immunologically active segment of an immunoglobulin molecule
  • Fab fragments of immunoglobulins
  • Fab' fragments of immunoglobulins
  • F (ab') 2 fragments of immunoglobulins
  • Fv fragments single-chain antibody molecules
  • single-chain antibody molecules or consist of one or A multispecific antibody formed by any fragment of an immunoglobulin molecule of a plurality of CDR regions.
  • the antibodies of the present invention may also be antibodies formed by binding one or more CDR regions of a particular human immunoglobulin to one or more different human immunoglobulin framework regions.
  • An antibody-associated "Fab" fragment refers to a portion of an antibody molecule comprising a variable region and a constant region of a light chain and a variable region and a constant region of a heavy chain that are disulfide-bonded.
  • a "Fab” fragment refers to a Fab fragment that contains a portion of the hinge region.
  • Fc of an antibody refers to a portion of an antibody that is disulfide-bonded by a second, third constant region of a first heavy chain and a second, third constant region of a second heavy chain.
  • the Fc portion of an antibody has many different functions but does not participate in antigen binding.
  • the "Fv” segment of an antibody refers to the smallest fragment of an antibody that binds to the entire antigen binding site.
  • An Fv fragment comprising a variable region (VL) of a light chain binds to a variable region (VH) of a heavy chain.
  • Single-chain antibody or “scFv” in the present invention refers to an engineered antibody in which a light chain variable region is directly linked to a heavy chain variable region or linked by a peptide chain ( Hous ton 1988).
  • the "single-chain antibody Fv-Fc" or “scFv-Fc” in the present invention also includes an engineered antibody formed by the Fc segment of the scFv-linked antibody.
  • antigenic determinant refers to the portion of the amino acid or atomic group in the antigen molecule that binds to the antibody.
  • the term "monoclonal antibody” as used in the present invention refers to a fragment of an antibody or antibody from a group of highly homologous antibody molecules, that is, a group of natural mutations that may occur in only a few cases. Identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on the antigen. Monoclonal antibody is different from polyclonal antibody, and polyclonal antibody contains recognition resistance. Antibody molecules of different epitopes on the original. Although the conventional monoclonal antibody is secreted by the hybridoma cells, the monoclonal antibody involved in the present invention is not limited to this preparation method.
  • the monoclonal antibodies involved in the present invention can be obtained by the hybridoma technique first reported by Kohler et al. (Nature, 256: 495, 1975), and can also be obtained by recombinant DNA techniques (see, for example, US P 4,816,567).
  • chimeric antibody refers to a portion of an antibody light chain or/and a heavy chain derived from a particular species or belonging to a particular antibody class or subclass having the same or homologous sequence.
  • An antibody while another portion of the antibody light chain or/and heavy chain is derived from another species or an antibody belonging to another antibody class or subclass having the same sequence or homology.
  • this antibody fragment retains its binding activity to the antigen of interest (US P 4,816, 567 to Cabi l ly et a l.; Morr i son et al., Proc. Nat l. Acad. Sc i USA, 81: 6851 6855 (1984)).
  • the term "humanized antibody” refers to the replacement of all or part of the CDR regions of a human immunoglobulin (receptor antibody) by the CDR regions of a non-human antibody (donor antibody).
  • An antibody or antibody fragment is obtained, wherein the donor antibody can be a mouse, rat or rabbit antibody having the desired specificity, affinity and reactivity.
  • the amino acid sequence of the framework region (FR) of the human immunoglobulin can also be replaced by the amino acid sequence of the corresponding non-human antibody.
  • the amino acid residues of the humanized antibody may also be derived from neither the recipient antibody nor the CDR region or framework region sequence of the donor antibody. The purpose of these artificial modifications is to further refine or optimize antibody performance.
  • a humanized antibody refers to a variable region containing at least one, usually two, nearly complete, wherein all or nearly all of the corresponding CDR regions are derived from a non-human antibody, wherein all or almost all of the FR regions are From human antibodies.
  • An ideal humanized antibody contains at least a portion of the Fc region of an immunoglobulin, typically the Fc region of a human immunoglobulin.
  • Jones et al. Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Pres ta, Curr. Op. Struct. Biol. , 2: 593 596 (1992); and Clark, Immunol.
  • isolated and isolated refers to that obtained by artificial means in a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed or that it has been separated from the natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide naturally present in a living animal, and the same high-purity polynucleotide or polypeptide isolated from this natural state is called Is separated.
  • the "separated” herein does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector" in the present invention refers to a nucleic acid delivery vehicle in which a polynucleotide encoding a protein is inserted and the protein is expressed.
  • the vector can be expressed by expression, transduction or transfection of the host cell by expression of the genetic material element carried therein in the host cell.
  • the vector includes: a plasmid; a phagemid; a cosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1 derived artificial chromosome (PAC); a phage such as ⁇ phage or M13 Phage and animal viruses.
  • the animal viruses used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, vesicular viruses (such as simple viral viruses), poxviruses, baculoviruses, papillomaviruses, papillomas. Vacuolating virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Alternatively, the vector may contain a replication initiation site.
  • the vector may also include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not just those.
  • the term "host cell” as used in the present invention refers to a cell into which a vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 flies. Cells or insect cells such as Sf9, or animal cells such as fibroblasts, CH0 cells, COS cells, NS0 cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the host cell can be ex vivo or in vivo, and can be a cultured cell or cell line.
  • neutralizing antibody refers to the ability to eliminate or significantly reduce target viral resistance. An antibody or antibody fragment that binds to virulence.
  • sequence percent in sequence refers to the percentage of nucleic acid or amino acid identity in a candidate nucleic acid or amino acid sequence to a corresponding nucleic acid or polypeptide sequence, respectively.
  • sequence similarity percentage as used herein in connection with a nucleic acid sequence or polypeptide sequence is defined as the similar percentage of a candidate nucleic acid sequence or amino acid residue sequence to a nucleic acid sequence or amino acid sequence of interest, respectively. For a sequence, compare it to the sequence of interest, skip the mutation gap if necessary to achieve the maximum percentage of gene similarity, without considering any conservative mutations of similar sequences.
  • a variety of alignment methods in the art can be used to determine the similarity of nucleic acid or amino acid sequences, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Mega lign (DNASTAR). Those skilled in the art will be familiar with setting the appropriate measurement parameters for comparison, including the comparison of full length sequences for some algorithms that use maximum comparability.
  • an antigen refers to a specific antibody with an affinity (KD) ⁇ 1 ( ⁇ 5 ⁇ ( eg 10 6 ⁇ , 10- 7 ⁇ , 10- 8 ⁇ , 10- 9 ⁇ , 10- 1 ⁇ ⁇ , etc.) binds to the antigen, wherein KD refers to the ratio of the dissociation rate to the binding rate (koff/kon), which can be determined by methods familiar to those skilled in the art.
  • KD refers to the ratio of the dissociation rate to the binding rate (koff/kon), which can be determined by methods familiar to those skilled in the art.
  • the monoclonal antibody and the humanized antibody of the present invention are capable of specifically binding to the H5 subtype avian influenza virus.
  • One aspect of the invention relates to monoclonal antibodies and humanized antibodies and their corresponding antigen-binding fragments which are capable of specifically binding to an H5 subtype avian influenza virus hemagglutinin protein.
  • the anti-H5 mAb of the present invention is preferably secreted by mouse hybridoma cell line 13D4.
  • the name of the monoclonal antibody is named after its corresponding hybridoma cell line. That is, this anti-H5 mAb was produced by the hybridoma cell line 13D4 and was named 13D4.
  • Monoclonal antibody 13D4 It can specifically bind to the H5 subtype avian influenza virus hemagglutinin protein.
  • the mouse hybridoma cell line 13D4 has been deposited with the China Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) under the accession number CCTCC - C200721 (deposited May 29, 2007)
  • the monoclonal antibody can be prepared using the hybridoma preparation method reported by Kohler et al., Nature 256: 495 (1975).
  • the immunogen (addition of an adjuvant if necessary) is first immunized with a mouse or other suitable host animal.
  • the immunogen or adjuvant is usually administered by subcutaneous injection or intraperitoneal injection.
  • Pre-linking of the immunogen to certain known proteins, such as serum albumin or soybean chymase inhibitors may help to enhance the immunogenicity of the antigen in the host.
  • the adjuvant may be a Freund's adjuvant or an MPL-TDM or the like. After the animal is immunized, lymphocytes secreting antibodies that specifically bind to the immunogen are produced in the body.
  • lymphocytes can also be obtained by in vitro immunization.
  • the lymphocytes of interest are harvested from osteomyeloma cells and fused with a suitable fusing agent, such as PEG, to obtain hybridoma cells (Coding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
  • the hybridoma cells prepared above may be inoculated into a suitable culture medium, and the culture solution preferably contains one or more substances capable of inhibiting the growth of unfused, maternal myeloma cells.
  • the culture solution preferably contains one or more substances capable of inhibiting the growth of unfused, maternal myeloma cells.
  • HGPRT hypoxanthine squamous acid transferase
  • the addition of hypoxanthine, aminoguanidine and thymine (HAT medium) to the culture medium can inhibit HGPRT.
  • HGPRT hypoxanthine squamous acid transferase
  • HAT medium aminoguanidine and thymine
  • Preferred myeloma cells should have a high fusion rate, stable antibody secretion capacity, and sensitivity to HAT culture.
  • murine myeloma is preferred for myeloma cells, such as MOP-21 and MC-11 mouse tumor derived rods (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2/0 or X63-Ag8. -653 cell line (American Type Culture Collection, Rockville, Md. USA).
  • human myeloma and human murine heterologous myeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001). (1984); Brodeur et al., Monoclona l Ant ibody Product ion Techniques and Appl icat ions, pp. 51-63, Marcel Dekker, Inc., New York, 1987).
  • a culture medium in which hybridoma cells are grown is used to detect the production of a monoclonal antibody against a specific antigen.
  • the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the target cell line can pass (Goding, Monoc lona l Ant ibodies: Pr inciples and Practice, pp. 59-103, Academic Press, 1996).
  • the standard limiting dilution method described is subcloned.
  • a suitable culture solution may be DMEM or RPMI-1640 or the like.
  • hybridoma cells can also grow in the form of ascites.
  • the traditional immunoglobulin purification method such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, can be used to secrete monoclonal antibodies from subcloned cells. Separated from cell culture fluid, ascites or serum.
  • the monoclonal antibody of the present invention may also be obtained by genetic engineering recombination techniques.
  • a DNA molecule encoding a monoclonal antibody heavy chain and a light chain gene can be isolated from a hybridoma cell by PCR amplification using a nucleic acid primer that specifically binds to the monoclonal antibody heavy chain and light chain genes.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into a host cell, such as E. col i cells, simian COS, CH0 cells, or other myeloma cells that do not produce immunoglobulin.
  • the transfected host cells are cultured under specific conditions and express the antibody of interest.
  • the antibodies of the invention and humanized antibodies have high specificity and high affinity for H5 hemagglutinin protein binding. These antibodies may have weak cross-reactivity to other subtypes of hemagglutinin proteins. Preferably, these antibodies are completely non-cross-reactive with other subtypes of hemagglutinin.
  • the KD value of the antibody of the invention and the humanized antibody binding to H5 hemagglutinin is less than 1 X 10" 5 M; preferably, the KD value is less than 1 X 10" 6 M; Alternatively, KD value of less than 1 x 10- 7 M, and most preferably, KD value of less than 1 x 10 _8 M.
  • the mAb and humanized antibodies of the invention may be antibodies of the traditional "Y" type structure comprising two heavy chains and two light chains.
  • the antibody may also be a Fab fragment, Fab', F(ab) 2 , Fv, or other type of partial fragment on an antibody having a conventional "Y"-type structure that retains affinity for hemagglutinin proteins.
  • the affinity of the hemagglutinin protein can be higher or lower than that of the transmitted type of antibody.
  • Antibody fragments of the invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Sc ience 229 : 81 (1985) ). In addition, these antibody fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Li tt le et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from E.
  • F (ab 2 fragments can be obtained by leucine zipper GCN4 ligation.
  • Fv, Fab or F (ab' ) 2 fragments can also be directly isolated directly from recombinant host cell culture broth. Other techniques for preparing antibody fragments are fully known to the person.
  • the invention relates to an encoding nucleic acid molecule that specifically binds to an antibody or humanized antibody or antibody fragment of an H5 hemagglutinin protein.
  • the nucleic acid molecule encoding the antibody can be isolated from hybridoma cells.
  • One of ordinary skill in the art is well aware of the nucleic acid sequences that can be determined using conventional techniques.
  • the antibody nucleic acid molecules to which the present invention relates can also be obtained by conventional genetic engineering recombinant techniques or chemical synthesis methods.
  • the sequences of the antibody nucleic acid molecules of the invention comprise a heavy chain variable region of an anti-H5 antibody or a partial nucleic acid sequence of an antibody molecule.
  • sequences of the antibody nucleic acid molecules of the invention also include a light chain variable region of an anti-H5 antibody or a partial nucleic acid sequence of an antibody molecule.
  • sequence of the antibody nucleic acid molecule of the invention further comprises the CDR sequence of the heavy or light chain variable region.
  • Mutual decision! 3 ⁇ 4 (complementary determinant The reg ion, CDR) is the site of binding to the epitope, and the CDR sequences in this study were determined by the Kabat method. However, the CDR sequences obtained by different partitioning methods are slightly different.
  • the present invention also provides a nucleic acid molecule capable of specifically binding to the coding sequence of an antibody fragment of the H5 subtype avian influenza virus.
  • the present invention still further relates to an isolated nucleic acid molecule encoding one of the heavy chain variable region amino acid sequences of SEQ ID NOs: 4-6.
  • the invention further relates to a nucleic acid molecule encoding one of the amino acid sequences of the light chain variable region of the antibody, SEQ ID NOs: 7-9.
  • the invention still further relates to encoding the humanized antibody heavy chain variable region amino acid sequence SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 3 2 of the isolated nucleic acid molecule.
  • the present invention also relates to the amino acid sequences encoding the antibody light chain variable region SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22.
  • the present invention relates to recombinant expression vectors containing the nucleic acid molecules of the present invention, and to host cells transformed with these molecules or vectors. Moreover, the present invention also relates to a method of culturing and isolating the antibody of the present invention by using a host cell comprising the nucleic acid molecule under specific conditions.
  • the monoclonal antibody 13D4 heavy and light chain variable region amino acid sequences can be deduced from the corresponding nucleic acid sequences.
  • the monoclonal antibody 13D4 heavy chain variable region amino acid sequence is SEQ ID NO: 1 or SEQ ID NO: 2, respectively.
  • the monoclonal antibody 13D4 light chain variable region amino acid sequence is SEQ ID NO: 3, respectively.
  • the invention also encompasses humanized antibodies which are grafted into a framework of human antibodies by one or more CDRs of murine monoclonal antibody 13D4 or variants thereof. Composed on.
  • amino acid sequence of the heavy chain variable region of the humanized antibody provided by the present invention is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • amino acid sequence of the light chain variable region of the humanized antibody provided herein is SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19. SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 3 3 .
  • the invention provides a variant of one or more amino acid substitutions of a heavy chain variable region of a humanized antibody, the amino acid sequence thereof and SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30
  • the sequence similarity of SEQ ID NO: 3 2 is at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%, still more preferably at least 90%, most preferably at least 95%.
  • the number of the substituted amino acids is 1 - 20, for example 1 - 10, preferably 1 - 5, including 1, 2, 3, 4, 5.
  • the invention provides a variant of one or more amino acid substitutions of a light chain variable region of a humanized antibody, the amino acid sequence of which is SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO : SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31
  • the sequence similarity of SEQ ID NO: 3 3 is at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%, still more preferably at least 90%, most preferably at least 95%.
  • the number of the substituted amino acids is 1 - 20, for example 1 - 10, preferably 1 - 5, including 1, 2, 3, 4, 5.
  • the amino acid sequences of the CDRs of the heavy and light chain variable regions of the monoclonal antibody 13D4 are determined as follows:
  • the amino acid sequences of the CDR1, CDR2 and CDR3 of the monoclonal 13D4 heavy chain are SEQ ID Nos: 4-6 o mAb 13D4, respectively.
  • Ammonia of CDR1, CDR2 and CDR3 of the light chain The base acid sequences are SEQ ID Nos: 7-9, respectively.
  • the invention provides a humanized antibody heavy chain or fragment that is resistant to H5, comprising the CDRs: one or more CDRs from SEQ ID NOs: 4-6.
  • the anti-H5 humanized mAb heavy chain or fragment comprises three CDRs having the amino acid sequence of SEQ ID NOs: 4-6.
  • the CDRs of the heavy chain or fragment of the anti-H5 humanized antibody may comprise an amino acid sequence which is a mutation or addition or deletion of one or more amino acids at SEQ ID NOs: 4-6.
  • the amino acid that is mutated or added or deleted does not exceed three amino acids. More preferably, the amino acid that is mutated or added or deleted does not exceed 2 amino acids. Most preferably, the amino acid that is mutated or added or deleted does not exceed 1 ammonia.
  • the invention provides an anti-H5 humanized antibody light chain or fragment comprising the CDRs: one or more CDRs from SEQ ID NOs: 7-9.
  • the anti-H5 humanized antibody light chain or fragment comprises three CDRs having the amino acid sequence of SEQ ID NOs: 7-9.
  • the CDRs of the light chain or fragment of the anti-H5 humanized antibody may comprise an amino acid sequence which is a mutation, addition or deletion of one or more amino acids at SEQ ID NOs: 7-9.
  • the amino acid that is mutated, added or deleted does not exceed three amino acids. More preferably, the amino acid that is mutated, added or deleted does not exceed 2 amino acids. Most preferably, the amino acid that is mutated, added or deleted does not exceed 1 ammonia.
  • Variants after mutation, addition or deletion of an amino acid of the above-described humanized antibody or CDR region or variable region still retain the ability to specifically bind to the H5 subtype avian influenza virus.
  • the invention also encompasses variants of such antigen-binding fragments.
  • the invention also provides nucleic acids encoding any of the above antibodies, heavy chains, light chains, variable regions, CDRs, fragments, or variants. Also provided are nucleic acids comprising a nucleotide sequence encoding any of the above antibodies, heavy chains, light chains, variable regions, CDRs, fragments, or variants.
  • Humanized antibody variants of the invention can be genetically engineered by conventional techniques The method is obtained. Those skilled in the art are fully aware of methods for engineering DNA molecules using nucleic acid mutations. In addition, nucleic acid molecules encoding heavy and light chain variants can also be obtained by chemical synthesis.
  • the invention provides a fusion protein comprising, in whole or in part, a humanized antibody of the invention, conjugated or fused to a molecule.
  • Both humanized antibodies and fusion proteins can be obtained using conventional genetic engineering techniques.
  • DNA encoding a monoclonal antibody can be engineered by mutation to replace the sequence of the constant region of the heavy and light chains of the human antibody with a homologous mouse sequence (Morr i son, et a l., Proc Nat. Acad. Sci. 81: 6851 (1984)), or by covalently ligating all or part of an immunoglobulin coding sequence to a non-immunoglobulin coding sequence to obtain a humanized antibody or fusion protein.
  • the invention provides an anti-H5 antibody capable of neutralizing the viral activity of an H5 subtype avian influenza virus.
  • the neutralizing antibody is capable of neutralizing at least 60%, or at least 70%, or preferably at least 75%, or preferably at least 80%, or preferably at least 85% of the viral activity of the H5 subtype avian influenza virus. Or preferably at least 90%, more preferably at least 95%, and most preferably at least 99%.
  • virus activity of an antibody neutralizing the H5 subtype avian influenza virus can be determined using conventional techniques.
  • the method of the neutralization test as described in Example 1 of the present invention can be used to determine the neutralizing activity of a specific H5 monoclonal antibody in the present invention.
  • the present invention provides a method for preventing and treating avian influenza virus-associated virus-infected patients, comprising administering to a patient an amount of a pharmaceutically active pharmaceutical composition comprising one or more monoclonal antibodies of the present invention. .
  • the invention also provides a pharmaceutical composition comprising one or more of the humanized antibodies or monoclonal antibodies of the invention or a salt drug obtained therefrom.
  • the intervention method of the drug group of the present invention may be a traditional intervention route, including oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic sulcus, groin. , intravesical, topical (eg, powder, bone or drops), or nasal route, but not limited to this.
  • a pharmaceutical composition suitable for parenteral injection may contain sterile aqueous or nonaqueous solutions, aerosols, suspensions or emulsions which are in accordance with the requirements for pharmaceutical preparation, and may be resuspended to injectable solutions or aerosols as appropriate.
  • Aseptic powder Suitable aqueous and non-aqueous vehicles, tools and various diluents such as water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol and the like), suitable mixtures, vegetable oils (such as Olive oil), and organic fats that can be used for injections, such as ethane oleic acid. If the lecithin capsid is used, the proper fluidity of the drug is maintained. Use aerosols, surfactants, etc. to maintain proper particle size.
  • the pharmaceutical composition of the present invention may further contain some protective, moisturizing, emulsifying and aerosolizing adjuvants, and may also contain instant ingredients for preventing microbial contamination, such as various antibacterial agents and antifungal agents. Such as parabens, chlorobutano l, benzoquinone, sorbic acid and the like. Agents that maintain osmotic pressure, such as sugars, NaCl, and the like, may also be included. Prolonged adsorption reagents can be used to prolong the adsorption time of the pharmaceutical composition for injection, such as monostearate and gel.
  • Oral solid phase dosage forms include capsules, tablets, powders, granules and the like.
  • the active ingredients in these solid phase dosage forms are admixed with at least one conventional inert pharmaceutical excipient (or carrier) such as sodium citrate, calcium phosphate, or (a) fillers or additives such as starch, lactose, sucrose, mannose and silicon.
  • binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;
  • C wetting agents such as glycerin;
  • fragmentation agents such as agar, Calcium carbonate, potato flour or tapioca flour;
  • a retarder such as paraffin;
  • an absorbent such as a tetraamino mixture;
  • a humectant such as cetyl alcohol and glyceryl monostearate;
  • adsorbents such as kaolin and bentonite;
  • lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or the like mixture.
  • talc calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or the like mixture.
  • talc calcium stearate
  • magnesium stearate solid polyethylene glycol
  • the solid phase dosage form may be formed by a good release or pulse dry release dosage form, and is added to various direct dry release excipients mentioned above to add an excipient which can change the dry release rate of the drug, and may be included in
  • the dosage form can also be in the form of a garment.
  • Rate release modifying agents include carboxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, cellulose ethane, cellulose acetate, polyethylene oxide, xanthan gum, amino acrylate copolymer, Hydrogenated seasoning oil, carnauba wax, paraffin wax, cellulose acetate phthalate, carboxypropyl methyl phthalate, methacrylic acid copolymer or a mixture of the above.
  • Modified release and pulsed dry release dosage forms may contain one or a group of excipients with improved release rates.
  • the pharmaceutical composition of the present invention may also be composed of a rapid atomizing agent or an anti-solvent (FDDFs), and may comprise the following components: aspartame, potassium sulfonamide, seed acid, croscarmel lose Sodium, crospovidone, ascorbic acid, ethane acrylate, ethane-based cellulose, gelatin, hydroxypropylmethylcellulose, magnesium stearate, mannitol, methylmethacrylate, flavored mint, poly Ethylene glycol, vaporized silica gel, silica, sodium starch glycolate, sodium stearate stearate, sorbitol, xylitol.
  • FDDFs rapid atomizing agent or an anti-solvent
  • atomization and dissolution depends on the solubility of the drug used. For example, if the drug is insoluble, it can be made into a rapid aerosolized form. If the drug is soluble, it can be made. Fast solvent type.
  • a similar form of solid phase composition is also formulated into a soft gelatin or hard gelatin filling form using, for example, lactose or milk sugar or other high molecular weight polyethylene glycol and similar excipients.
  • Solid dosage forms such as tablets, dragees, capsules, granules and the like can be made by means of an outer casing such as a casing or other known to those skilled in the art. It may also contain an opacifying agent, or it may contain similar ingredients which can slow, delay, and control the release of the active drug. It is also possible to use a component such as a polymer and a paraffin wax for embedding. If appropriate, the active ingredient may also be formulated in the form of a microterpene using one or more of the excipients described above. [0213] Liquid dosage forms for oral administration, including emulsions, solutions, suspensions, syrups, and elixirs, which meet pharmaceutical requirements.
  • the liquid dosage form may contain inert solutions such as water or other solvents, soluble agents and emulsifiers such as ethane alcohol, isopropyl alcohol, ethane carbonate, phenyl benzoic acid, which are commonly used in the art. Salt, propylene glycol, 1, 3-butene glycol, oil, in particular, cottonseed oil, groundnut oil, corn oil, olive oil, seasoning oil and sesame oil, glycerin, hydroquinone alcohol, polyethylene Alcohol and fatty acid sorbitol esters, and mixtures of the above or similar materials.
  • inert solutions such as water or other solvents, soluble agents and emulsifiers such as ethane alcohol, isopropyl alcohol, ethane carbonate, phenyl benzoic acid, which are commonly used in the art. Salt, propylene glycol, 1, 3-butene glycol, oil, in particular, cottonseed oil, groundnut oil, corn oil, olive oil, seasoning oil and sesam
  • the pharmaceutical compositions may also include adjuvants such as humectants, emulsifiers, suspending agents, saccharifying agents, flavoring agents, and flavoring agents.
  • adjuvants such as humectants, emulsifiers, suspending agents, saccharifying agents, flavoring agents, and flavoring agents.
  • the pharmaceutical composition may further comprise ethoxylated homopolyethanol, polyoxyethylene alkyl sorbitol and sorbitan lipid, microcrystalline cellulose, meta-alumina, bentonite, agar polymer and tragacanth, or these substances a suspending agent such as a mixture.
  • the pharmaceutical composition of the present invention is also formulated as a mixture suitable for veterinary use, or as a veterinary salt, or as a veterinary solvent or a preliminary drug, and is prepared according to the requirements of ordinary veterinary and veterinary practitioners.
  • a suitable dosage form that is most suitable for the dosage and route of administration of a particular animal.
  • the one or more of the transcribed antibodies of the present invention may be combined with other anti-viral agents for the prevention and or treatment of diseases associated with H5 avian influenza virus infection.
  • the monoclonal antibody can be administered simultaneously, separately or continuously with these antiviral agents.
  • Other antiviral agents include, but are not limited to, ribavirin, adamantane, carboxyurea, IL-2, IL-12 and pentacarboxylate.
  • the murine antibody 13D4 was humanized by the method of CDR grafting.
  • the Blas t search gene database (NCBI+GenBank+DDBJ+Kabat Database) was used to first find the human VH and VL germline gene variable region sequences with the highest homology to the FR region of the murine antibody 13D4. By homology analysis, it is determined that 1-69-04, 1-39-01 (Hwang WY, Almagro JC, Bus s TN, Tan P, Foote J. Use of vital germ in a genes in a CDR homo logy-based approach To ant ibody
  • the specific method comprises the following steps: analyzing the FR region of the human template and the different amino acids in the FR region of the murine antibody, and the amino acids close to the CDR region and the amino acids having different properties may have two possible alternative manners, that is, the mouse amino acid can be maintained, It can be a human amino acid.
  • Example 2 PCR primer design and PCR scheme of 13D humanized single-chain antibody gene library
  • the 13D4 humanized single-chain antibody library gene fragment was obtained by using overlap-extension PCR (S0E-PCR) method.
  • Amino acid substitutions in the FR region were amplified using degenerate primers.
  • the heavy and light chains were split into oligonucleotide sequences of about 50 bp in size, and the fragments overlap by about 20 bp.
  • the oligonucleotide primer sequences for amplifying the 13D4 humanized single-chain antibody are shown in Table 2.
  • the corresponding positions of the primers on the 13D4 humanized single-chain antibody are shown in Figure 2.
  • VhF12 5>CAGTCTGGAGCTGAGGTGAWGAAGCCTGGGTCCTCAGTGAAGGTCTCCTGCAAG ⁇ 3
  • VhF13 5>TTCCTTTCTATGCGGCCCAGCCGGCCCAGGTTCAGCTGGTG (CAG) CAGTCTGGAGCTGAGGTG ⁇ 3
  • Vh 2 5>TCCATGTAGGCTGTGCTCGTGGATKTATCTGCGGYGAWCGTGRCCYTGCCOTAAACTTC ⁇ 3
  • VhF3 5>ACGAGCACAGCCTACATGGAGCTCAGCAGCCTGASATCTGAGGACACTGCCGTCTAHAC ⁇ 3
  • VhR3 5>CCGCCTCCACCGCTACCACCCCCTCCAGATCCGCCACCTCCGGAGGACACGGTCACC ⁇ 3
  • plan3VHF2 5>CTCAAGCTTATGGGAAGGCTTACTTCTTCATTCCTGCTACTGAnGTCCC ⁇ 3
  • plan3VHR 5>TTrTGGTACCGGAGGACACGGTCACGGAG ⁇ 3
  • plan3VKF2 5>CGCGGATCCATGTCTGTGCCAACTCAGGTCCTGGGGnGCTGCTGCTGTGTGGCmC ⁇ 3
  • primers VhFl l/VhRl, VhF2/VhR2, VhF3/VhR3 were used for PCR amplification.
  • the PCR conditions were: 94 Denaturation 5 min, 94"C denaturation 30 s, 57 X annealing 30 s, 72: extension 30 s, repeat 10 cycles, then 72 reaction l Omin, analyze the amplified product by agarose gel electrophoresis, and use DNA purification recovery kit (TianGen, DP214-03) Recovered and purified the PCR product to obtain the first round of amplification products H1, H4, H5 fragments; using HI as a template, PCR amplification using primer VhF12/VhRl, PCR conditions are the same as above, obtaining fragment H2; using H2 as template PCR amplification was carried out using the primer VhF13/VhRl.
  • the PCR conditions were the same as above to obtain the fragment H3.
  • the S0E-PCR was performed on H3/H4. The specific steps were as follows: 5 ul fragment H3, H4, without primers, PCR amplification with the same conditions After 5 cycles, the amplified product 3ul was taken as a template, and the primer VhF13/VhR2 was added. The same conditions were used for PCR amplification for 8 cycles to obtain fragment H6.
  • S0E-PCR was performed on H5/H6, and the method was the same as above to obtain fragment H7.
  • primers VkFl l/VkRl, VkF2/VkR2, VkF3/VkR3 were used for the first round of PCR amplification to obtain the product Kl, ⁇ 3, ⁇ 4 fragments;
  • the template was amplified by PCR using primers VkF12/VkR1 to obtain fragment K2; S0E-PCR was performed on K3/K4 to obtain fragment K5; S0E-PCR was performed on K2/K5 to obtain fragment K6.
  • centrifugation at 6000 rpm for 5 min the supernatant was discarded, the cells were collected, resuspended in double resistant medium containing ampicillin and kanamycin (100 g/L), and cultured overnight with shaking at 37; 4 5000 rpm Centrifuge for 10 min, then transfer the supernatant to a clean EP tube, add 1/5 of the original volume (20% PEG8000 + 2.5 mol NaCl) to pellet the phage for 8 h, centrifuge at 4 X: 13700 rpm for 20 min, discard the supernatant, and re-precipitate with 1 ml TBS.
  • Humanized 13D4 single chain antibodies were selected from phage display libraries. Take the avian influenza virus hemagglutinin protein HA1 expressed by lug/mL yeast (prepared in our laboratory), coat the ELISA plate with CB (carbonate buffer, pH 9. 6 ), incubate for 2 h, wash with PBST 1 Then, add 200ulED blocking solution, 37 for 2h, 4 for storage. In addition, the monoclonal antibodies 2F2 and 3G4 (prepared by the laboratory) for avian influenza were mixed at a concentration of 10 ug/mL, and 100 ⁇ of coating solution was added per well, and after incubation at 37 °C for 2 hours, PBST was washed once. Then add 200ul ED blocking solution, incubate for 2h, pat dry, add lOOul 4HI YU22 virus dilution, 37 for 2h, wash with PBST 5 times, store for later use.
  • the above two antigenic plates coated with HA1 and YU22 viruses were used as solid phase screening media, and the 13D4 humanized single-chain antibody library was subjected to three rounds of "adsorption-competition-amplification" enrichment screening.
  • the 13D4 humanized single-chain antibody phage display library ( 1 X 10 11 ) was diluted 100 times with TBS, and then each well was added to 5 wells of HA1 and YU22 antigen plates, and OOul was added to each well, and an unrelated antigen plate 239 was set. 10 mg/ml) was a negative control, and after 37 hours of incubation, it was washed 5 times with TBS. The enriched phage antibody is then obtained using a competitive elution method.
  • the specific method is to dilute 13D4 mAb to lmg/ml with TBS, add 100ul per well, and incubate for 1h at 31 °C, collect 1ml of competitive supernatant, remove lul titration from it, and use the rest for infection logarithm
  • phage antibody amplification was carried out according to the method of Example 4, and then proceeded to the next round of screening; the second round of screening, the third round of screening method was the same as the first round.
  • Phage antibodies were prepared by randomly selecting 23 clones from the phage antibody titration plates after the second and third rounds of enrichment, respectively. The titration plates of the competing supernatants on the unrelated antigen plates showed no bacterial clonal growth, indicating that the HA1 and YU22 antigen plates specifically bind to the 13D4 humanized single-chain antibody library.
  • the above 46 clones were cultured with LB containing 100 g/L of ampicillin to OD - 0.5, plus M13K07 for 2 h, plus 100 g/L of kanamycin, shaking culture at 37 °C. 10h, centrifuged at 5000rpm for 10 minutes to collect the cells, and 4 to store the supernatant for testing.
  • the prepared HA1 and YU22 antigen plates were prepared, and 100 ⁇ l of the phage antibody supernatant to be tested was added to each well, and the double-well was repeated, and the M13K07 culture supernatant was used as a negative control. 37 C incubation lh After that, it was washed 5 times with TBS, then diluted with 1:5000 diluted M13-HRP 100 ul, 37 for 30 min, washed with TBS 5 times, and added with substrate TMB solution to develop color Omin, H 2 S0 4 to stop color reaction , at 0D 45 . /62 . The reading is determined.
  • a positive clone was obtained by reading more than 3 times the negative control, and the positive clone was subjected to gene sequencing.
  • the responses of the four phage antibodies of HA1-10, Yu22-6, Yu22-8 and S2K2-42 were positive.
  • the nucleic acids were extracted for sequencing.
  • the VH and VL amino acid sequence numbers of the four 13D4 humanized antibodies are shown in Table 3.
  • the mouse monoclonal antibody amino acids retained in their FR regions and the few mutant amino acids introduced are shown in Fig. 6.
  • 3% ⁇ The humanized 13D4 antibody is humanized to a degree between 88.3% - 89. 05%, if the four humanized VH and VL sequences and human constant region sequences are assembled into a full antibody molecule.
  • the 13D4 humanized single-chain antibody library was screened 7 times in total, and the results of each test are shown in Table 4. After 7 screenings, 524 clones were detected in this study, and 91 positive clones were selected, with a positive ratio of 17%. Sequencing results showed that there were 37 fully humanized single-chain antibody sequences, accounting for detection. 7% of the total.
  • Example 6 Construction of 13D4 humanized antibody expression vector and expression of humanized antibody
  • the humanized antibody was expressed using the Flp-In expression system of Invitrogen.
  • the double-priming eukaryotic expression vector P1AN3 was constructed, and the eukaryotic antibody expression vector PLAN3-CHCK containing human antibody CH and CK fragments was constructed based on PLAN3, as shown in Fig. 9.
  • CH is the human gamma-l heavy chain constant region
  • CK is the human kappa light chain constant region.
  • the humanized 13D4 VH fragment H13D4VH with the secretion signal peptide sequence was amplified by primers plan3VHF1/plan3VHR, plan3VHF2/plan3VHR (see Table 2 for specific sequence); primers3VKF/plan3VKR (see Table 2 for specific sequences)
  • a 13D4 humanized VK fragment H13D4VK with a secretion signal peptide sequence was amplified.
  • H13D4VH was digested with Hindin and Kpn l and ligated into the vector PLAN3 - CHCK digested with the same endonuclease; H13D4VK was digested with BamH I and EcoRV, ligated into the same endonuclease and digested with the same endonuclease. Confirmation of the vector PLAN3 - CHCK inserted into the H13D4VH fragment. Constructed eukaryotic expression vector containing 1304 humanized antibody? 1 ⁇ 3 - (:11( 1[ - 111304 is shown in Figure 10).
  • the eukaryotic expression vector PLAN3 - CHCK - H13D4 containing the 13D4 humanized antibody was constructed and co-transfected with the pOG4 4 plasmid into Flp-InTM-CH0 cells. The activity of the expressed humanized antibody was detected four days later.
  • Example 7 Hemagglutination inhibition assay to identify the activity of 13D4 humanized antibody
  • the reading value of the first hole is the dilution value of the starting sample (eg 1: 2), phase
  • the second hole should be 1:4, 1:8, 1:16, 1:32, etc.
  • the purified 13D4cAb (diluted to 2. Omg/L) was used as a positive control, and the humanized antibody and the representative strains of 11 strains of H5N1 viruses such as YU22, 115, 5, QH15, 999, 2439 and the hemagglutination inhibitory activity were broad.
  • the results of the spectral detection are shown in Table 6.
  • the 8 human-derived antibodies, like 13D4cAb, have different hemagglutination inhibitory activities against 11 H5N1 viruses such as YU22 (the number indicates the dilution factor), indicating that they all retain the broad-spectrum HI activity of 13D4cAb.
  • H1202-34 humanized antibody has the best HI broad-spectrum reactivity, basically Similar to chimeric antibodies.
  • Flp-in CH0 cells are stable cell lines with FRT integration sites after screening. Frozen and resuscitation do not require resistance to Zeocin resistance, but Zeocin is required for culture. Resistance, hygromyc in resistance is required during the selection process, as described in the Invi trogen system (www.invi trogen.com).
  • the Pla3 plasmid containing the humanized antibody gene sequence of interest is transfected into Flp-In CHO cells at a density of about 5-8 X 105, and the transfection plasmid concentration is greater than 0.3 ug/uL, and the RNA content (260/280) is required. Less than 2.0.
  • the plasmid of interest was transfected with P0G44 in a ratio of 1:9 in a six-well plate with a total amount of DNA of 4 ug.
  • a negative control containing no plasmid for POG44 was provided.
  • the fresh incubation medium was changed within 6-12 h of transfection and no longer contained Zeoc in resistance. After 48 hours of culture, the cells were transferred to a 10 mm large plate, and after 2-3 hours of cell adherence, 600 ug/ml of hygromycin resistance was added.
  • CH0 cells stably expressing the humanized antibody were cultured in 10 cm of cell culture under the following conditions: 1640 medium (10% FBS, 1% glutamine, 1% double antibody), 5% C02, 37 "C. When the cell density on the plate reaches 90% or more, it is passaged and amplified according to a ratio of 1: 2. When the antibody needs to be expressed, a plate with a density of about 95% is selected to directly replace the serum-free medium (SFM4CH0+1% glutamine, HyClone). Cultivate.
  • CH0 cells not integrated into the target gene were basically in a floating non-adherent state on the 11th day, leaving cells that could normally adhere to the wall. It may be that the stably expressed cells of the antibody gene of interest are successfully integrated, and the cells are continuously pressurized until the 15th day, and all the floating cells that do not adhere to the wall are all killed, and the area of adherent cells is expanded, indicating that they completely have hygromycin resistance. Sex. The transfected cells without the pOG44 plasmid were used as controls. After the 14th day of pressurization, all the cells in the control group died of poisoning.
  • the stable cell population obtained will be screened, and the cells of the first and second generations will be continuously expanded and frozen. Finally, a total of 7 cell lines stably expressing different humanized antibodies were obtained. These stable expression cell lines were first cultured using serum medium. When the cells were amplified to 20 plates, that is, 200 mL in total, all were replaced with serum-free medium for culture, and when cultured until the fifth day, the cell supernatant was collected and purification was started.
  • Example 9 Purification of 13D4 humanized antibody
  • the supernatant of the serum-free cultured cells was collected, and the antibody was purified using Prote in A. Specific steps are as follows: Cell culture supernatants were harvested about 200ml, 8000rpm, centrifugal l Omin, supernatant was saved by dry Na 2 HP0 4 adjusted PH value of 8.2 - Between 85 and then 0. 22 ⁇ ⁇ . ⁇ L diameter filter membrane filtration.
  • the Sepharose 4B medium coupled with Prote in A (Protein A is expressed and coupled in the laboratory) 10 ml column, connected to the AKTA Explorerl OO system, the A pump is connected to 0. 2M disodium hydrogen phosphate solution, B pump 0. 1M citric acid solution was added.
  • FIG. 12 shows the results of SDS-PAGE electrophoresis of the purified seven humanized antibodies. It can be seen from the results of silver staining that the purity of 34 after purification by Prote in A can reach 90 ° /. .
  • Example 10 Detection of binding ability of humanized antibody to antigen
  • Example 11 Determination of Broad-spectrum Hemagglutination Inhibitory Activity of Purified 13D4 Humanized Antibody According to the WHO flu test guide (http: //www.who. int/csr/resources/publicat ions/influenza/en/ whocdscsrncs20025rev.pdf), the 8HA virus is first deployed. The purified 13D4 humanized antibody and 13D4cAb were diluted at a concentration of 2. Omg/L. The second well to the last well of the hemagglutination plate was added with 25 ⁇ L of PBS in the first well.
  • the purified 13D4MAb (diluted to 2.0 mg/L) was used as a positive control, and the hemagglutination inhibitory activity and broad spectrum of each humanized antibody and 13D4 cAb and 14 strains of H5N1 virus such as YU22, 115, 5, QH15
  • the results of the test results are shown in Table 7.
  • the 7 human-derived antibodies and 13D4CAb have the same hemagglutination inhibitory activity as the 14 H5N1 viruses such as YU22 (the number indicates the dilution factor), indicating that they all retain the broad-spectrum HI of 13D4MAb.
  • DK/IDN/MS/2004 1600 800 800 800 400 200 800 800 800
  • VNM/1194/2004 400 400 1600 800 400 400 400 400 400 400
  • the neutralization titer was determined using the endpoint method.
  • the experimental period was about 5 days.
  • the specific steps were as follows: (1) Preparation of MDC cells: MDCK cells were seeded on 96-well plates 1 day earlier, and the cell density was about 2 10 4 /well. The next day, the cells were infected with the virus antibody mixture to determine the titer.
  • (6) HA method to determine the neutralization titer After 3 days, the above cell plate was taken out, and 50 ⁇ L of the infected MDCK cell supernatant was aspirated, added to a 96-well hemagglutination plate, and then 0.5% chicken blood 50 ⁇ was added. After reading for 30 min, the highest dilution of the antibody corresponding to the hemagglutination activity is the neutralization titer of the antibody.
  • S2K2-42 VH amino acid sequence SEQ ID NO: 16

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Abstract

La présente invention porte sur des anticorps humanisés qui se lient spécifiquement à l'hémagglutinine du sous-type H5 du virus de la grippe aviaire. De tels anticorps humanisés sont utiles dans la prévention et le traitement des maladies provoquées par le virus de la grippe aviaire, en particulier le sous-type H5 du virus de la grippe aviaire. L'invention porte également sur les séquences d'acides aminés apparentées, et sur des compositions pharmaceutiques contenant les anticorps humanisés.
PCT/CN2009/001129 2008-10-09 2009-10-09 Anticorps humanisés se liant à l'hémagglutinine du sous-type h5 du virus de la grippe aviaire et leur utilisation WO2010040281A1 (fr)

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CN111423507B (zh) * 2019-01-10 2022-04-15 中国科学院分子细胞科学卓越创新中心 广谱性中和流感病毒的全人抗体
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