WO2010040281A1 - Humanized antibodies binding to avian influenza virus subtype h5 haemagglutinin and uses thereof - Google Patents

Humanized antibodies binding to avian influenza virus subtype h5 haemagglutinin and uses thereof Download PDF

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Publication number
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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French (fr)
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

Abstract

The present invention provides humanized antibodies that specifically bind to the hemagglutinin of avian influenza virus subtype H5. Such humanized antibodies are useful in the prevention and treatment of the diseases caused by avian influenza virus, particularly the avian influenza subtype H5. Also provided herein are the related amino acid sequences, and pharmaceutical compositions containing the humanized antibodies.

Description

H5亚型禽流感病毒血凝素蛋白的人源化抗体  Humanized antibody to hemagglutinin protein of H5 subtype avian influenza virus
及其用途 发明领域  And its use
[0002]本发明涉及可特异性结合 H5 亚型禽流感病毒血凝素 ( HA )蛋白之人源化抗体， 及其保守性变异体或活性片段， 及应用 该人源化抗体或片段用于预防与治疗的方法和用途。 背景技术  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
自 H5型禽流感于 1996年首先在我国广东省农场的鹅群暴发以 来（Xu X et a l.， 1999, Virology) , 由此病毒演生出来的另一株 Η5病毒在香港的禽鸟农场（1997年 4月）及市场（1997年 11月）暴 发， 引起了历史上禽流感病毒第一次直接由禽传人事件，前后共 18 例确诊病例中有 6人死亡。 2003年以来， Η5病毒株在整个东亚及 东南亚国家的相继暴发， WHO及流感专家预测 H5禽流感病毒将最有 可能成为下一次人类大流感暴发的流行株。 2004年初， H5型高致 病性禽流感也先后在我国十几个省暴发， 并在中国香港、 泰国、 荷 兰等发现 H5型禽流感传染鸡、 鸭、 苍鹭、 虎、 猫等多种动物的事 件。 更令人担忧的是， 在泰国已经出现疑似人传染人事件， 马来西 亚也有多例报道。 进入 2005年， 罗马尼亚、 俄罗斯、 土耳其等欧 洲国家相继发现禽类感染 H5型禽流感病毒致死事件， 专家认为此 乃带毒候鸟迁移的结果， 这使得控制高致病性 H5型禽流感病毒的 进一步扩散传播变得更为困难。 专家预测， 随着侯鸟的传播， H5 型禽流感病毒有可能通过欧亚、亚非大陆桥进一步传播到卫生条件 极差的非洲国家， 使得 H5型禽流感病毒获得和其它人流感病毒充 分重组的机会和时间，届时将很可能形成一种对人类高度致命的全 新流感病毒， 其给人类带来的各种损失将难以估计。 根据 WHO统计 结果， 截止 2006年 1月 19 日， 全球因感染 H5N1病毒死亡的人类 个案已达 80例， 给全球公共卫生安全带来极大的考验。 Since the outbreak of the H5 avian flu in 1996, the first outbreak of geese in farms in Guangdong Province, China (Xu X et al., 1999, Virology), another Η5 virus that was derived from this virus was farmed in Hong Kong. The outbreak (April 1997) and the market (November 1997) caused the first bird flu virus in history to be directly transmitted from birds. Six of the 18 confirmed cases were reported. Since 2003, Η5 strains have been outbreaks throughout East and Southeast Asian countries. WHO and influenza experts predict that the H5 avian flu virus will most likely be the next epidemic of the human outbreak of human influenza. In early 2004, 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. In 2005, 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. Experts predict that with the spread of migratory birds, 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. According to 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.
目前经过长期使用确定有效的两种的流感病毒治疗药物主要 为两个类型： M2离子通道抑制剂金刚烷胺（amantadine)和金刚乙胺 (rimantadine ) ，神经酰胺酶抑制剂奥司他韦 （oseltamivir) 和 扎那米韦（zanamivir ) (Monto, A. S. Vaccine, 2003. 21 (16): 1796-800)。前两者以抑制病毒离子通道蛋白 M2而发挥抗病毒效果； 后者能选择性地抑制病毒表面神经氨酸酶的活性， 阻止子代病毒颗 粒在宿主细胞的复制和释放， 有效地预防感冒和緩解症状， 使目前 比较有效的抗流感病毒药物， 但目前已不断发现新的 H5N1 病毒变 异株对其有抗药性。 这些药物如果在发病的最初 2天内服用， 将会 缩短病程甚至拯救病人的生命。 虽然离子通道抑制剂对于流感病毒 的一些亚型十分有用（Dolin, R. et al.， N Engl J Med, 1982. 307 (10) : 580-4)， 但是它却有很严重的副作用，并且病毒正在很快 的进化出了耐药株（Shiraishi, K. et al. , J Infect Dis, 2003， 188 (1)： 57-61)， 而且这些耐药株的传播性和致病性并没有减弱。  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. Although ion channel inhibitors are useful for some subtypes of influenza viruses (Dolin, R. et al., N Engl J Med, 1982. 307 (10): 580-4), they have serious side effects, and The virus is rapidly evolving resistant strains (Shiraishi, K. et al., J Infect Dis, 2003, 188 (1): 57-61), and the spread and pathogenicity of these resistant strains are not Weakened.
感染过 H5N1病毒而得到恢复的病人拥有在体外实验能中和病 毒的抗体， 提示抗体可作为治疗方法之一 (de Jong, M. D. et al.， Nat Med., 2006, 12: 1203-1207 ) 。 在临床上， 多克隆及单克隆抗 体可有效地用于预防曱肝、 乙肝、 狂犬、 呼吸道合胞病毒（RSV) 感染 （Sawyer, L. A. , Antiviral Res. , 2000, 47: 57-77 ) 。 在 1918年西班牙流感期间亦用人恢复期血清治疗，使流行期的死亡率 下降了 50% ( Luke, T. C. et al. , Ann Intern Med. , 2006,  Patients who have been infected with the H5N1 virus have antibodies that neutralize the virus in vitro, suggesting that antibodies can be used as a treatment (de Jong, M. D. et al., Nat Med., 2006, 12: 1203-1207). Clinically, polyclonal and monoclonal antibodies are effective in the prevention of sputum, hepatitis B, rabies, and respiratory syncytial virus (RSV) infection (Sawyer, L. A., Antiviral Res., 2000, 47: 57-77). In the 1918 Spanish flu period, human serum was also used to restore the mortality rate during the epidemic period by 50% (Luke, T. C. et al., Ann Intern Med., 2006,
145: 599-609 )。 我国 SARS病人和 H5N1感染病人的成功治疗经验， 也发现利用病人恢复性血清能够很好的抑制病毒在体内的复制，从 而使生命垂危的患者恢复健康。在小鼠动物模型中， H5N1特异的人 源化鼠单抗、 全人源单抗和 F(ab， )2片段已被证明能有效地预防 和治疗 H5N1 ( Lu, J. et al. , Respir Res. , 2006， 7: 43; Simmons, C.P. et al. , PLOS Medicine, 2007, 4 (5) : 928-936; Hanson, B. J. et a l. , Respir Res. , 2006, 7: 126 ) „ 高致病性禽流感病毒 H5N1 在受体结合位点的持续抗原突变即抗原漂变 （ant igenic dr if t ) ， 对这些抗体的广谱抗病毒治疗能力提出了挑战。 因此具有广谱中和 能力的单抗的获得将成为 H5N1治疗的希望， 利用高质量的抗 H5N1 广谱中和性单抗治疗 H5N1病毒感染患者可能会有抑制 H5N1病毒在 机体内的复制并获得理想的治疗效果，这是一种全新的抗病毒治疗 方式。 145: 599-609). The successful treatment experience of SARS patients and H5N1 infected patients in China has also found that the use of the patient's restorative serum can effectively inhibit the replication of the virus in the body, thereby restoring the life-threatening patients to health. In mouse animal models, 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. , 2006, 7: 126 ) „ Highly pathogenic avian influenza virus H5N1 sustained antigenic mutation at the receptor binding site, ie ant igenic dr if t , for these antibodies The ability to broad-spectrum antiviral therapy poses challenges. Therefore, the acquisition of monoclonal antibodies with broad-spectrum neutralization capacity will be the hope of H5N1 treatment. Patients with H5N1 virus infection may be treated with high-quality anti-H5N1 broad-spectrum neutralizing monoclonal antibody. It inhibits the replication of H5N1 virus in the body and achieves the desired therapeutic effect. This is a new antiviral treatment.
本实验室利用不同时间、 不同地点、 不同宿主分离到的多个禽 流感病毒 H5N1代表株免疫小鼠， 持续进行单克隆抗体的制备， 已 制备出了包含 400多株具有血凝抑制 （ HI )活性的 H5特异性单克 隆抗体的 H5单抗库。  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.
根据对近千株全球各地 H5N1 病毒基因组序列进行的***进化 分析， 我们筛选出 34株不同地域、 不同进化分支、 不同宿主来源 的 H5N1代表株， 加上 2006年最新分离出的 7个毒株共 41个毒株 组成代表性 H5N1病毒库。对这 41株 H5N1病毒进行各单抗的 HI活 性测定， 可根据 HI活性的差异将这 400多株单抗分为 10个类型， 同时可将这 41株代表性病毒分为八个 HI活性分支。初步 选出 17 株单抗对 2002以来的 H5N1病毒株的抗原多样性进行分析。根据血 凝抑制实验和细胞中和实验的结果， 可将 7种 H5N1亚型分成 4个 抗原群。 其中 13D4、 16F13等单抗均可对绝大部分毒株具有很高的 中和活性， 为治疗性抗体研究奠定了基础 （Wu, W. L. , Chen, Y.， Wang, P.， Song, W. , Lau, S. Y. , Rayner, J. M. , Smi th, G. J. , Webs ter, R. G.， Pe i r i s, J. S. , L in, T.， Xia, N.， Guan, Y. and Chen, H. 2008， J Vi ro l . 82 (4)， 1798-1807 ) 。  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. According to the results of the hemagglutination inhibition test and the cell neutralization experiment, seven H5N1 subtypes can be divided into four antigenic groups. Among them, 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).
[0011]本发明的根本目的在于，获得 13D4治疗性单抗的人源 化抗体， 及其保守性变异体或活性片段， 以期用于 H5N1 病毒治疗 性药物的研制及相关研究。 发明内容 [0011] 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
[0012] 本发明提供了可特异性结合 H5亚型禽流感病毒血凝素 ( HA )蛋白之人源化抗体， 该人源化抗体可用于预防和 /或治疗禽 流感病毒， 特别是 H5亚型禽流感病毒引起的疾病. 本发明也提供 了相关的核酸分子，以及其含该人源化抗体的药物组合物。 附图说明  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. DRAWINGS
图 1是 13D4鼠单抗人源化的模板及人源化抗体 FR区部分氨基 酸的替换. 其中下划线斜体为 CDR区； 括号中的两个氨基酸表示该 位点有两种氨基酸的替换方式.  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.
图 2显示 13D4人源化单链抗体库片段的 PCR扩增策略。  Figure 2 shows the PCR amplification strategy for the 13D4 humanized single-chain antibody library fragment.
图 3显示 13D4人源化单链抗体库 DM片段的 PCR扩增结果。 图 4显示带有 13D4人源化单链抗体的噬菌粒载体 pCANTAB 5E。 图 5显示噬菌体抗体在 HA和 YU22抗原上进行 ELISA检测的结 果。  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.
图 6显示四株 13D4人源化抗体 VH， VL中保留的鼠抗体氨基酸位 点。  Figure 6 shows the murine antibody amino acid positions retained in four 13D4 humanized antibodies VH, VL.
图 7 显示八株 13D4人源化抗体 VH, VL中保留的鼠抗体氨基酸 位点.  Figure 7 shows the amino acid sites of the murine antibody retained in eight strains of 13D4 humanized antibody VH, VL.
图 8显示 8株 13D4人源化噬菌体单链抗体与 HA1及 YU22病毒 的 ELISA反应结果. M13噬菌体为阴性对照. 二抗为 HRP-ant i- M13 抗体.  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.
图 9显示含有人抗体 CH和 CK片段的表达载体 PLAN3-CHCK 图 10 显 示 13D4 人 源 化 抗体 的 真核表达载体 Figure 9 shows the expression vector containing the human antibody CH and CK fragments PLAN3-CHCK Figure 10 shows the eukaryotic expression vector of 13D4 humanized antibody
PLAN3-CHCK-H13D4. PLAN3-CHCK-H13D4.
图 11显示潮霉素抗性筛选表达人源化抗体的稳定细胞株 图 12 显示经 Protein A纯化后人源化抗体的 SDS-PAGE结果. 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.
A. H1108-18; B. Y1213-21 ; C. H1121-29; D. H1108-18; E. 37; F. H1121-37; G， H1126- 08 A. H1108-18; B. Y1213-21; C. H1121-29; D. H1108-18; E. 37; F. H1121-37; G, H1126- 08
图 13显示 13D4人源化抗体与 HA1反应的 ELISA结果。 具体实施方式  Figure 13 shows the results of ELISA for the reaction of 13D4 humanized antibody with HA1. detailed description
[0040]本发明申请中涉及的有关术语的定义如下：  [0040] The definitions of related terms involved in the present application are as follows:
[0041] 本发明中的 "血凝素" 一词指禽流感病毒的包膜糖蛋 白。 血凝素蛋白介导流感病毒针对宿主细胞的吸附和进入。 禽流感 病毒的血凝素蛋白有 16个血清学亚型， HA1 - HA16,分别对应于 16 个病毒亚型， 即 Hl - H16。  The term "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.
[0042] 本发明中的 "抗体" 一词指任何免疫球蛋白， 包括能 结合特异性抗原的单抗、 多抗、 双特异性或多特异性抗体。 一个完 整的抗体包含两条重链和两条轻链。每条重链含有一个可变区和第 一、 第二、 第三三个恒定区； 每条轻链包含一个可变区和一个恒定 区。 抗体呈 "Y" 型， "Y" 型结构的颈部含有两条重链的第二和第 三恒定区， 其通过二硫键结合形成。 "Y" 型结构的每条臂含有其 中一条重链的第一恒定区和可变区（VH)，和一条轻链的可变区（VL) 和恒定区。 轻链和重链的可变区决定抗原的结合； 每条链的可变区 均含有三个高变区， 称互补决定区（CDR ) (轻链（L )的 CDR包含 LCDRU LCDR2、 LCDR3, 重链（H)的 CDR包含 HCDR1、 HCDR2、 HCDR3 ( 其由 Kabat 等人命名 ， 见 Sequences of Proteins of Immunologica l Interes t, Fif th Edi t ion (1991)， 第 1-3卷， NIH Publ icat ion 91-3242, Bethesda Md ) 0 其中， 三个 CDR由构架区 ( FR )间隔开。 构架区比 CDR区更保守并形成一个架子状结构支撑 超变区。 重链和轻链的恒定区与抗原结合无关， 但具有多种效应功 能。抗体依据重链恒定区的氨基酸序列可以分成几类，主要是： IgA、 IgD、 IgE、 IgG和 IgM， 其中有些类还进一步分成亚类， 如 IgGl、 IgG2、 IgG3、 IgG4、 IgAl或 IgA2等。 The term "antibody" as used in the present invention 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. The 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, The 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). 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.
[0043] 本发明中的 "抗体" 一词， 除特指完整的免疫球蛋白 外， 也指免疫球蛋白的片段（如至少是免疫球蛋白分子的一个免疫 活性区段） ， 如 Fab、 Fab'、 F (ab' ) 2、 Fv片段、 单链抗体分子或 由含有一个或多个 CDR区的免疫球蛋白分子的任意片段形成的多特 异性抗体。 另外， 本发明涉及的抗体也可以是由一个特定的人免疫 球蛋白中的一个或多个 CDR区结合一个或多个不同的人免疫球蛋白 的构架区形成的抗体。 [0043] The term "antibody" in the present invention, except the specific immunoglobulin In addition, it also refers to fragments of immunoglobulins (eg, at least one immunologically active segment of an immunoglobulin molecule), such as Fab, Fab', F (ab') 2, Fv fragments, 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. In addition, 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.
[0044]抗体相关的 "Fab" 片段是指含有一条轻链的可变区和 恒定区和一条重链的可变区和恒定区经二硫键结合起来的抗体分 子的一部分。  [0044] 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.
[0045] "Fab' " 片段是指包含了部分铰链区的 Fab片段。  [0045] A "Fab" fragment refers to a Fab fragment that contains a portion of the hinge region.
[0046] F (ab 2 指的是 Fab， 的二聚体。  F (ab 2 refers to a dimer of Fab,.
[0047]抗体的 "Fc" 指的是第一重链的第二、 第三恒定区与第 二重链的第二、 第三恒定区经二硫键结合成的抗体的一部分。 抗体 的 Fc段有多种不同的功能， 但不参与抗原的结合。  [0047] "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.
[0048]抗体的 "Fv"段指的是能结合完整的抗原结合位点的抗 体的最小片段。 一个 Fv片段包括一条轻链的可变区（VL)结合到一 条重链的可变区（VH)。  [0048] 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.
[0049]本发明中的 "单链抗体" 或 "scFv" 指的是由轻链可变 区与重链可变区直接相连或通过一个肽链连接而成的工程抗体 ( Hous ton 1988 )  [0049] "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).
[0050] 本发明中的 "单链抗体 Fv-Fc" 或 " scFv-Fc" 也包括 scFv连接抗体的 Fc段形成的工程抗体。  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.
[0051] 本发明中的 "抗原决定簇" （或称表位）指的是抗原 分子中与抗体结合的那部分氨基酸或原子基团。  The "antigenic determinant" (or epitope) in the present invention refers to the portion of the amino acid or atomic group in the antigen molecule that binds to the antibody.
[0052] 本发明中的 "单抗" 一词指的是来自一群高度同源的 抗体分子中的一个抗体或抗体的一个片断，也即除仅在少数情况下 可能出现的自然突变外， 一群完全相同的抗体分子。 单抗对抗原上 的单一表位具有高特异性。 单抗与多抗不同， 多抗是包含了识别抗 原上的不同表位的抗体分子。 虽然传统的单抗是由杂交瘤细胞分泌 的， 但本发明涉及的单抗并不仅限于此制备方法。 如， 本发明涉及 的单抗可釆用 Kohler 等首次报道的杂交瘤技术获得 （Nature, 256: 495 , 1975 ) ， 也可釆用重组 DNA 技术获得（如参见 U. S. P 4, 816, 567)。 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. For example, 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).
[0053] 本发明中的 "嵌合抗体"一词指的是抗体轻链或 /和重 链的一部分源自某一特定物种或属于某一特定抗体类或亚类的序 列相同或同源的抗体， 而抗体轻链或 /和重链的另一部分源自另一 物种或属于另一抗体类或亚类的序列相同或同源的抗体。 不管怎 样， 这种抗体片段仍保留了对目标抗原的结合活性（U. S. P 4， 816, 567 to Cabi l ly et a l.； Morr i son et a l.， Proc. Nat l. Acad. Sc i. USA, 81: 6851 6855 (1984) )。  The term "chimeric antibody" as used in the present invention 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. In any case, 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)).
[0054]本申请中， "人源化抗体" 一词指的是人源免疫球蛋白 (受体抗体）的全部或部分 CDR区被一非人源抗体（供体抗体）的 CDR区替换后得到抗体或抗体片段， 其中的供体抗体可以是有预期 特异性、 亲和性和反应性的小鼠、 大鼠或兔抗体。 另外， 人源免疫 球蛋白的构架区（FR )的氨基酸序列也可被相应的非人源抗体的氨 基酸序列所替换。 而且， 人源化抗体的氨基酸残基也可以既非来源 于受体抗体， 也非来源于供体抗体的 CDR区或构架区序列。 这些人 工修饰的目的是进一步完善或优化抗体性能。 总之， 人源化抗体是 指含有至少一个、 通常是两个几乎完整的可变区， 其中对应的所有 或几乎所有的 CDR区是来自非人源抗体，其中的全部或几乎全部的 FR 区是来自人源抗体。 理想的人源化抗体至少含有免疫球蛋白的 Fc区的一部分， 通常是人源免疫球蛋白的 Fc区。 更多详细内容请 参阅： Jones et a l. , Nature, 321: 522 525 (1986) ; Reichmann et a l. , Nature, 332: 323 329 (1988) ; Pres ta, Curr. Op. Struct. Biol. , 2: 593 596 (1992) ; and Clark, Immunol. Today 21: 397 402 (2000) . [0055]本申请涉及的 "被分离的" 、 "分离的" ， 指的是天然 状态下经人工手段获得的。 如果自然界中出现某一种 "被分离" 的 物质或成分，那么可能是其所处的天然环境发生了改变或从天然环 境下离分出该物质， 或二者情况均有发生。 比如， 某一活体动物体 内天然存在的某种未被分离的多聚核苷酸或多肽， 而从这种天然状 态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为被分 离。 这里的 "被分离" 不排除混有人工或合成的物质， 也不排除存 在不影响物质活性的其它不纯物质。 [0054] In the present application, 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. In addition, 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. Moreover, 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. In general, 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. For more details, please refer to: 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. Today 21: 397 402 (2000) . [0055] The term "isolated" and "isolated" as used in this application 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.
[0056]本发明中 "载体" 一词指的是， 可将编码某蛋白的多聚 核苷酸***其中并使蛋白获得表达的一种核酸运载工具。载体可通 过转化、 转导或转染宿主细胞， 使其携带的遗传物质元件在宿主细 胞内表达得以表达。 举例来说， 栽体包括： 质粒； 噬菌粒； 柯斯质 粒； 人工染色体如酵母人工染色体（YAC )、 细菌人工染色体（BAC ) 或 P1来源的人工染色体（PAC )； 噬菌体如 λ噬菌体或 M13噬菌体 及动物病毒等。 用作载体的动物病毒种类有逆转录酶病毒（包括慢 病毒） 、 腺病毒、 腺相关病毒、 疱療病毒（如单纯疾奢病毒） 、 痘 病毒、 杆状病毒、 ***瘤病毒、 ***多瘤空泡病毒（如 SV40 )。 一 种载体可能含有多种控制表达的元件， 包括启动子序列、 转录起始 序列、 增强子序列、 选择元件及报告基因。 另外， 载体还可含有复 制起始位点。 载体还有可能包括有协助其进入细胞的成分， 如病毒 颗粒、 脂质体或蛋白外壳， 但不仅仅只有这些物质。  [0056] 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. For example, 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 λ 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.
[0057] 本发明中 "宿主细胞" 一词指的是导入载体的细胞， 包括如下许多细胞类型， 如大肠杆菌或枯草菌等原核细胞， 如酵母 细胞或曲霉菌等真菌细胞， 如 S2果蝇细胞或 Sf9等昆虫细胞， 或 者如纤维原细胞， CH0细胞， COS细胞， NS0细胞， HeLa细胞， BHK 细胞， HEK 293细胞或人细胞的动物细胞。 宿主细胞可以是离体的 或者在体的， 可以是培养的细胞或者细胞系。  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.
[0058] "中和抗体"一词指的是能清除或显著降低目标病毒抗 原结合毒力的抗体或抗体片段。 [0058] The term "neutralizing antibody" refers to the ability to eliminate or significantly reduce target viral resistance. An antibody or antibody fragment that binds to virulence.
[0059] "序列相同百分比"一词指的是候选序列中的核酸或氨 基酸分别与对应的核酸或多肽序列中核酸或氨基酸相同性的百分 比。 这里涉及到的与核酸序列或者多肽序列有关的术语 "序列相似 百分比"定义为候选核酸序列或氨基酸残基序列分别与目的核酸序 列或氨基酸序列的相似百分比。 对于某一个序列， 将它和目的序列 进行比对，必要时可跳过突变缺口，以达到最大的基因相似百分比， 而不去考虑相似序列的任意保守性突变。本领域的多种比对方法可 用于确定核酸或氨基酸序列的相似性， 如可用的计算机软件包括 BLAST, BLAST-2, ALIGN, ALIGN- 2 或 Mega l ign (DNASTAR)等。 熟 悉本领域技术的工作人员懂得给比对设定合适的测量参数， 包括为 使用最大可比性的一些运算法则达到而全长序列的比较。  The term "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. The term "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.
[0060] "特异性结合" 一词指的是， 指两分子间的非随机结合 反应， 如抗体和产生该抗体的抗原间的反应。 此处， 结合第一种抗 原的抗体对第二种抗原的结合亲和力是检测不到或很弱。在某些实 施方式中， 一个某抗原特异性抗体是指以亲和力（KD ) < 1 (Γ5 Μ (如 10 6 Μ、 10—7 Μ、 10— 8 Μ、 10— 9 Μ、 10— 1ϋ Μ等）结合该抗原， 其中 KD 指解离率与结合率的比值（koff/kon )， 其可以采用本领域技术人 员熟悉的方法进行测定。 本发明在如下段落中进行举例说明： [0060] The term "specifically binds" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen that produces the antibody. Here, the binding affinity of the antibody that binds the first antigen to the second antigen is undetectable or weak. In certain embodiments, an antigen refers to a specific antibody with an affinity (KD) <1 (Γ 5 Μ ( eg 10 6 Μ, 10- 7 Μ, 10- 8 Μ, 10- 9 Μ, 10- 1ϋ Μ, 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. The invention is illustrated in the following paragraphs:
[0061]抗体  [0061] Antibody
[0062]本发明所述单抗和人源化抗体能够特异性结合 H5 亚型 禽流感病毒。 本发明的一个方面涉及能特异性结合 H5亚型禽流感 病毒血凝素蛋白的单抗和人源化抗体及其相应的抗原结合片段。  [0062] 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.
[0063]本发明所述抗 H5单抗优选是由小鼠杂交瘤细胞株 13D4 所分泌的。单抗的名称以其相应的杂交瘤细胞株进行命名。也就是， 此抗 H5单抗由杂交瘤细胞株 13D4产生，并命名为 13D4。单抗 13D4 能特异性结合 H5亚型禽流感病毒血凝素蛋白。 小鼠杂交瘤细胞株 13D4 已在中国典型培养物保藏中心（CCTCC, Wuhan University, Wuhan, China) 进行保藏， 保藏号是 CCTCC - C200721 (保藏时间 2007年 5月 29 日） [0063] 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)
[0066]可以采用 Kohler 等在 Nature 256: 495 (1975)中报 道的杂交瘤制备方法来制备单抗。 首先将免疫原（必要时候添加佐 剂）免疫注射小鼠或其它合适的宿主动物。 免疫原或佐剂的注射方 式通常为皮下多点注射或腹腔注射。免疫原预先藕联到某些已知蛋 白， 如血清白蛋白或大豆姨酶抑制剂上， 可能会有助于增强抗原在 宿主内的免疫原性。 佐剂可以利用福氏佐剂或 MPL- TDM等。 动物受 免疫后， 体内会有分泌特异性结合免疫原的抗体的淋巴细胞产生。 另外， 淋巴细胞也可以利用体外免疫获得。 收集目的淋巴细胞与骨 髓瘤细胞并用合适的融合剂， 如 PEG, 进行融合以获得杂交瘤细胞 (Coding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996)。  [0066] 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. In addition, 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).
[0067]上述制备的杂交瘤细胞可以接种到合适的培养液中生 长， 培养液中最好含有一种或多种能够抑制未融合的、 母体骨髓瘤 细胞生长的物质。例如，对缺乏次黄嘌呤鸟嘌呤鱗酸转移酶（HGPRT or HPRT) 的母体骨髓瘤细胞， 在培养液中添加次黄嘌呤、 氨基喋 呤和胸腺嘧啶（HAT培养基）等物质将可以抑制 HGPRT-缺陷细胞的 生长。  [0067] 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. For example, in the case of maternal myeloma cells lacking hypoxanthine squamous acid transferase (HGPRT or HPRT), the addition of hypoxanthine, aminoguanidine and thymine (HAT medium) to the culture medium can inhibit HGPRT. - Growth of defective cells.
[0068]优选的骨髓瘤细胞应该具有融合率高，抗体分泌能力稳 定，对 HAT培养液敏感等能力。其中，骨髓瘤细胞首选鼠源骨髓瘤， 如 MOP- 21 and MC-11小鼠肿瘤衍生棒 (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA) ， 和 SP-2/0 or X63-Ag8-653 细胞株 ( American Type Culture Collection, Rockville, Md. USA) 。 另外也有研究报道利用人骨髓瘤和人鼠异 源骨髓瘤细胞株制备人单抗（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)。 [0068] Preferred myeloma cells should have a high fusion rate, stable antibody secretion capacity, and sensitivity to HAT culture. Among them, 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). In addition, studies have reported the use of 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).
[0069]杂交瘤细胞生长的培养液用于检测针对特异抗原的单 抗的产生。测定杂交瘤细胞产生的单抗的结合特异性有免疫沉淀或 体外结合试验，如放射免疫试验（ RIA )、酶联免疫吸附试验（ ELISA )。 例如， 利用 Munson 等在 Anal. Biochem. 107: 220 (1980)描述的 Scatchard分析法可用来测定单抗的亲和力。  [0069] 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). For example, the Scatchard analysis described by Munson et al., Anal. Biochem. 107: 220 (1980) can be used to determine the affinity of a monoclonal antibody.
[0070]当杂交瘤产生的抗体的特异性、亲和力和反应性确定之 后， 目 的细胞株可以通过 (Goding, Monoc lona l Ant ibodies: Pr inciples and Pract ice, pp. 59-103, Academic Press, 1996) 所描述的标准的有限稀释法进行亚克隆化。 合适的培养液可以是 DMEM或 RPMI-1640等。另外，杂交瘤细胞还可以腹水瘤的形式在动 物体内生长。  [0070] After the specificity, affinity, and reactivity of the antibody produced by the hybridoma are determined, 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. In addition, hybridoma cells can also grow in the form of ascites.
[0071]利用传统的免疫球蛋 ^纯化方法，如蛋白 A琼脂糖凝胶、 羟基磷灰石层析、 凝胶电泳、 透析或亲和层析等， 可以将亚克隆细 胞分泌的单抗从细胞培养液、 腹水或血清中分离出来。  [0071] 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.
[0072]本发明所述单抗还可以是通过基因工程重组技术获得。 利用特异性结合单抗重链和轻链基因的核酸引物进行 PCR扩增，可 以从杂交瘤细胞中分离得到编码单抗重链和轻链基因的 DNA分子。 所得 DNA分子***表达载体内， 然后转染宿主细胞， 如 E. col i 细 胞、猿猴 COS 、 CH0细胞、或其它不产生免疫球蛋白的骨髓瘤细胞。 转染后的宿主细胞在特定条件下培养并表达目标抗体。  [0072] 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.
[0073]本发明的抗体和人源化抗体对 H5血凝素蛋白结合具有 高特异性和高亲和力。这些抗体对其它亚型的血凝素蛋白可能会有 弱的交叉反应性， 优选地， 这些抗体对其它亚型的血凝素蛋白完全 没有交叉反应性。 一方面， 本发明的抗体和人源化抗体结合 H5血 凝素的 KD值小于 1 X 10"5M; 优选地， KD值小于 1 X 10"6M; 更优 选地， KD值小于 1 x 10— 7M， 最优选地， KD值小于 1 x 10_8M。 [0073] 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. In one aspect, 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.
[0074]本发明的单抗和人源化抗体可以是包含两条重链和两 条轻链的传统的 "Y" 型结构状的抗体。 另外， 所述抗体也可以是 保持了对血凝素蛋白亲和力的传统的 "Y"型结构状的抗体上的 Fab 片段、 Fab' 、 F (ab) 2 、 Fv、 或其它类型的部分片段， 其结合血凝 素蛋白的亲和力可以高于或低于传 的 型结构状的抗体。 [0074] 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. In addition, 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.
[0075]本发明的抗体片段可以利用水解完整的抗体分子获得 (参见 Mor imoto et a l. , J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et a l. , Sc ience 229: 81 (1985) )。 另外， 这些抗体片段也可以直接由重组宿主细胞产生（reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999) ; Li t t le et a l. , Immunol. Today, 21: 364-370 (2000) )。 比如， Fab'片段可 以直接从 E. col i 细胞中获得或化学藕联形成 F (ab' ) 2 片段 (Carter et a l. , Bio/Technology, 10: 163-167 (1992) )。 再如， F (ab 2 片段可以用亮氨酸拉链 GCN4 连接获得。 另外， Fv、 Fab 或 F (ab' ) 2 片段也可以直接从重组宿主细胞培养液中直接分离得 到。 本领域的普通技术人员完全知晓制备抗体片段的其它技术。 [0075] 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. col i cells or chemically linked to form F(ab')2 fragments (Carter et al., Bio/Technology, 10: 163-167 (1992)). For example, F (ab 2 fragments can be obtained by leucine zipper GCN4 ligation. In addition, 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.
[0076]抗体核酸序列  [0076] Antibody nucleic acid sequence
[0077]本发明涉及特异性结合 H5血凝素蛋白的抗体或人源化 抗体或抗体片段的编码核酸分子。编码抗体的核酸分子可以从杂交 瘤细胞中分离得到。本领域的普通技术人员完全知晓利用常规技术 可以测定这些分子的核酸序列。本发明涉及的抗体核酸分子也可以 利用传统的基因工程重组技术或化学合成方法获得。 一方面， 本发 明涉及的抗体核酸分子的序列包含了抗 H5抗体的重链可变区或抗 体分子的部分核酸序列。 另一方面， 本发明涉及的抗体核酸分子的 序列也包括抗 H5抗体的轻链可变区或抗体分子的部分核酸序列。 另一方面，本发明涉及的抗体核酸分子的序列还包括重链或轻链可 变区的 CDR 序歹 'j。 互 卜决定！ ¾ ( complementary determinant reg ion, CDR )是与抗原表位结合的部位， 本研究中的 CDR序列通 过 Kabat 方法进行确定。但是不同的划分方法得到的 CDR序列稍有 不同。 [0077] 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. In one aspect, 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. In another aspect, the 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. In another aspect, the 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.
[0080] 本发明还提供能特异性结合 H5亚型禽流感病毒的抗体 片段的编码序列的核酸分子。  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.
本发明更进一步还涉及编码抗体重链可变区氨基酸序列 SEQ ID NOs: 4- 6之一的被分离的核酸分子。 本发明还涉及编码抗体轻 链可变区氨基酸序列 SEQ ID NOs: 7-9之一的核酸分子。  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.
本发明更进一步还涉及编码人源化抗体重链可变区氨基酸序 列 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之 一的被分离的核酸分子。本发明还涉及编码抗体轻链可变区氨基酸 序列 SEQ ID NO: 11、 SEQ ID NO: 13、 SEQ ID NO: 15和 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、 SEQ ID NO: 3 3之 一的核酸分子。  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. A nucleic acid molecule of one of SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33.
[0082] 本发明涉及含有本发明所述核酸分子的重组表达载 体， 也涉及转化了这些分子或者载体的宿主细胞。 而且， 本发明还 涉及利用包含了所述核酸分子的宿主细胞在特定条件下培养并分 离得到发明所述抗体的方法。  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.
[0083] 人源化抗体和抗体多肽序列  Humanized antibody and antibody polypeptide sequences
[0084]单抗 13D4重链和轻链可变区氨基酸序列可以从对应的 核酸序列中推导得到。单抗 13D4重链可变区氨基酸序列分别是 SEQ ID NO: 1或 SEQ ID NO: 2。 单抗 13D4轻链可变区氨基酸序列分别 是 SEQ ID NO: 3。  [0084] 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.
[0095] 另一方面， 本发明也包括了人源化抗体， 它们是由鼠 源单抗 13D4或其变异体的一个或多个 CDR嫁接到人源抗体的框架 上组成的。 In another aspect, 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.
一方面，本发明提供的人源化抗体的重链可变区氨基酸序列为 In one aspect, the amino acid sequence of the heavy chain variable region of the humanized antibody provided by the present invention is
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、 或 SEQ ID NO: 3 2。 另一方面， 本发明提供的人源化抗体的轻链可变区氨基酸序列 为 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或 SEQ ID NO: 3 3。 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, or SEQ ID NO: 3 2 . In another aspect, the 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 .
[0085] 另一方面， 本发明提供人源化抗体的重链可变区的一 个或多个氨基酸替代的变异体，其氨基酸序列与 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或 SEQ ID NO: 30、 SEQ ID NO: 3 2的序列相似性至少达 70 % , 优选是至少 75 %， 优选是至少 80 %， 优选是 85 %， 再优选是至少 90 % , 最好的是至少 95 %。 优选地， 所述替代的氨基酸个数为 1 - 20个， 例如 1 - 10个， 优选 1 - 5个， 包括 1、 2、 3、 4、 5个。  In another aspect, 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%. Preferably, the number of the substituted amino acids is 1 - 20, for example 1 - 10, preferably 1 - 5, including 1, 2, 3, 4, 5.
[0086] 另一方面， 本发明提供人源化抗体的轻链可变区的一 个或多个氨基酸替代的变异体，其氨基酸序列与 SEQ ID N0: 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 或 SEQ ID NO: 3 3的序列相似性至少达 70 % , 优选是至少 75 %， 优选是至少 80 %， 优选是 85 % , 再优选是至少 90 % , 最好的是至少 95 % 。 优选地， 所述替代的氨基酸个数为 1 - 20个， 例如 1 - 10个， 优选 1 - 5个， 包括 1、 2、 3、 4、 5个。  In another aspect, 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 Or 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%. Preferably, the number of the substituted amino acids is 1 - 20, for example 1 - 10, preferably 1 - 5, including 1, 2, 3, 4, 5.
[0087] 单抗 13D4的重链和轻链可变区的 CDR的氨基酸序列确 定如下： 单抗 13D4重链的 CDR1、 CDR2 和 CDR3的氨基酸序列分别 为 SEQ ID Nos: 4-6 o 单抗 13D4轻链的 CDR1、 CDR2 和 CDR3的氨 基酸序列分别为 SEQ ID Nos: 7-9。 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.
[0088] 另一方面， 本发明提供了抗 H5 的人源化抗体重链或 片段， 其含有如下 CDR: 来自 SEQ ID NOs: 4-6的一个或多个 CDR。 一实施方式中， 抗 H5人源化单抗重链或片段含有三个其氨基酸序 列为 SEQ ID NOs: 4-6的 CDR。  In another aspect, 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. In one embodiment, the anti-H5 humanized mAb heavy chain or fragment comprises three CDRs having the amino acid sequence of SEQ ID NOs: 4-6.
[0089]另一方面， 抗 H5人源化抗体的重链或片段的 CDR含有 的氨基酸序列可能是在 SEQ ID NOs: 4-6上出现一个或多个氨基酸 的突变或增添或缺失。 优选的是， 突变或添加或缺失的氨基酸不超 过 3个氨基酸。 更优选的是， 突变或添加或缺失的氨基酸不超过 2 个氨基酸。 最优选的是， 突变或添加或缺失的氨基酸不超过 1个氨  [0089] In another aspect, 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. Preferably, 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.
[0090] 另一方面，本发明提供了抗 H5人源化抗体轻链或片段 含有如下 CDR: 来自 SEQ ID NOs: 7-9的一个或多个 CDR。 在一实 施方式中， 抗 H5人源化抗体轻链或片段含有三个其氨基酸序列为 SEQ ID NOs: 7-9的 CDR。 In another aspect, 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. In one embodiment, the anti-H5 humanized antibody light chain or fragment comprises three CDRs having the amino acid sequence of SEQ ID NOs: 7-9.
[0091]另一方面， 抗 H5人源化抗体的轻链或片段的 CDR 含有 的氨基酸序列可能是在 SEQ ID NOs: 7-9上出现一个或多个氨基酸 的突变、 增添或缺失。 优选的是， 突变、 添加或缺失的氨基酸不超 过 3个氨基酸。 更优选的是， 突变、 添加或缺失的氨基酸不超过 2 个氨基酸。 最优选的是， 突变、 添加或缺失的氨基酸不超过 1个氨  [0091] In another aspect, 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. Preferably, 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.
[0092]上述人源化抗体或 CDR区或可变区的氨基酸发生突变、 添加或缺失之后的变异体仍然保留特异性结合 H5亚型禽流感病毒 的能力。 本发明也包含这样的抗原结合片段的变异体。 [0092] 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.
本发明还提供编码任何上述抗体、 重链、 轻链、 可变区、 CDR、 片段、 或变异体的核酸。 还提供包含编码任何上述抗体、 重链、 轻 链、 可变区、 CDR、 片段、 或变异体的核苷酸序列的核酸。  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.
[0093] 本发明的人源化抗体变异体可以通过传统的基因工程 方法获得。本领域的技术人员完全知晓利用核酸突变改造 DNA分子 的方法。 另外， 编码重链和轻链变异体的核酸分子也可以通过化学 合成获得。 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.
[0094]融合蛋白  [0094] fusion protein
[0096]另一方面， 本发明还提供了藕联或融合了某种分子的 完全或部分含有本发明所述人源化抗体的融合蛋白。  [0096] In another aspect, the invention provides a fusion protein comprising, in whole or in part, a humanized antibody of the invention, conjugated or fused to a molecule.
[0097]人源化抗体和融合蛋白都可以利用传统的基因工程技 术获得。 如， 编码单抗的 DNA可以通过突变的方法把人源抗体的重 链和轻链的恒定区的序列替换成同源性的鼠源序列而改造成 (Morr i son, et a l.， Proc. Nat. Acad. Sci. 81: 6851 (1984) )， 或通过把整个或部分免疫球蛋白编码序列与非免疫球蛋白编码序 列进行共价藕联而获得人源化抗体或融合蛋白。  [0097] Both humanized antibodies and fusion proteins can be obtained using conventional genetic engineering techniques. For example, 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.
[0098]中和抗体  Neutralizing antibody
[0099] 另一方面，本发明提供了能够中和 H5亚型禽流感病毒 之病毒活性的抗 H5抗体。 在一实施方式中， 这种中和抗体能够中 和 H5亚型禽流感病毒之病毒活性的至少 60%， 或至少 70%, 或优选 至少 75%, 或优选至少 80%， 或优选至少 85%， 或优选至少 90%, 更 优选至少 95%, 最优选至少 99%。  In another aspect, the invention provides an anti-H5 antibody capable of neutralizing the viral activity of an H5 subtype avian influenza virus. In one embodiment, 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%.
[0100] 本领域普通技术人员完全知晓利用传统的技术方法可 以测定抗体中和 H5 亚型禽流感病毒的病毒活性。 如本发明的实施 例 1中所描述的中和试验的方法即可用于测定本发明中的某一个特 定 H5单抗的中和活性。  It is well known to those of ordinary skill in the art that the 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.
[0203] 治疗方法和药物组合物  Treatment method and pharmaceutical composition
[0204] 本发明提供了一种预防和治疗禽流感病毒相关病毒感 染患者的方法， 包括对患者干预一定量的包含了一种或多种本发明 所述单抗的有药物活性的药物组合物。本发明还提供了一种含有本 发明所述的一种或多种人源化抗体或单抗的药物组合物或在此基 础上得到的盐类药物。 [0205] 本发明所述药物组^ ^物的干预方式可以是传统的干预 途径， 包括口服、 口腔、 舌下、 眼球、 局部、 肠胃外、 直肠、 叶鞘 内、 内胞浆网槽内、 腹股沟、 膀胱内、 局部（如， 粉剂、 药骨或滴 剂） ， 或鼻腔途径， 但不仅局限于此。 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. [0205] 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.
[0206] 适合肠胃外途径注射的药物组合物可能含有符合药物 制备要求的无菌水或非水溶液、 气雾剂、 悬浮液或乳剂， 可在临用 时重悬成可注射的溶液或气雾剂的无菌粉剂。如适合的水性和非水 性载体， 工具和各种稀释液如水、 乙醇、 多羟基化合物（如丙烯乙 二醇、聚乙烯二醇、 丙三醇及其类似物），合适的混合物， 菜油（如 橄榄油） ， 和可用于注射的有机脂， 如乙烷油酸。 如使用卵磷脂衣 壳维持药物的合适流动性。 如使用气雾剂、 表面活性剂以维持合适 的颗粒尺寸。  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.
[0207] 本发明所述的药物组合物还可含有一些起保护性、 保 湿、乳化和气雾化的佐剂，也可以含有预防微生物污染的速溶成分， 如各种抗细菌试剂、抗真菌试剂，如 parabens, chlorobutano l, 苯 酴， 山梨酸及类似物。 也可以包括维持渗透压的试剂， 如糖、 NaCl 及其类似物。可使用延长吸附的试剂来延长注射用药物组合物吸附 时间， 如单硬脂酸盐和凝胶等。  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.
[0208] 口服固相剂型包括胶襞、 片剂、 粉剂、 颗粒剂等。 这 些固相剂型中的活性成分至少混有一种传统的惰性药物赋形剂（或 载体）如柠檬酸钠、 磷酸钙， 或 （a )填充剂或添加剂如淀粉、 乳 糖、 蔗糖、 甘露糖和硅酸； （b ) 粘合剂， 如羧曱基纤维素、 藻酸 盐、 明胶、 聚乙烯吡咯烷酮、 蔗糖和***树胶； （C ) 湿润剂， 如甘油； （ d )碎裂剂， 如琼脂， 碳酸钙， 马铃薯粉或木薯粉； （ e ) 緩凝剂， 如石蜡； （ f )促吸收剂， 如四氨基混合物； （ g )保湿剂， 如十六烷基醇和单硬脂酸甘油酯； （h )吸附剂， 如高岭土和斑脱 土； （ i )润滑剂， 如滑石， 硬脂酸钙， 硬脂酸镁， 固体聚乙二醇， 硫酸十二烷醇钠， 或其上述物质的混合物。 在片剂和胶嚢剂型中， 可能还含有緩冲剂。 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. (b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (C) wetting agents such as glycerin; (d) fragmentation agents such as agar, Calcium carbonate, potato flour or tapioca flour; (e) a retarder such as paraffin; (f) an absorbent, such as a tetraamino mixture; (g) a humectant such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin and bentonite; (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or the like mixture. In tablets and capsules, It may also contain a buffer.
[0209] 固相剂型可以通过做 良释放或脉冲幹放剂型， 是 在上述提到的各种直接幹放赋形剂中添加一些能改变药物幹放速 率的赋形剂而形成， 可以包含在剂型中也可以做成外衣的形式。 速 率释放改造剂包括羧丙基甲基纤维素， 甲基纤维素， 碳甲基纤维素 钠， 纤维素乙烷， 醋酸纤维素， 聚乙烯氧化物， 黄原胶糖， 异丙烯 酸氨共聚物， 氢化调味油， 巴西棕榈蜡， 石蜡， 邻苯二酸醋酸纤维 素， 邻苯二曱酸羧丙基甲基纤维素， 曱基丙烯酸共聚物或上述物质 的混合物。 改良释放和脉沖幹放剂型可能含有一种或一组具有改良 释放速率的赋形剂。  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.
[0210] 本发明所述药物组合物还可由快速的雾化剂或消溶剂 ( FDDFs )组成， 可以包含如下成分： 天冬氨酰苯丙氨酸甲酯， 磺 胺钾， 种樣酸， croscarmel lose sodium, crospovidone, 抗坏血 酸， 乙烷基丙烯酸盐， 乙烷基纤维素， 明胶， 氢氧基丙基甲基纤 维素， 硬脂酸镁， 甘露醇， 甲基乙丁烯酸盐， 调味薄荷， 聚乙二醇， 气化硅胶， 二氧化硅， 乙醇酸淀粉钠，硬脂酸延胡索酸钠， 山梨醇， 木糖醇。 这里用于描述 FDDFs的 "雾化和消溶" 一词， 依赖于所用 药物的溶解性， 如药物是不可溶的， 可制成快速的雾化剂型， 如药 物是可溶的， 则可制成快速的溶剂型。  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. The term "atomization and dissolution" as used herein to describe the FDDFs 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.
[0211] 类似形式的固相组合物也使用诸如乳糖或牛奶糖或其 它高分子量的聚乙二醇及类似的赋形剂制成软明胶或硬明胶的填 充剂型。  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.
[0212] 诸如片剂、 糖衣剂、 胶嚢剂和颗粒剂等之类的固体剂 型可以通过诸如肠衣或其它本领域普通人员均知晓的外包衣壳的 方式制成。 也可以是含有乳浊剂、 也可以是含有能起緩慢、 延迟、 控制活性药物释放的类似的成分。也可以使用多聚物和石蜡等成分 进行包埋。 如果合适， 也可用上述一种或多种赋形剂把活性成分制 成微嚢的形式的剂型。 [0213] 用于口服的液体剂型， 包括符合药物要求的乳状剂、 溶液、 悬浮液、 糖浆和西也剂等。 除了活性成分， 液体剂型也可含 有本领域常用的一些惰性溶液， 如水或其它溶剂， 可溶性试剂和乳 化剂， 如乙烷基醇， 异丙基醇， 乙烷基碳酸盐， 苯基安息香酸盐， 丙稀乙二醇， 1， 3-丁烯乙二醇， 油， 特别是， 棉籽油， 落花生油， 玉米油， 橄榄油， 调味油和芝麻油， 甘油， 氢糠基醇， 聚乙二醇和 脂肪酸山梨醇酯， 以及上述物质的混合物或类似的物质。 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. In addition to the active ingredient, 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.
[0214] 除了这些惰性稀释液， 药物组合物也可包括保湿剂、 乳化剂、 悬浮剂、 糖化剂、 调味剂和香味剂等佐剂。 另外， 药物组 合物还可包括乙氧基化均聚乙醇， 聚氧乙烯烷基山梨醇和山梨聚糖 脂， 微晶纤维素， 间氢氧化铝， 膨润土， 琼脂聚合物和黄芪胶， 或 这些物质的混合物之类的悬浮剂。  In addition to these inert diluents, the pharmaceutical compositions may also include adjuvants such as humectants, emulsifiers, suspending agents, saccharifying agents, flavoring agents, and flavoring agents. In addition, 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.
[0215] 本发明所述药物组合物也制成适合兽用治疗的混合 物， 或符合兽用的盐类， 或符合兽用的溶剂或初成药， 并根据普通 兽医和兽医从业者的要求制成一种最适合某种特定动物的给药剂 量和途径药物的合适剂型。  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.
[0216] 本发明所述一个或多个 ^源化抗体可以结合其它抗病 毒试剂用于预防和或治疗 H5禽流感病毒感染相关疾病。 单抗可以 和这些抗病毒试剂同时、 分开或连续给药。 其它抗病毒试剂包括利 巴韦林， 金刚烷， 羧基脲， IL-2， IL-12 和五羧链胞酸， 但不仅 限于这些。 实施例  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. Example
[0220] 下面结合具体实施例与附图， 对本发明进一步加以描 述， 但这些描述并不构成对本发明的限制。 实施例 1 13D4抗体序列分析及人源模板的选择  The present invention is further described in the following with reference to the accompanying drawings and the accompanying drawings. Example 1 Sequence analysis of 13D4 antibody and selection of human template
我们已克隆出 13D4鼠单抗的基因可变区氨基酸序列， 重链可 变区氨基^^列见 SEQ ID NO: 1或 SEQ ID NO: 2， 轻链可变区氨 基酸序列见 SEQ ID NO: 3。 釆用 Kabat方法， 找出其中的 CDR序列， 如表 1所示。 We have cloned the amino acid sequence of the variable region of 13D4 murine mAb, and the heavy chain can The amino acid sequence of the variable region is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3. Using the Kabat method, find the CDR sequences therein, as shown in Table 1.
表 1 通过 Kabat方法确定的 13D4单克隆抗体 CDR氨基酸序列  Table 1 13D4 monoclonal antibody CDR amino acid sequence determined by Kabat method
Figure imgf000022_0001
通过 CDR移植的方法对鼠源抗体 13D4进行人源化。通过 Blas t 搜索基因数据库（NCBI+GenBank+DDBJ+Kabat Database) , 首先找 出与鼠源抗体 13D4的 FR区同源性最高的人 VH和 VL胚系基因可变 区序列。 经同源性分析， 确定以 1-69-04， 1-39-01 (Hwang WY, Almagro JC， Bus s TN, Tan P, Foote J. Use of human germl ine genes in a CDR homo logy-based approach to ant ibody
Figure imgf000022_0001
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
human izat ion. Methods, 2005， 36 (1) : 35-42)及其胚系基因序列作 为人抗体的模板， 在 NCBI , Kabat或 GenBank中均有 4艮道。 再将鼠 源抗体 13D4的重链和轻链 CDR区分别移植到人源模板 VH和 VL的 FR框架上。然后通过构建噬菌体单链抗体库的方式来优化 选人源 化抗体的 FR区氨基酸回复突变。 具体方法为， 分析人模板的 FR区 与鼠抗体的 FR区中的不同氨基酸， 靠近 CDR区的氨基酸以及性质 差别大的氨基酸均可存在两种可能的替换方式， 即可以保持鼠源氨 基酸， 也可以是人源的氨基酸。 总体而言，在 FR区有 21个位点有 两种氨基酸的替换，因此构建的 13D4人源化单链抗体库的氨基酸序 列多样性理论上为 1X1 οδ。 实施例 2 13D 人源化单链抗体基因库的 PCR引物设计及 PCR方案 采用重叠延伸 PCR (spl ic ing over lapping extens ion-PCR, S0E-PCR)方法获得 13D4人源化单链抗体库基因片段。位于 FR区的 氨基酸替换采用简并引物方式来扩增。分别将重链和轻链拆分为大 小约 50 bp的寡核苷酸序列， 片段之间重叠约 20 bp。 扩增 13D4 人源化单链抗体的寡核苷酸引物序列见表 2 ,引物在 13D4人源化单 链抗体上的相应位置见图 2 。 Human izat ion. Methods, 2005, 36 (1): 35-42) and its germline gene sequence as a template for human antibodies, 4 lanes in NCBI, Kabat or GenBank. The heavy and light chain CDR regions of murine antibody 13D4 were then transplanted into the FR framework of human template VH and VL, respectively. The FR region amino acid back mutation of the selected humanized antibody is then optimized by constructing a phage single-chain antibody library. 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. Overall, there are 21 amino acid substitutions in 21 sites in the FR region, thus constructing the amino acid sequence of the 13D4 humanized single-chain antibody library. The column diversity is theoretically 1X1 ο δ . 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.
表 2: 13D4人源化单链抗体库基因扩增引物  Table 2: 13D4 Humanized Single-Chain Antibody Library Gene Amplification Primers
引物名称 引物序列 Primer name primer sequence
VhFll 5>AGTGAAGGTCTCCTGCAAGGCTOTGGAGGC (TAC) ACATTCAGTGGGCACTGG<3  VhFll 5>AGTGAAGGTCTCCTGCAAGGCTOTGGAGGC (TAC) ACATTCAGTGGGCACTGG<3
VhRl 5>CACTCAAGTCCTTGTCCAGGGSCCTGCTTTACCCACTCTATCCAGTG<3  VhRl 5>CACTCAAGTCCTTGTCCAGGGSCCTGCTTTACCCACTCTATCCAGTG<3
VhF12 5>CAGTCTGGAGCTGAGGTGAWGAAGCCTGGGTCCTCAGTGAAGGTCTCCTGCAAG<3  VhF12 5>CAGTCTGGAGCTGAGGTGAWGAAGCCTGGGTCCTCAGTGAAGGTCTCCTGCAAG<3
VhF13 5>TTCCTTTCTATGCGGCCCAGCCGGCCCAGGTTCAGCTGGTG (CAG) CAGTCTGGAGCTGAGGTG< 3 VhF13 5>TTCCTTTCTATGCGGCCCAGCCGGCCCAGGTTCAGCTGGTG (CAG) CAGTCTGGAGCTGAGGTG< 3
VhF2 5>CTGGACAAGGACHGAGTGGATSGGAGAGATTTTACCTGG<3 VhF2 5>CTGGACAAGGACHGAGTGGATSGGAGAGATTTTACCTGG<3
Vh 2 5>TCCATGTAGGCTGTGCTCGTGGATKTATCTGCGGYGAWCGTGRCCYTGCCOTAAACTTC<3 Vh 2 5>TCCATGTAGGCTGTGCTCGTGGATKTATCTGCGGYGAWCGTGRCCYTGCCOTAAACTTC<3
VhF3 5>ACGAGCACAGCCTACATGGAGCTCAGCAGCCTGASATCTGAGGACACTGCCGTCTAHAC<3VhF3 5>ACGAGCACAGCCTACATGGAGCTCAGCAGCCTGASATCTGAGGACACTGCCGTCTAHAC<3
VhR3 5>CCGCCTCCACCGCTACCACCCCCTCCAGATCCGCCACCTCCGGAGGACACGGTCACC<3 VhR3 5>CCGCCTCCACCGCTACCACCCCCTCCAGATCCGCCACCTCCGGAGGACACGGTCACC<3
VkFll 5>GTCTCCATCCTCCCTGTCCGCATCAGTAGGAGACAGGGTCWCCRTCACCTGCAAGGCCAG<3 VkFll 5>GTCTCCATCCTCCCTGTCCGCATCAGTAGGAGACAGGGTCWCCRTCACCTGCAAGGCCAG<3
VkRl 5>CTGTACCGGTAGGATGCCGAGTAAATCAGGAG (GGC) TTTCGGGGCTTTCCCTGGTTTCTGm< 3VkRl 5>CTGTACCGGTAGGATGCCGAGTAAATCAGGAG (GGC) TTTCGGGGCTTTCCCTGGTTTCTGm< 3
VkF12 5>GGTGGTAGCGGTGGAGGCGGGAGTGACATCCAG (GTG) ATGACCCAGTCTCCATCCTCCCTGTC<3VkF12 5>GGTGGTAGCGGTGGAGGCGGGAGTGACATCCAG (GTG) ATGACCCAGTCTCCATCCTCCCTGTC<3
VkF2 5>CTCGGCATCCTACCGGTACAGTGGAGTCCCATCC (GAC) CGCTOAGTGGCAGTGGATCTGG<3VkF2 5>CTCGGCATCCTACCGGTACAGTGGAGTCCCATCC (GAC) CGCTOAGTGGCAGTGGATCTGG<3
VkR2 5>GCAAAATCnCAGGTOCAGACTGCTGATGGTGAGAGTG<3 VkR2 5>GCAAAATCnCAGGTOCAGACTGCTGATGGTGAGAGTG<3
VkF3 5>CTGCAACCTGAAGATTTTGCAACC (GAC) TACTTC (TAC) TGTCAGCAATATAACAAC<3  VkF3 5>CTGCAACCTGAAGATTTTGCAACC (GAC) TACTTC (TAC) TGTCAGCAATATAACAAC<3
VkR3 5>TTTGCGGCCGCCCGTTTTATTTCCACCTTGGTGCCGCC (GGC) ACCGAACGTGAGCGG<3 plan3VHFl 5>Τ(ΠΌ( ΤΑ(:Τ0Απ6Τ( Π ^ΑΤΑΤ。Τ(ηΌΤ( Α00Τ :Α0(^Τ0。Α&< 3  VkR3 5>TTTGCGGCCGCCCGTTTTATTTCCACCTTGGTGCCGCC (GGC) ACCGAACGTGAGCGG<3 plan3VHFl 5>Τ(ΠΌ( Τ(:Τ0Απ6Τ( Π ^ΑΤΑΤ.Τ(ηΌΤ( Α00Τ :Α0(^Τ0.Α&< 3
plan3VHF2 5>CTCAAGCTTATGGGAAGGCTTACTTCTTCATTCCTGCTACTGAnGTCCC<3 plan3VHR 5>TTrTGGTACCGGAGGACACGGTCACGGAG<3 plan3VHF2 5>CTCAAGCTTATGGGAAGGCTTACTTCTTCATTCCTGCTACTGAnGTCCC<3 plan3VHR 5>TTrTGGTACCGGAGGACACGGTCACGGAG<3
plan3VKFl 5>GCTGCTGCTGTGGCnACAGATGCAAGATGTGACATCCAGATGACCCAGTC<3 plan3VKFl 5>GCTGCTGCTGTGGCnACAGATGCAAGATGTGACATCCAGATGACCCAGTC<3
plan3VKF2 5>CGCGGATCCATGTCTGTGCCAACTCAGGTCCTGGGGnGCTGCTGCTGTGGCmC<3 plan3VKF2 5>CGCGGATCCATGTCTGTGCCAACTCAGGTCCTGGGGnGCTGCTGCTGTGGCmC<3
plan3V R 5>TTTTGATATCCCGTTTTATTTCCAGCTTG<3 以鼠源抗体 13D4 的重链可变区基因为模板， 利用引物 VhFl l/VhRl, VhF2/VhR2, VhF3/VhR3进行笫一轮 PCR扩增， PCR条 件为： 94 预变性 5min， 94"C变性 30s，57 X退火 30s， 72 :延伸 30s , 重复 10个循环， 再 72 反应 l Omin, 通过琼脂糖凝胶电泳分 析扩增产物， 并用 DNA纯化回收试剂盒（TianGen，DP214-03 )回收 纯化 PCR产物， 获得第一轮扩增产物 H1，H4, H5片段；以 HI为模板， 利用引物 VhF12/VhRl进行 PCR扩增， PCR条件同上， 获得片段 H2; 以 H2为模板， 利用引物 VhF13/VhRl进行 PCR扩增， PCR条件同上， 获得片段 H3; 对 H3/H4进行 S0E-PCR, 具体步骤为： 各取 5ul片段 H3, H4 , 不加引物，以同上条件进行 PCR扩增 5个循环后， 取出扩 增产物 3ul为模板， 加引物 VhF13/VhR2， 同上条件进行 PCR扩增 8 个循环， 获得片段 H6; 对 H5/H6进行 S0E-PCR, 方法同上， 获得片 段 H7。 同样， 以鼠源抗体 13D4轻链可变区基因为模板， 利用引物 VkFl l/VkRl, VkF2/VkR2， VkF3/VkR3进行第一轮 PCR扩增， 获得 产物 Kl， Κ3, Κ4片段； 以 K1为模板，利用引物 VkF12/VkRl进行 PCR 扩增，获得片段 K2;对 K3/K4进行 S0E-PCR,获得片段 K5;对 K2/K5 进行 S0E-PCR, 获得片段 K6。 将 Η7/Κ6进行 S0E-PCR, PCR条件为: 94 'C预变性 5min， 94 变性 30s， 57 Ό退火 30s， 延伸 50s， 重 复 12个循环， 再 72 反应 l Omin, 纯化回收后便获得 13D4的人源 化单链抗体库片段 H - K。 各 PCR扩增片段结果如图 3所示。 实施例 3: 13D4人源化单链抗体库的构建 plan3V R 5>TTTTGATATCCCGTTTTATTTCCAGCTTG<3 Using the heavy chain variable region gene of murine antibody 13D4 as a template, 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. Similarly, using the murine antibody 13D4 light chain variable region gene as a template, primers VkFl l/VkRl, VkF2/VkR2, VkF3/VkR3 were used for the first round of PCR amplification to obtain the product Kl, Κ3, Κ4 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. Η7/Κ6 was subjected to SOE-PCR , PCR conditions are: 94 'C pre-denaturation 5min, 94 denaturation 30s, 57 Ό annealing 30s, extension 50s, repeat 12 cycles, then 72 reaction l Omin, purification and recovery to obtain 13D4 humanized single-chain antibody library fragment H - K. The results of each PCR amplified fragment are shown in Figure 3. Example 3: Construction of 13D4 humanized single-chain antibody library
将 13D4的人源化单链抗体库 DNA片段 H - K用 Sfi 、 Not l 双酶切后， 按片段与载体 pCANTAB5E(Amersham公司） 2： 1的摩 尔比进行连接。 连接物用乙醇纯化后， 经电穿孔（电转条件： 25 μ F、 2.5KV、 200Ω )转化入电转感受态大肠杆菌 ER2738 (感受态效 率为 8x 108)。 S0C培养基恢复 45min后， 取出 1 0 Oul用于检测 库容量， 余下的菌液涂布 LB (含 100 g/L的氨苄青霉素 ）平板， 37 静置培养过夜。 菌斑长满整个平板后， 加 8ml LB (含 100 g/L 的氨苄青霉素 ）+2ml 50%的甘油， 用涂布器轻轻刮下菌落， 分装 于 1.5ml小管， - 80 保存备用。 最终确认库容量为 3 X 107。 随机 挑取 10个克隆测序， 结果表明，***片段均为 13D4人源化的单链 抗体片段， 并且在序列多样性符合设计要求。 质粒的构建如图 4所 示。 实施例 4: 13D4人源化单链抗体噬菌体展示库的扩增 13D4 humanized single-chain antibody library DNA fragment H - K with Sfi, Not l After double digestion, the fragments were ligated in a molar ratio of 2:1 to the vector pCANTAB5E (Amersham). After the linker was purified with ethanol, it was transformed into electroporation competent E. coli ER2738 by electroporation (electrical conversion conditions: 25 μF, 2.5 KV, 200 Ω) (competitive efficiency was 8 ×10 8 ). After recovery of the S0C medium for 45 min, 10 Oul was taken out for the detection of the reservoir capacity, and the remaining bacterial solution was coated with LB (100 g/L ampicillin) plate, and 37 was allowed to stand overnight. After the plaque has grown over the entire plate, add 8 ml of LB (containing 100 g/L of ampicillin) + 2 ml of 50% glycerol, gently scrape the colony with a coater, dispense into 1.5 ml small tubes, and store at -80. The final confirmation library capacity is 3 X 10 7 . Ten clones were randomly picked and sequenced. The results showed that the inserts were all 13D4 humanized single-chain antibody fragments, and the sequence diversity met the design requirements. The construction of the plasmid is shown in Figure 4. Example 4: Amplification of 13D4 humanized single chain antibody phage display library
200ul含单链抗体库的细菌转接于 70ml LB中， 37 C振荡培养 至 OD=0.5， 加氨苄青霉素 （100 g/L) ， 再振荡培养至 0D= 0.8， 加辅助噬菌体 M13K07, 37°C进行超感染 2h后， 6000rpm离心 5min, 弃去上清， 收集细胞， 用含氨苄青霉素和卡那霉素 （100 g/L) 的 双抗性培养基重新悬起， 37 振荡培养过夜； 4 5000rpm 离心 lOmin, 然后将上清转移至干净的 EP管中， 加原体积 1/5 的（20% PEG8000+2.5mol NaCl)沉淀噬菌体 8h， 4 X： 13700rpm 离心 20min, 弃上清， 沉淀用 lmlTBS重新悬起， 再加原体积 1/5的（20% PEG8000+2.5mol NaCl)进行二次沉淀， 4TC13700rpm离心 20min， 弃上清， 沉淀用 200ul TBS重新吹溶， 便为 13D4人源化单链抗体 库一次扩增产物； 取出 lOul稀释至 1(Γ5用于滴定， 从滴定板挑单 克隆用于下一步检测。 实施例 5: 13D4人源化单链抗体噬菌体展示库的筛选 200ul bacteria containing single-chain antibody library were transferred to 70ml LB, cultured at 37 C to OD=0.5, ampicillin (100 g/L), and cultured to 0D=0.8, plus helper phage M13K07, 37 °C After 2 h of superinfection, 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. Suspended, add 1/5 of the original volume (20% PEG8000 + 2.5mol NaCl) for secondary precipitation, centrifuge at 4TC13700rpm for 20min, discard the supernatant, and re-dissolve the pellet with 200ul TBS, which is 13D4 humanized single-chain antibody. a library amplification product; lOul withdrawn diluted to 1 (Γ 5 for titration picked from plates used for the next detection monoclonal Example 5: 13D4 humanized single chain antibody screening of phage display libraries.
从噬菌体展示库中 选人源化 13D4单链抗体。 取 lug/mL酵母表达的禽流感病毒血凝素蛋白 HA1(本实验室制 备） ， 用 C B (碳酸盐緩冲液， pH 9. 6 ) 包被 ELISA板， 温育 2h后， PBST洗涤 1次， 再加入 200ulED封闭液， 37 温育 2h， 4 储存备用。 另外， 将针对禽流感的单克隆抗体 2F2和 3G4 (本实 验室制备） 以 10ug/mL的浓度混合包板， 每孔加 l OOul 包被液， 37 °C温育 2h后， PBST洗涤 1次，再加入 200ul ED封闭液， 温育 2h, 拍干后， 加入 l OOul 4HI的 YU22病毒稀释液， 37 温育 2h, PBST洗涤 5次， 储存备用。 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 Å 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.
以上述包被了 HA1和 YU22病毒的两种抗原板为固相筛选介质， 对 13D4人源化单链抗体库进行 3轮 "吸附 -竟争-扩增" 富集筛 选。取 13D4人源化单链抗体噬菌体展示库（ 1 X 1011)用 TBS稀释 100 倍后，分别向 HA1和 YU22抗原板的各 5孔， 每孔加入 l OOul , 同时 设非相关抗原板 239 ( 10mg/ml )为阴性对照， 37 温育 lh后， 用 TBS洗涤 5次。 然后采用竟争洗脱法获得富集的噬菌体抗体。 具体 方法是， 用 TBS稀释 13D4 mAb至 lmg/ml， 每孔加入 100ul， 31 °C 温育 lh后， 收集竟争上清液共 1ml , 从中取出 lul滴定， 剩下的全 部用于感染对数期的 ER2738菌， 按照实施例 4的方法进行噬菌体 抗体扩增， 然后进入下一轮筛选； 第二轮筛选， 第三轮筛选方法同 第一轮。 分别从第二轮和第三轮富集后的噬菌体抗体滴定板上，随 机各挑选 23个克隆制备噬菌体抗体。 非相关抗原板上的竟争上清 液的滴定板没有细菌克隆生长，说明 HA1和 YU22抗原板与 13D4人 源化单链抗体库有特异性的结合。 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 For the ER2738 strain, 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.
将上述 46个克隆， 用含 100 g/L的氨苄青霉素抗性的 LB培养 至 OD - 0. 5 , 加 M13K07辅助超感染 2h后加 100 g/L的卡那霉素， 37 °C振荡培养 10h， 5000rpm离心 l Omin收集菌体， 4 保存上清用 于检测。  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.
取准备好的 HA1和 YU22抗原板， 每孔加入 l OOul待测噬菌体 抗体上清，双孔重复， 以 M13K07培养上清为阴性对照。 37 C温育 lh 后，用 TBS洗涤 5次，然后加以 1： 5000稀释的 M13 - HRP 100ul， 37 温育 30min后， 用 TBS洗涤 5次， 加底物 TMB溶液显色 l Omin, H2S04终止显色反应， 于 0D45。/62。测定读值。 取读值大于阴性对照 3 倍以上者为阳性克隆， 将阳性克隆进行基因序列测定。 由图 5 的 ELISA检测结果可以看出， HA1-10, Yu22-6， Yu22-8和 S2K2-42四 个噬菌体抗体的反应为阳性。 提取核酸进行测序， 四株 13D4人源 化抗体的 VH和 VL氨基酸序列编号见表 3， 它们的 FR区保留的鼠单 抗氨基酸及带入的少数突变氨基酸见图 6。 如果将这四种人源化的 VH和 VL序列与人恒定区序列组装成完整抗体分子，那么人源化的 13D4抗体的人源化程度在 88. 3%- 89. 05%之间。 The prepared HA1 and YU22 antigen plates were prepared, and 100 μl 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. As can be seen from the ELISA test results of Fig. 5, 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%之间之间之间。 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.
13D4人源化抗体的 VH和 VL氨基酸序列编号 VH and VL amino acid sequence numbers of 13D4 humanized antibodies
Figure imgf000028_0001
按照以上同样的方法， 本研究对 13D4人源化单链抗体库共进 行了 7次的筛选， 每次检测情况如表 4所示。 经过 7次的筛选， 本 研究一共检测了 524个克隆， 挑选出了 91个阳性克隆， 阳性比例 为 17%; 测序结果表明， 其中完全正确的人源化单链抗体序列有 37 个， 占检测总数的 7%。
Figure imgf000028_0001
According to the same method as above, 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.
从筛选获得的 37个阳性克隆中， 挑选出与抗原结合活性比较 高，人源化程序比较好的 8个人源化序列进行序列分析（见图 7 )， 其他的人源化序列保留备用。这 8个人源化单链抗体的噬菌体表达 上清与 HA1及 YU22抗原板的 ELISA反应结果， 如图 8所示。 这 8 个人源化抗体的 VH和 VL氨基酸序列编号见表 3, 以理论上完全人 源化的 13D4mAb， 即框架区完全为人源氨基酸残基的 H13D4为参照 (人源化程度为： 90. 61%)， 它们的 FR区保留的鼠单抗氨基酸个数 及构建成完整抗体后的人源化程度见表 5。 O 2010/040281 人源化程度 - (抗体的全部氨基酸个数—可变区上保留的氣源氨 基酸个数) /抗体的全部氨基酸个数 X 100%。 From the 37 positive clones obtained by screening, 8 humanized sequences with higher antigen binding activity and better humanization procedures were selected for sequence analysis (see Figure 7), and other humanized sequences were reserved. The ELISA reaction results of the phage expression supernatants of the eight humanized single-chain antibodies and the HA1 and YU22 antigen plates are shown in Fig. 8. The VH and VL amino acid sequence numbers of these 8 humanized antibodies are shown in Table 3, with reference to the theoretically fully humanized 13D4 mAb, ie, the framework region is completely H13D4 of human amino acid residues (humanization degree: 90.61) %), the number of murine monoclonal antibodies retained in their FR regions and the degree of humanization after constructing intact antibodies are shown in Table 5. O 2010/040281 Degree of humanization - (the total number of amino acids in the antibody - the number of amino acids remaining in the variable region) / the total number of amino acids in the antibody X 100% .
13D4人源化单链抗体库所有摔选结果 13D4 humanized single-chain antibody library all the results of the election
筛选次数 检测克隆 阳性克隆 正确序列 Number of screenings Detection clones Positive clones Correct sequence
1 46 5 41 46 5 4
--
2 46 5 2 2 46 5 2
1  1
3 40 6 4 3 40 6 4
4 40 5 44 40 5 4
5 160 30 125 160 30 12
6 100 20 16 100 20 1
7 92 20 107 92 20 10
Total 524 91(17%) 37 (7%) 表 5 13D4人源化抗体部分阳性克隆的人源化程度分析 Total 524 91 (17%) 37 (7%) Table 5 Analysis of humanization degree of 13D4 humanized antibody partial positive clone
FR区保留鼠源 FR area retains mouse source
人源化抗体 人源化程度  Humanized antibody
氨基酸个数  Number of amino acids
H13D4 0 90. 61% H13D4 0 90. 61%
Figure imgf000030_0001
Figure imgf000030_0001
H1126-08 15 88. 38%  H1126-08 15 88. 38%
H1213-21 11 88. 97%  H1213-21 11 88. 97%
H1202-34 8 89. 42% H1202-34 8 89. 42%
Figure imgf000030_0002
Figure imgf000030_0002
H1121-37 14 88. 52%  H1121-37 14 88. 52%
10 89. 12%  10 89. 12%
37 17 88. 08%  37 17 88. 08%
实施例 6: 13D4人源化抗体表达载体的构建和人源化抗体的表达 采用 Invitrogen公司的 Flp-In表达***来表达人源化抗体。 以商品化的表达载体 PCDM5. 1 为基础， 构建双启动真核表达载体 P1AN3, 并以 PLAN3为基础， 构建含有人抗体 CH和 CK片段的真核 抗体表达栽体 PLAN3 - CHCK, 如图 9所示。 CH为人 gamma-l重链恒 定 区 ， CK 为 人 kappa 轻链 恒 定 区 。 分别 以 引 物 plan3VHFl/plan3VHR, plan3VHF2/plan3VHR (具体序列见表 2) , 扩 增获得带有分泌信号肽序列的人源化 13D4的 VH片段 H13D4VH; 以 引物 plan3VKF/plan3VKR (具体序列见表 2)， 扩增获得带有分泌信 号肽序列的 13D4人源化的 VK片段 H13D4VK。 H13D4VH以 Hindin和 Kpn l双酶切， 连入以同样内切酶双酶切的载体 PLAN3 - CHCK 中； H13D4VK以 BamH I和 EcoRV双酶切，连入以同样内切酶双酶切的并 且已经确认连入 H13D4VH片段的载体 PLAN3 - CHCK中。构建好的含 有1304人源化抗体的真核表达载体？1^^3 - (：11( 1[ - 111304如图 1 0 所示。 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. Based on the commercial expression vector PCDM5.1, 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. Show. CH is the human gamma-l heavy chain constant region and 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).
构建好的含有 13D4人源化抗体的真核表达栽体 PLAN3 - CHCK - H13D4 , 与 pOG4 4质粒共转染 Flp-In™-CH0细胞。 四天后检测分 泌表达的人源化抗体的活性。 实施例 7. 血凝抑制实验鉴定 13D4人源化抗体的活性  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
按 照 WHO 的 流 感 试 验 指 南 According to the WHO Sense Test Guide
( hup: //www. who. int/csr/resources/publ i cat ions/ inf luenza /en/whocdscsrncs20025rev. pdf )进行， 先调配 8AD的病毒。 然后 将人源化抗体的细胞表达上清倍比稀释：在血凝板的第 2个孔到最 后一个孔中加入 25 μ L的 PBS, 在第 1孔中加入 50 μ L人源化抗体 的细胞上清，然后从第 1孔中吸取 25 μ ί 到第 2孔中混匀后再吸取 25 L到第 3个孔中， 如此向后倍比稀释至最后一孔并弃去最后的 25 μ ί; 每孔加入 25 8AD病毒， 然后加入 0. 75 %的鸡红细胞悬 液 50 L， 轻轻混匀， 静置 30 min后， 观察结果； 读取血凝抑制值( hup: //www. who. int/csr/resources/publ i cat ions/ inf luenza /en/whocdscsrncs20025rev. pdf ), first deploy 8AD virus. The cell expression supernatant of the humanized antibody is then diluted by dilution: 25 μL of PBS is added from the second to the last well of the hemagglutination plate, and 50 μL of the humanized antibody is added to the first well. Cell supernatant, then pipet 25 μί from well 1 into well 2 and mix and absorb 25 L into the third well, so that the dilution is repeated to the last well and the last 25 μ is discarded. ί; Add 25 8AD virus per well, then add 0.75 % chicken red blood cell suspension 50 L, mix gently, let stand for 30 min, observe the results; read the hemagglutination inhibition value
( HI值）时， 第 1孔的读值为起始样品的稀释度值（如 1: 2 ) , 相 应的第 2孔开始为 1: 4、 1: 8、 1: 16、 1: 32等。 (HI value), 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.
以纯化后的 13D4cAb (稀释至 2. Omg/L)为阳性对照， 各人源化 抗体与 YU22, 115， 5, QH15, 999， 2439等 11株 H5N1病毒的代表 株的血凝抑制活性及广谱性检测结果如表 6所示， 8个人源抗体与 13D4cAb一样对 YU22等 11株 H5N1病毒都有不同（数字表示稀释倍 数）的血凝抑制活性， 说明他们都保留了 13D4cAb的广谱 HI活性， 但他们对其中的一些代表株病毒的血凝活性比 13D4cAb低，说明人 源化的改造对他们的活性有影响， 其中 H1202-34人源化抗体的 HI 广谱反应性最好， 基本上与嵌合抗体相似。  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. However, they have lower hemagglutination activity than some of the representative strains of the virus than 13D4cAb, indicating that humanized transformation has an effect on their activity, and H1202-34 humanized antibody has the best HI broad-spectrum reactivity, basically Similar to chimeric antibodies.
表 6. 13D4人源化抗体的广谱血凝抑制活性测定  Table 6. Determination of broad-spectrum hemagglutination inhibition activity of 13D4 humanized antibody
H5N1病毒株 H1124- H112 Y121 H120 H110 H112 Y112 37 13D4  H5N1 strain H1124- H112 Y121 H120 H110 H112 Y112 37 13D4
38 6-08 3-21 2-34 8-18 1-37 1-29 cAb 38 6-08 3-21 2-34 8-18 1-37 1-29 cAb
IDN/2A/04 2 4 2 4 2 <2 2 2 2IDN/2A/04 2 4 2 4 2 <2 2 2 2
A/Ck/HK/Yu22/02 8 8 8 8 4 4 8 8 8A/Ck/HK/Yu22/02 8 8 8 8 4 4 8 8 8
CK/YN/ 115 / 04 8 4 4 4 2 2 4 8 16CK/YN/ 115 / 04 8 4 4 4 2 2 4 8 16
Indones ia/5/05 64 32 16 64 32 32 16 64 64Indones ia/5/05 64 32 16 64 32 32 16 64 64
BhGs/QH15/05 16 8 16 16 2 4 4 16 64BhGs/QH15/05 16 8 16 16 2 4 4 16 64
Ck/HN/999/05 - 32 64 64 16 16 16 64 32Ck/HN/999/05 - 32 64 64 16 16 16 64 32
Ck/GX/2439/04 0 2 2 2 2 2 1 2 2Ck/GX/2439/04 0 2 2 2 2 2 1 2 2
D /VNM/283/05 2 4 2 4 2 4 4 4 4D /VNM/283/05 2 4 2 4 2 4 4 4 4
VNM/1194/04 2 1 2 2 0 2 2 2 2VNM/1194/04 2 1 2 2 0 2 2 2 2
MDK/JX/1653/05 8 8 2 4 2 1 2 8 8MDK/JX/1653/05 8 8 2 4 2 1 2 8 8
Jogjakata/BVet/ 64 64 64 64 32 32 64 64 64Jogjakata/BVet/ 64 64 64 64 32 32 64 64 64
04 实施例 8. 13D4人源化抗体稳定表达细胞株的筛选 04 Example 8. Screening of 13D4 humanized antibody stably expressing cell lines
Flp-in CH0细胞为经过筛选后带有 FRT整合位点的稳定细胞 株，冻存和复苏不需要抗性 Zeocin抗性，但是在培养时需有 Zeocin 抗性， 在 选过程中需要潮霉素 （ Hygromyc in ) 抗性， 详见 Invi trogen ***介绍 (www. invi trogen. com)。 将含有目的人源化 抗体基因序列的 Plan3质粒， 转染 Flp-In CHO细胞， 密度大约为 5-8 X 105 , 转染质粒浓度需大于 0. 3ug/uL， RNA含量（ 260/280)需 小于 2. 0。将目的质粒与 P0G44以 1 : 9的比例在六孔板中进行转染， DNA总量为 4ug。设不含 POG44只带有质粒的阴性对照。转染 6-12h 内换新鲜温育培养基并不再含有 Zeoc in抗性。 培养 48h后将细胞 传至 10mm大板，待 2-3h细胞贴壁后加入 600ug/ml的潮霉素抗性。 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.
3-4天换一次新鲜培养基并加入潮霉素抗性， 仔细观察细胞生长状 态。 12天后稳定表达细胞株克隆开始出现， 用 Marker笔标注出， 并观察其随天数增加的形态变化。 14-15天后吸出培养基，用 ve r s on 洗一遍， 再用灭菌过后剪成方块的滤纸沾胰酶挑出克隆株入 96孔 板培养。 从 96孔板到 6孔板， 再到 10cm培养板， 不断加压并扩大 培养， 待细胞量足够时就冻存初^后的细胞， 并继续扩大培养。 取 稳定表达人源化抗体的 CH0细胞， 在 10cm细胞培养 中培养， 培 养条件为： 1640培养基（ 10%FBS, 1%谷氨酰胺， 1%双抗）， 5% C02 , 37 "C。待板上细胞密度达到 90%以上时按 1 : 2比例传代扩增。待需 要表达抗体时， 选取密度达到 95%左右的板直接更换无血清培养基 ( SFM4CH0+1%谷氨酰胺， HyClone )进行培养。 Change the fresh medium for 3-4 days and add hygromycin resistance. Carefully observe the cell growth status. After 12 days, stable expression cell clones began to appear, marked with a Marker pen, and observed for morphological changes with increasing days. After 14-15 days, the medium was aspirated and washed with ve r s on, and the cloned strain was extracted with a trypsin which was cut into squares after sterilization and cultured into a 96-well plate. From the 96-well plate to the 6-well plate, and then to the 10 cm plate, the cells were continuously pressurized and expanded, and when the amount of cells was sufficient, the cells after the initial cells were frozen and the culture was further expanded. 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.
如图 11 所示， 在潮霉素抗性的选择下， 没有整合入目的基因 的 CH0细胞在第 11天时， 基本上处于漂浮不贴壁生长状态， 剩下 还能正常贴壁生长的细胞， 就可能是成功整合入目的抗体基因的稳 定表达细胞， 继续加压至第 15天， 不贴壁生长的漂浮细胞全部死 亡， 贴壁生长的细胞面积反而扩大， 说明它们完全具有了潮霉素抗 性。 以不加 pOG44质粒的转染细胞为对照， 在加压第 14天后， 对 照组细胞出全部中毒死亡。 将筛选获得的稳定细胞群落， 不断扩大 培养并冻存第 1， 2代的细胞。 最后共获得 7株稳定表达不同人源 化抗体的细胞林. 先利用有血清培养基培养这些稳定表达细胞株， 等扩增至 20板， 即共 200mL时， 全部换成无血清培养基进行培养， 培养至第 5天时， 收集细胞上清， 开始纯化。 实施例 9. 13D4人源化抗体的纯化 As shown in Figure 11, under the selection of hygromycin resistance, 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
收集无血清培养的细胞上清， 利用 Prote in A纯化抗体。 具体 步骤如下： 收获细胞培养上清约 200ml， 8000rpm, 离心 l Omin, 保 留上清， 利用干粉 Na2HP04调节其 PH值为 8. 2 - 8 . 5之间， 然后 用 0. 22 μ πι孑 L径滤膜过滤。 取偶联上 Prote in A的 Sepharose 4B 介质（Protein A为本实验室表达并偶联） 10ml装柱， 连接至 AKTA Explorerl OO***， 将 A泵接入 0. 2M磷酸氢二钠溶液， B泵接入 0. 1M柠檬酸溶液。选取检测波长 UV280nm，流速 6ml /min，调节 A/B 泵进样比例， 先取 100%B ( pH2. 3 ) 冲洗柱子出去杂蛋白， 取 10%B ( pH 8. 0 )平衡 pH， 待检测波长稳定后归零， 上样， 待穿透峰过后 取 10%B继续平衡至检测波长降至零点并稳定后， 用 70%B ( pH4. 0 ) 洗脱， 收取洗脱峰， 进行 SDS PAGE纯度检测。 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 μ πι.孑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. Select the detection wavelength UV280nm, flow rate 6ml / min, adjust the A / B pump injection ratio, first take 100% B (pH 2.3) wash the column out of the protein, take 10% B (pH 8. 0) balance pH, wavelength to be detected After stabilization, return to zero, load, after the peak is over, take 10% B and continue to equilibrate until the detection wavelength drops to zero and stabilize. Then elute with 70% B (pH 4.0), collect the elution peak, and perform SDS PAGE purity. Detection.
最后获得 7种纯化的人源化抗体， 贮存浓度均为 2 mg/ml。 图 12为纯化的 7种人源化抗体的 SDS-PAGE电泳结果， 从银染结果可 以看出， 经 Prote in A纯化过后的 34纯度可以达到 90°/。。 实施例 10 人源化抗体与抗原结合能力的检测  Finally, seven purified humanized antibodies were obtained at a storage concentration of 2 mg/ml. Figure 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
先将各人源化抗体浓度调整到同一水平 （25ug/mL ) ， 然后取包被 好的抗原板 HAl (200ng/孔），分别加入不同的人源化抗体， 反应 lh， PBST洗板 5次， 加 GAH- HRP酶标（ 1 : 2000 )抗体， 37 反应 30min, PBST洗板 5次， 加底物 TMB溶液显色 15min, 112504终止显色反应， 于 OD450/620测定读值， 分析结果。 如图 13所示， 各人源化抗体与 HA1 的结合能力差别不大， 其中 37和 H1126-08与 HA1的结合能力较好。 实施例 11 纯化 13D4人源化抗体的广谱血凝抑制活性测定 按 照 WHO 的 流 感 试 验 指 南 ( http: //www. who. int/csr/resources/publicat ions/ influenza/en/ whocdscsrncs20025rev.pdf )进行， 先调配 8HA的病毒。 纯化后的 13D4 人源化抗体和 13D4cAb在 2. Omg/L浓度基础上， 再倍比稀释： 在血凝板 的第 2个孔到最后一个孔中加入 25 μ L的 PBS, 在第 1孔中加入 50 μ L 原倍的人源化抗体，然后从第 1孔中吸取 25μί 到第 2孔中混匀后再吸 取 25 μ L到第 3个孔中，如此向后倍比稀释至最后一孔并弃去最后的 25 μί; 每孔加入 25 8ΗΑ病毒， 轻轻混匀， 静置 30min后， 加入 0.75 %的鸡红细胞悬液 50μΙ 轻轻混匀， 静置 30min后， 观察结果； 读取 血凝抑制值（HI值）时，第 1孔的读值为起始样品的稀释度值（如 1: 1)， 相应的第 2孔开始为 1:2、 1:4、 1:8、 1: 16等。 First adjust the concentration of each humanized antibody to the same level (25ug/mL), then take the coated antigen plate HAl (200ng/well), add different humanized antibodies, react for 1h, wash the plate 5 times with PBST. Add GAH-HRP enzyme label (1: 2000) antibody, 37 reaction for 30 min, wash plate with PBST for 5 times, add substrate TMB solution for 15 min, 11 2 50 4 to terminate the color reaction, and read the value at OD450/620. Analysis results. As shown in Figure 13, the binding ability of each humanized antibody to HA1 was not much different, and 37 and H1126-08 had better binding ability to HA1. 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 μL of PBS in the first well. Add 50 μL of the original humanized antibody, then take 25 μί from the first well to the second well and mix and then absorb 25 μL into the third well, so that the mixture is diluted to the last one. Hole and discard the last 25 μί; add 25 8 每 virus per well, mix gently, let stand for 30 min, add 0.75 % chicken red cell suspension 50 μΙ, mix gently, let stand for 30 min, observe the results; read When the blood coagulation inhibition value (HI value), the reading value of the first hole is the dilution value of the starting sample (for example, 1:1), and the corresponding second hole starts with 1:2, 1:4, 1:8. 1:16 and so on.
以纯化后的 13D4MAb (稀释至 2. Omg/L)为阳性对照， 各人源化抗体 及 13D4 cAb与 YU22, 115, 5, QH15等 14株 H5N1病毒的代表株的血 凝抑制活性及广谱性检测结果如表 7所示， 7个人源抗体及 13D4CAb与 13D4MAb一样对 YU22等 14株 H5N1病毒都有不同（数字表示稀释倍数） 的血凝抑制活性， 说明他们都保留了 13D4MAb的广谱 HI活性， 但他们 对其中的一些代表株病毒的血凝活性比 13D4MAb低，说明人源化的改造 对他们的活性有影响， 其中人源化抗体 37、 H1126-08的 HI广傅反应性 最好， 基本上与 13D4MAb相似， 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. Active, but they have lower hemagglutination activity than some of the representative strains of virus than 13D 4 MAb, indicating that humanized transformation has an effect on their activity, among which humanized antibody 37, H1126-08 HI Guangfu reactivity Best, basically similar to 13D4MAb,
表 7. 13D 4人源化抗体的广谱血凝抑制活性测定 (2.0m g/m L) Table 7. Determination of broad-spectrum hemagglutination inhibition activity of 13D 4 humanized antibody (2.0m g/m L)
H5N1病毒株 13D4MAb 13D4CAb HI 126-08 37 H1121-37 H1202-34 H1213-21 Y1121-29 H1108-18H5N1 strain 13D4MAb 13D4CAb HI 126-08 37 H1121-37 H1202-34 H1213-21 Y1121-29 H1108-18
CK/GY/3570/2005 800 1600 800 1600 1600 400 800 800 1600CK/GY/3570/2005 800 1600 800 1600 1600 400 800 800 1600
C /IDN/2A/2004 6400 6400 6400 3200 6400 1600 1600 6400 3200C /IDN/2A/2004 6400 6400 6400 3200 6400 1600 1600 6400 3200
Indonesia/5/2005 1600 1600 1600 1600 1600 400 800 1600 1600Indonesia/5/2005 1600 1600 1600 1600 1600 400 800 1600 1600
DK/IDN/MS/2004 1600 800 800 800 400 200 800 800 800DK/IDN/MS/2004 1600 800 800 800 400 200 800 800 800
BHGS/QH/ 15/2005 1600 1600 3200 1600 3200 800 1600 1600 1600BHGS/QH/ 15/2005 1600 1600 3200 1600 3200 800 1600 1600 1600
VNM/1194/2004 400 400 400 1600 800 400 400 400 400VNM/1194/2004 400 400 400 1600 800 400 400 400 400
CK/VNM/568/2005 1600 1600 800 3200 1600 400 800 1600 1600CK/VNM/568/2005 1600 1600 800 3200 1600 400 800 1600 1600
HK/213/2003 6400 3200 6400 〉6400 1600 1600 3200 1600 3200HK/213/2003 6400 3200 6400 〉6400 1600 1600 3200 1600 3200
DK/VN /283/2005 800 800 800 1600 1600 400 800 400 800DK/VN /283/2005 800 800 800 1600 1600 400 800 400 800
CK/Bantul/BBVet-1/2005 800 800 800 1600 400 400 800 800 800CK/Bantul/BBVet-1/2005 800 800 800 1600 400 400 800 800 800
MDK/JX/1653/2005 800 400 800 800 800 400 400 400 400MDK/JX/1653/2005 800 400 800 800 800 400 400 400 400
CK/YN/115/2004 1600 800 1600 3200 800 800 800 400 800 shenzhen/406H/2006 1600 800 1600 1600 800 800 800 400 400CK/YN/115/2004 1600 800 1600 3200 800 800 800 400 800 shenzhen/406H/2006 1600 800 1600 1600 800 800 800 400 400
A/CK/HK/YU22/2002 800 800 800 1600 800 400 400 400 400 A/CK/HK/YU22/2002 800 800 800 1600 800 400 400 400 400
实施例 12 13D4人源化抗体的中和活性测定 Example 12 Determination of Neutralizing Activity of 13D4 Humanized Antibody
细胞微孔中和实验 Cell microporous neutralization experiment
中和滴度测定使用终点判定法，实验周期约 5天具体步骤如下： ( 1 ) MDC 细胞的准备：提前 1天在 96孔板上接种 MDCK细胞， 细胞密度约为 2 104 个 /孔。 第二天进行使用病毒抗体混合液感 染细胞测定滴度。 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.
( 2 )抗体的准备： 在 96孔细胞板上进行抗体的稀释， 在第 A 排微孔中各加入 80 按 1: 100稀释的单抗， 第 B H排的微孔中 各加入 40 μ L MEM, 然后从 A孔中吸取 40 液体到 B孔中， 依 次到 H孔， 做倍比稀释， 最终抗体稀释度为 1: 100 1: 12800。  (2) Preparation of antibody: The antibody was diluted in a 96-well cell plate, and 80 cells of 1:100 dilution were added to each of the A-row microwells, and 40 μL of MEM was added to each well of the BH row. Then, draw 40 liquid from the A well into the B well, and then go to the H well, and do the dilution. The final antibody dilution is 1: 100 1: 12800.
( 3 )抗体 病毒混合液制备： 在上述加有单抗的细胞孔中， 分别加入 40 的 200 TCID50 ( 2 x 102 TCID50/100 L ) 病毒 液， 稍稍混匀， 置 37 孵育培 2 h。  (3) Preparation of antibody virus mixture: Add 40 TCID50 (2 x 102 TCID50/100 L) virus solution to the cell hole with the monoclonal antibody, mix it slightly, and incubate for 37 hours.
( 4 ) MDCK细胞的感染： 取出 MDCK细胞培养板， 先用 200 μ L无血清 MEM培养基洗涤 MDCK细胞一次； 然后每个孔加入 35 M L 病毒抗体混合液， 置 37 5 % C02培养箱孵育 1 h。 (5) 细胞换液： 取出感染病毒的上述细胞板， 吸去上清， 在 每个孔中加入 200 含 10%FBS的 MEM培养基， 置 37 5% C02培养箱培养 72 h。 (4) Infection of MDCK cells: Remove the MDCK cell culture plate, first wash the MDCK cells once with 200 μL of serum-free MEM medium; then add 35 M L of virus antibody mixture to each well and incubate in a 37 5 % C02 incubator. 1 h. (5) Cell exchange: The above-mentioned cell plates infected with the virus were taken out, and the supernatant was aspirated. 200 MEM medium containing 10% FBS was added to each well, and cultured in a 37 5% C02 incubator for 72 hours.
(6) HA方法判定中和滴度： 3天后， 取出上述细胞板， 吸取 50 μ L感染病毒后的 MDCK细胞上清，加入到 96孔血凝板中，然后 加入 0.5%的鸡血 50 μΐ, 孵育 30 min后读值， 血凝活性呈现阴 性孔对应的抗体最高稀释度即为此抗体的中和滴度。 (6) HA method to determine the neutralization titer: After 3 days, the above cell plate was taken out, and 50 μL of the infected MDCK cell supernatant was aspirated, added to a 96-well hemagglutination plate, and then 0.5% chicken blood 50 μΐ 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.
13D4 VH氨基酸序列： SEQ ID NO: 1
Figure imgf000038_0001
13D4 VH amino acid sequence: SEQ ID NO: 1
Figure imgf000038_0001
DVWGQGTSVTVSS  DVWGQGTSVTVSS
13D4 VH氨基酸序列： SEQ ID NO: 2
Figure imgf000038_0002
13D4 VH amino acid sequence: SEQ ID NO: 2
Figure imgf000038_0002
DVWGQGTLVTVSS  DVWGQGTLVTVSS
13D4 VL氨基酸序列： SEQ ID NO: 3
Figure imgf000038_0003
13D4 VL amino acid sequence: SEQ ID NO: 3
Figure imgf000038_0003
HAl-10 VH氨基酸序列: SEQ ID NO: 10 HAl-10 VH amino acid sequence: SEQ ID NO: 10
QVQLQQSGAEVMKPGSSVKVSCKASGYTFSG GSGNIHYNE FKGRATITAD STSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS  QVQLQQSGAEVMKPGSSVKVSCKASGYTFSG GSGNIHYNE FKGRATITAD STSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS
HAl-10 VL氨基酸序列： SEQ ID NO: 11
Figure imgf000038_0004
HAl-10 VL amino acid sequence: SEQ ID NO: 11
Figure imgf000038_0004
Yu22-6 VH氨基酸序列： SEQ ID NO: 12 Yu22-6 VH amino acid sequence: SEQ ID NO: 12
IHYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS Yu22-6 VL氨基酸序列： SEQ ID NO: 13
Figure imgf000039_0001
IHYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS Yu22-6 VL amino acid sequence: SEQ ID NO: 13
Figure imgf000039_0001
Yu22-8 VH氨基酸序列： SEQ ID NO: 14 Yu22-8 VH amino acid sequence: SEQ ID NO: 14
QVQLVQSGAEVMKPGSSVKVSCKASGGTFSGHWIE IHYNEKFKGRATIAADKSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS QVQLVQSGAEVMKPGSSVKVSCKASGGTFSGHWIE IHYNEKFKGRATIAADKSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS
Yu22-8 VL氨基酸序列： SEQ ID NO: 15
Figure imgf000039_0002
Yu22-8 VL amino acid sequence: SEQ ID NO: 15
Figure imgf000039_0002
S2K2-42 VH氨基酸序列： SEQ ID NO: 16 S2K2-42 VH amino acid sequence: SEQ ID NO: 16
QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHWIE^ QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHWIE^
IHYNEKFKGRATFTADTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS IHYNEKFKGRATFTADTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS
S2K2-42 VL氨基酸序列： SEQ ID NO: 17
Figure imgf000039_0003
S2K2-42 VL amino acid sequence: SEQ ID NO: 17
Figure imgf000039_0003
H1202-34 VH氨基^ 列： SEQ ID NO: 18 H1202-34 VH amino group: SEQ ID NO: 18
QVQLQQSGAEVKKPGSLVKVSCKASGYTFSGHWIEW1 QVQLQQSGAEVKKPGSLVKVSCKASGYTFSGHWIEW 1
IHYNEKFKG VTITADTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS H1202-34 VL氨基^ ^列： SEQ ID NO: 19
Figure imgf000040_0001
IHYNEKFKG VTITADTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDVWG QGTLVTVSS H1202-34 VL amino ^ ^ column: SEQ ID NO: 19
Figure imgf000040_0001
H1124-38 VH
Figure imgf000040_0002
SEQ ID NO: 20 H1124-38 VH
Figure imgf000040_0002
SEQ ID NO: 20
QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHW SGNIHYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYFDV WGQGTLVTVSS  QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHW SGNIHYNEKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYFDV WGQGTLVTVSS
H1124-38 VL氨基酸序列： SEQ ID NO: 21
Figure imgf000040_0003
H1124-38 VL amino acid sequence: SEQ ID NO: 21
Figure imgf000040_0003
H1126-08 VH氨基^ 列： SEQ ID NO: 22 H1126-08 VH Amino^ Column: SEQ ID NO: 22
QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHWIEW1 QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHWIEW 1
GSGNIHYNEKFKGKVTFTADKSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS GSGNIHYNEKFKGKVTFTADKSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS
H1126-08 VL氨基^ ^列： SEQ ID NO: 23
Figure imgf000040_0004
H1126-08 VL amino ^ ^ column: SEQ ID NO: 23
Figure imgf000040_0004
H1213-21 VH氨基^ ^列： SEQ ID NO: 24 H1213-21 VH amino ^ ^ column: SEQ ID NO: 24
QVQLQQSGAEVMKPGSSVKVSCKASGYTFSGHWIEW1 QVQLQQSGAEVMKPGSSVKVSCKASGYTFSGHWIEW 1
GSGNIHYNEKFKGKATFTANKSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS GSGNIHYNEKFKGKATFTANKSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS
H1213-21 VL氨基^ 列： SEQ ID NO: 25 H1108-18 VH氨基 列： SEQ ID NO: 26 H1213-21 VL amino group column: SEQ ID NO: 25 H1108-18 VH amino acid column: SEQ ID NO: 26
QVQLQQSGAEVMKPGSSVKVSCKASGYTFSGHWIEW GSGNIHYNEKFKSRVTFAANTSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS  QVQLQQSGAEVMKPGSSVKVSCKASGYTFSGHWIEW GSGNIHYNEKFKSRVTFAANTSTSTAYMELSSLRSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS
HI 108-18 VL氨基^ ^列： SEQ ID NO: 27
Figure imgf000041_0001
HI 108-18 VL amino ^ ^ column: SEQ ID NO: 27
Figure imgf000041_0001
H1121-37 VH氨基^ ^列： SEQ ID NO: 28 H1121-37 VH amino ^ ^ column: SEQ ID NO: 28
QVQLQQSGAEVMKPGSSVKVSCKASGYTFSGHWIEW1 QVQLQQSGAEVMKPGSSVKVSCKASGYTFSGHWIEW 1
GSGNIHYNEKFKDKATFTADTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS GSGNIHYNEKFKDKATFTADTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS
H1121-37 VL氨基^ ^列： SEQ ID NO: 29
Figure imgf000041_0002
H1121-37 VL amino ^ ^ column: SEQ ID NO: 29
Figure imgf000041_0002
Y1121-29 VH氨基^ ^列： SEQ ID NO: 30 Y1121-29 VH amino ^ ^ column: SEQ ID NO: 30
QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHWIEWV SGNIHYNEKFKGKVTFTADKSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDV WGQGTLVTVSS  QVQLQQSGAEVKKPGSSVKVSCKASGYTFSGHWIEWV SGNIHYNEKFKGKVTFTADKSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYFDV WGQGTLVTVSS
Y1121-29 VL氨基^ ^列： SEQ ID NO: 31
Figure imgf000041_0003
Y1121-29 VL amino ^ ^ column: SEQ ID NO: 31
Figure imgf000041_0003
37 VH氨基酸序列： SEQ ID NO: 32 GSGNIHYNEKFKGKVTFTANTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS 37 VH amino acid sequence: SEQ ID NO: 32 GSGNIHYNEKFKGKVTFTANTSTSTAYMELSSLTSEDTAVYYCARLGTTAVERDWYF DVWGQGTLVTVSS
37 VL氨基酸序列： SEQ ID NO: 33 37 VL amino acid sequence: SEQ ID NO: 33
关于保藏的微生物的说明 Description of preserved microorganisms
Figure imgf000043_0001
本栏由受理局填写 本栏由国际局填写 口本页和国际申请一起收到 口国际局于以下日期收到本页:
Figure imgf000043_0001
This column is filled in by the receiving Office. This column is filled in by the International Bureau. This page is received with the international application. The International Bureau received this page on the following dates:
年 月 日 授权官员 授权官员  Year Month Day Authorized Official Authorized Officer
PCT/RO/134表 PCT/RO/134 form

Claims

1、 能与鼠单克隆抗体 13D4竟争结合 H5亚型禽流感病毒血凝 素蛋白的人源化抗体， 所述鼠单克隆抗体 13D4 为杂交瘤细胞株 13D4所产生的单克隆抗体， 杂交瘤细胞株 13D4保藏在中国典型培 养物保藏中心， 保藏号是 CCTCC一 C20072L 1. A humanized antibody capable of binding to a murine monoclonal antibody 13D4 in combination with an H5 subtype avian influenza virus hemagglutinin protein, wherein the murine monoclonal antibody 13D4 is a monoclonal antibody produced by a hybridoma cell line 13D4, a hybridoma The cell line 13D4 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC-C20072L.
2、 权利要求 1 所述的人源化抗体， 其是衍生自鼠单抗 13D4 的人源化抗体。 2. The humanized antibody of claim 1, which is a humanized antibody derived from murine monoclonal antibody 13D4.
3、 权利要求 1或 2所述的人源化抗体， 包含： 含有氨基酸序 列为 SEQ ID NOs: 4-6的 CDR中的一个或多个（如 1、 2或 3个） CDR的重链可变区，或其含有一个或多个（例如 1 - 10个， 1、 2、 3、 4或 5个）氨基酸替代的变异体。 3. The humanized antibody of claim 1 or 2, comprising: a heavy chain comprising one or more (eg 1, 2 or 3) CDRs of the CDRs having the amino acid sequence of SEQ ID NOs: 4-6 A variable region, or a variant thereof comprising one or more (eg, 1 - 10, 1, 2, 3, 4, or 5) amino acid substitutions.
4、 权利要求 1或 2所述的人源化抗体， 包含： 含有氨基酸序 列为 SEQ ID NOs: 7-9的 CDR中的一个或多个（如 1、 2或 3个） CDR的轻链可变区， 或其含有一个或多个（例如 1 - 10个， 1、 2、 3、 4或 5个）氨基酸替代的变异体。 4. The humanized antibody of claim 1 or 2, comprising: a light chain comprising one or more (eg 1, 2 or 3) CDRs of the CDRs having the amino acid sequence of SEQ ID NOs: 7-9 A variable region, or a variant thereof containing one or more (eg, 1 - 10, 1, 2, 3, 4, or 5) amino acid substitutions.
5、 如权利要求 3所述的人源化抗体， 其中， 所述的重链可变 区包括选自如下一组的氨基酸序列： The humanized antibody according to claim 3, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of:
SEQ ID NO: 10、 SEQ ID NO: 12， SEQ ID NO: 14、 SEQ ID NO: 16、 SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:
SEQ ID NO: 18、 SEQ ID NO: 20, SEQ ID NO: 22、 SEQ ID NO: 24、SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
SEQ ID NO: 26， SEQ ID NO: 28、 SEQ ID NO: 30、 SEQ ID NO: 32或 其变异体或片段。 SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or a variant or fragment thereof.
6、 如权利要求 4所述的人源化抗体， 其中， 所述的轻链可变 The humanized antibody according to claim 4, wherein the light chain is variable
42 42
更正 _页 （细则第 91条) 区包括选自如下一组的氨基酸序列： Correction_page (Article 91) The region includes an amino acid sequence selected from the group consisting of:
SEQ ID NO: 11、 SEQ ID NO: 13、 SEQ ID NO: 15和 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、 SEQ ID NO: 33或 其变异体或片段。  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, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 or a variant or fragment thereof.
7、 权利要求 1 - 6任一项所迷的人源化抗体， 其包括非 -CDR 区， 该非- CDR区来自不是鼠类的物种。 7. The humanized antibody of any of claims 1 to 6 which comprises a non-CDR region from a species other than a murine.
8、 权利要求 7所述的人源化抗体， 其中， 所述的非- CDR区来 自人类的抗体。 The humanized antibody according to claim 7, wherein the non-CDR region is derived from an antibody of a human.
9、 权利要求 8所述的人源化抗体， 其中， 所述的人类非 - CDR 区具有一个或多个（例如〗- 20个， 优选 1 - 10个， 1 - 5个）保守 氨基酸替代。 9. The humanized antibody of claim 8, wherein said human non-CDR region has one or more (e.g., - 20, preferably 1 - 10, 1 - 5) conservative amino acid substitutions.
10、 权利要求 1 - 9任一项所述的人源化抗体， 其中， 所述的 抗体为 Fab、 Fab' 、 F (ab) 2 、 scFv或 Fv。 The humanized antibody according to any one of claims 1 to 9, wherein the antibody is Fab, Fab', F(ab) 2 , scFv or Fv.
11、 权利要求 1 - 10任一项所述的人源化抗体， 其结合 H5亚 型禽流感病毒血凝素的 KD值小于 1 X 10"5Mo The humanized antibody according to any one of claims 1 to 10, which binds to the H5 subtype avian influenza virus hemagglutinin having a KD value of less than 1 X 10" 5 M o
12、 如权利要求 11所述的抗体， 其结合 H5亚型禽流感病毒 血凝素的 KD值小于 1 X 10— fiM。 12. The antibody of claim 11 which binds to the H5 subtype avian influenza virus hemagglutinin having a KD value of less than 1 X 10 - fi M.
13、 一种能特异性结合 H5亚型愈流感病毒血凝素蛋白的单克 隆抗体， 其包含： 含有氨基酸序列为 SBQ ID NOs: 4-6的 CDR中的 一个至三个 CDR 的重链可变区， 和 /或含有氨基酸序列为 SEQ ID 13. A monoclonal antibody capable of specifically binding to an H5 subtype influenza virus hemagglutinin protein, comprising: a heavy chain comprising one to three CDRs of a CDR having an amino acid sequence of SBQ ID NOs: 4-6 The variable region, and/or the amino acid sequence is SEQ ID
43 43
更正-页 （细则第 91条) NOs: 7-9的 CDR中的一个至三个 CDR的轻链可变区。 Correction - Page (Article 91) NOs: one to three light chain variable regions of the CDRs of 7-9.
14、 权利要求 13所述的单克隆抗体， 其中， 所述的单克隆抗 体为杂交瘤细胞株 13D 所产生的单克隆抗体， 杂交瘤细胞株 13D4 的保藏号是 CCTCC - C200721. The monoclonal antibody according to claim 13, wherein the monoclonal antibody is a monoclonal antibody produced by the hybridoma cell line 13D, and the hybridoma cell line 13D4 is deposited with CCTCC-C200721.
15、 权利要求 13或 14的单克隆抗体的人源化抗体， 其能特 异性结合 H5亚型禽流感病毒血凝素蛋白， 优选地其基本上保持所 迷单克隆抗体的特异性、亲和性和反应性， 例如保持至少 70 %、 80 %、 优选 90 %的特异性、 亲和性和反应性。 15. A humanized antibody to a monoclonal antibody according to claim 13 or 14 which specifically binds to an H5 subtype avian influenza virus hemagglutinin protein, preferably which substantially retains the specificity and affinity of the monoclonal antibody. Sex and reactivity, for example maintaining at least 70%, 80%, preferably 90% specificity, affinity and reactivity.
16、 编码权利要求 1 - 15任一项的抗体的重链、 轻链、 重链 可变区、 或轻链可变区的核酸分子。 16. A nucleic acid molecule encoding a heavy chain, a light chain, a heavy chain variable region, or a light chain variable region of an antibody of any of claims 1-15.
17、 一种核酸分子， 其包含能够编码抗体重链可变区的核酸 序列， 而所述的抗体重链可变区则包含选自如下一组的氨基酸序 列： SEQ ID NOs: 4 - 6。 17. A nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, and wherein said antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6.
18、 一种核酸分子， 其包含能够编码抗体轻链可变区的核酸 序列， 而所述的抗体轻链可变区则包含选自如下一组的氣基酸序 列： SEQ ID NOs: 7-9。 18. A nucleic acid molecule comprising a nucleic acid sequence encoding a variable region of an antibody light chain, and wherein said antibody light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 7- 9.
19、 权利要求 17所述的核酸分子， 其中， 所述的重链可变区 包含选自如下一组的氨基酸序列： The nucleic acid molecule according to claim 17, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of:
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 .  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 .
44 44
更正 _页 （细则第 91条) Correction_page (Article 91)
20、 权利要求 18所述的核酸分子， 其中， 所述的轻链可变区 包含选自如下一组的氨基酸序列： The nucleic acid molecule according to claim 18, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of:
SEQ ID NO: 11、 SEQ ID NO: 13、 SEQ ID NO: 15和 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、 SEQ ID NO: 3 3。  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, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 3 3.
21、 一种包含如权利要求 16-20任一项所述的核酸分子的表 达载体。 An expression vector comprising the nucleic acid molecule of any one of claims 16-20.
22、 一种包含如权利要求 21所述的表达载体的宿主细胞。 22. A host cell comprising the expression vector of claim 21.
23、 一种药物组合物， 其包含如权利要求 1-15任一项所述的 抗体或其在药学上可接受的盐。 A pharmaceutical composition comprising the antibody of any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.
24、 权利要求 23所述的药物组合物， 其进一步包括其它抗病 毒成分或其在药学上可接受的盐。 24. The pharmaceutical composition of claim 23, further comprising other anti-viral ingredients or a pharmaceutically acceptable salt thereof.
25、权利要求 24所迷的药物組合物，其中，所迷的抗体为 Fab、 Fab' 、 F (ab) 2 、 scFv或 Fv0 The pharmaceutical composition according to claim 24, wherein the antibody is Fab, Fab', F(ab) 2 , scFv or Fv 0
26、 一种预防或治疗禽流感病毒感染或由禽流感病毒感染在 人或动物上引发的疾病的方法，其包括向所述的人或动物给药有效 剂量的、 权利要求 1 - 15任一项的抗体、 权利要求 16 - 20的核酸 分子、 权利要求 21的表达载体、 或权利要求 23-25任一项所述的 药物组合物。 26. A method of preventing or treating avian influenza virus infection or a disease caused by avian influenza virus infection in a human or an animal, comprising administering to said human or animal an effective amount of any of claims 1-15 An antibody of the invention, a nucleic acid molecule according to claims 16-20, an expression vector according to claim 21, or a pharmaceutical composition according to any one of claims 23-25.
27、 权利要求 1-15任一项所述的人源化抗体用于禽流感疾病 的治疗药物筛选的用途。 27. Use of the humanized antibody of any of claims 1-15 for the screening of therapeutic drugs for avian influenza diseases.
45 45
更正 _页 （细则第 91条） Correction_page (Article 91)
28、 权利要求 1-15任一项所述的人源化抗体用于制备预防或 治疗禽流感病毒感染或禽流感疾病的疫苗的用途。 28. Use of the humanized antibody of any of claims 1-15 for the preparation of a vaccine for the prevention or treatment of avian influenza virus infection or avian influenza disease.
29、 权利要求 1-15任一项的抗体、 权利要求 16 -20的核酸 分子、 权利要求 21的表达载体、 或权利要求 23 25任一项所述的 药物組合物用于制备药物的用途，所述药物用于预防或治疗禽流感 病毒感染或由禽流感病毒感染在人或动物上引发的疾病。 The use of the antibody of any one of claims 1 to 15, the nucleic acid molecule of claims 16 to 20, the expression vector of claim 21, or the pharmaceutical composition according to any one of claims 23 to 25 for the preparation of a medicament, The medicament is for preventing or treating an avian influenza virus infection or a disease caused by an avian influenza virus infection on a human or an animal.
30、 权利要求 1-15任一项中定义的 CDR、 重链、 轻链、 重链 可变区、 或轻链可变区。 30. A CDR, heavy chain, light chain, heavy chain variable region, or light chain variable region as defined in any one of claims 1-15.
46 46
更正 _页 （细则第 91条）  Correction _ page (Article 91)
PCT/CN2009/001129 2008-10-09 2009-10-09 Humanized antibodies binding to avian influenza virus subtype h5 haemagglutinin and uses thereof WO2010040281A1 (en)

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