WO2023103788A1 - 特异性结合肺炎克雷伯菌o2抗原和o1抗原的双特异性抗体以及组合物 - Google Patents

特异性结合肺炎克雷伯菌o2抗原和o1抗原的双特异性抗体以及组合物 Download PDF

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WO2023103788A1
WO2023103788A1 PCT/CN2022/133880 CN2022133880W WO2023103788A1 WO 2023103788 A1 WO2023103788 A1 WO 2023103788A1 CN 2022133880 W CN2022133880 W CN 2022133880W WO 2023103788 A1 WO2023103788 A1 WO 2023103788A1
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seq
amino acid
acid sequence
sequence
antigen
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PCT/CN2022/133880
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French (fr)
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牛玉强
李平
李忠
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北京三诺佳邑生物技术有限责任公司
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Publication of WO2023103788A1 publication Critical patent/WO2023103788A1/zh

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    • 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
    • A61K39/02—Bacterial antigens
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial

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  • the present application relates to an antibody specifically binding to Klebsiella pneumoniae (Klebsiella pneumoniae; K.pneumoniae) O2 antigen, a bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae O1 antigen, and comprising Pharmaceutical composition of antibody specifically binding to Klebsiella pneumoniae O2 antigen and antibody and/or bispecific antibody specifically binding to Klebsiella pneumoniae O1 antigen, and preparation method and use thereof, including prevention and treatment thereof Methods of Klebsiella infection.
  • Klebsiella pneumoniae Klebsiella pneumoniae
  • K.pneumoniae a bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae O1 antigen
  • Pharmaceutical composition of antibody specifically binding to Klebsiella pneumoniae O2 antigen and antibody and/or bispecific antibody specifically binding to Klebsiella pneumoniae O1 antigen and preparation method and use thereof, including prevention and treatment thereof Methods of Kle
  • Klebsiella belongs to Gram-negative bacteria, which is a kind of nosocomial opportunistic pathogen causing diseases such as pneumonia, meningitis, liver abscess, urinary system inflammation, wound infection or sepsis.
  • Klebsiella infections are mainly caused by Klebsiella pneumoniae, which is the most important species in the genus Klebsiella in medicine (Podschun, R, and U Ullmann. Clinical microbiology reviews vol. 11, 4( 1998):589-603.).
  • strains of Klebsiella pneumoniae have become increasingly drug-resistant and the rate of drug-resistant strains has increased year by year, making this bacterial infection very difficult to treat.
  • Factors related to Klebsiella virulence include capsular polysaccharide (CPS), lipopolysaccharide (LPS), adhesion factors, siderophore system and other virulence factors. These virulence factors play an important role in the process of bacterial adhesion to host cells, evasion of host immune response/immune killing, etc. Among them, lipopolysaccharide (LPS) plays a very important role in bacterial pathogenicity, and is an important molecule that causes systemic inflammatory response of cells. LPS is a major and essential component of the outer leaflet of the cell membrane of all Gram-negative bacteria.
  • LPS LPS
  • lipid A lipid A
  • core oligosaccharide core oligosaccharide
  • O antigen O antigen
  • the application provides an isolated antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen, which comprises: (i) V H , which comprises the amino acid sequence shown in SEQ ID NO:17 HC-CDR1, HC-CDR2 and HC-CDR3 contained in the V H ; and V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in the V L as shown in the amino acid sequence SEQ ID NO:21; (ii) V H comprising HC-CDR1, HC-CDR2 and HC-CDR3 contained in V H as shown in the amino acid sequence of SEQ ID NO: 18; and V L comprising the amino acid sequence of SEQ ID NO: 22 LC-CDR1, LC-CDR2 and LC-CDR3 contained in the V L shown; (iii) V H , which comprises HC-CDR1, HC-CDR2 and HC contained in the V H shown in the amino acid sequence S
  • an isolated antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen comprising: (i) V H , said V H comprising: HC-CDR1 comprising an amino acid sequence SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant of said VH comprising in its HC-CDRs A substitution of up to about 5 amino acids; and a VL comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:8, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 , which comprises the amino acid sequence of SEQ ID NO: 15, or a variant of said V L comprising a substitution of up to about 5 amino acids in its LC-CDRs; (ii) V H , said V H comprising: HC-CDR1, It comprises
  • an isolated antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprising: (i) VH comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof, The variant has at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 17; and V L comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof, said variant having the amino acid sequence of SEQ ID NO 21 has at least about 80% sequence homology; (ii) VH comprising the amino acid sequence SEQ ID NO: 18 or a variant thereof having at least about 80% sequence with the amino acid sequence SEQ ID NO: 18 homology; and V L , which comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 22; (iii) V H , It comprises the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least about 80% sequence homo
  • an isolated antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen is provided that specifically binds to the D-galactan I domain within the side chain of the Klebsiella pneumoniae LPS antibodies or antigen-binding fragments.
  • an isolated antibody that specifically binds to the Klebsiella pneumoniae O2 antigen is provided that competes with any of the isolated antibodies described above with Klebsiella pneumoniae K. pneumoniae having a D-galactan I domain. Rabsiella specific binding.
  • an isolated antibody specifically binding to Klebsiella pneumoniae O2 antigen is provided, which binds to the same epitope as any of the above-mentioned isolated antibodies specifically binding to Klebsiella pneumoniae O2 antigen.
  • any of the isolated antibodies described above that specifically binds Klebsiella pneumoniae O2 antigen comprise an Fc region.
  • the isolated antibody that specifically binds the Klebsiella pneumoniae O2 antigen is a full-length IgG antibody.
  • the isolated antibody that specifically binds the Klebsiella pneumoniae O2 antigen is a full-length IgG1 or IgG4 antibody.
  • the isolated antibody that specifically binds the Klebsiella pneumoniae O2 antigen is a chimeric, fully human or humanized antibody.
  • the isolated antibody that specifically binds to the Klebsiella pneumoniae O2 antigen is an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab)' 2 , Fab'-SH , single chain Fv (scFv), Fv fragments, dAb, Fd, nanobody (nanobody), double chain antibody (diabody) and linear antibody.
  • an isolated nucleic acid molecule encoding an antibody that specifically binds to any of the above-described Klebsiella pneumoniae O2 antigens.
  • a vector comprising any one of the nucleic acid molecules described above.
  • a host cell comprising any one of the above-mentioned antibodies that specifically bind to Klebsiella pneumoniae O2 antigen, any of the above-mentioned nucleic acid molecules, or any of the above-mentioned kind of carrier.
  • a method for preparing an antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprising: a) culturing under conditions that can effectively express the antibody specifically binding to the Klebsiella pneumoniae O2 antigen any of the host cells described above; and b) obtaining the expressed antibody from the host cell.
  • compositions, kits, and articles of manufacture comprising any one of the above-described antibodies that specifically bind to the Klebsiella pneumoniae O2 antigen are provided.
  • a method of treating a disease or condition in an individual in need thereof comprising administering to said individual an effective amount of any one of the above-described antibodies that specifically bind to the Klebsiella pneumoniae O2 antigen or comprising pharmaceutical composition.
  • any one of the above-mentioned antibodies that specifically bind to Klebsiella pneumoniae O2 antigen in the preparation of a pharmaceutical composition for treating a disease or condition in an individual in need thereof use of any one of the above-mentioned antibodies that specifically bind to the O2 antigen of Klebsiella pneumoniae or a pharmaceutical composition comprising the same in the preparation of a medicament for treating a disease or a disease is provided.
  • the disease or condition comprises a condition associated with Klebsiella infection.
  • the disease or condition includes pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis, diarrhea, soft tissue infection, post-organ transplant infection, Surgical infection, wound infection, pulmonary infection, suppurative liver abscess, lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or Spondyloarthropathy.
  • the application provides a bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and specifically binds to Klebsiella pneumoniae O1 antigen, and a bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen Antibody and pharmaceutical composition of antibody specifically binding to Klebsiella pneumoniae O1 antigen. Also provided are methods for the prevention and treatment of Klebsiella pneumoniae infection.
  • the application provides a bispecific antibody, which includes a first antigen-binding domain that specifically binds Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds Klebsiella pneumoniae O1 antigen , wherein the first antigen binding domain comprises: (a) heavy chain variable region ( VH ), said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence SEQ ID NO:4; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and a light chain variable region (V L ), said V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; or (b) a heavy chain variable region (V H ), said V H comprising: V
  • the second antigen binding domain comprises: (a) heavy chain variable region (V H ), said V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; HC- CDR2, which comprises the amino acid sequence of SEQ ID NO:37; and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:39; and a light chain variable region (V L ), said V L comprising: LC-CDR1, which comprises The amino acid sequence of SEQ ID NO:41; LC-CDR2 comprising the amino acid sequence of SEQ ID NO:43; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:45; or (b) heavy chain variable region (V H ) , the VH comprises: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO:36; HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:38; and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:40; and a
  • the first antigen-binding domain comprises: VH , said VH comprises: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC- CDR2, which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:11 , LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and a second antigen-binding domain comprising: V H comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO: 35, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 37, and HC - CDR3, which comprises the amino acid sequence of SEQ ID NO: 39; and V L , which comprises:
  • the first antigen binding domain comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and VL , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:11, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and a second antigen binding domain comprising: V H comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 36 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:42, LC-CDR2
  • the first antigen binding domain comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 7; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO: 10, and LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 10; ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a second antigen binding domain comprising: V H comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 35 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:39; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:41
  • the first antigen binding domain comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 7; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO: 10, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 10; ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:15; and a second antigen binding domain comprising: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO:36 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:42
  • the application provides a bispecific antibody, which includes a first antigen-binding domain that specifically binds Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds Klebsiella pneumoniae O1 antigen , wherein the first antigen binding domain comprises: (a) VH comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising a variant having at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28 and V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or comprising a variant sequence having at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 24 or 33; or V H comprising Comprising HCDR1, HCDR2 and HCDR3 contained in V H as shown in amino acid sequence SEQ ID NO: 19 or 28; and V L comprising LCDR1, LCDR2 contained in V L as shown in amino acid sequence SEQ ID NO: 24 or 33 and LCDR3; or (b)
  • the second antigen binding domain comprises: (a) a VH comprising, or comprising at least about 80% identical to, the amino acid sequence of SEQ ID NO: 47 or 51 Variant sequences of sequence homology; and V L comprising the amino acid sequence of SEQ ID NO: 49 or 53, or comprising a variant sequence having at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 49 or 53 or V H , which comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in amino acid sequence SEQ ID NO: 47 or 51; and V L , which comprises V shown in amino acid sequence SEQ ID NO: 49 or 53 L comprises LCDR1, LCDR2 and LCDR3; or (b) VH comprising the amino acid sequence of SEQ ID NO: 48 or 52, or comprising at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 48 or 52 Variant sequences; and V L comprising the amino acid sequence of SEQ ID NO: 50 or 54, or comprising a variant
  • the first antigen binding domain comprises: a VH comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28 and V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or comprising a variant sequence having at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 24 or 33; and its second The antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 47 or 51, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 47 or 51; and VL comprising Comprising the amino acid sequence of SEQ ID NO: 49 or 53, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 49 or 53.
  • the first antigen binding domain comprises: a VH comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28 and V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or comprising a variant sequence having at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 24 or 33; and its second The antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 48 or 52, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 48 or 52; and VL comprising Comprising the amino acid sequence of SEQ ID NO: 50 or 54, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 50 or 54.
  • said first antigen binding domain comprises: a VH comprising, or comprising at least 80% sequence homology to, the amino acid sequence of SEQ ID NO: 20 or 29 and V L comprising the amino acid sequence of SEQ ID NO: 25 or 34, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 25 or 34; and its second The antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 47 or 51, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 47 or 51; and VL comprising Comprising the amino acid sequence of SEQ ID NO: 49 or 53, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 49 or 53.
  • said first antigen binding domain comprises: a VH comprising, or comprising at least 80% sequence homology to, the amino acid sequence of SEQ ID NO: 20 or 29 and V L comprising the amino acid sequence of SEQ ID NO: 25 or 34, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 25 or 34; and its second The antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 48 or 52, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 48 or 52; and VL comprising Comprising the amino acid sequence of SEQ ID NO: 50 or 54, or comprising a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 50 or 54.
  • the structure of the bispecific antibody is selected from structural forms such as DVD-Ig, Bs4Ab, Hetero H, CrossMab, IgG-(scFv) 2 or scFv-Fab IgG.
  • the bispecific antibody adopts a DVD-Ig structure.
  • the bispecific antibody comprises four polypeptide chains: two of the polypeptide chains both comprise a V H 1 -LV H 2 -CH 1 structure, wherein V H 1 specifically binds to Klebsiella pneumoniae The heavy chain variable region of the O2 antigen; VH2 is the heavy chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen; L is the connecting peptide; CH1 is the heavy chain constant region CH1 domain; wherein The polypeptide chain further comprises Fc, and the Fc comprises CH2 and CH3 domains; the other two polypeptide chains both comprise VL1 - LVL2 - CL structure, wherein VL1 is a protein that specifically binds pneumoniae The light chain variable region of the Lebsiella O2 antigen; VL2 is the light chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen; L is the connecting peptide; CL
  • the bispecific antibody comprises four polypeptide chains: wherein two polypeptide chains both comprise a V H 1-LV H 2-CH 1 structure, wherein V H 1 specifically binds pneumonia The heavy chain variable region of Klebsiella O1 antigen; VH2 is the heavy chain variable region that specifically binds Klebsiella pneumoniae O2 antigen; L is the connecting peptide; CH1 is the heavy chain constant region CH1 domain; wherein the polypeptide chain further comprises Fc, and the Fc comprises CH 2 and CH 3 domains; the other two polypeptide chains both comprise V L 1-LV L 2- CL structure, wherein V L 1 is specific V L 2 is the light chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen; L is the connecting peptide; CL is the light chain constant region; Among them, VH1 and VL1 constitute the antigen-binding domain (Fv) that can specifically bind to the O1 antigen of Klebsiella pneumoniae, and VH2 - CH1 and
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO: 61, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 61; and/or Or the amino acid sequence of SEQ ID NO:62, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:62; or (b) the amino acid sequence of SEQ ID NO:63, or comprising the amino acid sequence of SEQ ID NO:63 A variant sequence having at least 80% sequence homology to ID NO:63; and/or the amino acid sequence SEQ ID NO:64, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence SEQ ID NO:64 or (c) the amino acid sequence of SEQ ID NO: 65, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 65; and/or the amino acid sequence of SEQ
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO:86, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO:86; and/or Or the amino acid sequence of SEQ ID NO:62, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:62; or (b) the amino acid sequence of SEQ ID NO:87, or comprising the amino acid sequence of SEQ ID NO:87 A variant sequence having at least 80% sequence homology to ID NO:87; and/or the amino acid sequence SEQ ID NO:64, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence SEQ ID NO:64 or (c) the amino acid sequence of SEQ ID NO: 88, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 88; and/or the amino acid sequence of SEQ ID NO: 66, or comprising and A variant sequence having at least 80% sequence having at least
  • the bispecific antibody adopts the Bs4Ab structure.
  • the bispecific antibody comprises four polypeptide chains: wherein two polypeptide chains comprise a VH1 - CH1 -L1- VH2 -L3- VL2 structure from N-terminus to C-terminus, Wherein V H 1 is a heavy chain variable region specifically binding to the Klebsiella pneumoniae O2 antigen; V H 2 is a heavy chain variable region specifically binding to the Klebsiella pneumoniae O1 antigen; L1 and L3 are connecting peptides; CH1 is a heavy chain constant region CH1 domain; wherein said polypeptide chain further comprises Fc comprising CH2 and CH3 domains; and the other two polypeptide chains comprise V from N-terminus to C-terminus L 1- CL structure, wherein V L 1 is the light chain variable region that specifically binds to the Klebsiella pneumoniae O2 antigen, and CL is the light chain constant region.
  • V H 1- CH 1 and V L 1- CL constitute the antigen-binding domain (Fab) that specifically binds to the O2 antigen of Klebsiella pneumoniae
  • V H 2-L3-V L 2 constitutes the antigen-binding domain (Fab) that specifically binds to the Klebsiella pneumoniae O2 antigen.
  • the bispecific antibody comprises four polypeptide chains: wherein two polypeptide chains comprise a VH1 - CH1 -L1- VH2 -L3- VL2 structure from N-terminus to C-terminus, Wherein VH1 is a heavy chain variable region specifically binding to the Klebsiella pneumoniae O1 antigen; VH2 is a heavy chain variable region specifically binding to the Klebsiella pneumoniae O2 antigen; L1 and L3 are connecting peptides; CH1 is a heavy chain constant region CH1 domain; wherein said polypeptide chain further comprises Fc comprising CH2 and CH3 domains; and the other two polypeptide chains comprise V from N-terminus to C-terminus L 1- CL structure, wherein V L 1 is the light chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen, and CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL constitute the antigen-binding domain (Fab) that specifically binds to the O1 antigen of Klebsiella pneumoniae
  • VH2 -L3- VL2 constitutes the antigen-binding domain (Fab) that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO: 69, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 69; and/or Or the amino acid sequence of SEQ ID NO:70, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:70; or (b) the amino acid sequence of SEQ ID NO:71, or comprising the amino acid sequence of SEQ ID NO:71 A variant sequence of ID NO:71 having at least 80% sequence homology; and/or an amino acid sequence of SEQ ID NO:72, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:72 or (c) the amino acid sequence of SEQ ID NO: 73, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 73; and/or the amino acid sequence of SEQ ID NO: 74, or comprising a variant sequence with A
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO:90, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO:90; and/or Or the amino acid sequence of SEQ ID NO:70, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:70; or (b) the amino acid sequence of SEQ ID NO:91, or comprising the amino acid sequence of SEQ ID NO:91 A variant sequence having at least 80% sequence homology to ID NO:91; and/or the amino acid sequence SEQ ID NO:72, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence SEQ ID NO:72 or (c) the amino acid sequence of SEQ ID NO:92, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:92; and/or the amino acid sequence of SEQ ID NO:74, or comprising the amino acid sequence of SEQ ID NO:74, or
  • the bispecific antibody adopts the structure of Hetero H, CrossMab.
  • the bispecific antibody comprises four polypeptide chains: one of the polypeptide chains comprises a VH1 - CH1 structure from the N-terminus to the C-terminus, wherein VH1 specifically binds to Klebsiella pneumoniae The heavy chain variable region of the O2 antigen; CH1 is the heavy chain constant region CH1 domain; wherein the polypeptide chain further comprises Fc, the Fc comprises CH2 and CH3 domains; and one of the polypeptide chains From the N-terminal to the C-terminal comprises V L 1- CL , wherein V L 1 is a light chain variable region that specifically binds to the Klebsiella pneumoniae O2 antigen, and CL is a light chain constant region; and one of the polypeptide chains is from N-terminal to C-terminal comprises V H 2- CL , wherein V H 2 is a heavy chain variable region that specifically binds to the Klebsiella pneumonia
  • V H 1- CH 1 and V L 1- CL constitute the antigen-binding domain (Fab) that specifically binds to Klebsiella pneumoniae O2 antigen
  • V H 2- CL and V L 2- CH 1 consist of Antigen-binding domain (Fab) that specifically binds Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody comprises four polypeptide chains: one of the polypeptide chains comprises a VH1 - CH1 structure from the N-terminus to the C-terminus, wherein VH1 specifically binds to Klebsiella pneumoniae The heavy chain variable region of the O1 antigen; CH1 is the heavy chain constant region CH1 domain; wherein the polypeptide chain further comprises Fc, the Fc comprises CH2 and CH3 domains; and one of the polypeptide chains From the N-terminal to the C-terminal comprises V L 1- CL , wherein V L 1 is a light chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen, and CL is a light chain constant region; and one of the polypeptide chains is from N-terminal to C-terminal comprises V H 2- CL , wherein V H 2 is a heavy chain variable region that specifically binds to the Klebsiella pneumoniae O2 antigen; CL is a light chain constant region; wherein the polypeptide chain further comprises
  • V H 1- CH 1 and V L 1- CL constitute the antigen-binding domain (Fab) specifically binding to the Klebsiella pneumoniae O1 antigen
  • V H 2- CL and V L 2- CH 1 consist of Antigen-binding domain (Fab) that specifically binds Klebsiella pneumoniae O2 antigen.
  • the bispecific antibody comprises: (a); the amino acid sequence of SEQ ID NO: 76, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 76; and /or the amino acid sequence of SEQ ID NO:77, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:77; and/or the amino acid sequence of SEQ ID NO:78, or comprising the amino acid sequence of SEQ ID NO:78
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO:94, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO:94; and/or Or the amino acid sequence of SEQ ID NO:77, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:77; and/or the amino acid sequence of SEQ ID NO:95, or comprising the amino acid sequence of SEQ ID NO:95 NO:95 has a variant sequence with at least 80% sequence homology; and/or the amino acid sequence of SEQ ID NO:74, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:74; or (b) the amino acid sequence of SEQ ID NO:96, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:96; and/or the amino acid sequence of SEQ ID NO:80, or comprising the amino acid sequence of SEQ ID NO:80
  • the bispecific antibody adopts an IgG-(scFv) 2 structure.
  • the bispecific antibody comprises four polypeptide chains: wherein two polypeptide chains comprise VH1 -CH1- CH2 - CH3 - LVH2 - L3 from N-terminus to C-terminus -VL2 structure, wherein VH1 is the heavy chain variable region that specifically binds to the Klebsiella pneumoniae O2 antigen; VH2 is the heavy chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen; V L2 is the light chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen; L and L3 are connecting peptides; CH1 is the CH1 domain of the heavy chain constant region; CH2 is the heavy chain constant region C H2 domain; CH3 is the CH3 domain of the heavy chain constant region; and two of the polypeptide chains comprise VL1 - CL structure from N-terminus to C-terminus,
  • V H 1- CH 1 and V L 1- CL constitute the antigen-binding domain (Fab) that specifically binds to Klebsiella pneumoniae O2 antigen;
  • V H 2-L3-V L 2 constitutes the antigen-binding domain (Fab) that specifically binds to the Antigen binding domain (scFv) of the Lebsiella 01 antigen.
  • the bispecific antibody comprises four polypeptide chains: wherein two polypeptide chains comprise VH1 -CH1- CH2 - CH3 - LVH2 - L3 from N-terminus to C-terminus -VL2 structure, wherein VH1 is the heavy chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen; VH2 is the heavy chain variable region that specifically binds to the Klebsiella pneumoniae O2 antigen; V L2 is the light chain variable region that specifically binds to Klebsiella pneumoniae O2 antigen; L and L3 are connecting peptides; CH1 is the CH1 domain of the heavy chain constant region; CH2 is the heavy chain constant region C H2 domain; CH3 is the CH3 domain of the heavy chain constant region; and two of the polypeptide chains comprise VL1 - CL structure from N-terminus to C-terminus, wherein VL1 is the one that specifically binds pneumoniae The light chain variable region of the Lebsi
  • V H 1- CH 1 and V L 1- CL constitute the antigen-binding domain (Fab) that specifically binds to the Klebsiella pneumoniae O1 antigen;
  • V H 2-L3-V L 2 constitutes the antigen-binding domain (Fab) that specifically binds to the Antigen binding domain (scFv) of the Lebsiella O2 antigen.
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO:97, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO:97; and/or Or the amino acid sequence of SEQ ID NO:83, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:83; or (b) the amino acid sequence of SEQ ID NO:98, or comprising the amino acid sequence of SEQ ID NO:98 A variant sequence having at least 80% sequence homology of ID NO:98; and/or an amino acid sequence of SEQ ID NO:85, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:85 .
  • the bispecific antibody adopts a scFv-Fab IgG structure.
  • the bispecific antibody comprises three polypeptide chains: one of the polypeptide chains comprises a VH1 - CH1 structure from the N-terminus to the C-terminus, wherein VH1 specifically binds to Klebsiella pneumoniae The heavy chain variable region of the O2 antigen; CH1 is the heavy chain constant region CH1 domain; wherein the polypeptide chain further comprises Fc, the Fc comprises CH2 and CH3 domains; and one of the polypeptide chains From the N-terminal to the C-terminal comprises V L 1- CL , wherein V L 1 is a light chain variable region that specifically binds to the Klebsiella pneumoniae O2 antigen, and CL is a light chain constant region; and one of the polypeptide chains is from N-terminal to C-terminal contains V H 2-L3-V L 2, wherein V H 2 is the heavy chain variable region that specifically binds Klebsiella
  • V H 1- CH 1 and V L 1- CL constitute the antigen-binding domain (Fab) that specifically binds to Klebsiella pneumoniae O2 antigen;
  • V H 2-L3-V L 2 constitutes the antigen-binding domain (Fab) that specifically binds to the Antigen binding domain (scFv) of the Lebsiella 01 antigen.
  • the bispecific antibody comprises three polypeptide chains: one of the polypeptide chains comprises a VH1 - CH1 structure from the N-terminus to the C-terminus, wherein VH1 specifically binds to Klebsiella pneumoniae The heavy chain variable region of the O1 antigen; CH1 is the heavy chain constant region CH1 domain; wherein the polypeptide chain further comprises Fc, the Fc comprises CH2 and CH3 domains; and one of the polypeptide chains From the N-terminal to the C-terminal comprises V L 1- CL , wherein V L 1 is a light chain variable region that specifically binds to the Klebsiella pneumoniae O1 antigen, and CL is a light chain constant region; and one of the polypeptide chains is from N-terminal to C-terminal contains V H 2-L3-V L 2, wherein V H 2 is the heavy chain variable region that specifically binds Klebsiella pneumoniae O2 antigen; V L 2 is the heavy chain variable region that specifically binds Klebsiella pneumoniae O
  • V H 1- CH 1 and V L 1- CL constitute the antigen-binding domain (Fab) that specifically binds to the Klebsiella pneumoniae O1 antigen;
  • V H 2-L3-V L 2 constitutes the antigen-binding domain (Fab) that specifically binds to the Antigen binding domain (scFv) of the Lebsiella O2 antigen.
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO: 81, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 81; and/or Or the amino acid sequence of SEQ ID NO:82, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:82; and/or the amino acid sequence of SEQ ID NO:83, or comprising the amino acid sequence of SEQ ID NO:83 NO:83 has a variant sequence of at least 80% sequence homology; or (b) the amino acid sequence SEQ ID NO:84, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence SEQ ID NO:84 and/or the amino acid sequence of SEQ ID NO:82, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:82; and/or the amino acid sequence of SEQ ID NO:85, or comprising the amino acid sequence of SEQ ID NO:85 Vari
  • the bispecific antibody comprises: (a) the amino acid sequence of SEQ ID NO:99, or a sequence comprising a variant having at least 80% sequence homology to the amino acid sequence of SEQ ID NO:99; and/or Or the amino acid sequence of SEQ ID NO: 100, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 100; and/or the amino acid sequence of SEQ ID NO: 83, or comprising the amino acid sequence of SEQ ID NO: 83 NO:83 has a variant sequence with at least 80% sequence homology; or (b) the amino acid sequence of SEQ ID NO:101, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:101 and/or the amino acid sequence of SEQ ID NO:100, or a variant sequence comprising at least 80% sequence homology with the amino acid sequence of SEQ ID NO:100; and/or the amino acid sequence of SEQ ID NO:85, or comprising the amino acid sequence of SEQ ID NO:
  • the bispecific antibody comprises Fc, wherein Fc is selected from Fc of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • Fc comprises an Fc variant.
  • the Fc is glycosylated.
  • the Fc is deglycosylated.
  • the Fc is reduced-fucosylated or afucosylated.
  • the application provides a pharmaceutical composition comprising: (i) an antibody or an antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae and (ii) an antibody or antigen-binding fragment that specifically binds to the O1 antigen of Klebsiella pneumoniae Antigen-binding fragment; wherein the antibody or antigen-binding fragment specifically in conjunction with Klebsiella pneumoniae O2 antigen comprises: (a) heavy chain variable region (V H ), and said V H comprises: heavy chain complementarity determining region (HC- CDR)1, which comprises the amino acid sequence of SEQ ID NO:1; HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:4; and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and the light chain variable region ( V L ), said V L comprising: light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11; LC-CDR2 comprising: light
  • the application provides a method for treating and/or preventing a disease or disorder in an individual in need thereof, comprising administering to the individual an effective amount of (i) an antibody or antigen specifically binding to the Klebsiella pneumoniae O2 antigen A binding fragment and (ii) an antibody or an antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen; wherein an antibody or an antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen comprises: (a) a heavy chain variable region (V H ), said V H comprising: heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and HC- CDR3 comprising the amino acid sequence of SEQ ID NO:6; and a light chain variable region (V L ), said V L comprising: light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: (a) V H , and the V H comprises: HC-CDR1, which comprising the amino acid sequence of SEQ ID NO: 35; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37 ; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and V L comprising : LC- CDR1, which comprises the amino acid sequence of SEQ ID NO:41; LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:43; and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:45; or (b) V H , the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:36; HC-CDR2 comprising the amino acid sequence of SEQ ID NO:38; and HC-CDR3 comprising the amino acid sequence of SEQ ID
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH , and the VH comprises: HC-CDR1, which comprises an amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:7; and V L comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and specifically binding to the Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 35, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and HC-CDR3
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH , and the VH comprises: HC-CDR1, which comprises an amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:7; and V L comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and specifically binding to the Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 36, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and HC-CDR3
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH , and the VH comprises: HC-CDR1, which comprises an amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and V L comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 11, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and specifically binding to the Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 35, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and HC-CDR3
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH , and the VH comprises: HC-CDR1, which comprises an amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and V L comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 11, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and specifically binding to the Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 36, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and HC-CDR3
  • the application provides a pharmaceutical composition comprising: (i) an antibody or an antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae and (ii) an antibody or antigen-binding fragment that specifically binds to the O1 antigen of Klebsiella pneumoniae Antigen-binding fragment; wherein the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: (a) V H comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising the amino acid sequence of SEQ ID NO: 19 or 28 has a variant sequence of at least about 80% sequence homology; and VL , which comprises the amino acid sequence SEQ ID NO: 24 or 33, or comprises at least about 80% with the amino acid sequence SEQ ID NO: 24 or 33 Variant sequences of sequence homology; or V H , which comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in the amino acid sequence SEQ ID NO: 19 or
  • the application provides a method for treating and/or preventing a disease or a disease in an individual in need thereof, comprising administering to the individual an effective amount of (i) an antibody or an antigen specifically binding to the Klebsiella pneumoniae O2 antigen Binding fragments and (ii) antibodies or antigen-binding fragments that specifically bind to the Klebsiella pneumoniae O1 antigen; wherein the antibodies or antigen-binding fragments that specifically bind to the Klebsiella pneumoniae O2 antigen comprise: (a) V H , which comprises the amino acid sequence of SEQ ID NO: 19 or 28, or a variant sequence comprising at least about 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28; and V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or a variant sequence comprising at least about 80% sequence homology with the amino acid sequence of SEQ ID NO: 24 or 33; or V H comprising a V H comprising the amino acid sequence of SEQ ID NO
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: (a) V H comprising the amino acid sequence of SEQ ID NO: 47 or 51 , or comprising a variant sequence having at least about 80% sequence homology to the amino acid sequence SEQ ID NO:47 or 51; and V L comprising the amino acid sequence SEQ ID NO:49 or 53, or comprising the amino acid sequence SEQ ID NO: 49 or 53 has a variant sequence of at least about 80% sequence homology; or V H , which comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in the amino acid sequence SEQ ID NO: 47 or 51; and V L , which comprises LCDR1, LCDR2 and LCDR3 contained in V L as shown in amino acid sequence SEQ ID NO: 49 or 53; or (b) V H , which comprises amino acid sequence SEQ ID NO: 48 or 52, or comprises and amino acid A variant sequence having at least about 80% sequence homo
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH comprising the amino acid sequence of SEQ ID NO: 20 or 29, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 20 or 29; and a V L comprising the amino acid sequence of SEQ ID NO: 25 or 34, or comprising the amino acid sequence of SEQ ID NO: 25 or 34 variant sequences having at least 80% sequence homology; and an antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen comprising: VH comprising the amino acid sequence of SEQ ID NO: 47 or 51, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 47 or 51; and a V L comprising the amino acid sequence of SEQ ID NO: 49 or 53, or comprising the amino acid sequence of SEQ ID NO: 49 or 53 variant sequences with at least 80% sequence identity.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH comprising the amino acid sequence of SEQ ID NO: 20 or 29, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 20 or 29; and a V L comprising the amino acid sequence of SEQ ID NO: 25 or 34, or comprising the amino acid sequence of SEQ ID NO: 25 or 34 variant sequences having at least 80% sequence homology; and the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen comprising: VH comprising the amino acid sequence SEQ ID NO: 48 or 52, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 48 or 52; and a V L comprising the amino acid sequence of SEQ ID NO: 50 or 54, or comprising the amino acid sequence of SEQ ID NO: 50 or 54 variant sequences with at least 80% sequence identity.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28; and a V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or comprising the amino acid sequence of SEQ ID NO: 24 or 33 variant sequences having at least 80% sequence homology; and an antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen comprising: VH comprising the amino acid sequence of SEQ ID NO: 47 or 51, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 47 or 51; and a V L comprising the amino acid sequence of SEQ ID NO: 49 or 53, or comprising the amino acid sequence of SEQ ID NO: 49 or 53 variant sequences with at least 80% sequence identity.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28; and a V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or comprising the amino acid sequence of SEQ ID NO: 24 or 33 variant sequences having at least 80% sequence homology; and an antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen comprising: VH comprising the amino acid sequence SEQ ID NO: 48 or 52, or comprising A variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 48 or 52; and a V L comprising the amino acid sequence of SEQ ID NO: 50 or 54, or comprising the amino acid sequence of SEQ ID NO: 50 or 54 variant sequences with at least 80% sequence identity.
  • the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen and the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen are administered simultaneously. In some embodiments, the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen and the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen are administered sequentially.
  • a method for treating and/or preventing a disease or disorder in an individual in need thereof comprising administering to the individual an effective amount of any antibody or antigen-binding fragment described herein and/or any drug Compositions and/or bispecific antibodies.
  • the disease or condition comprises one or more symptoms caused by Klebsiella infection.
  • the Klebsiella is Klebsiella pneumoniae.
  • the symptoms include pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis, diarrhea, soft tissue infection, infection after organ transplantation, surgery Infection, wound infection, lung infection, suppurative liver abscess, lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spinal joint disease.
  • an isolated nucleic acid molecule encoding any of the bispecific antibodies described above is provided.
  • a vector comprising any of the nucleic acid molecules described above is provided.
  • a host cell comprising any bispecific antibody, any nucleic acid molecule or any vector as described above.
  • a method for preparing a bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen comprising: a) effectively expressing and specifically binding to Klebsiella pneumoniae O2 antigen and cultivating any one of the above-mentioned host cells under the condition of the bispecific antibody to the O1 antigen; and b) obtaining the expressed bispecific antibody in the host cell.
  • compositions, kits and articles of manufacture comprising any of the bispecific antibodies, nucleic acid molecules, vectors, or host cells described above.
  • Fig. 1 is a schematic diagram showing the structures of KP19173 strain LPS and KP19213 strain LPS of Klebsiella pneumoniae O1 serotype.
  • Fig. 2 is a schematic diagram showing the structure of Klebsiella pneumoniae O2 serotype KP19180 strain LPS and KP19203 strain LPS.
  • Figure 3A shows the ELISA binding results of antibodies K2, K3 or K5 that specifically bind to Klebsiella pneumoniae O2 antigen and KP19180 strain O2 type LPS
  • Figure 3B shows the antibody that specifically binds to Klebsiella pneumoniae O2 antigen The ELISA binding results of K2, K3 or K5 and KP19203 strain O2 type LPS
  • Figure 3C shows the ELISA binding results of antibodies K2, K3 or K5 that specifically bind to Klebsiella pneumoniae O2 antigen and KP19173 strain O1 type LPS
  • 3D shows the ELISA binding result of the antibody K2, K3 or K5 specifically binding to the O2 antigen of Klebsiella pneumoniae and the O1 type LPS of the KP19213 strain.
  • Figure 4A is the ELISA binding result of the antibody specifically binding to Klebsiella pneumoniae O2 antigen and the KP19002O2 type LPS of Klebsiella pneumoniae
  • Figure 4B is the binding result of the antibody specifically binding to Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae strain The ELISA binding result of KP19003O2 type LPS
  • Figure 4C is the ELISA binding result of the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen and the KP19005O2 type LPS of the Klebsiella pneumoniae strain
  • Figure 4D is the specific binding to the Klebsiella pneumoniae O2 antigen
  • Figure 7A shows the schematic structure of DVD-Ig (Dual-variable domain-Ig) bispecific antibody
  • Figure 7B shows the schematic structure of Bs4Ab bispecific antibody
  • Figure 7C shows the structure of Hetero H, CrossMab bispecific antibody Schematic diagram
  • Figure 7D shows a schematic diagram of the structure of an IgG-(scFv) 2 bispecific antibody
  • Figure 7E shows a schematic diagram of the structure of a scFv-Fab IgG bispecific antibody.
  • FIG. 8A-8C show the ELISA binding results of exemplary bispecific antibodies with DVD-Ig structure and different serotypes of Klebsiella pneumoniae LPS.
  • Fig. 8A is the binding result of O1 type LPS of KP19173 strain;
  • Fig. 8B is the binding result of KP19213 strain O1 type LPS;
  • Fig. 8C is the binding result of KP19180 strain O2 type LPS.
  • Figures 8D-8F show the ELISA binding results of exemplary bispecific antibodies with Bs4Ab structure and different serotypes of Klebsiella pneumoniae LPS.
  • Fig. 8D is the binding result of O1 type LPS of KP19173 strain;
  • Fig. 8E is the binding result of KP19213 strain O1 type LPS;
  • Fig. 8F is the binding result of KP19180 strain O2 type LPS.
  • Figure 8G- Figure 8I show the ELISA binding results of exemplary bispecific antibodies of Hetero H and CrossMab structures to LPS of Klebsiella pneumoniae strains of different serotypes, respectively.
  • Fig. 8G is the binding result of O1 type LPS of KP19173 strain;
  • Fig. 8H is the binding result of KP19213 strain O1 type LPS;
  • Fig. 8I is the binding result of KP19180 strain O2 type LPS.
  • Figure 8J- Figure 8L show the ELISA binding results of exemplary bispecific antibodies with IgG-(scFv) 2 structure and different serotypes of Klebsiella pneumoniae LPS respectively.
  • Fig. 8J is the binding result of O1 type LPS of KP19173 strain;
  • Fig. 8K is the binding result of KP19213 strain O1 type LPS;
  • Fig. 8L is the binding result of KP19180 strain O2 type LPS.
  • Figure 8M- Figure 8O show the ELISA binding results of exemplary bispecific antibodies with scFv-Fab IgG structure and different serotypes of Klebsiella pneumoniae LPS respectively.
  • Fig. 8M is the binding result of O1 type LPS of KP19173 strain;
  • Fig. 8N is the binding result of KP19213 strain O1 type LPS;
  • Fig. 8O is the binding result of KP19180 strain O2 type LPS.
  • Figures 9A-9C show the neutralizing activity results of exemplary bispecific antibodies of DVD-Ig structure against different serotypes of Klebsiella pneumoniae LPS.
  • Fig. 9A is the neutralizing activity of KP19173 strain O1 type LPS;
  • Fig. 9B is the result of neutralizing activity of KP19213 strain O1 type LPS;
  • Fig. 9C is the result of neutralizing activity of KP19180 strain O2 type LPS.
  • FIGD-9F show the neutralizing activity results of exemplary bispecific antibodies with Bs4Ab structure against different serotypes of Klebsiella pneumoniae strain LPS.
  • Figure 9D is the result of neutralizing activity against KP19173 strain O1 type LPS;
  • Figure 9E is the result of neutralizing activity against KP19213 strain O1 type LPS;
  • Figure 9F is the result of neutralizing activity against KP19180 strain O2 type LPS.
  • Figure 9G- Figure 9I show the results of neutralizing activity of exemplary bispecific antibodies of Hetero H and CrossMab structures to different serotypes of Klebsiella pneumoniae strain LPS, respectively.
  • Figure 9G is the result of neutralizing activity against KP19173 strain O1 type LPS;
  • Figure 9H is the result of neutralizing activity against KP19213 strain O1 type LPS;
  • Figure 9I is the result of neutralizing activity against KP19180 strain O2 type LPS.
  • Figure 9J- Figure 9L show the neutralizing activity results of exemplary bispecific antibodies of IgG-(scFv) 2 structure to different serotypes of Klebsiella pneumoniae strain LPS respectively.
  • Figure 9J is the result of neutralizing activity against KP19173 strain O1 type LPS;
  • Figure 9K is the result of neutralizing activity against KP19213 strain O1 type LPS;
  • Figure 9L is the result of neutralizing activity against KP19180 strain O2 type LPS.
  • Figure 9M- Figure 9O show the neutralizing activity results of exemplary bispecific antibodies of scFv-Fab IgG structure to different serotypes of Klebsiella pneumoniae strain LPS respectively.
  • Figure 9M is the result of neutralizing activity against KP19173 strain O1 type LPS;
  • Figure 9N is the result of neutralizing activity against KP19213 strain O1 type LPS;
  • Figure 9O is the result of neutralizing activity against KP19180 strain O2 type LPS.
  • Figure 10A shows the results of the promotion of opsonophagocytosis and bactericidal activity of the bispecific antibody K5-G2-Ig1 in the form of DVD-Ig against the KP19173 strain
  • Figure 10B shows the results of the bispecific antibody K5-G7-Ig1 in the form of DVD-Ig against The results of the opsonophagocytosis and bactericidal activity of the KP19173 strain
  • Figure 10C shows the results of the opsonophagocytosis and bactericidal activity of the bispecific antibody K5-G2scFv-Ig1 in the form of Bs4Ab against the KP19173 strain
  • Figure 10D shows the results of the bispecific antibody in the form of Bs4Ab Results of the opsonophagocytosis-promoting bactericidal activity of K5-G7scFv-Ig1 against KP19173 strain.
  • Figure 11A shows the results of the serum bactericidal activity of the bispecific antibody K5-G2-Ig1 in the form of DVD-Ig against the KP19173 bacterial strain
  • Figure 11B shows the results of the bispecific antibody K5-G7-Ig1 in the form of DVD-Ig against the KP19173 bacterial strain
  • Figure 11C shows the results of serum bactericidal activity of the bispecific antibody K5-G2scFv-Ig1 in the form of Bs4Ab against the KP19173 strain
  • Figure 11D shows the results of the bispecific antibody K5-G7scFv-Ig1 in the form of Bs4Ab against Serum bactericidal activity results of the KP19173 strain.
  • Figure 12A shows the therapeutic effect of the bispecific antibody K5-G2-Ig1 on pneumonia caused by Klebsiella pneumoniae KP19180 infection in the mouse pneumonia treatment model
  • Figure 12B shows the bispecific antibody K5-G2- Therapeutic effect of Ig1 on pneumonia caused by Klebsiella pneumoniae KP19173 infection in mice.
  • the present application provides an antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen, and on the other hand provides a bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen.
  • One aspect provides a composition comprising an antibody or antigen-binding fragment that specifically binds an O2 antigen of Klebsiella pneumoniae and an antibody or antigen-binding fragment that specifically binds an O1 antigen of Klebsiella pneumoniae.
  • nucleic acid encoding an antibody or an antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen, or a bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen, comprising The composition of the antibody of the primary bacteria O2 antigen and the antibody specifically binding to the Klebsiella pneumoniae O1 antigen, and the preparation and use of the antibody specifically binding to the Klebsiella pneumoniae O2 antigen or specifically binding to the Klebsiella pneumoniae O1 antigen Antibodies or bispecific antibodies and methods comprising compositions thereof.
  • O antigen refers to one of the components of Klebsiella LPS, which together with lipid A and core oligosaccharides constitute LPS.
  • the "O1 antigen” refers to the O antigen part of the O1 serotype Klebsiella LPS
  • O2 antigen refers to the O antigen part of the O2 serotype Klebsiella LPS
  • the O antigen comprises D-galactan-I disaccharide units.
  • treatment is a method of obtaining a beneficial or desired result, including a clinical result.
  • the beneficial or desired clinical results include, but are not limited to, one or more of the following: Alleviation of one or more symptoms caused by the disease, reduction of the degree of the disease, stabilization of the disease (for example, prevention or delay the progression of the disease), prevent or delay the spread of the disease (e.g., systemic spread of pathogenic bacteria), prevent or delay the recurrence of the disease, delay or slow the progression of the disease, improve the disease state, alleviate the disease (partially or completely), reduce the need for treatment of the disease dose of one or more other drugs, delay disease progression, improve or enhance quality of life, increase body weight, and/or prolong survival.
  • treatment also includes the reduction of the pathological consequences of infection (eg, host cell lysis or necrosis). The methods of the present application contemplate any one or more aspects of these treatments.
  • prevent and similar words, such as “prevented”, “preventing”, “prevention” or “prophylactic” mean a condition that prevents, inhibits or reduces A measure of the likelihood of occurrence or recurrence of a disease or condition, such as a pathogenic infection. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, “prevention” and like words also include lessening the intensity, effects, symptoms and/or burden of a disease or condition before its onset or recurrence. As used herein, “prevention” and like words also include reducing the risk and susceptibility to occurrence or recurrence of a disease or condition, such as infection by a pathogen.
  • Antibody or antigen-binding fragment As described in this application, the term “antibody” is broad and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding.
  • variable regions in the two chains usually include three hypervariable loops, called complementarity determining regions (CDRs), the light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, the heavy chain (HC ) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3).
  • CDRs complementarity determining regions
  • the light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3
  • the heavy chain (HC ) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3
  • the CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold to support the hypervariable loops.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are classified based on the amino acid sequence of the constant region of their heavy chains.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, characterized by heavy chains of the alpha, delta, epsilon, gamma and mu types, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain n
  • IgA2 ⁇ 2 heavy chain
  • the term "antigen-binding fragment” includes an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide bond stabilized Fv Fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody (scFv), scFv dimer (bivalent Diabodies), multispecific antibodies consisting of antibody fragments comprising one or more CDRs, single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody capable of binding to an antigen but not comprising a complete antibody structure Antibody fragments.
  • the Fab fragment antigen-binding
  • Antigen-binding fragments also include fusion proteins comprising the antibody fragments described above.
  • An antigen-binding fragment is capable of binding the same antigen as a parent antibody or a fragment of a parent antibody (eg, a parent scFv).
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted into framework regions from one or more different human antibodies.
  • bispecific antibody refers to binding specificities for two different antigens or epitopes in one antibody molecule.
  • the production process of bispecific antibodies includes the design of the complete molecule, the synthesis and cloning of the nucleotide sequence of each domain, the expression in mammalian cells and the purification of the final product.
  • Exemplary bispecific antibody structures include structures known in the art, for example, DVD-Ig structure, Bs4Ab structure, Hetero H, CrossMab structure, IgG-(scFv) 2 structure or scFv-Fab IgG structure, etc. (e.g., See review literature Labrijn AF, et al. Nat Rev Drug Discov. 2019 Aug; 18(8):585-608).
  • DVD-Ig (“Dual-variable domain-Ig) bispecific antibody, its structure is that the N-terminus of the light chain and heavy chain of a normal IgG antibody is connected to the VL and VH domains of another antibody respectively, through which two antibodies The VH and VL interact to form an antigen-binding domain (Fv), which can simultaneously bind to the corresponding antigen to achieve bispecificity.
  • the bispecific antibody of the exemplary DVD-Ig structure is in the literature Wu C, et al. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules.MAbs.2009Jul-Aug; 1(4):339-47 as well.
  • the Bs4Ab structure bispecific antibody is a bispecific 4-valent antibody, which includes a full-length IgG1 structure, and achieves bispecificity by inserting another binding unit scFv in its hinge region.
  • the bispecific antibody about the structure of Bs4Ab is in the literature Bezabeh B, et al.Insertion of scFv into the hinge domain of full-length IgG1monoclonal antibody results in tetravalent bispecific molecule with robust properties.MAbs.2017Feb/Mar;9(2):240 It is also documented in -256.
  • CrossMab structure bispecific antibody that is, a knobs-in-holes (KIH) structure (knobs-in-holes, KIH) was designed in the Fc region, and two Cys residue mutations (S354C on the "knob” side) were introduced to form a stabilized disulfide bridge , and Y349C is on the "hole” side).
  • KIH knobs-in-holes
  • S354C Cys residue mutations
  • CrossMab technology is based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, which can be the exchange of the complete Fab domain (CrossMAb Fab), or the exchange of only the variable region in the Fab domain (CrossMAb V H -V L ) or exchange of only the constant region (CrossMAb CH 1- CL ).
  • the bispecific antibody about Hetero H, CrossMab structure is also in the document Klein C, et al.The use of CrossMAb technology for the generation of bi-and multispecific antibodies.MAbs.2016Aug-Sep;8(6):1010-20 recorded.
  • IgG-(scFv) 2 structure bispecific antibody, that is, the scFv fragment of another antibody is connected to the Fc ends of the two heavy chains of an IgG antibody to achieve bispecificity.
  • Bispecific antibodies with IgG-(scFv) 2 structure are also described in Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2): 159-63.
  • the scFv-Fab IgG bispecific antibody is a heterodimeric antibody with an IgG antibody structure, in which one Fab arm is replaced with a scFv structure, in which the first monomer contains scFv and IgG Fc, and the scFv is linked to IgG FcC through a linker peptide N-terminal to the H2 domain, the second monomer contains Fab and IgG Fc.
  • the term "antigen-binding domain” refers to the portion of an antigen-binding molecule that specifically binds to an antigen. More specifically, the term “antigen-binding domain” refers to a portion of an antibody that includes a region that specifically binds and is complementary to a portion or all of an antigen. In the case of large antigens, the antigen-binding molecule may only bind to a specific part of the antigen, called an epitope.
  • an antigen binding domain may be provided by one or more variable regions (also referred to as variable domains).
  • the antigen binding domain comprises an antibody light chain variable region (V L ) and an antibody heavy chain variable region (V H ).
  • an antigen binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.
  • Antigen-binding domains that specifically bind Klebsiella pneumoniae O2 antigen or O1 antigen include antibodies and antigen-binding fragments further defined in this application.
  • epitope refers to a specific atom or group of amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope on an antigen if they appear to compete for binding to an antigen.
  • the primary antibody when the first antibody inhibits the binding of the second antibody to the Klebsiella pneumoniae O2 antigen target by at least 50% (such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), the primary antibody "competes" with the secondary antibody for binding to the Klebsiella pneumoniae O2 antigen target, and vice versa.
  • PCT publication WO 03/48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.
  • the term “specifically binds”, “specifically recognizes” or “is specific for” refers to a measurable and reproducible interaction, for example binding of a target to an antibody can The presence of the target is determined in a heterogeneous population of molecules, including biomolecules.
  • the ability of an antibody to specifically recognize a certain target means that the antibody binds to the target with higher affinity, avidity, easier and/or longer duration than other targets.
  • an antibody that specifically recognizes an antigen reacts with one or more epitopes of the antigen with a binding affinity at least 10 times greater than its binding affinity for other targets.
  • an "isolated" antibody is one that is (1) not related to a naturally occurring protein, (2) free of other proteins of the same origin, (3) produced from a cell of a different species Expressed, or (4) does not exist in nature.
  • isolated nucleic acid refers to nucleic acid of genomic, cDNA or synthetic origin or combinations thereof. Depending on its source, said "isolated nucleic acid” (1) is not related to all or part of the polynucleotide found in nature in the “isolated nucleic acid", (2) may be related to a polynucleotide to which it is not naturally associated is operably linked, or (3) does not occur in nature as part of a longer sequence.
  • CDR complementarity determining region
  • chimeric antibody means that a part of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, and the (s) chain The remaining part is identical or homologous to the corresponding sequence in antibodies from another genus or belonging to other antibody classes or subclasses, and fragments of such antibodies, as long as they have biological activity in this application (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • Fv is the smallest antibody fragment that contains the complete antigen recognition and binding site. This fragment is a dimer formed by the tight non-covalent linkage of one heavy chain variable domain and one light chain variable domain. Six hypervariable loops (3 loops each in the light and heavy chains) are derived from the folding of these two domains, which provide the antibody with amino acid residues for binding to the antigen and endow the antibody with the antigen binding specificity. However, even a single variable domain (or half of an Fv fragment, which contains only the 3 CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the full binding site.
  • Single-chain Fv also abbreviated “sFv” or “scFv”
  • sFv is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain.
  • the scFv polypeptide further includes a linking polypeptide between the VH and VL domains , which allows the scFv to form a desired structure for antigen binding.
  • diabodies refers to a small antibody fragment prepared by constructing a scFv fragment (see the above paragraph) using a short linker (for example, 5-10 residues) between V H and V L , This allows for interchain rather than intrachain pairing of the variable regions, resulting in a bivalent fragment, ie, a fragment with two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VH and VL domains of the two antibodies are located on different polypeptide chains.
  • Diabodies are fully described in EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • Humanized forms of non-human (eg, rodent) antibodies are chimeric antibodies, which include minimal sequence derived from the non-human antibody.
  • humanized antibodies are human immunoglobulins (recipient antibodies) in which the hypervariable region (HVR) residues of the recipient antibody are derived from non-human species such as mouse, rat, rabbit or Substitution of non-human primate hypervariable region residues with desired antibody specificity, affinity and performance (donor antibody).
  • HVR hypervariable region residues of the recipient antibody
  • donor antibody residues in the framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can include residues that are found neither in the recipient antibody nor in the donor antibody. These modifications can further improve antibody performance.
  • a humanized antibody will comprise substantially at least one, and usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin.
  • the framework regions are human immunoglobulin sequences.
  • the human antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • amino acid sequence homology percentage (%) or “homology” of the polypeptide and antibody sequences identified in the present application is defined as a sequence comparison where conservative substitutions are considered to be part of the sequence homology, The percentage of identical amino acid residues in the candidate sequence and the polypeptide sequence to be compared. Percent amino acid sequence identity can be determined by various alignment methods that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • amino acid sequence identity percent values are calculated using the sequence alignment computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5( 1): 113, 2004) generated.
  • Fc (fragment crystallizable)" or “Fc region” refers to a polypeptide comprising an entire antibody constant region, excluding the CH 1 domain, and in some cases comprising a partial hinge, whether in monomeric or multimeric form .
  • the original immunoglobulin source of native Fc is preferably of human origin, and may be any immunoglobulin, for example, IgG1, IgG2, IgG3 or IgG4.
  • Native Fcs are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (ie, disulfide bonds) and non-covalent associations.
  • the Fc region of an immunoglobulin typically comprises the CH2 and CH3 domains of the heavy chain constant region, and optionally the CH4 domain.
  • each of the two Fc monomers in the Fc dimer comprises an amino acid substitution that promotes heterodimerization of the two monomers.
  • heterodimerization of Fc monomers can be facilitated by introducing different but compatible substitutions such as "knob-into-hole” residue pairs in the two Fc monomers.
  • the "knob-into-hole” technique is also disclosed in US Patent Publication No. 8,216,805.
  • one Fc monomer comprises the knob mutation T366W and the other Fc monomer comprises the hole mutations T366S, L358A and Y407V.
  • two Cys residues (S354C on the "knob” side and Y349C on the "hole” side) that form a stabilizing disulfide bridge are introduced.
  • Fc receptor or "FcR” is used to describe a receptor that binds the Fc region of an antibody.
  • the FcR described herein is an FcR that binds an IgG antibody (a gamma receptor), including receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants of these receptors and available into spliced form.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (activating receptor) and Fc ⁇ RIIB (inhibiting receptor), which have similar amino acid sequences and differ mainly in the cytoplasmic domain.
  • the cytoplasmic domain of the activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine activation motif (ITAM).
  • the cytoplasmic domain of the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997)).
  • ITAM immunoreceptor tyrosine activation motif
  • ITIM immunoreceptor tyrosine inhibition motif
  • FcR in this application encompasses other types of FcRs, including FcRs identified in the future.
  • FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the neonate (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994 )).
  • FcRn refers to the neonatal Fc receptor (FcRn).
  • FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an alpha chain non-covalently bound to beta2 microglobulin.
  • MHC major histocompatibility complex
  • FcRn plays an important role in the passive transport of immunoglobulin IgGs from mother to neonate and in the regulation of serum IgG levels.
  • FcRn acts as a salvage receptor that binds and transports endocytosed IgG in intact form within and between cells and saves them from undergoing the default degradation pathway.
  • CHI domain of the human IgG heavy chain constant region typically extends from amino acid 118 to amino acid 215 (EU numbering system).
  • a "hinge region” is generally defined as extending from Glu 216 to Pro 230 of human IgGl (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG subtypes can be aligned with the IgGl sequence by placing the first and last cysteine residues that form inter-heavy chain disulfide bonds in the same position as IgGl.
  • the " CH2 domain" of a human IgG Fc region generally extends from amino acid 231 to amino acid 340.
  • the CH2 domain is unique in that it does not pair tightly with another region. Instead, two N-terminally linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It has been speculated that sugars may act as a surrogate for domain-to-domain pairing, helping to keep the CH2 domain stable. Burton, Molec Immunol. 22:161-206 (1985).
  • the " CH3 " domain encompasses the extension within the Fc region from the C-terminal residue to the CH2 domain (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 of IgG).
  • a “functional Fc fragment” has the "effector function” that a native Fc region sequence has.
  • exemplary “effector functions” include Clq binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; Such as B cell receptor; BCR) and so on.
  • effector functions typically require binding of the Fc region to a binding domain (eg, antibody variable region) and can be assessed using a variety of assays well known in the art.
  • Antibodies of IgG Fc variants having "altered" FcR binding affinity or ADCC activity have increased or decreased FcR binding activity and/or ADCC activity compared to a parent polypeptide or a polypeptide comprising a native Fc sequence.
  • An Fc variant exhibiting "enhanced binding" to an FcR has a higher binding affinity (e.g., lower apparent Kd or IC50 value) for at least one FcR than the parental polypeptide or a polypeptide comprising a native IgG Fc sequence .
  • the binding ability is increased by 3 times, such as 5, 10, 25, 50, 60, 100, 150, 200, even up to 500 times or the binding ability is increased by 25% to 1000% compared with the parental polypeptide.
  • An Fc variant that exhibits "reduced binding" to an FcR has a lower affinity for at least one FcR (eg, a higher apparent Kd or IC50 value) than a parental polypeptide.
  • the binding ability is reduced by 40% or more compared to the parental polypeptide.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as natural killer cells (NK), neutrophils, and Binding to Fc receptors (FcRs) on macrophages) enables these cytotoxic effector cells to specifically bind antigen-bearing target cells and subsequently kill the target cells using cytotoxins.
  • Antibodies "arm” the cytotoxic cells and are required for this killing.
  • NK cells only express Fc ⁇ RIII, while monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • an in vitro ADCC assay can be performed, as described in US Patent No. 5,500,362 or 5,821,337. Suitable effector cells for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK).
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • the ADCC activity of a molecule of interest can also be assessed in vivo, for example as described in animal models as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
  • the polypeptide is substantially the same amount as the wild-type IgG Fc polypeptide (or parental polypeptide) in the experiment, it can mediate ADCC more effectively in vitro or in vivo.
  • Such variants are typically identified using any in vitro ADCC assay known in the art, eg, assays or methods for identifying ADCC activity, eg, in animal models and the like. In some embodiments, such variants mediate ADCC 5 to 100 fold more efficiently, eg 25 to 50 fold, compared to wild type Fc (or parental polypeptide).
  • “Complement-dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (subclasses of appropriate structure) that bind cognate antigens.
  • Clq first component of the complement system
  • a CDC assay can be performed as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
  • Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in US Patent No. 6,194,551B1 and WO99/51642. The contents of these patent publications are expressly incorporated by reference into this application. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are mutually degenerate forms and encode the same amino acid sequence.
  • a nucleotide sequence encoding a protein or RNA may also include introns, eg, a nucleotide sequence encoding a protein may, in some forms, include introns.
  • operably linked refers to a functional linkage between a regulatory sequence and a heterologous nucleotide sequence such that the latter is expressed.
  • a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, when necessary, join two protein coding regions in the same reading frame.
  • “Homologous” refers to sequence similarity or sequence homology between two polypeptides or between two nucleic acid molecules. If the same position in the two compared sequences is the same base or amino acid monomer subunit, for example, the same position in the two DNA molecules is adenine, then the two DNA molecules are homologous at this position.
  • the percent homology between two sequences is a function of the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the homology of the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally speaking, when comparing two sequences, the comparison is performed with the aim of obtaining the maximum homology.
  • an “effective amount” of an antibody (including bispecific antibody) or composition disclosed in the present application refers to an amount sufficient to achieve a specific purpose.
  • An “effective amount” can be determined empirically and by methods known in relation to the purpose.
  • a therapeutically effective amount refers to the amount of an antibody (including bispecific antibody) or composition described herein effective to "treat" a disease or condition in an individual.
  • a therapeutically effective amount of an antibody or composition disclosed herein can reduce the number of infected cells, inhibit (i.e. slow and preferably stop to some extent) the spread of the infection, and/or To relieve to some extent one or more symptoms associated with the infection.
  • the antibodies or compositions disclosed herein can inhibit the growth of Klebsiella pneumoniae and/or kill Klebsiella pneumoniae, and the antibodies can be cytostatic and/or cytotoxic.
  • a therapeutically effective amount is an amount that inhibits infection in a patient.
  • a therapeutically effective amount is an amount that completely clears the infection in the patient.
  • pharmaceutically acceptable refers to a material that has no biological activity or other undesired properties, for example, the material can be added to a pharmaceutical composition administered to a patient, without causing significant adverse biological reactions, or interacting in a deleterious manner with any other components contained in the composition.
  • Pharmaceutically acceptable carriers or excipients preferably meet the required criteria for toxicology or manufacturing testing and/or are included in the inactive ingredient guidelines prepared by the US Food and Drug Administration.
  • Embodiments of the present application described in this application should be understood to include “consisting of” and/or “consisting essentially of” embodiments.
  • reference to "not" a value or parameter generally means and describes "other than” a value or parameter.
  • the method cannot be used to treat type X infections, meaning that the method is generally used to treat other types than type X infections.
  • Antibody that specifically binds Klebsiella pneumoniae O2 antigen
  • the application provides antibodies or antigen-binding fragments that specifically bind to the Klebsiella pneumoniae O2 antigen, including, but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and the present application Antibody molecules comprising heavy and/or light chain CDRs as described above.
  • the antibody or antigen-binding fragment is an isolated antibody that binds the O2 antigen.
  • Expected antibodies or antigen-binding fragments that specifically bind to the Klebsiella pneumoniae O2 antigen include all or fragments of full-length antibodies (such as full-length IgG1, IgG2 or IgG4) that specifically bind to the Klebsiella pneumoniae O2 antigen, Single-chain antibodies that specifically bind to Klebsiella pneumoniae O2 antigen, multispecific (such as bispecific) antibodies that bind to Klebsiella pneumoniae O2 antigen, immunoconjugates that specifically bind to Klebsiella pneumoniae O2 antigen, and whatnot.
  • full-length antibodies such as full-length IgG1, IgG2 or IgG4
  • Single-chain antibodies that specifically bind to Klebsiella pneumoniae O2 antigen Single-chain antibodies that specifically bind to Klebsiella pneumoniae O2 antigen
  • multispecific (such as bispecific) antibodies that bind to Klebsiella pneumoniae O2 antigen immunoconjugates that specifically bind to Klebsi
  • the antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen is Fab, Fab', F(ab)'2, Fab'-SH, single chain antibody (scFv), Fv fragment, dAb, Fd, Nanobody or diabody.
  • an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen refers to an antibody or antigen-binding fragment that binds to the Klebsiella pneumoniae O2 antigen with an affinity at least 10 times greater than the binding affinity to a non-target above (including, for example, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 times).
  • non-target refers to an antigen that is not the Klebsiella pneumoniae O2 antigen.
  • Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation analysis (RIA).
  • Kd values can be determined by methods known in the art, such as surface plasmon resonance (SPR) techniques or biolayer interferometry (BLI) techniques.
  • the antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen can (a) promote, mediate or enhance the opsonophagocytosis (OPK) of Klebsiella pneumoniae, and/or ( b) promoting, mediating or enhancing complement-mediated serum bactericidal activity (SBA) against Klebsiella pneumoniae.
  • OPK opsonophagocytosis
  • SBA complement-mediated serum bactericidal activity
  • the non-human antibody comprises the human CDR sequence and the non-human framework region sequence of the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen described herein, and in some embodiments, the non-human Framework region sequences include any sequence useful for generating heavy and/or light chain variable regions using one or more human CDR sequences as described herein, including for example mammals such as mouse, rat, rabbit , pigs, cattle (eg, cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (eg, marmosets, macaques), and the like.
  • mammals such as mouse, rat, rabbit , pigs, cattle (eg, cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (eg, marmosets, macaques), and the like.
  • the non-human antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen comprises grafting one or more of the human CDR sequences described herein into a non-human framework region (e.g., mouse or chicken framework region sequence) to specifically bind to the Klebsiella pneumoniae O2 antigen antibody or antigen-binding fragment.
  • a non-human framework region e.g., mouse or chicken framework region sequence
  • the antibody or antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae binds to the I domain of D-galactan in LPS of Klebsiella pneumoniae.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen is specific to the Klebsiella pneumoniae O2 antigen and has no species cross-reactivity or is not compatible with other types of non-pneumonia Klebsiella O2 antigen cross-reacts.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen cross-reacts with other types of non-Klebsiella pneumoniae O2 antigens.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: (i) V H , which comprises the HC-CDR1 contained in the V H as shown in the amino acid sequence of SEQ ID NO: 17 , HC-CDR2 and HC-CDR3; and V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in V L as shown in the amino acid sequence SEQ ID NO: 21; or (ii) V H comprising HC-CDR1, HC-CDR2 and HC-CDR3 comprising V H as shown in amino acid sequence SEQ ID NO:18; and V L comprising LC contained in V L as shown in amino acid sequence SEQ ID NO:22 -CDR1, LC-CDR2 and LC-CDR3; or (iii) VH , which comprises HC-CDR1, HC-CDR2 and HC-CDR3 comprised by VH as shown in the amino acid sequence SEQ ID NO: 19; and
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: VH , the VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant of said VH comprising up to about 5 amino acid substitutions in its HC-CDRs; and VL , the VL comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, Or a variant of said VL comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: V H , and the V H comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprising the amino acid sequence of SEQ ID NO: 3, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or variants of said VH comprising up to about 5 amino acid substitutions in the HC-CDRs; and VL , the VL comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, Or a variant of said VL comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: V H , and the V H comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant of said VH comprising up to about 5 amino acid substitutions in its HC-CDRs; and VL , the VL comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, Or a variant of said VL comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: VH , the VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant of said VH comprising up to about 5 amino acid substitutions in its HC-CDRs; and VL , the VL comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, Or a variant of said VL comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: VH , the VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or variants of said VH comprising up to about 5 amino acid substitutions in its HC-CDRs; and VL , the VL comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, Or a variant of said VL comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof, which is identical to the amino acid sequence of SEQ ID NO: 17 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology; and a VL comprising the amino acid sequence SEQ ID NO: 21 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of SEQ ID NO: 21 sequence homology.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof, which is identical to the amino acid sequence of SEQ ID NO: 18 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and V L comprising the amino acid sequence SEQ ID NO: 22 or a variant thereof having at least about 80% (eg at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of SEQ ID NO: 22 sequence homology.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof, which is identical to the amino acid sequence of SEQ ID NO: 19 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology; and a VL comprising the amino acid sequence SEQ ID NO: 23 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of SEQ ID NO: 23 sequence homology.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof, which is identical to the amino acid sequence of SEQ ID NO: 19 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology; and a VL comprising the amino acid sequence SEQ ID NO: 24 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of SEQ ID NO: 24 sequence homology.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof, which is identical to the amino acid sequence of SEQ ID NO: 20 has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology; and a VL comprising the amino acid sequence SEQ ID NO: 25 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of SEQ ID NO: 25 sequence homology.
  • amino acid substitutions described above are limited to the "exemplary substitutions” shown in Table 10 of the present application. In some embodiments, amino acid substitutions are limited to the "preferred substitutions” shown in Table 10 of this application.
  • the present application provides an antibody that can competitively bind to Klebsiella pneumoniae having a D-galactan I domain with any of the antibodies that specifically bind to the O2 antigen of Klebsiella pneumoniae Antibody. In some embodiments, the present application provides an antibody that competitively binds to the same epitope as any of the above-mentioned antibodies that specifically bind to the O2 antigen of Klebsiella pneumoniae.
  • competition experiments can be used to identify antibodies that specifically bind to Klebsiella pneumoniae O2 antigens described herein that compete with Klebsiella pneumoniae having a D-galactan I domain. Monoclonal antibodies. Competition experiments can determine whether two antibodies bind to the same epitope by recognizing identical or spatially overlapping epitopes or by one antibody competitively inhibiting binding of the other antibody to the antigen. In certain embodiments, such competing antibodies bind to the same epitope as an antibody described herein. Some exemplary competition assays include, but are not limited to, conventional assays as mentioned in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • each antibody is said to bind the same epitope if it blocks 50% or more of the binding of the other antibody.
  • the antibody that competes with the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen described herein is a chimeric antibody, a humanized antibody, or a fully human antibody.
  • Exemplary sequences of antibodies specifically binding to the O2 antigen of Klebsiella pneumoniae are shown in Table 2, wherein the CDRs are numbered according to the Kabat definition. Those skilled in the art will recognize that there are a number of known algorithms (Kabat's definition) to predict the location of CDRs and define antibody light and heavy chain variable regions. Also within the scope of this application are antibodies comprising the CDRs, VH and/or VL sequences of the antibodies described herein, but based on prediction algorithms other than those exemplified in the table below.
  • Klebsiella infections are associated with hospitalization. As opportunistic pathogens, Klebsiella primarily attack immunocompromised individuals with severe underlying diseases such as diabetes or chronic pulmonary obstruction. With the passage of time and the continuous development of taxonomy, the classification of Klebsiella has been continuously revised, and three main classifications have emerged: Cowan, Bascomb and in Classify Klebsiella into 5 categories, including Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella terrestris, Klebsiella phytogenes and Klebsiella ornithinolyticus, and Klebsiella pneumoniae Also included are Klebsiella stinkynasopneumoniae and Klebsiella rhinosclerosis subsp.
  • Klebsiella pneumoniae (Podschun, R, and U Ullmann. Clinical microbiology reviews vol. 11, 4(1998): 589-603.).
  • Klebsiella granulomatosis etc. appearing according to other classification/nomenclature are also within the scope of consideration of this application.
  • Klebsiella pneumoniae is the species that causes the most infections and is the most important species in the genus Klebsiella. Klebsiella pneumoniae can cause sepsis, pneumonia, urinary tract infection, rickets, etc. (Podschun, R, and U Ullmann. Clinical microbiology reviews vol. 11, 4(1998): 589-603.).
  • LPS Lipopolysaccharide
  • O antigen or O polysaccharide
  • RU oligosaccharide repeating units
  • O1 and O2 serotype strains contains O antigen composed of galactose (galactans, gal) homopolymer.
  • O1 serotype expresses D-galactan-I (D-gal-I) consisting of ⁇ 3)- ⁇ -D-Galf-(1 ⁇ 3)- ⁇ -D-Galp-(1 ⁇ as a repeating unit D-galactan-II (D-gal-II )(Whitfield, C et al.Journal of bacteriology vol.173,4(1991):1420-31.; Kol, O et al.Carbohydrate research vol.236(1992):339-44.).
  • D-gal- II is a structure specific to O1-type LPS (Pennini, Principle E et al. Nature communications vol.8,1 1991.8Dec.2017).
  • O2-type LPS consists only of D-gal-I (Whitfield, C et al .Journal of bacteriology vol.174,15(1992):4913-9.).
  • the synthesis of D-gal-I is encoded by his linked rfb (wb) operon (Clarke, B R, and C Whitfield. Journal of bacteriology vol.174,14(1992):4614-21.; Kelly, R F, and C Whitfield.
  • the O1 serotype strain carries a non-linked locus (wbbYZ) responsible for the synthesis of D-gal-II (Hsieh, Pei-Fang et al. Frontiers in microbiology vol. 5 608.19 Nov. 2014).
  • D-gal-I of the O2 serotype can be modified by stoichiometric or non-stoichiometric addition of O-acetyl or terminal D-galactose (Kelly et al., 1995).
  • D-gal-II can combine with D-gal-I or D-gal-III homopolymers, resulting in the presence of both in the O1 serotype.
  • the O2 serotype consists only of D-gal-I or D-gal-III homopolymers (Stojkovic, Katarina et al. Frontiers in microbiology vol.8 684.25Apr.2017).
  • the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen is a full-length antibody.
  • the full-length antibody specifically binding to Klebsiella pneumoniae O2 antigen is IgA, IgD, IgE, IgG or IgM.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises an antibody heavy chain constant region and an antibody light chain constant region.
  • the full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises an IgG constant region, such as the constant region of IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises an IgG1 type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises an IgG2 type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae comprises an IgG3 type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae comprises an IgG4 type heavy chain constant region.
  • the IgG refers to human IgG.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57. In some embodiments, the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen comprises a lambda light chain constant region.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen comprises an antibody heavy chain variable region and an antibody light chain variable region.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG1 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:5; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:8, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 12, and LC-CDR3, it comprises aminoacid sequence SEQ ID NO:15.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG1 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:6; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:9, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 12, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG1 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:6; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 13, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG1 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:6; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:11, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 14, and LC-CDR3, it comprises aminoacid sequence SEQ ID NO:16.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG1 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:7; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 12, and LC-CDR3, it comprises aminoacid sequence SEQ ID NO:15.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG4 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:5; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:8, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 12, and LC-CDR3, it comprises aminoacid sequence SEQ ID NO:15.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG4 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:6; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:9, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 12, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG4 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:6; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 13, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG4 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:6; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:11, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 14, and LC-CDR3, it comprises aminoacid sequence SEQ ID NO:16.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • a full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprising an IgG4 constant region wherein the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: a) a heavy chain Variable region, the heavy chain variable region comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:7; and b) a light chain variable region comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence SEQ ID NO: 12, and LC-CDR3, it comprises aminoacid sequence SEQ ID NO:15.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • Antibodies that specifically bind to the O1 antigen of Klebsiella pneumoniae that specifically bind to the O1 antigen of Klebsiella pneumoniae
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen includes, but is not limited to, humanized antibody, chimeric antibody, mouse antibody, human antibody, and the present application Antibody molecules comprising heavy and/or light chain CDRs as described above.
  • the antibody or antigen-binding fragment is an isolated antibody that binds the Klebsiella pneumoniae O1 antigen.
  • Contemplated antibodies or antigen-binding fragments that specifically bind to the Klebsiella pneumoniae O1 antigen include, all or fragments of full-length antibodies (such as full-length IgG1, IgG2 or IgG4) that specifically bind to the Klebsiella pneumoniae O1 antigen, specific Single-chain antibodies that specifically bind to Klebsiella pneumoniae O1 antigen, multispecific (such as bispecific) antibodies that bind to Klebsiella pneumoniae O1 antigen, immunoconjugates that specifically bind to Klebsiella pneumoniae O1 antigen, and whatnot.
  • full-length antibodies such as full-length IgG1, IgG2 or IgG4
  • Single-chain antibodies that specifically bind to Klebsiella pneumoniae O1 antigen
  • multispecific (such as bispecific) antibodies that bind to Klebsiella pneumoniae O1 antigen immunoconjugates that specifically bind to Klebsiella pneumoniae O1 antigen, and whatnot.
  • the antibody that specifically binds Klebsiella pneumoniae O1 antigen is Fab, Fab', F(ab)'2, Fab'-SH, single chain antibody (scFv), Fv fragment, dAb, Fd, Nanobodies or diabodies.
  • an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen refers to an antibody or antigen-binding fragment that binds to the Klebsiella pneumoniae O1 antigen with an affinity at least 10 times greater than the binding affinity to a non-target above (including, for example, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 times).
  • non-target refers to an antigen that is not the Klebsiella pneumoniae O1 antigen.
  • Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation analysis (RIA).
  • Kd values can be determined by methods known in the art, such as surface plasmon resonance (SPR) techniques or biolayer interferometry (BLI) techniques.
  • the anti-antibody or antigen-binding fragment (a) that specifically binds to the Klebsiella pneumoniae O1 antigen promotes, mediates or enhances the opsonophagocytosis (OPK) activity of Klebsiella pneumoniae, and/ or (b) promoting, mediating or enhancing complement-mediated serum bactericidal activity (SBA) against Klebsiella pneumoniae.
  • OPK opsonophagocytosis
  • SBA complement-mediated serum bactericidal activity
  • non-human antibodies or antigen-binding fragments comprising human sequences (eg, human heavy and light chain variable regions comprising human CDR sequences), non-human antibodies or antigen-binding fragments are also contemplated.
  • the non-human antibody or antigen-binding fragment comprises human CDR sequences and non-human framework region sequences of the antibodies or antigen-binding fragments described herein, and in some embodiments, the non-human framework region sequences comprise any Sequences for generating heavy and/or light chain variable regions using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (eg, marmosets, macaques), etc.
  • mammals such as mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats,
  • non-human antibodies or antigen-binding fragments include antibodies produced by grafting one or more of the human CDR sequences described herein into non-human framework regions (e.g., mouse or chicken framework region sequences) or antigen-binding fragments.
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen described herein specifically binds to an epitope in the Klebsiella pneumoniae O1 antigen.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen is specific to the Klebsiella pneumoniae O1 antigen and has no species cross-reactivity or is not compatible with other types of non-pneumonia Klebsiella O1 antigen cross-reacts.
  • the antibody or antigen-binding fragment that specifically binds to a Klebsiella pneumoniae O1 antigen cross-reacts with other types of non-Klebsiella pneumoniae O1 antigens.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen comprises an IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to the O1 antigen of Klebsiella pneumoniae comprises an IgG2 type heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment that specifically binds to the O1 antigen of Klebsiella pneumoniae comprises an IgG3 type heavy chain constant region.
  • the antibody or antigen-binding fragment that specifically binds to the O1 antigen of Klebsiella pneumoniae comprises an IgG4 type heavy chain constant region.
  • the IgG refers to human IgG.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen comprises a kappa light chain constant region.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:57.
  • the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen comprises a lambda light chain constant region.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:58.
  • the anti-O1 antigen antibody or antigen-binding fragment comprises an antibody heavy chain variable region and an antibody light chain variable region.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen can be selected from the antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen described in Chinese patent application 202110980272.2.
  • the application provides a bispecific antibody, which includes a first antigen-binding domain that specifically binds Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds Klebsiella pneumoniae O1 antigen .
  • the first antigen-binding domain specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO:2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:5; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:8, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:15.
  • the first antigen-binding domain specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO:3, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:9, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.
  • the first antigen-binding domain specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.
  • the first antigen-binding domain specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:11, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.
  • the first antigen-binding domain specifically binding to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO:2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:7; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:15.
  • the second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO:37; and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:39; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:41; LC-CDR2 comprising the amino acid sequence of SEQ ID NO:43; and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:45.
  • the second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO:38, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:40; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:42, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:44, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46.
  • the first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 17 or 26, or comprising the same amino acid sequence of SEQ ID NO: 17 or 26 a variant sequence having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and a VL comprising an amino acid sequence SEQ ID NO: 21 or 30, or comprising at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 21 or 30) %) sequence homology variant sequence; or V H , which comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in the amino acid sequence SEQ ID NO: 17 or 26; and V L , which comprises the amino acid sequence SEQ ID NO: 17 or 26;
  • the V L which comprises
  • the first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 18 or 27, or comprising the same amino acid sequence of SEQ ID NO: 18 or 27 a variant sequence having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and a VL comprising an amino acid sequence SEQ ID NO: 22 or 31, or comprising at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 22 or 31) %) sequence homology variant sequence; or V H , it comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in the amino acid sequence SEQ ID NO: 18 or 27; and V L , it comprises the amino acid sequence SEQ ID NO: 18 or 27;
  • the V L comprises the
  • the first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising the same amino acid sequence of SEQ ID NO: 19 or 28 a variant sequence having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and a VL comprising an amino acid sequence SEQ ID NO: 23 or 32, or comprising at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 23 or 32) %) sequence homology variant sequence; or VH , which comprises HCDR1, HCDR2 and HCDR3 contained in VH as shown in the amino acid sequence SEQ ID NO: 19 or 28; and VL , which comprises the amino acid sequence SEQ ID NO: 19 or 28;
  • VH which comprises
  • the first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 19 or 28, or comprising the same amino acid sequence of SEQ ID NO: 19 or 28 a variant sequence having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and a VL comprising an amino acid sequence SEQ ID NO: 24 or 33, or comprising at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 24 or 33) %) sequence homology variant sequence; or VH , which comprises HCDR1, HCDR2 and HCDR3 contained in VH as shown in the amino acid sequence SEQ ID NO: 19 or 28; and VL , which comprises the amino acid sequence SEQ ID NO: 19 or 28;
  • VH which comprises
  • the first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 20 or 29, or comprising the same amino acid sequence of SEQ ID NO: 20 or 29 a variant sequence having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and a VL comprising an amino acid sequence SEQ ID NO: 25 or 34, or comprising at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 25 or 34) %) sequence homology variant sequence; or V H , which comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in the amino acid sequence SEQ ID NO: 20 or 29; and V L , which comprises the amino acid sequence SEQ ID NO: 20 or 29;
  • V H comprising
  • the second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 47 or 51, or comprising the same amino acid sequence of SEQ ID NO: 47 or 51 a variant sequence having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and VL comprising an amino acid sequence SEQ ID NO: 49 or 53, or comprising at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 49 or 53) %) sequence homology variant sequence; or V H , which comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in the amino acid sequence SEQ ID NO: 47 or 51; and V L , which comprises the amino acid sequence SEQ ID NO: 47 or 51;
  • the V L which comprises
  • the second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 48 or 52, or comprising the same amino acid sequence of SEQ ID NO: 48 or 52 a variant sequence having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence homology; and a VL comprising an amino acid sequence SEQ ID NO: 50 or 54, or comprising at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 50 or 54) %) sequence homology variant sequence; or V H , which comprises HCDR1, HCDR2 and HCDR3 contained in V H as shown in the amino acid sequence SEQ ID NO: 48 or 52; and V L , which comprises the amino acid sequence SEQ ID NO: 48 or 52;
  • the application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • the first antigen binding domain comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 11, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16; and wherein the second antigen binding domain comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 35, HC -CDR2 comprising the amino acid sequence of SEQ ID NO:37, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:39; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 41.
  • the application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • the first antigen binding domain comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 11, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16; and wherein the second antigen binding domain comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 36, HC -CDR2 comprising the amino acid sequence of SEQ ID NO:38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 42.
  • the application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • An antigen binding domain wherein the first antigen binding domain comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 15; and wherein the second antigen binding domain comprises: V H , said V H comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 35, HC -CDR2 comprising the amino acid sequence of SEQ ID NO:37, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:39; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 41.
  • the application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • An antigen binding domain wherein the first antigen binding domain comprises: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 15; and wherein the second antigen binding domain comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 36, HC -CDR2 comprising the amino acid sequence of SEQ ID NO:38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 42.
  • the application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • an antigen binding domain wherein the first antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 19 or 28, or a variant comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28 sequence; and V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 24 or 33; and wherein said second antigen The binding domain comprises: a VH comprising the amino acid sequence of SEQ ID NO: 47 or 51, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 47 or 51; and a VL comprising The amino acid sequence of SEQ ID NO: 49 or 53, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 49 or 53.
  • the application provides a bispecific antibody, which includes a first antigen-binding domain that specifically binds the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds the Klebsiella pneumoniae O1 antigen.
  • an antigen binding domain wherein the first antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 19 or 28, or a variant comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 19 or 28 sequence; and V L comprising the amino acid sequence of SEQ ID NO: 24 or 33, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 24 or 33; and wherein the second antigen binding domain Comprising: VH comprising the amino acid sequence of SEQ ID NO: 48 or 52, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 48 or 52; and VL comprising the amino acid sequence SEQ ID NO: 50 or 54, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 50 or 54.
  • the application provides a bispecific antibody, which includes a first antigen-binding domain that specifically binds the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds the Klebsiella pneumoniae O1 antigen.
  • an antigen binding domain wherein the first antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 20 or 29, or a variant comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 20 or 29 sequence; and V L comprising the amino acid sequence of SEQ ID NO: 25 or 34, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 25 or 34; and wherein the second antigen binding domain Comprising: VH comprising the amino acid sequence of SEQ ID NO: 47 or 51, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 47 or 51; and VL comprising the amino acid sequence SEQ ID NO: 49 or 53, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 49 or 53.
  • the application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen, and a second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • an antigen binding domain wherein the first antigen binding domain comprises: VH comprising the amino acid sequence of SEQ ID NO: 20 or 29, or a variant comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 20 or 29 sequence; and V L comprising the amino acid sequence of SEQ ID NO: 25 or 34, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 25 or 34; and wherein the second antigen binding domain Comprising: VH comprising the amino acid sequence of SEQ ID NO: 48 or 52, or a variant sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 48 or 52; and VL comprising the amino acid sequence SEQ ID NO: 50 or 54, or a variant sequence comprising at least 80% sequence homology to the amino acid sequence of SEQ ID NO: 50 or 54.
  • any bispecific antibody described in the present application it has a dual variable region immunoglobulin molecule (DVD-Ig) structure, and the structure is N of the V L and V H of a normal IgG antibody.
  • the ends are connected to the VL and VH domains of another antibody, and the VH and VL of the two antibodies interact to form an antigen-binding domain, which can simultaneously bind to the corresponding antigen to achieve bispecificity.
  • DVD-Ig dual variable region immunoglobulin molecule
  • the DVD-IgG structure is a homodimer structure consisting of two identical monomers, each monomer includes two antigen-binding domains, one of which is Fv and the other The antigen-binding domain is Fab, and the above two domains are connected in series by a linker peptide (L).
  • the DVD-Ig structure further includes Fc.
  • An exemplary DVD-Ig structure is shown in Figure 7A.
  • one of the antigen-binding domains specifically binds to the Klebsiella pneumoniae O1 antigen, and the other antigen-binding domain specifically binds to the Klebsiella pneumoniae O2 antigen.
  • the bispecific antibody can simultaneously bind to the Klebsiella pneumoniae O2 antigen and the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody is composed of two identical monomers, and each monomer includes two polypeptide chains, namely a heavy chain and a light chain, for a total of four polypeptide chains.
  • the heavy chain includes V H 1 -LV H 2 -CH 1 from N-terminal to C-terminal
  • the light chain includes V L 1-LV L 2- CL from N-terminal to C-terminal.
  • said heavy chain further comprises Fc
  • the Fc comprises CH2 and CH3 domains.
  • the heavy chain comprises VH1 - LVH2 -CH1- CH2 - CH3 from N- terminus to C-terminus, and the light chain comprises VL1 from N-terminus to C-terminus -LV L 2-C L .
  • VH1 and VL1 are the heavy chain variable region and light chain variable region that specifically bind to one antigen
  • VH2 and VL2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, respectively Light chain variable region
  • L is linker peptide
  • CH1 is heavy chain constant region CH1 domain
  • CL is light chain constant region.
  • V H 1 and V L 1 constitute one antigen-binding domain (Fv) of the bispecific antibody
  • V H 2- CH 1 and V L 2- CL constitute the other antigen-binding domain (Fab ).
  • the V H 1 and V L 1 are respectively the heavy chain variable region and the light chain variable region that specifically bind to the Klebsiella pneumoniae O1 antigen, and the V H 1 and V L 1 constitute specificity.
  • V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to Klebsiella pneumoniae O2 antigen, respectively
  • V H 2- CH 1 and V L 2- CL constitute the antigen-binding domain (Fab) that specifically binds the Klebsiella pneumoniae O2 antigen.
  • the V H 1 and V L 1 are respectively the heavy chain variable region and the light chain variable region that specifically bind to the Klebsiella pneumoniae O2 antigen, and the V H 1 and V L 1 constitute specificity Antigen-binding domain (Fv) that binds to Klebsiella pneumoniae O2 antigen;
  • V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to Klebsiella pneumoniae O1 antigen, respectively,
  • V H 2- CH 1 and V L 2- CL constitute the antigen-binding domain (Fab) that specifically binds the Klebsiella pneumoniae O1 antigen.
  • the connecting peptide (or linker) sequence can be a single amino acid or polypeptide sequence.
  • the connecting peptide (or linker) comprises or consists of a glycine-serine linker.
  • glycine-serine linker refers to a peptide consisting of glycine and serine residues.
  • Exemplary glycine-serine linkers include amino acid sequences of the general formula (Gly 4 Ser)n, where n is a positive integer (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • a preferred glycine-serine linker is (Gly 4 Ser) 2 , ie GGGGSGGGGS (SEQ ID NO: 102) and (Gly 4 Ser) 4 , ie GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 103).
  • Another preferred glycine-serine linker is (Gly 4 Ser) 3 , ie GGGGSGGGGSGGGGS (SEQ ID NO: 104).
  • two or more glycine-serine linkers are tandem in a linker peptide.
  • the connecting peptide includes at least a portion of the hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, e.g., (Gly 4 Ser) n ).
  • the connecting peptide can also be selected to comprise the amino acid sequence ASTKGP (SEQ ID NO: 105) or the amino acid sequence TVAAP (SEQ ID NO: 106).
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:61, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:62, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:63, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:64, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:65, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:66, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:67, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:68, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:61, or has at least 80% (such as at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:61. %, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO:62, or with amino acid sequence SEQ ID NO:62 has at least 80% (for example Variant sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:63, or has at least 80% (such as at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:63. %, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO:64, or with amino acid sequence SEQ ID NO:64 has at least 80% (for example Variant sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:65, or has at least 80% (such as at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:65. %, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO:66, or with amino acid sequence SEQ ID NO:66 has at least 80% (for example Variant sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:67, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 68, or comprising at least 80% with the amino acid sequence SEQ ID NO: 68 Variant sequences with (eg at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:86, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO:62, or comprising at least Variant sequences having 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:87, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO:64, or comprising at least Variant sequences having 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:88, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO:66, or comprising at least Variant sequences having 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:89, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO:68, or comprising at least Variant sequences having 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO: 115, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO:66, or comprising at least Variant sequences having 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.
  • the bispecific antibody according to the present application has a Bs4Ab structure, which comprises two identical monomers, and each monomer comprises two antigen-binding domains, one of which is Fab, another antigen binding domain is scFv.
  • the bispecific antibody further comprises Fc
  • the Fc comprises CH2 and CH3 domains.
  • the scFv is linked to Fab via a first linker peptide (L1), and linked to Fc via a second linker peptide (L2).
  • An exemplary Bs4Ab structure is shown in Figure 7B.
  • one of the antigen binding domains specifically binds to the Klebsiella pneumoniae O1 antigen
  • the other antigen binding domain specifically binds to the Klebsiella pneumoniae Primary bacteria O2 antigen binding.
  • the bispecific antibody can simultaneously bind to the Klebsiella pneumoniae O2 antigen and the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody according to the present application is composed of two identical monomers, and each monomer comprises two polypeptide chains, namely a heavy chain and a light chain, for a total of four polypeptide chains.
  • the heavy chain of the bispecific antibody comprises from N-terminus to C-terminus: VH1 - CH1 -L1- VH2 -L3- VL2 structure.
  • the heavy chain of the bispecific antibody comprises from N-terminus to C-terminus: VH1 - CH1 -L1- VL2 -L3- VH2 structure.
  • the heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the heavy chain of the bispecific antibody comprises from N-terminus to C-terminus: VH1 - CH1 -L1- VH2 -L3- VL2 -L2- CH2 - CH3 structure. In other embodiments, the heavy chain of the bispecific antibody comprises from N-terminus to C-terminus: VH1 - CH1 -L1- VL2 -L3- VH2 -L2- CH2 - CH 3 structures. In some embodiments, the light chain of the bispecific antibody comprises, from the N-terminus to the C-terminus: a VL1 - CL structure.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, respectively, and VH2 and VL2 are the heavy chain variable region that specifically binds to another antigen. region and the light chain variable region, CH1 is the heavy chain constant region CH1 domain, CL is the light chain constant region, L1, L2 and L3 are connecting peptides.
  • V H 1- CH 1 and V L 1CL constitute one of the antigen-binding domains (Fab) of the bispecific antibody, and V H 2-L3-V L 2 or V L 2-L3-V H 2 constitute the bispecific Another antigen-binding domain (scFv) of an antibody.
  • V H 1 and V L 1 are heavy chain variable regions and light chain variable regions that specifically bind to Klebsiella pneumoniae O1 antigen, respectively, V H 1- CH 1 and V L 1- CL constitutes the antigen-binding domain (Fab) that specifically binds to the Klebsiella pneumoniae O1 antigen; VH2 and VL2 are the heavy chain variable region and light chain variable region that specifically bind to the Klebsiella pneumoniae O2 antigen The variable region, VH2 -L3- VL2 or VL2 -L3- VH2 constitutes the antigen-binding domain (scFv) that specifically binds the Klebsiella pneumoniae O2 antigen.
  • Fab antigen-binding domain
  • V H 1 and V L 1 are heavy chain variable regions and light chain variable regions that specifically bind to Klebsiella pneumoniae O2 antigen respectively, V H 1- CH 1 and V L 1C L constitutes the antigen-binding domain (Fab) that specifically binds to the Klebsiella pneumoniae O2 antigen; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to the Klebsiella pneumoniae O1 antigen Region, VH2 -L3- VL2 or VL2 -L3- VH2 constitutes the antigen-binding domain (scFv) that specifically binds the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody comprises CH1 comprising the amino acid sequence of SEQ ID No:60. In some embodiments, the bispecific antibody comprises CH2 - CH3 comprising the amino acid sequence of SEQ ID NO:59.
  • the antigen-binding domain scFv that specifically binds Klebsiella pneumoniae O2 antigen or O1 antigen includes a genetically engineered cysteine mutation, through VH and Two cysteine mutations were introduced into the VL interface to obtain disulfide bond-stabilized bispecific antibodies.
  • Linking peptides can be used to link the domains and/or structural regions of the chimeric heavy chains of bispecific antibodies into one continuous molecule.
  • the bispecific antibody comprises at least two linker peptides, L1 and L2.
  • the bispecific antibody may comprise an additional linker, such as a flexible linker linking the variable heavy and light chains of the scFv.
  • the bispecific antibody may include additional linkers, such as flexible linkers linking the variable heavy and light chains of the scFv and other linkers for linking other binding units to the core structure of the bispecific antibody. connector.
  • a typical, non-limiting example of a connecting peptide is a polypeptide chain comprising at least 4 residues.
  • Such linker sites may be flexible, hydrophilic, and form little or no secondary structure themselves (linker sites or flexible linker sites).
  • Linkers of at least 4 amino acids can be used to link domains and/or regions close to each other after molecular assembly is complete. Longer connectors can also be used.
  • linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 , 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues.
  • the linkers can be the same or different (eg, the same or different lengths and/or amino acid sequences).
  • the connecting peptide comprises or consists of a glycine-serine linker.
  • glycine-serine linker refers to a peptide consisting of glycine and serine residues.
  • Exemplary glycine-serine linkers include amino acid sequences of the general formula (Gly 4 Ser)n, where n is a positive integer (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • a preferred glycine-serine linker is (Gly 4 Ser) 2 , ie GGGGSGGGGS (SEQ ID NO: 102) and (Gly 4 Ser) 4 , ie GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 103).
  • Another preferred glycine-serine linker is (Gly 4 Ser) 3 , ie GGGGSGGGGSGGGGS (SEQ ID NO: 104).
  • two or more glycine-serine linkers are tandem in a linker peptide.
  • the connecting peptide includes at least a portion of the hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, e.g., (Gly 4 Ser) n ).
  • L1 and/or L2 comprise a hinge portion and a linker portion, eg, a linker portion comprising a glycine-serine linker.
  • L1 and/or L2 include only a hinge portion or only a linker portion, eg, a glycine-serine linker.
  • L1 and L2 include a glycine-serine linker.
  • the glycine-serine linker portions of L1 and L2 are the same length, while in other aspects, the glycine-serine linker portions of LI and L2 are different in length.
  • the heavy and light chains of the scFv can be linked by a flexible linker.
  • the flexible linker generally does not include a hinge portion, but is instead a glycine-serine linker or other flexible linker.
  • the length and amino acid sequence of the flexible linker interconnecting the scFv domains can be selected and optimized.
  • the connecting peptide (eg, L1 and/or L2) comprises a glycine-serine or all glycine linker and a portion or a modified portion of the hinge region.
  • the linker peptide (L1) linking one of the antigen binding domains (such as Fab or scFv) and the other antigen binding domain (such as scFv or Fab) in the bispecific antibody comprises the amino acid sequence EPKSDKTGGGGSGGGGS (SEQ ID NO: 107 ) or EPKSCGKTGGGGSGGGGS (SEQ ID NO: 108) or EPKSCGGGGSGGGGS (SEQ ID NO: 109).
  • the connecting peptide (L2) connecting the scFv and Fc of the binding antigen binding domain of the bispecific antibody comprises the amino acid sequence GGGGSGGGGSEPKSDKTHTCPPCP (SEQ ID NO: 110) or GGGGSGGGGSCPPCP (SEQ ID NO: 111) or GGGGSGGGGSDKTHTCPPCP (SEQ ID NO: 111) or GGGGSGGGGSDKTHTCPPCP (SEQ ID NO: :112).
  • the bispecific antibody is bispecific except for a linker peptide linking one antigen-binding domain to another antigen-binding domain or a linker peptide linking one of the antigen-binding domains to Fc (eg, L1 and L2).
  • Antibodies can optionally contain additional linker peptides. The length and sequence of these additional linker peptides are independently chosen.
  • the bispecific antibody may also include a flexible linker peptide (L3) linking the variable heavy and light chains (V HSCFV and V LSCFV ) in the scFv of the antigen-binding domain.
  • a flexible linking peptide may include a glycine-serine linker.
  • the flexible linker peptide (L3) linking the variable heavy and light chains of the scFv comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 103).
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:69, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:70, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:71, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:72, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:73, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:74, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:75, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:69, or has at least 80% (such as at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:69. %, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 70, or with amino acid sequence SEQ ID NO: 70 has at least 80% (for example Variant sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO: 71, or has at least 80% (such as at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO: 71 %, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 72, or with amino acid sequence SEQ ID NO: 72 has at least 80% (for example Variant sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:73, or has at least 80% (such as at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:73. %, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 74, or with amino acid sequence SEQ ID NO: 74 has at least 80% (for example Variant sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:75, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 74, or have at least 80% ( For example variant sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • the chimeric heavy chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: VH1 - CH1 -L1- VH2 -L3- VL2 -L2-C H2 - CH3 .
  • the light chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: V L 1- CL .
  • the bispecific antibody comprises or has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%) of the amino acid sequence of SEQ ID NO: 90 , 97%, 98% or 99%) sequence homology variant sequence; and/or comprising or having at least 80% (for example at least 80%, 85%) of the amino acid sequence SEQ ID NO:70 with the amino acid sequence SEQ ID NO:70 %, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • the chimeric heavy chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: VH1 - CH1 -L1- VH2 -L3- VL2 -L2-C H2 - CH3 .
  • the light chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: V L 1- CL .
  • the bispecific antibody comprises or has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%) of the amino acid sequence of SEQ ID NO: 91 , 97%, 98% or 99%) sequence homology variant sequence; and/or comprising or having at least 80% (for example at least 80%, 85%) of the amino acid sequence SEQ ID NO:72 with the amino acid sequence SEQ ID NO:72 %, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • the chimeric heavy chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: VH1 - CH1 -L1- VH2 -L3- VL2 -L2-C H2 - CH3 .
  • the light chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: V L 1- CL .
  • the bispecific antibody comprises or has at least 80% (eg, at least 80%, 85%, 90%, 95%, 96%) of the amino acid sequence of SEQ ID NO: 92 , 97%, 98% or 99%) sequence homology variant sequence; and/or comprising or having at least 80% (for example at least 80%, 85%) of the amino acid sequence SEQ ID NO:74 with the amino acid sequence SEQ ID NO:74 %, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • the chimeric heavy chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: VH1 - CH1 -L1- VH2 -L3- VL2 -L2-C H2 - CH3 .
  • the light chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: V L 1- CL .
  • the bispecific antibody comprises or has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%) of the amino acid sequence of SEQ ID NO: 93 , 97%, 98% or 99%) sequence homology variant sequence; and/or comprising or having at least 80% (for example at least 80%, 85%) of the amino acid sequence SEQ ID NO:74 with the amino acid sequence SEQ ID NO:74 %, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody described in the present application it has a Hetero H, CrossMab structure, which is a bivalent bispecific antibody composed of a heterodimer, comprising two antigen-binding domains Fab .
  • the bispecific antibody further comprises two Fcs comprising CH2 and CH3 domains.
  • amino acid residues in the CH3 domain in one of the Fc are replaced with amino acid residues with larger side chain volumes to form a "knob”
  • amino acid residues in the CH3 domain in the other Fc Residues are replaced by amino acid residues with smaller side chain volumes to form "holes", which can promote the mutual association of heterodimers.
  • the positions of the light chain constant region ( CL ) and the heavy chain constant region CH1 domain ( CH1 ) can be replaced; or the heavy chain variable region (V H ) and the light chain variable region (V L ) can be interchanged; or light chain constant region ( CL ) and heavy chain constant region CH 1 domain ( CH 1) and heavy chain variable region (V H ) and light chain variable region (V L ) replace each other simultaneously.
  • An exemplary Hetero H, CrossMab structure is shown in Figure 7C.
  • the Fc is derived from wild-type human IgG1 Fc.
  • the CH3 domain of the Fc comprises, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and/or Y407V, wherein the numbering is according to the EU index such as Kabat .
  • one of the antigen-binding domains specifically binds to the Klebsiella pneumoniae O2 antigen, and the other antigen-binding domain specifically binds to the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody can simultaneously bind to the Klebsiella pneumoniae O2 antigen and the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody described in the present application has a Hetero H, CrossMab structure, which is a heterodimer composed of two different monomers, and each monomer contains two polypeptides chain.
  • the first monomer comprises a first heavy chain and a first light chain of an antibody that specifically binds one of the antigens
  • the second monomer comprises a second heavy chain and a second light chain of an antibody that specifically binds the other antigen.
  • the first heavy chain of the bispecific antibody includes from the N-terminus to the C-terminus: a V H 1- CH 1 structure; the first light chain includes a V L 1-C from the N-terminus to the C-terminus L structure.
  • the first heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the first heavy chain of the bispecific antibody includes from the N-terminal to the C-terminal: V H 1- CH 1- CH 2- CH 3 structure; the first light chain from the N-terminal to the The C-terminus includes a VL1 - CL structure.
  • VH1 and VL1 are the heavy chain variable region and the light chain variable region that specifically bind to one of the antigens, respectively, CH1 is the CH1 domain of the heavy chain constant region, and CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL constitute an antigen-binding domain (Fab) that binds one of the antigens.
  • the second heavy chain of the bispecific antibody includes from N-terminus to C-terminus: V H 2- CL structure; the second light chain includes from N-terminus to C-terminus: V L 2-C H1 structure. In some embodiments, the second heavy chain further comprises an Fc comprising CH2 and CH3 domains. In some embodiments, the second heavy chain of the bispecific antibody comprises from the N-terminus to the C-terminus: VH2 - CL - CH2 - CH3 structure; the first light chain from the N-terminus to the C-terminal The end includes a V L 2- CH 1 structure.
  • VH2 and VL2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, respectively, CH1 is the CH1 domain of the heavy chain constant region, and CL is the light chain constant region.
  • VH2 - CL and VL2 - CH1 constitute an antigen-binding domain (Fab) that binds another antigen.
  • Fab antigen-binding domain
  • the positions of CL and CH1 in the first monomer or the second monomer of the bispecific antibody are replaced with each other.
  • the positions of VH1 and VL1 are interchangeable.
  • the positions of VH2 and VL2 are interchangeable.
  • amino acid residues in the CH3 domain of the first heavy chain are replaced with amino acid residues with a larger side chain volume to form a "knob”
  • amino acid residues in the CH3 domain of the second heavy chain Residues are replaced with amino acid residues of smaller side chain volume to form "holes”.
  • amino acid residues in the CH3 domain of the second heavy chain are replaced with amino acid residues with larger side chain volumes to form a "knob”
  • Amino acid residues are replaced with amino acid residues of smaller side chain volume to form "holes”.
  • the CH3 domain comprises, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A and/or Y407V, wherein the numbering is according to the EU index such as Kabat.
  • V H 1 and V L 1 are the heavy chain variable region and the light chain variable region that specifically bind to the Klebsiella pneumoniae O1 antigen, respectively, and V H 2 and V L 2 are the The heavy chain variable region and the light chain variable region of the Klebsiella pneumoniae O2 antigen, V H 1- CH 1 and V L 1- CL form the antigen-binding domain ( Fab), VH2 - CL and VL2 - CH1 constitute the antigen-binding domain (Fab) that specifically binds the Klebsiella pneumoniae O2 antigen.
  • Fab antigen-binding domain
  • V H 1 and V L 1 are the heavy chain variable region and the light chain variable region that specifically bind to the Klebsiella pneumoniae O2 antigen, respectively, and V H 2 and V L 2 are the specific Binding heavy chain variable region and light chain variable region of Klebsiella pneumoniae O1 antigen, V H 1- CH 1 and V L 1- CL form an antigen-binding domain that specifically binds Klebsiella pneumoniae O2 antigen (Fab), VH2 - CL and VL2 - CH1 constitute the antigen-binding domain (Fab) that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • Fab antigen-binding domain
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:76, or comprises at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody according to the present application comprises an amino acid sequence of SEQ ID NO: 77, or comprises an amino acid sequence of SEQ ID NO: 77 having at least 80%, such as at least 80%, 85%, 90% %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:78, or comprises at least 80%, such as at least 80%, 85%, or 90%, of the amino acid sequence of SEQ ID NO:78. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody according to the present application comprises an amino acid sequence of SEQ ID NO: 74, or comprises an amino acid sequence of SEQ ID NO: 74 having at least 80%, such as at least 80%, 85%, 90%. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody according to the present application comprises an amino acid sequence of SEQ ID NO:79, or comprises an amino acid sequence of SEQ ID NO:79 having at least 80%, such as at least 80%, 85%, or 90%. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody according to the present application comprises an amino acid sequence of SEQ ID NO:80, or comprises an amino acid sequence of SEQ ID NO:80 having at least 80%, such as at least 80%, 85%, or 90%. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:76, or has at least 80%, such as at least 80%, 85%, or 90%, of the amino acid sequence of SEQ ID NO:76 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO:77, or have at least 80% such as at least 80% with the amino acid sequence SEQ ID NO:77 %, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO:78, or with the amino acid sequence SEQ ID NO:78 A variant sequence having at least 80%, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and the amino acid sequence of SEQ ID NO: 74, or with A variant sequence having at least 80%, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:79, or has at least 80%, such as at least 80%, 85%, or 90%, of the amino acid sequence of SEQ ID NO:79 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 80, or have at least 80% such as at least 80% with the amino acid sequence SEQ ID NO: 80 %, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO:78, or with the amino acid sequence SEQ ID NO:78 A variant sequence having at least 80%, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and the amino acid sequence of SEQ ID NO: 74, or with A variant sequence having at least 80%, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:94, or has at least 80%, such as at least 80%, 85%, or 90%, of the amino acid sequence of SEQ ID NO:94 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO: 77, or have at least 80% with amino acid sequence SEQ ID NO: 77 such as Variant sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology;/or the amino acid sequence of SEQ ID NO: 95, or with the amino acid sequence of SEQ ID NO:95 has at least 80% such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; / or amino acid sequence SEQ ID NO: 74, or a variant sequence having at least 80%, such as at least 80%, 85%, 90%, 95%, 9
  • any bispecific antibody according to the present application comprises the amino acid sequence of SEQ ID NO:96, or has at least 80%, such as at least 80%, 85%, or 90%, of the amino acid sequence of SEQ ID NO:96 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO: 80, or have at least 80% with the amino acid sequence SEQ ID NO: 80 for example Variant sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology;/or the amino acid sequence of SEQ ID NO: 95, or with the amino acid sequence of SEQ ID NO:95 has at least 80% such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; / or amino acid sequence SEQ ID NO: 74, or a variant sequence having at least 80%, such as at least 80%, 85%, 90%, 95%, 96%
  • the bispecific antibody described in the present application has an IgG-(scFv) 2 structure, which is composed of two identical monomers, and each monomer contains two antigen-binding domains, wherein One antigen binding domain is Fab and the other antigen binding domain is scFv.
  • the bispecific antibody further comprises Fc
  • the Fc comprises CH2 and CH3 domains.
  • the scFv is linked to the carboxyl terminus of Fc via a linker peptide (L).
  • L linker peptide
  • one of the antigen binding domains specifically binds to the Klebsiella pneumoniae O1 antigen
  • the other antigen binding domain specifically binds to the Klebsiella pneumoniae Primary bacteria O2 antigen binding.
  • the bispecific antibody can simultaneously bind to the Klebsiella pneumoniae O2 antigen and the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibodies disclosed herein comprise two identical monomers, each monomer comprising one heavy chain and one light chain.
  • the heavy chain of the bispecific antibody comprises from N-terminus to C-terminus: VH1 - CH1 - CH2 - CH3 structure.
  • the light chain of the bispecific antibody comprises from N-terminus to C-terminus: V L 1- CL .
  • the heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the heavy chain of the bispecific antibody comprises from N-terminus to C-terminus: VH1 - CH1 -CH2- CH3 - LVH2 - L3 - VL2 structure.
  • the heavy chain of the bispecific antibody comprises from the N-terminus to the C-terminus: VH1 - CH1 -CH2- CH3 - LVL2 - L3 - VH2 structure.
  • the light chain of the bispecific antibody comprises, from the N-terminus to the C-terminus: a VL1 - CL structure.
  • VH1 and VL1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, respectively
  • VH2 and VL2 are the heavy chain variable region that specifically binds to another antigen.
  • V H 1- CH 1 and V L 1- CL constitute one of the antigen-binding domains (Fab) of the bispecific antibody, and V H 2-L3-V L 2 or V L 2-L3-V H 2 constitute the bispecific antibody Another antigen-binding domain (scFv) of a specific antibody.
  • Fab antigen-binding domain
  • scFv antigen-binding domain
  • V H 1 and V L 1 are heavy chain variable regions and light chain variable regions that specifically bind to Klebsiella pneumoniae O1 antigen, respectively, V H 1- CH 1 and V L 1- CL constitutes the antigen-binding domain (Fab) that can specifically bind to Klebsiella pneumoniae O1 antigen; VH2 and VL2 are the heavy chain variable region and light chain that specifically bind to Klebsiella pneumoniae O2 antigen Chain variable region, VH2 -L3- VL2 or VL2 -L3- VH2 constitutes an antigen-binding domain (scFv) capable of specifically binding to Klebsiella pneumoniae O2 antigen.
  • Fab antigen-binding domain
  • scFv antigen-binding domain
  • V H 1 and V L 1 are heavy chain variable regions and light chain variable regions that specifically bind to the Klebsiella pneumoniae O2 antigen, respectively, V H 1- CH 1 and V L 1- CL constitutes the antigen-binding domain (Fab) that can specifically bind to Klebsiella pneumoniae O2 antigen; VH2 and VL2 are the heavy chain variable region and light chain that specifically bind to Klebsiella pneumoniae O1 antigen The chain variable region, VH2 -L3- VL2 or VL2 -L3- VH2 constitutes an antigen-binding domain (scFv) capable of specifically binding to the Klebsiella pneumoniae O1 antigen.
  • the bispecific antibody comprises CH1 comprising the amino acid sequence of SEQ ID No:60. In some embodiments, the bispecific antibody comprises CH2 - CH3 comprising the amino acid sequence of SEQ ID NO:59.
  • Linking peptides can be used to link the domains and/or regions of the chimeric heavy chains of bispecific antibodies into one continuous molecule.
  • the bispecific antibody may comprise an additional linker, such as a flexible linker linking the variable heavy and light chains of the scFv.
  • the bispecific antibody may include additional linkers, such as flexible linkers linking the variable heavy and light chains of the scFv and other linkers for linking other binding units to the core structure of the bispecific antibody. connector.
  • linker is a polypeptide chain comprising at least 4 residues.
  • Such linker sites may be flexible, hydrophilic, and form little or no secondary structure themselves (linker sites or flexible linker sites).
  • Linkers of at least 4 amino acids can be used to link domains and/or regions close to each other after molecular assembly is complete. Longer connectors can also be used.
  • linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 , 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues.
  • the linkers can be the same or different (eg, the same or different lengths and/or amino acid sequences).
  • the connecting peptide comprises or consists of a glycine-serine linker.
  • glycine-serine linker refers to a peptide consisting of glycine and serine residues.
  • Exemplary glycine-serine linkers include amino acid sequences of the general formula (Gly 4 Ser)n, where n is a positive integer (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • a preferred glycine-serine linker is (Gly 4 Ser) 2 , ie GGGGSGGGGS (SEQ ID NO: 102) and (Gly 4 Ser) 4 , ie GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 103).
  • Another preferred glycine-serine linker is (Gly 4 Ser) 3 , ie GGGGSGGGGSGGGGS (SEQ ID NO: 104).
  • two or more glycine-serine linkers are tandem in a linker peptide.
  • the connecting peptide includes at least a portion of the hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, e.g., (Gly 4 Ser) n ).
  • the hinge region e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule
  • a series of glycine-serine residues e.g., a glycine-serine linker, e.g., (Gly 4 Ser) n .
  • the connecting peptide comprises a glycine-serine or all glycine linker and a portion or a modified portion of the hinge region.
  • the linking peptide (eg, L) linking the antigen-binding domain to the carboxy-terminal CH3 of the Fc region in the bispecific antibody comprises the amino acid sequence GGGGSGGGGTGGGGS (SEQ ID NO: 114).
  • the bispecific antibody in addition to the linker peptide (such as L) linking the antigen-binding domain to Fc, can optionally contain additional linker peptides.
  • the length and sequence of these additional linker peptides are independently chosen.
  • the bispecific antibody may also include a flexible linker peptide (L3) that links the variable heavy and light chains (V HSCFV and V LSCFV ) of the scFv.
  • a flexible linking peptide may include a glycine-serine linker. Typically, such joints do not include a hinge portion.
  • the flexible linker peptide (L3) linking the variable heavy and light chains of the scFv comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 103).
  • the chimeric heavy chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: VH1 - CH1 - CH2 -CH3- LVH2 - L3 -V L2 .
  • the light chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: V L 1- CL .
  • the bispecific antibody comprises or has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%) of the amino acid sequence of SEQ ID NO: 97 , 97%, 98% or 99%) sequence homology variant sequence; and/or comprising or having at least 80% (for example at least 80%, 85%) of the amino acid sequence SEQ ID NO:83 with the amino acid sequence SEQ ID NO:83 %, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • the chimeric heavy chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: VH1 - CH1 - CH2 -CH3- LVH2 - L3 -V L2 .
  • the light chain of the bispecific antibody comprises the following polypeptide sequence from N-terminus to C-terminus: V L 1- CL .
  • the bispecific antibody comprises or has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%) of the amino acid sequence of SEQ ID NO: 98 , 97%, 98% or 99%) sequence homology variant sequence; and/or comprising or having at least 80% (for example at least 80%, 85%) of the amino acid sequence SEQ ID NO:85 with the amino acid sequence SEQ ID NO:85 %, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology of the variant sequence.
  • any bispecific antibody described herein has a scFv-Fab IgG structure, which is in the form of a heterodimer.
  • the heterodimeric antibody comprises a first monomer comprising an antigen-binding domain (Fab) capable of binding one of the antigens and a second monomer comprising an antigen-binding domain (Fab) capable of binding the other antigen. antigen-binding domain (scFv).
  • the bispecific antibody further comprises two Fcs comprising CH2 and CH3 domains, and the two Fcs further comprise amino acid substitutions, which can promote mutual binding of heterodimers.
  • Variable Regions An exemplary scFv-Fab IgG structure of the variable region is shown in Figure 7E.
  • one of the antigen binding domains specifically binds to the Klebsiella pneumoniae O2 antigen
  • the other antigen binding domain specifically binds to the Klebsiella pneumoniae Bacterial O1 antigen binding.
  • the bispecific antibody can simultaneously bind to the Klebsiella pneumoniae O2 antigen and the Klebsiella pneumoniae O1 antigen.
  • any bispecific antibody described herein has a scFv-Fab IgG structure, which is in the form of a heterodimer.
  • the heterodimeric antibody comprises a first monomer and a second monomer, the first monomer comprising two polypeptide chains: a first heavy chain and a light chain, wherein the first heavy chain comprises from the N-terminus to the C-terminus : V H 1- CH 1 structure.
  • the light chain comprises a VL1 - CL structure.
  • said first heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the first heavy chain comprises from N-terminus to C-terminus: VH1 - CH1 - CH2 - CH3 structure
  • the light chain comprises VL1 - CL structure
  • VH1 and VL1 are the heavy chain variable region and the light chain variable region that specifically bind to one of the antigens, respectively
  • CH1 is the CH1 domain of the heavy chain constant region
  • CL is the light chain constant region.
  • VH1 - CH1 and VL1 - CL constitute one of the antigen-binding domains (Fab).
  • the second monomer comprises one polypeptide chain: the second heavy chain.
  • the second heavy chain comprises a VH2 -L3- VL2 structure or a VL2 -L3- VH2 structure from N-terminus to C-terminus.
  • the second heavy chain further comprises an Fc comprising CH2 and CH3 domains.
  • the first heavy chain comprises from N-terminus to C-terminus: VH2 -L3- VL2 - CH2 - CH3 structure or VL2 -L3- VH2 - CH2 -CH3 structure.
  • VH2 and VL2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, respectively, and L3 is the connecting peptide.
  • VH2 -L3- VL2 or VL2 -L3- VH2 constitutes another antigen binding domain (scFv).
  • V H 1 and V L 1 are heavy chain variable regions and light chain variable regions that specifically bind to Klebsiella pneumoniae O1 antigen, respectively, V H 1- CH 1 and V L 1- CL constitutes the antigen-binding domain (Fab) that specifically binds the Klebsiella pneumoniae O1 antigen.
  • V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to Klebsiella pneumoniae O2 antigen, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitute the antigen-binding domain (scFv) that specifically binds to Klebsiella pneumoniae O2 antigen.
  • V H 1 and V L 1 are heavy chain variable regions and light chain variable regions that specifically bind to the Klebsiella pneumoniae O2 antigen, respectively, V H 1- CH 1 and V L 1- CL constitutes the antigen-binding domain (Fab) that specifically binds the Klebsiella pneumoniae O2 antigen.
  • V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to the Klebsiella pneumoniae O1 antigen, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitute the antigen-binding domain (scFv) that specifically binds to the Klebsiella pneumoniae O1 antigen.
  • the Fc is derived from human wild-type IgG1.
  • the Fc in one monomer contains, but is not limited to, the following amino acid substitutions E357Q and S364K relative to human wild-type IgG1; the Fc in the other monomer contains, but not limited to, relative to human wild-type IgG1
  • Amino acid substitutions Q295E, L368D, K370S, N384D, Q418E and N421D were as follows, wherein the numbering is according to the EU index as Kabat.
  • an exemplary linker peptide (eg, L3) linking VH1 and VL1 in a scFv comprises GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 113).
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO:81, or comprises at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:81. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO:82, or comprises at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:82. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO:83, or comprises at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:83. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO:84, or comprises at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:84. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO:85, or comprises at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:85. %, 95%, 96%, 97%, 98% or 99%) sequence homology of variant sequences.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO: 81, or has at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO: 81 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 82, or have at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 83, or comprising the same amino acid sequence SEQ ID NO :83 A variant sequence having at least 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO:84, or has at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO:84 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 82, or have at least 80% (such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and amino acid sequence SEQ ID NO: 85, or comprising the same amino acid sequence SEQ ID NO :85 A variant sequence having at least 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO: 99, or has at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO: 99 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO: 100, or have at least 80% ( For example, a variant sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and/or the amino acid sequence of SEQ ID NO: 83, or comprising and amino acid A variant sequence having at least 80% (eg at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the sequence SEQ ID NO: 83.
  • any bispecific antibody as described herein comprises the amino acid sequence of SEQ ID NO: 101, or has at least 80% (e.g., at least 80%, 85%, 90%) of the amino acid sequence of SEQ ID NO: 101 , 95%, 96%, 97%, 98% or 99%) sequence homology variant sequence; and/or amino acid sequence SEQ ID NO: 100, or have at least 80% ( For example, a variant sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence homology; and/or the amino acid sequence of SEQ ID NO: 85, or comprising the amino acid sequence A variant sequence having at least 80% (eg at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the sequence SEQ ID NO: 85.
  • Exemplary antibody sequences are shown in Table 2-Table 10, wherein the CDR numbering is based on the EU index in Kabat. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the location of CDRs and defining antibody light and heavy chain variable regions. Comprising an antibody that specifically binds to the Klebsiella pneumoniae O2 antigen, an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, and the CDRs, VH and/or VL sequences of the bispecific antibody as described in the present application, but Antibodies based on predictive algorithms other than those exemplified in the table below are also within the scope of this application.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen can be selected from the anti-O1 antigen antibodies described in Chinese patent application 202110980272.2, which is incorporated in this application by reference.
  • Table 2 Exemplary CDR sequences of antibodies specifically binding to Klebsiella pneumoniae O2 antigen
  • Table 3-1 Exemplary antibody V H V L sequences that specifically bind to the Klebsiella pneumoniae O2 antigen
  • Table 3-2 Exemplary antibody V H V L cysteine variant sequences that specifically bind to the Klebsiella pneumoniae O2 antigen
  • Table 4 Exemplary CDR sequences of antibodies specifically binding to the Klebsiella pneumoniae O1 antigen
  • Table 5-1 Exemplary V H V L sequences of antibodies specifically binding to the Klebsiella pneumoniae O1 antigen
  • Table 5-2 Exemplary antibody V H V L cysteine variant sequences that specifically bind to the Klebsiella pneumoniae O1 antigen
  • Table 7-1 Partial heavy chain and light chain sequences of exemplary bispecific antibodies that specifically bind O2 antigen and O1 antigen in DVD-Ig structure
  • Table 7-2 Partial heavy chain and light chain sequences of exemplary bispecific antibodies that Bs4Ab structure specifically binds O2 antigen and O1 antigen
  • Table 7-3 Partial heavy chain and light chain sequences of exemplary bispecific antibodies of Hetero H, CrossMab structure specifically binding O2 antigen and O1 antigen
  • Table 7-4 Partial heavy chain and light chain sequences of exemplary bispecific antibodies of scFv-Fab IgG structure specifically binding O2 antigen and O1 antigen
  • Table 8-1 Full-length sequences of heavy and light chains of exemplary bispecific antibodies that specifically bind O2 antigen and O1 antigen in DVD-Ig structure
  • Table 8-2 Full-length sequences of heavy chain and light chain of exemplary bispecific antibodies that Bs4Ab structure specifically binds O2 antigen and O1 antigen
  • Table 8-3 Hetero H, the full-length sequence of the heavy chain and light chain of an exemplary bispecific antibody that specifically binds O2 antigen and O1 antigen in the structure of CrossMab
  • Table 8-4 Full-length sequences of heavy and light chains of exemplary bispecific antibodies whose IgG-(scFv) 2 structure specifically binds O2 antigen and O1 antigen
  • Table 8-5 Full-length sequences of heavy and light chains of exemplary bispecific antibodies whose scFv-Fab IgG structures specifically bind O2 antigen and O1 antigen
  • GKPGSGKPGSGKPGSGKPGS 114 GGGGSGGGGTGGGGS
  • the application provides a pharmaceutical composition, comprising: (i) an antibody or an antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen and (ii) an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen Antibodies or antigen-binding fragments.
  • a pharmaceutical composition comprising: (i) an antibody or an antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae and (ii) an antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae Antibody or antigen-binding fragment, wherein: the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: (a) heavy chain variable region (V H ), and said V H comprises: HC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or variants of said VH , the HC- CDRs comprising up to about 5 amino acid substitutions; and a V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:8, LC-CDR2 comprising the amino acid sequence
  • a pharmaceutical composition comprising: (i) an antibody or an antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae and (ii) an antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae Antibody or antigen-binding fragment, wherein: the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen comprises: (a) V H , and said V H comprises: HC-CDR1, which comprises the amino acid sequence SEQ ID NO: 35.
  • HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39, or a variant of said VH comprising at most about 5 of its HC-CDRs amino acid substitutions; and a VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:41, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and LC-CDR3 comprising the amino acid sequence Sequence SEQ ID NO: 45, or variants of said V L comprising up to about 5 amino acid substitutions in its LC-CDRs; or (b) V H comprising: HC- CDR1 comprising amino acids The sequence of SEQ ID NO:36, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:40, or variants of said VH , in which HC-CDRs comprising
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen in the pharmaceutical composition comprises: V H comprising the amino acid sequence of SEQ ID NO: 17, and V L comprising the amino acid Sequence SEQ ID NO:21.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1, HC in VH having the amino acid sequence of SEQ ID NO: 17 - CDR2 and HC-CDR3; and a VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a VL having the amino acid sequence of SEQ ID NO: 21.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen in the pharmaceutical composition comprises: V H comprising the amino acid sequence of SEQ ID NO: 18, and V L comprising amino acids Sequence SEQ ID NO:22.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1, HC in VH having the amino acid sequence of SEQ ID NO: 18 - CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO: 22.
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen in the pharmaceutical composition comprises: V H comprising the amino acid sequence of SEQ ID NO: 19, and V L comprising the amino acid Sequence SEQ ID NO:23.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1, HC in VH having the amino acid sequence of SEQ ID NO: 19 - CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO: 23.
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen in the pharmaceutical composition comprises: V H comprising the amino acid sequence of SEQ ID NO: 19, and V L comprising the amino acid Sequence SEQ ID NO:24.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1, HC in VH having the amino acid sequence of SEQ ID NO: 19 - CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO:24.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen in the pharmaceutical composition comprises: V H comprising the amino acid sequence of SEQ ID NO: 20, and V L comprising amino acids Sequence SEQ ID NO:25.
  • the antibody or antigen - binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1, HC in VH having the amino acid sequence of SEQ ID NO:20 - CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO: 25.
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen in the pharmaceutical composition comprises: V H comprising the amino acid sequence of SEQ ID NO: 47, and V L comprising the amino acid Sequence SEQ ID NO:49.
  • the antibody or antigen - binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: VH comprising HC-CDR1, HC in VH having the amino acid sequence of SEQ ID NO:47 - CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO:49.
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen in the pharmaceutical composition comprises: V H comprising the amino acid sequence of SEQ ID NO: 48, and V L comprising the amino acid Sequence SEQ ID NO:50.
  • the antibody or antigen - binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: VH comprising HC-CDR1, HC in VH having the amino acid sequence of SEQ ID NO:48 - CDR2 and HC-CDR3; and a VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a VL having the amino acid sequence of SEQ ID NO:50.
  • a pharmaceutical composition comprising: an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen, wherein
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:11, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumonia pneumonia
  • a pharmaceutical composition comprising: an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen, wherein
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:11, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumonia pneumonia
  • a pharmaceutical composition comprising: an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen, wherein
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:7; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumonia pneumonia
  • a pharmaceutical composition comprising: an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen, wherein
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:7; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumonia pneumonia
  • Binding affinity can be expressed using Kd, Koff, Kon or Ka.
  • Koff refers to the rate constant for the dissociation of an antigen binding domain from an antigen binding domain/antigen complex, as determined by a kinetic selection device.
  • Kon refers to the association rate constant for the binding of an antibody to an antigen to form an antigen-binding domain/antigen complex.
  • the dissociation constant "Kd” used in this application refers to the dissociation constant when a specific antibody-antigen interacts, and refers to the antigen concentration required when the antigen occupies half of all antibody-binding domains and reaches equilibrium in the antibody molecule solution, equal to Koff/Kon. The determination of Kd assumes that all bound molecules are in solution.
  • the corresponding dissociation rate constant is expressed in terms of EC50, which is a good approximation of Kd.
  • the affinity association constant Ka is the reciprocal of the dissociation constant Kd.
  • the equilibrium dissociation constant (Kd) can be used as an indicator of the affinity of the antigen-binding domain portion with the antigen.
  • Kd equilibrium dissociation constant
  • simple analysis can be performed by the Scatchard method using antibodies labeled with various markers, and a Biacore instrument (manufactured by Amersham Biosciences), and the interaction between biomolecules can be analyzed by surface plasmon resonance according to the user's manual or the attached kit. . Kd values obtained using these methods are expressed in units of M.
  • An antibody that specifically binds a target may have, for example, ⁇ 10 ⁇ 7 M, ⁇ 10 ⁇ 8 M, ⁇ 10 ⁇ 9 M, ⁇ 10 ⁇ 10 M, ⁇ 10 ⁇ 11 M, ⁇ 10 ⁇ 12 M, or ⁇ 10 ⁇ Kd value of 13M .
  • the binding specificity of an antibody can be determined experimentally by methods known in the art. These methods include, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore tests, and peptide scans, among others.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen specifically binds to the Klebsiella pneumoniae O2 antigen target has a Kd value of 10 ⁇ 7 M to 10 ⁇ 13 M ( For example, 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, or 10 -10 M to 10 -12 M).
  • the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae and the O2 antigen of Klebsiella pneumoniae is 10 ⁇ 7 M to 10 ⁇ 13 M, 1 ⁇ 10 -7 M to 5 ⁇ 10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 - 9 M, 10 -8 M to 10 -13 M, 1 ⁇ 10 -8 M to 5 ⁇ 10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -13 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -12 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -11 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -10 M, 10 -9 M to 10 -13 M
  • the Kd value of the binding between an antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen and a non-target is higher than that between an antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen and
  • the Kd value of the target and in some embodiments cited in this application, the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen has a higher binding affinity to the target (for example, Klebsiella pneumoniae O2 antigen) than Binding affinity of antibodies or antigen-binding fragments that specifically bind the Klebsiella pneumoniae O2 antigen to non-targets.
  • non-target refers to non-Klebsiella pneumoniae O2 antigen.
  • the Kd value of an antibody or antigen-binding fragment that specifically binds to a Klebsiella pneumoniae O2 antigen for binding to a non-Klebsiella pneumoniae O2 antigen target is an antibody or an antigen-binding fragment that specifically binds to a Klebsiella pneumoniae O2 antigen.
  • the Kd of binding between the antigen-binding fragment and the target Klebsiella pneumoniae O2 antigen is at least 10 times, for example, 10-100 times, 100-1000 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 - 10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen specifically binds to the Klebsiella pneumoniae O1 antigen target has a Kd value of 10 ⁇ 7 M to 10 ⁇ 13 M ( For example, 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, or 10 -10 M to 10 -12 M).
  • the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen and the Klebsiella pneumoniae O1 antigen is 10 ⁇ 7 M to 10 ⁇ 13 M, 1 ⁇ 10 -7 M to 5 ⁇ 10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 - 9 M, 10 -8 M to 10 -13 M, 1 ⁇ 10 -8 M to 5 ⁇ 10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -13 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -12 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -11 M, 5 ⁇ 10 -9 M to 1 ⁇ 10 -10 M, 10 -9 M to 10 -13 M, 10
  • the Kd value of the binding between the antibody or the antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen and the non-target is higher than that between the antibody or the antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen
  • the Kd value of the target and in some embodiments cited in this application, the binding affinity of the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen to the target (for example, the Klebsiella pneumoniae O1 antigen) is higher than Binding affinity of antibodies or antigen-binding fragments that specifically bind the Klebsiella pneumoniae O1 antigen to non-targets.
  • non-target refers to non-Klebsiella pneumoniae O1 antigen.
  • the Kd value of the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen for non-Klebsiella pneumoniae O1 antigen target binding is the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen
  • the Kd of binding between the antigen-binding fragment and the target Klebsiella pneumoniae O1 antigen is at least 10 times, for example, 10-100 times, 100-1000 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 - 10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.
  • nucleic acid molecules encoding antibodies or antigen-binding fragments that specifically bind Klebsiella pneumoniae O2 antigens, antibodies or antigen-binding fragments that specifically bind Klebsiella pneumoniae O1 antigens, and bispecific antibodies.
  • one (or a group) encoding full-length antibody that specifically binds Klebsiella pneumoniae O2 antigen or an antibody that specifically binds Klebsiella pneumoniae O1 antigen or that specifically binds Klebsiella pneumoniae The nucleic acid of the bispecific antibody of Klebsiella pneumoniae O2 antigen and O1 antigen, including any full-length antibody specifically binding to Klebsiella pneumoniae O2 antigen described in this application, or the antibody specifically binding to Klebsiella pneumoniae O1 antigen Full-length antibodies or full-length bispecific antibodies that specifically bind to Klebsiella pneumoniae O2 antigen and O1 antigen.
  • the nucleic acid (or a set of nucleic acids) of the antibody or antigen-binding fragment or bispecific antibody described herein may also include a nucleic acid sequence encoding a polypeptide tag (such as a protein purification tag, His-tag, HA tag ).
  • a polypeptide tag such as a protein purification tag, His-tag, HA tag
  • the present application also considers antibodies that specifically bind to Klebsiella pneumoniae O2 antigen, antibodies that specifically bind to Klebsiella pneumoniae O1 antigen, or bispecific antibodies that specifically bind to Klebsiella pneumoniae O2 antigen and O1 antigen or a nucleic acid molecule encoding said antibody; or an isolated host cell comprising a vector carrying said nucleic acid molecule.
  • a variant includes a nucleotide sequence that hybridizes to a nucleic acid sequence encoding an antibody or antigen-binding fragment or bispecific antibody of the application at least under moderately stringent hybridization conditions.
  • the present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.
  • a natural or synthetic nucleic acid encoding an antibody or antigen-binding fragment or bispecific antibody is inserted into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, a promoter promoter (such as a lymphocyte-specific promoter) and 3' untranslated region (UTR), can express antibody or antigen-binding fragment or bispecific antibody.
  • the vectors are suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences and promoters to regulate the expression of the nucleic acid sequence of interest.
  • nucleic acids described herein can also be used in nucleic acid immunization and gene therapy by using standard gene delivery protocols. Methods of nucleic acid delivery are known in the art. See, eg, U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, the entire contents of which are incorporated herein by reference. In some embodiments, the present application also provides gene therapy vectors.
  • Nucleic acids can be cloned into many types of vectors.
  • nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector technology is well known in the art and described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), among other virology or molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • suitable vectors include an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus is then isolated and delivered to cells of the subject either in vivo or in vitro.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • Vectors derived from retroviruses are suitable tools for long-term gene transfer because they allow long-term stable integration of the transgene and propagation in progeny cells.
  • Lentiviral vectors have an additional advantage over tumor-derived retroviruses such as murine leukemia virus in that they can transduce non-dividing cells such as hepatocytes. At the same time, it has the added advantage of low immunogenicity.
  • promoter elements such as enhancers, regulate transcription initiation frequency. Typically they are located 30-110 bp upstream of the initiation site, although it has recently been discovered that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is usually flexible so that promoter function is maintained when elements are swapped or moved relative to each other. In the thymidine kinase (tk) promoter, activity does not begin to decline until the spacing between promoter elements increases to 50 bp.
  • tk thymidine kinase
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence, which can drive high-level expression of any polynucleotide sequence operably linked to it.
  • Another example of a suitable promoter is the elongation factor 1 alpha (EF-1 alpha) promoter.
  • constitutive promoters can also be used, including but not limited to, Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter promoters, MoMuLV promoters, avian leukemia virus promoters, Epstein-Barr virus immediate early promoters, Rous sarcoma virus promoters, and human gene promoters, such as but not limited to, actin promoter, myosin promoter promoter, hemoglobin promoter and creatine kinase promoter.
  • the present application should not be limited to the use of only constitutive promoters. Inducible promoters are also contemplated as part of this application.
  • an inducible promoter provides a molecular switch that turns on expression of a polynucleotide sequence to which it is operably linked when such expression is desired and turns off expression when it is not.
  • Inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
  • expression of the antibody or antigen-binding fragment or bispecific antibody is inducible.
  • the nucleic acid sequence encoding the antibody or antigen-binding fragment or bispecific antibody is operably linked to an inducible promoter, including any inducible promoter described herein.
  • an inducible promoter provides a molecular switch that turns on expression of a polynucleotide sequence to which it is operably linked when expression is desired and turns off expression when expression is not desired.
  • exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone regulatory elements (see, e.g., Mader, S. and White, J.H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607 ), synthetic ligand regulatory elements (seeing Spencer, D.M.et al (1993) Science 262:1019-1024), and ionizing radiation regulatory elements (seeing Manome, Y.et al.
  • the inducible promoter system used to express the antibody or antigen-binding fragment or bispecific antibody is the Tet system.
  • the inducible promoter system for expressing the antibody or antigen-binding fragment or bispecific antibody is the E. coli lac suppression system.
  • an exemplary inducible promoter system employed herein is the Tet system.
  • the system is based on the Tet system described by Gossen et al. (1993).
  • the polynucleotide of interest is controlled by a promoter comprising one or more Tet operator (TetO) sites.
  • TetO Tet operator
  • TetR Tet repressor
  • the activated state e.g., in the presence of inducers such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or their active analogs, the inducer releases TetR from TetO, resulting in transcription .
  • inducers such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or their active analogs
  • Doxycycline is a member of the tetracycline antibiotic family with the chemical name 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a , 11,11a,12,12a-Hexahydrotetraene-3-carboxamide.
  • TetR is codon optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, resulting in a large number of variants in the sequence of a given nucleic acid without any change in the amino acid sequence it encodes. However, many organisms differ in their codon usage, also known as "codon bias" (ie, the preference for a given amino acid to use a particular codon). Codon bias is often associated with the presence of dominant tRNA species for specific codons, which in turn increases the efficiency of mRNA translation. A coding sequence derived from a particular species (eg, prokaryotes) can thus be tailored by codon optimization to enhance its expression in a different species (eg, eukaryotes).
  • Tet-Off transcription is inactivated in the presence of Tc or Dox.
  • a tetracycline-regulated transcriptional activator consisting of TetR fused to the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of target nucleic acids under the transcriptional control of a tetracycline-responsive promoter element (TRE).
  • the TRE element consists of a TetO sequence fused in tandem to a promoter (usually a minimal promoter sequence derived from the human cytomegalovirus immediate early promoter).
  • Tc or Dox binds TRE and activates transcription of target genes.
  • tTA cannot bind TRE and target genes cannot be expressed.
  • rtTA is a fusion protein consisting of the TetR repressor and the VP16 transactivation domain.
  • a change of 4 amino acids in the DNA-binding region of TetR altered the binding properties of rtTA such that it could only recognize the tetO sequence on the target transgenic TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can activate the transcription of TRE-regulated target genes only in the presence of Dox.
  • lac repressor system of E. coli (see Brown et al., Cell 49:603-612 (1987)).
  • the Lac repressor system functions by regulating the transcription of a polynucleotide of interest operably linked to a promoter comprising the lac operator (lacO).
  • lacO lac operator
  • lacR lac repressor
  • lacR lacR
  • lacR lacR
  • Expression of the polynucleotide of interest is induced by a suitable inducer, for example, isopropyl- ⁇ -Dthiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -Dthiogalactopyranoside
  • the expression vector to be introduced into the cells may also contain a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells transfected or infected with the viral vector.
  • selectable markers can be carried on separate DNA fragments and used in co-transfection experiments. Either a selectable marker gene or a reporter gene can be flanked by appropriate regulatory sequences to enable its expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes can be used to identify potentially transfected cells and evaluate the function of regulatory sequences.
  • a reporter gene is a gene not present in or expressed by a recipient organism or tissue that encodes a polypeptide whose expression is manifested by some readily detectable property, such as enzymatic activity. After the DNA is introduced into the recipient cells, the expression of the reporter gene is detected at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tel et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are known and can be prepared by known techniques or obtained commercially.
  • the construct with the smallest 5' flanking region capable of showing the highest expression level of the reporter gene is considered a promoter.
  • Such a promoter region can be linked to a reporter gene and used to assess the ability of certain substances to regulate transcription driven by the promoter.
  • nucleic acid encoding any of the antibodies or antigen-binding fragments or bispecific antibodies described herein is provided.
  • the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the antibody or antigen-binding fragment or bispecific antibody.
  • each of the one or more nucleic acid sequences is contained in a separate vector.
  • at least some of the nucleic acid sequences are contained within the same vector.
  • all nucleic acid sequences are contained within the same vector.
  • Vectors may be selected, for example, from mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses).
  • mammalian expression vectors such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art.
  • expression vectors can be introduced into host cells by physical, chemical or biological methods.
  • polynucleotides are introduced into host cells by calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, among others. See, eg, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, magnetic beads and lipid-based systems including oil-in-water emulsions, micelles, mixed gels clumps and liposomes.
  • colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, magnetic beads and lipid-based systems including oil-in-water emulsions, micelles, mixed gels clumps and liposomes.
  • An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (eg, an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be bound to a lipid.
  • Lipid-bound nucleic acids can be encapsulated into the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules that bind liposomes and oligonucleotides, and include Buried in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, suspended in lipids, contained in or mixed with micelles , or otherwise bind to lipids.
  • Lipid, lipid/DNA or lipid/expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist in bilayer structures, in micelles or in "collapsed" structures.
  • Lipids are fatty substances, either naturally occurring or synthetic.
  • lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.
  • experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cell.
  • Such experiments include, for example, "molecular biology” experiments well known to those skilled in the art. For example, Southern and Northern blotting, RT-PCR and PCR; "biochemical” experiments, such as detecting the presence or absence of a specific polypeptide, such as by immunological methods (ELISAs and Western blots) or by the experiments described in this application. Identification is within the scope of this application.
  • the antibody or antigen-binding fragment (eg, an antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen or O1 antigen) is a monoclonal antibody.
  • the antibody or antigen-binding fragment or bispecific antibody is derived from a monoclonal antibody.
  • the antibody or antigen-binding fragment or bispecific antibody comprises the VH and VL from a monoclonal antibody, or a variant thereof.
  • said antibody or antigen-binding fragment or bispecific antibody further comprises CH1 and CL regions from a monoclonal antibody, or a variant thereof.
  • Monoclonal antibodies can be prepared using, for example, methods known in the art, including hybridoma cell methods, yeast display, phage display methods, or using recombinant DNA methods. Additionally, exemplary yeast display and phage display methods are described in this application and in the Examples below. Bispecific antibodies can be produced by methods known in the art, including chemical conjugation, hybridoma methods, and genetic engineering methods.
  • lymphocytes In the hybridoma method, hamsters, mice, or other suitable host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent.
  • lymphocytes can be immunized in vitro. Immunizing agents may include polypeptides or fusion proteins of the protein of interest.
  • PBLs peripheral blood lymphocytes
  • splenocytes or lymph node cells are used if cells of non-human mammalian origin are desired.
  • Lymphocytes are fused with an immortal cell line using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells.
  • Immortal cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Typically rat or mouse myeloma cell lines are used.
  • Hybridoma cells can be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells.
  • a suitable medium which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells.
  • the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT)
  • the hybridoma cell culture medium usually includes hypoxanthine, aminopterin, and thymidine (HAT medium), which can Prevents the growth of HGPRT-deficient cells.
  • the immortalized cell line fuses efficiently, ensures high level and stable expression of antibody by the selected antibody-producing cells, and is sensitive to certain medium, such as HAT medium.
  • the immortal cell line is a mouse myeloma cell line available from, eg, the Salk Cell Collection, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for the production of human monoclonal antibodies.
  • the medium in which the hybridoma cells are grown can then be assayed for the presence of monoclonal antibodies directed against the polypeptide.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art.
  • the binding affinity of monoclonal antibodies can be determined by Scatchard analysis as described, for example, in Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • hybridoma cells After the desired hybridoma cells have been identified, the clones of interest can be subcloned by limiting dilution and cultured by standard methods. Suitable media for this purpose include, for example, Modified Eagle's Medium (DMEM) and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in ascites in mammals.
  • DMEM Modified Eagle's Medium
  • RPMI-1640 medium RPMI-1640
  • Monoclonal antibodies secreted by subclones can be isolated or purified from culture medium or ascitic fluid by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • said antibody or antigen-binding fragment or bispecific antibody comprises an antibody selected from an antibody library (eg, a scFv or Fab fragment displaying phage library) cloned sequences.
  • an antibody library eg, a scFv or Fab fragment displaying phage library
  • Such clones can be identified by screening a combinatorial library of antibody fragments having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding properties.
  • the repertoires of the VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phage, as in Winter et al. ., Ann. Rev. Immunol., 12:433-455 (1994).
  • Phage typically display antibody fragments as scFv fragments or as Fab fragments.
  • Immune-derived library phages provide high-affinity antibodies against the immunogen without the need to construct hybridoma cells.
  • natural repertoires e.g.
  • the antibody or antigen-binding fragment or bispecific antibody is prepared by phage display screening the antigen-binding part of the library that can specifically bind to the target (eg, Klebsiella pneumoniae O2 antigen or O1 antigen).
  • the library can be a human scFv phage display library with at least 1 ⁇ 10 9 (e.g. at least 1 ⁇ 10 9 , 2.5 ⁇ 10 9 , 5 ⁇ 10 9 , 7.5 ⁇ 10 9 , 1 ⁇ 10 10 , 2.5 ⁇ 10 10 , 5 ⁇ 10 10 , 7.5 ⁇ 10 10 , or 1 ⁇ 10 11 ) diversity of unique human antibody fragments.
  • the library is a natural human library constructed from DNA extracted from PMBCs and spleens of healthy subjects, comprising all human heavy and light chain subfamilies.
  • the library is a human natural library constructed by DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients and patients with infectious diseases.
  • the library is a semi-synthetic human library in which the heavy chain CDR3s are completely randomized, with all amino acids (except cysteine) present at any given position with equal probability. (See, eg, Hoet, RM et al., Nat. Biotechnol. 23(3):344-348, 2005).
  • the semi-synthetic human library has a heavy chain CDR3 length of 5 to 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23 or 24) amino acids.
  • the library is a fully synthetic phage display library.
  • the library is a non-human phage display library.
  • Phage clones with high affinity for target antigens can be screened by iterative binding of phage to target antigens bound to a solid support (e.g., for solution panning beads or mammalian cells for cell panning), followed by removal of unbound phage and elution of specifically bound phage. Subsequently, bound phage clones are eluted and used to infect suitable host cells, such as E.coli XL1-Blue, for expression and purification.
  • target antigens e.g., Klebsiella pneumoniae O2 antigen or O1 antigen
  • a solid support e.g., for solution panning beads or mammalian cells for cell panning
  • bound phage clones are eluted and used to infect suitable host cells, such as E.coli XL1-Blue, for expression and purification.
  • Multiple rounds of panning eg, 2, 3, 4, 5, 6 or more rounds
  • solution panning cell panning, or a combination of both
  • Specific binding of the enriched phage clones to the target antigen can be detected by any method known in the art, including, for example, ELISA and FACS.
  • Monoclonal antibodies can also be prepared by recombinant DNA methods, such as described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies described in this application can be easily isolated and sequenced by conventional methods (eg, by oligonucleotide probes that can specifically bind to genes encoding the light and heavy chains of murine antibodies).
  • Hybridoma cells as described above or the antigen-specific phage clones of the present application can be used as a source of such DNA.
  • the DNA can be placed in an expression vector, which is then transfected into host cells, such as simian COS cells, Chinese hamster ovarian cancer (CHO) cells, or non-immunoglobulin-producing myeloma cells, to obtain Monoclonal antibodies synthesized in cells.
  • host cells such as simian COS cells, Chinese hamster ovarian cancer (CHO) cells, or non-immunoglobulin-producing myeloma cells.
  • the DNA may also be modified, for example, by substituting human heavy and light chain constant structure and/or framework region coding sequences for homologous non-human sequences (U.S. Patent No. 4,816,567; Morrison et al., supra), or by replacing non-human All or part of the coding sequence for an immunoglobulin polypeptide is covalently linked to the immunoglobulin coding sequence.
  • This non-immunoglobulin polypeptide can replace the constant region of the antibody of the present application, or can replace an antigen-binding site in the variable region of the antibody of the present application to form a chimeric bivalent antibody.
  • additional variable regions directed against different epitopes or antigens can be included to generate chimeric bispecific antibodies.
  • the antibody can be a monovalent antibody.
  • Methods of making monovalent antibodies are known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. Heavy chains are typically truncated anywhere in the Fc region to prevent cross-linking of the heavy chains to each other. Alternatively, the relevant cysteine residues are substituted with other amino acid residues or deleted to prevent cross-linking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.
  • Chemical Conjugation is the earliest technique used to prepare bispecific antibodies.
  • Brennan used the chemical combination of two monoclonal antibody G1 fragments to prepare bispecific antibodies for the first time (Brennan M, et al.Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments[J].Science,1985,229 (4708):81-83).
  • the advantages of the chemical coupling method are fast, easy to operate, and high recovery rate, but it is also easy to destroy the antigen-binding domain of the antibody, affect the activity of the antibody, and easily form multimers.
  • hybridoma cell lines to prepare bispecific antibodies refers to the fusion of two different hybridoma cell lines through cell fusion technology, and then the identification and isolation of cells that can produce specific therapeutic antibodies (Kohler, G. , et al.Continuous cultures of fused cells secreting antibody of predefined specificity[J].J Immunol.,2005,174(5):2453-2455). Because two hybridoma cells can produce two different light-heavy chains, and these light-heavy chains can be randomly combined, the bispecific antibody prepared by this method has greater randomness and low production efficiency.
  • KiH KiH into hole
  • CrossMab Two common techniques currently being applied to improve the light-heavy chain pairing problem. KiH technology introduces an asymmetric mutation structure in the CH3 domain (“knob” mutation refers to replacing a smaller residue with a large amino acid residue in the CH3 domain, while “hole” mutation refers to Use smaller amino acid residues to replace larger residues).
  • the Fc region of the engineered bispecific antibody is more prone to heterodimerization than homodimerization due to steric hindrance (Ridgway J B, et al. "Knobs-into-holes" engineering of antibody CH 3 domains for heavy chain heterodimerization [J]. Protein Eng. 1996, 9(7):617-621).
  • the introduction of a Y349C mutation in the glycosylated CH3 domain can form a disulfide bond between the glycosylated heavy chains and enhance the stability of KiH (Kuglstatter A, et al.
  • charge effects of amino acid residues have also been used to enhance heterodimerization between the two heavy chains of bispecific antibodies.
  • charge effects of amino acid residues have also been used to enhance heterodimerization between the two heavy chains of bispecific antibodies.
  • Mutations of the two chains, K409D and D399K, K409D/K392D and D399K/E356K, or E356K/E357K/D399K and K370E/K409D/K439E, etc. can increase heterodimer formation to a certain extent (IGAWA T, et al .Methods for producing polypeptides by regulating polypeptide; association: US, 20100015133A1[P].2006). Combining KiH steric hindrance effect and charge effect is also one of the strategies to improve heterodimerization.
  • CrossMab technology is a new antibody pairing technology developed by Roche based on KiH technology, by swapping the light chain and heavy chain of one Fab of a bispecific antibody, while the other does not.
  • the exchanged light chain will contain a part of the homologous heavy chain fragment, so that it cannot pair with the unexchanged heavy chain, thus ensuring the correct combination between the light chain and the heavy chain (Schaefer W, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies[J].Proc Natl Acad Sci USA, 2011,108(27):11187-11192).
  • Antibody variable regions with the desired binding specificity can be fused to immunoglobulin constant regions.
  • the fusion is preferably to an immunoglobulin heavy chain constant region, which includes at least part of the hinge, CH2 and CH3 domains.
  • the CH1 domain of the heavy chain constant region comprising the site necessary for light chain binding is present in at least one fusion.
  • DNA encoding the fusion of the immunoglobulin heavy chain and, if desired, the DNA encoding the light chain of the immunoglobulin is inserted into a separate expression vector and co-transfected into a suitable host organism.
  • antibody variable regions directed against different epitopes or different antigens can be fused to immunoglobulin constant region sequences to generate chimeric bispecific antibodies.
  • the antibody or antigen-binding fragment or bispecific antibody may be a humanized antibody or a fully human antibody.
  • Humanized forms of non-human (e.g., mouse) antibody portions are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab') 2 , scFv, or other fragments of antibodies).
  • Antigen-binding subsequences which generally include minimal sequence derived from non-human immunoglobulins.
  • Humanized antibodies include human immunoglobulins, immunoglobulin chains or fragments thereof (recipient antibodies) in which residues from the recipient CDRs are replaced by non-human (donor antibody) CDRs having the desired specificity, affinity and properties.
  • Residue substitutions such as mouse, rat or rabbit CDRs.
  • human immunoglobulin Fv framework region residues are substituted by corresponding non-human residues.
  • Humanized antibodies may also comprise amino acid residues that are found neither in the recipient antibody nor in imported CDR or framework region sequences.
  • a humanized antibody will comprise at least one, and usually two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are human immunoglobulin consensus sequences .
  • humanized antibodies typically contain one or more amino acid residues that have been introduced from a non-human source. Those non-human amino acid residues are often referred to as "imported” residues, usually from an "imported” variable region.
  • humanization can basically be carried out according to the following method of Winter and its colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 ( 1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of human antibodies.
  • such "humanized” antibody portions U.S. Patent No.
  • a humanized antibody portion is that portion of a typically human antibody in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.
  • Producing human antibody portions is an alternative to humanization.
  • transgenic animals eg, mice
  • mice that are capable of producing a fully human antibody library upon immunization but not endogenous immunoglobulins are currently produced.
  • JH antibody heavy-chain joining region
  • Fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (eg, mice in which endogenous immunoglobulin genes have been partially or fully silenced). After antigen stimulation, it can be found that the production of fully human antibodies is very similar to that in humans in all aspects, including gene rearrangement, assembly and antibody library. This method is described, for example, in U.S. Patent Nos.
  • Human antibodies or human antibody portions can also be produced by in vitro activation of B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991); Marks et al., J.Mol.Biol., 222:581 (1991). Techniques of Cole et al. and Boerner et al. It can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991).
  • the antibody or antigen-binding fragment provided by the application for example, an antibody that specifically binds Klebsiella pneumoniae O2 antigen or O1 antigen, or a double antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen Amino acid sequences of variants of specific antibodies
  • an antibody that specifically binds Klebsiella pneumoniae O2 antigen or O1 antigen or a double antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen
  • Amino acid sequences of variants of specific antibodies are also contemplated.
  • the amino acid sequences of antigen-binding entity variants can be prepared by introducing appropriate modifications in the nucleotide sequence encoding the antigen-binding entity or by peptide synthesis.
  • Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antigen-binding entity.
  • the final construction can be accomplished by any combination of amino acid residue deletions, insertions, and substitutions to impart the desired characteristics. For example, antigen binding.
  • variants of antibodies or antigen-binding fragments having one or more amino acid substitutions are provided.
  • Target sites for substitution mutations include hypervariable regions (HVRs) and framework regions (FRs).
  • Amino acid substitutions can be introduced in the target antibody to screen for products with desired activity, for example, improved biological activity, maintained/improved antigen binding ability, reduced immunogenicity, reduced pathogen binding or improved opsonophagocytic killing of pathogens (OPK ), for example, Klebsiella pneumoniae.
  • the amino acid substitutions described herein are limited to the "exemplary substitutions" in Table 10 of this application.
  • the amino acid substitutions are limited to the "preferred substitutions" in Table 10 of the present application.
  • Amino acids are divided into different classes according to their side chain properties:
  • Acidic amino acids aspartic acid Asp, glutamic acid Glu;
  • Aromatic amino acids tryptophan Trp, tyrosine Tyr, phenylalanine Phe.
  • Substitution of non-conservative amino acids involves substitution of one of the above classes for another class.
  • An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated, variant antibody portions are displayed on phage, and variants are screened for specific biological activity (eg, based on RBC cell lysis inhibition assay or binding affinity). Alterations (eg, substitutions) can be made in regions of the HVRs to achieve improved RBC lysis-based inhibition assays or antibody affinity. Alterations can be made in HVR "hotspots", i.e. residues encoded by codons that are highly mutated during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol.
  • variable genes selected for affinity maturation are introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis) . Secondary libraries are then created. This library is screened to identify antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-mediated approach, in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Often the CDR-H3 and CDR-L3 regions in particular are key targets.
  • HVR-mediated approach in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Often the CDR-H3 and CDR-L3 regions in particular are key targets.
  • substitutions, insertions, or deletions may occur within one or more of the HVRs, so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes eg, conservative substitutions provided herein
  • HVRs that do not substantially reduce binding affinity. These changes may occur outside of HVR "hot spots" or SDRs.
  • each HVR is either unchanged or comprises no more than 1, 2 or 3 amino acid substitutions.
  • alanine scanning mutagenesis One method by mutagenesis for identifying amino acids or regions of an antibody capable of binding a target is called “alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085.
  • target residues e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid
  • neutral or negatively charged amino acids e.g., , alanine or glutamic acid
  • Further substitutions can be introduced at the amino acid position to demonstrate that the position is functionally sensitive to the initial substitution.
  • the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants are screened to determine whether they possess the desired properties.
  • Amino acid sequence insertions including amino-terminal and/or carboxy-terminal fusions, ranging in length from 1 residue to polypeptides containing 100 or more residues, also include intrasequence insertions of 1 or more amino acid residues base.
  • terminal insertions include antigen-binding moieties having a methionyl residue at the N-terminus.
  • Other insertional variants of the antigen-binding moiety include fusions to the N- or C-terminus of the antigen-binding moiety with an enzyme (eg, ADEPT) or a polypeptide that increases the serum half-life of the antigen-binding moiety.
  • one or more amino acid modifications are introduced into an antibody or antigen-binding fragment described herein (e.g., a full-length antibody that specifically binds Klebsiella pneumoniae O2 antigen, a full-length antibody that specifically binds Klebsiella pneumoniae A full-length antibody to O1 antigen, a full-length bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen, or a fusion protein comprising said antibody or antigen-binding fragment or bispecific antibody), Fc variants are thereby generated.
  • the Fc variants have enhanced ADCC potency, generally associated with Fc-binding receptors (FcRs).
  • the Fc variant has reduced ADCC potency.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • a specific antigen-binding moiety eg, an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or Bispecific antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen
  • NK cells activated by antibodies. NK cells express the Fc receptor CD16.
  • This receptor recognizes and binds the Fc portion of an antibody molecule bound to the surface of a target cell.
  • the most common Fc receptors on the surface of NK cells are CD16 or Fc ⁇ RIII. Binding of Fc receptors to the Fc region of an antibody results in activation of NK cells, release of cytolytic granules, and subsequent apoptosis of target cells.
  • the present application also provides an antibody specifically binding to Klebsiella pneumoniae O2 antigen, an antibody specifically binding to Klebsiella pneumoniae O1 antigen, or an antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen Bispecific antibody variants (e.g., full-length antibodies that specifically bind K. pneumoniae O2 antigen, full-length antibodies that specifically bind K. pneumoniae O1 antigen, or that specifically bind K.
  • pneumoniae O2 antigen and O1 A variant of a full-length bispecific antibody to an antigen) that contains an Fc region with one or more effector functions, which makes it an ideal candidate antibody for applications that specifically bind to the Klebsiella pneumoniae O2 antigen
  • the in vivo half-life of antibodies, antibodies that specifically bind K. pneumoniae O1 antigen, or bispecific antibodies that specifically bind K. pneumoniae O2 ) is unnecessary or harmful.
  • the reduction/elimination of CDC and/or ADCC activity is confirmed by performing in vitro and/or in vivo cytotoxicity assays.
  • Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks Fc ⁇ R binding (and thus likely lacks ADCC activity), but retains FcRn binding ability.
  • NK cells express only Fc ⁇ RIII
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assessment of ADCC activity of target molecules are described in US Pat. )) and Hellstrom, I et al., Proc. Nat'l Acad. Sci.
  • nonradioactive assays can be used (see, e.g., the ACTI TM Flow Cytometry Nonradioactive Cytotoxicity Assay (Cell Technology, Inc. Mountain View, Calif.) and the CYTOTOX 96 TM Nonradioactive Cytotoxicity Assay (Promega, Madison, Wis. )). Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK).
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • the ADCC activity of the molecule of interest is tested in vivo, eg, in an animal model as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).
  • a C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q, thus lacking CDC activity. See, eg, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402.
  • a CDC assay can be performed (see, eg, Gazzano-Santoro et al., J. Immunol.
  • Antibodies with reduced effector function include substitutions of one or more residues at residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Pat. No. 6,737,056). These Fc variants include Fc variants with substitutions of two or more residues at positions 265, 269, 270, 297, and 327, including Fc variants known as "DANA", which have residues at positions 265 and 297 The base is substituted with alanine (U.S. Pat. No. 7,332,581).
  • alterations in the Fc region result in altered (ie, enhanced or decreased) opsonization, as described in Moore et al., MAbs. 2(2):181-189 (2010).
  • an antibody specifically binding to Klebsiella pneumoniae O2 antigen, an antibody specifically binding to Klebsiella pneumoniae O1 antigen or a double antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen is provided.
  • Specific antibodies (such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigens, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigens, or full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigens and O1 antigens Long bispecific antibody) variants comprising Fc variants with one or more amino acid substitutions capable of increased half-life and/or enhanced binding to Fc receptors (FcRn).
  • Antibodies with extended half-life and improved FcRn binding are described in US2005/0014934A1 (Hinton et al.). These antibody Fc regions contain one or more amino acid substitutions that enhance the binding of the Fc region to FcRn.
  • These Fc variants comprise 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or One or more substitutions in residue 434, eg, substitution of residue 434 in the Fc region (U.S. Pat. No. 7,371,826).
  • Antibodies that specifically bind to the Klebsiella pneumoniae O2 antigen, antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen, or antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen comprising any of the Fc variants described in the present application or combinations thereof are also contemplated.
  • Bispecific antibodies to O2 antigen and O1 antigen of Klebsiella pneumoniae (such as full-length antibodies that specifically bind to Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind to Klebsiella pneumoniae O1 antigen, or antibodies that specifically bind to Klebsiella pneumoniae O1 antigen full-length bispecific antibody to the O2 and O1 antigens of Lebsiella).
  • the antibody specifically binding to Klebsiella pneumoniae O2 antigen provided by the application, the antibody specifically binding to Klebsiella pneumoniae O1 antigen or the antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen
  • Bispecific antibodies such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigens, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigens, or that specifically bind Klebsiella pneumoniae O2 antigens and O1 antigens full-length bispecific antibody
  • the antibody that specifically binds to Klebsiella pneumoniae O2 antigen By changing the antibody that specifically binds to Klebsiella pneumoniae O2 antigen, the antibody that specifically binds to Klebsiella pneumoniae O1 antigen, or the bispecific antibody or its polypeptide part that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen
  • the amino acid sequence can be used to add or remove one or more glycosylation sites, which can easily add or delete antibodies that specifically bind to the Klebsiella pneumoniae O2 antigen and antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen.
  • the glycosylation site on the antibody or the bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen The glycosylation site on the antibody or the bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen.
  • the antibody specifically binding to Klebsiella pneumoniae O2 antigen, the antibody specifically binding to Klebsiella pneumoniae O1 antigen or the bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen contains an Fc region, which can be Change the sugar attached to it.
  • Native antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, usually N-linked to the Fc region CH2 domain Asn297, see e.g. Wright et al., TIBTECH 15:26-32( 1997).
  • the oligosaccharides may comprise a variety of sugars such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • sugars such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • the antibody that specifically binds Klebsiella pneumoniae O2 antigen, the antibody that specifically binds Klebsiella pneumoniae O1 antigen or the antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen of the present application can be Bispecific antibodies undergo oligosaccharide modification to generate antibodies that specifically bind Klebsiella pneumoniae O2 antigen, antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or antibodies that specifically bind Klebsiella pneumoniae with certain improved properties Bispecific antibody variants of bacterial O2 antigen and O1 antigen.
  • N-glycans linked to the CH2 domain of the Fc region are heterogeneous.
  • Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity, see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83.
  • a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum.
  • N-glycosylation of the Fc region is important for its binding to Fc ⁇ Rs; non-fucosylated N-glycans enhance the binding ability of Fc to Fc ⁇ RIIIa.
  • Enhanced binding to Fc ⁇ RIIIa results in enhanced ADCC effect, which is advantageous in certain antibody therapeutic applications where cytotoxicity is required.
  • enhanced effector functions may be detrimental when Fc-mediated cytotoxicity is not desired.
  • the Fc fragment or CH2 domain is aglycosylated.
  • glycosylation is prevented by mutating the N-glycosylation site in the CH2 domain.
  • antibodies specifically binding to Klebsiella pneumoniae O2 antigens, antibodies specifically binding to Klebsiella pneumoniae O1 antigens, or bispecific antibodies specifically binding to Klebsiella pneumoniae O2 antigens and O1 antigens are provided.
  • Antibodies (such as full-length antibodies that specifically bind to Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind to Klebsiella pneumoniae O1 antigen, or full-length double antibodies that specifically bind to Klebsiella pneumoniae O2 antigen and O1 antigen Specific antibody) variants comprising an Fc region wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may enhance ADCC function.
  • the present application provides an antibody specifically binding to Klebsiella pneumoniae O2 antigen, an antibody specifically binding to Klebsiella pneumoniae O1 antigen or a bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen Variant relative to the same antibody that specifically binds Klebsiella pneumoniae O2 antigen, antibody that specifically binds Klebsiella pneumoniae O1 antigen, or that specifically binds Klebsiella pneumoniae O2 antigen produced by wild-type CHO cells
  • the bispecific antibody to the O1 antigen has reduced fucose.
  • the antibodies are characterized by having a lower amount of fucose than antibodies produced by native CHO cells (eg, CHO cells producing native glycosylated forms, CHO cells containing the native FUT8 gene).
  • native CHO cells eg, CHO cells producing native glycosylated forms, CHO cells containing the native FUT8 gene.
  • the N-linked glycans of the antibody have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose.
  • the antibody that specifically binds to the Klebsiella pneumoniae O2 antigen may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%.
  • the antibody specifically binding to Klebsiella pneumoniae O2 antigen, the antibody specifically binding to Klebsiella pneumoniae O1 antigen, or the bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen N-linked glycans of antibodies that do not contain fucose i.e., antibodies that specifically bind Klebsiella pneumoniae O2 antigen, antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or antibodies that specifically bind Klebsiella pneumoniae
  • the bispecific antibody against bacterial O2 antigen and O1 antigen does not contain fucose at all, or has no fucose or is fucosylated.
  • the content of fucose was calculated by calculating the average content of fucose in the sugar chain attached to Asn297 relative to the total of all sugar structures (such as complex, hybrid or mannose structures) attached to Asn297 measured by MALDI-TOF mass spectrometry. Quantities are determined as described in WO 2008/077546. Asn297 refers to the asparagine residue located at position 297 of the Fc region (EU Fc region residue numbering system). However, Asn297 can also be located ⁇ 3 amino acids upstream or downstream of position 297, ie between positions 294 and 300, due to minor sequence variations of the antibody. These fucosylated variants may have enhanced ADCC function. See, eg, US Patent Publication Nos.
  • Cell lines capable of producing afucosylated antibodies include Lec13CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003 /0157108A1, Presta, L; and WO 2004/056312A1, Adams et al., especially Example 11), and gene knockout cell lines, such as ⁇ -1,6-fucosyltransferase gene, FUT8 gene knockout Removed CHO cells (see Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO2003 /085107).
  • the full-length antibody of Klebsiella pneumoniae O2 antigen, the full-length antibody specifically binding to Klebsiella pneumoniae O1 antigen or the full-length bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen) variant is further Provides bisected oligosaccharides, e.g., linked to an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or that specifically binds Klebsiella pneumoniae O2 antigen and O1
  • the biantennary oligosaccharides of the bispecific antibody Fc region of the antigen are
  • Such antibodies that specifically bind to Klebsiella pneumoniae O2 antigen, antibodies that specifically bind to Klebsiella pneumoniae O1 antigen, or bispecific antibodies that specifically bind to Klebsiella pneumoniae O2 antigen and O1 antigen (such as specific Full-length antibody binding to Klebsiella pneumoniae O2 antigen, full-length antibody specifically binding to Klebsiella pneumoniae O1 antigen or full-length bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen)
  • the body may have reduced fucosylation and/or enhanced ADCC function. Examples of such antibody variants are in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.
  • antibodies specifically binding to the Klebsiella pneumoniae O2 antigen antibodies specifically binding to the Klebsiella pneumoniae O1 antigen, or bispecific antibodies specifically binding to the Klebsiella pneumoniae O2 antigen and the O1 antigen (e.g., specific Full-length antibody binding to Klebsiella pneumoniae O2 antigen, full-length antibody specifically binding to Klebsiella pneumoniae O1 antigen or full-length bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen)
  • a body having at least one galactose residue in the oligosaccharide linked to the Fc region is provided.
  • Such antibodies that specifically bind Klebsiella pneumoniae O2 antigen, antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or bispecific antibody variants that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen may have Enhanced CDC function.
  • Such variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • Antibodies (such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen Bispecific antibody) variants comprise an Fc region capable of binding Fc ⁇ RIII.
  • the antibody that specifically binds Klebsiella pneumoniae O2 antigen, the antibody that specifically binds Klebsiella pneumoniae O1 antigen or the antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 comprising an Fc region
  • Bispecific antibodies to antigens e.g., full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or that specifically bind Klebsiella pneumoniae O2 antigen and O1
  • variants have ADCC activity in the presence of human effector cells (such as T cells), or with other antibodies that specifically bind Klebsiella pneumoniae O2 antigen with human wild-type IgG1 Fc region
  • Antibodies that specifically bind Klebsiella pneumoniae O1 antigen or bispecific antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen such as full-length antibodies that specifically
  • cysteine-engineered antibodies that specifically bind to Klebsiella pneumoniae O2 antigen, antibodies that specifically bind to Klebsiella pneumoniae O1 antigen, or antibodies that specifically bind to Klebsiella pneumoniae O2 Antigen and bispecific antibody to O1 antigen (such as a full-length antibody that specifically binds to Klebsiella pneumoniae O2 antigen, a full-length antibody that specifically binds to Klebsiella pneumoniae O1 antigen, or a full-length antibody that specifically binds to Klebsiella pneumoniae O2 antigen Antigen and a full-length bispecific antibody to the O1 antigen) in which one or more amino acid residues are substituted by cysteine residues.
  • O1 antigen such as a full-length antibody that specifically binds to Klebsiella pneumoniae O2 antigen, a full-length antibody that specifically binds to Klebsiella pneumoniae O1 antigen, or a full-length antibody that specifically binds to Klebsi
  • the substituted residue occurs in an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or an antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen The accessible sites of bispecific antibodies.
  • reactive sulfhydryl groups are located on antibodies that specifically bind Klebsiella pneumoniae O2 antigen, antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or antibodies that specifically bind Klebsiella pneumoniae O1 antigen
  • the accessible site of the bispecific antibody of O2 antigen and O1 antigen of Klebsiella pneumoniae can be used for the antibody that specifically binds to the O2 antigen of Klebsiella pneumoniae, the antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae, or the specific Bispecific antibodies that specifically bind Klebsiella pneumoniae O2 antigens and O1 antigens are coupled with other moieties, such as drug moieties or linker-drug moieties, to prepare specific binding Klebsiella pneumoniae O2 antibodies as further described in this application.
  • Antibodies e.g., full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen Bispecific antibodies
  • the bispecific antibody provided by the application specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen (for example, a full-length bispecific antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen Antibodies) can be further modified to include other non-protein moieties known in the art and readily available.
  • Moieties suitable for derivatizing bispecific antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-Dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof.
  • PEG polyethylene glycol
  • ethylene glycol/propylene glycol copolymers carboxymethylcellulose
  • dextran polyvinyl alcohol
  • polyvinylpyrrolidone poly-1, 3-Dioxolane
  • Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water.
  • the polymers can be of any molecular weight and can be branched or unbranched.
  • the number of polymers attached to the bispecific antibody that specifically binds K. pneumoniae O2 antigen and O1 antigen can vary, and if more than one polymer is attached, they can be the same or different molecules.
  • the amount and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the need to improve the properties of bispecific antibodies that specifically bind Klebsiella pneumoniae O2 and O1 antigens Or function, whether the bispecific antibody derivatives that specifically bind to Klebsiella pneumoniae O2 antigen and O1 antigen are used for treatment under specific conditions, etc.
  • the present application also provides an antibody that specifically binds to Klebsiella pneumoniae O2 antigen, an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, or a bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen.
  • Antibodies such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen bispecific antibody), nucleic acid encoding an antibody, a vector comprising a nucleic acid encoding an antibody, or a composition (eg, a pharmaceutical composition, also referred to herein as a formulation) of a host cell comprising a nucleic acid or vector as described herein.
  • a composition eg, a pharmaceutical composition, also referred to herein as a formulation
  • a pharmaceutical composition comprising any antibody specifically binding to the O2 antigen of Klebsiella pneumoniae, an antibody specifically binding to the O1 antigen of Klebsiella pneumoniae, or a specific antibody described in this application.
  • a bispecific antibody combined with Klebsiella pneumoniae O2 antigen and O1 antigen and a pharmaceutically acceptable carrier.
  • Antibodies that specifically bind to the O2 antigen of Klebsiella pneumoniae, antibodies that specifically bind to the O1 antigen of Klebsiella pneumoniae, or bispecific antibodies that specifically bind to the O2 and O1 antigens of Klebsiella pneumoniae can be mixed with the desired purity.
  • Antibodies and optional pharmaceutically acceptable carriers, excipients or stabilizers Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed.
  • the antibody, the antibody specifically binding to Klebsiella pneumoniae O1 antigen or the bispecific antibody preparation specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen is prepared in the form of freeze-dried preparation or liquid preparation.
  • Acceptable carriers, excipients, or stabilizers that are nontoxic to recipients at the dosages and concentrations employed include buffers such as: phosphates, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives such as Octadecyldimethylbenzylammonium chloride; Hexamethylammonium chloride; Benzalkonium chloride; Benzethonium chloride; Phenol; Butanol or benzyl alcohol; Alkylparabens such as p-hydroxybenzoate Methyl formate or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins such as serum Albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine,
  • Lyophilized formulations suitable for subcutaneous administration are described in WO97/04801. Such freeze-dried preparations can be reconstituted into high-protein-concentration preparations with appropriate diluents, and the reconstituted preparations can be administered to individuals to be treated in this application through subcutaneous administration.
  • Cationic liposomes or liposomes can be used to specifically bind the antibody of the present application to the Klebsiella pneumoniae O2 antigen, the antibody specifically to the Klebsiella pneumoniae O1 antigen or the specificity to the Klebsiella pneumoniae Bispecific antibodies to O2 antigen and O1 antigen are delivered to cells.
  • the preparation described in the present application includes an antibody specifically binding to Klebsiella pneumoniae O2 antigen, an antibody specifically binding to Klebsiella pneumoniae O1 antigen or a bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen Antibodies (such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen In addition to bispecific antibodies), it may also contain one or more other active substances necessary for the treatment of specific diseases, preferably substances with complementary activities and no adverse reactions to each other.
  • an antibody that specifically binds Klebsiella pneumoniae O2 antigen in addition to an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or a bispecific antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen, It may be desirable to further include other therapeutically active substances, eg antibiotics. These molecules are present in combination in amounts effective for the intended purpose.
  • the effective amount of other active substances depends on the antibody that specifically binds to Klebsiella pneumoniae O2 antigen, the antibody that specifically binds to Klebsiella pneumoniae O1 antigen or the antibody that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen in the preparation
  • These drugs are generally used at the same doses and routes of administration as described in this application, or at 1% to 99% of the currently used doses.
  • the antibody specifically binding to Klebsiella pneumoniae O2 antigen, the antibody specifically binding to Klebsiella pneumoniae O1 antigen or the bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen (for example, specific full-length antibody that specifically binds to the O2 antigen of Klebsiella pneumoniae, a full-length antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae, or a full-length bispecific antibody that specifically binds to the O2 and O1 antigens of Klebsiella pneumoniae)
  • microcapsules prepared for example by coacervation techniques and interfacial polymerization, for example in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively Hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules.
  • Sustained release formulations can be prepared
  • Antibodies specifically binding to Klebsiella pneumoniae O2 antigens antibodies specifically binding to Klebsiella pneumoniae O1 antigens, or bispecific antibodies specifically binding to Klebsiella pneumoniae O2 antigens and O1 antigens (for example, specific full-length antibody that specifically binds to the O2 antigen of Klebsiella pneumoniae, a full-length antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae, or a full-length bispecific antibody that specifically binds to the O2 and O1 antigens of Klebsiella pneumoniae) sustained-release preparations.
  • bispecific antibodies specifically binding to Klebsiella pneumoniae O2 antigens and O1 antigens for example, specific full-length antibody that specifically binds to the O2 antigen of Klebsiella pneumoniae, a full-length antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae, or a full-length bispecific antibody that specifically binds to the O2 and O
  • sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody (or fragment thereof) in the form of shaped articles, eg, films or microcapsules.
  • sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No.
  • L-glutamic acid And L-glutamate ethyl ester copolymer non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer such as LUPRON DEPOT TM (composed of lactic acid-glycolic acid copolymer and leuprolide acetate can be injection microspheres) and poly-D(-)-3-hydroxybutyrate. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, certain hydrogels can release proteins for a shorter period of time.
  • the mechanism of aggregation is found to be intermolecular SS bond formation through thiodisulfide exchange, it may be possible to modify sulfhydryl residues, lyophilize in acidic solutions, control water content, use appropriate additives, and develop specific polymers. matrix composition for stabilization.
  • Antibodies (such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigen, or full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen Bispecific antibodies) are formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof.
  • an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or a bispecific antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen Formulated in a buffer containing about 50 mM to about 100 mM NaCl.
  • an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or a bispecific antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen Formulated in a buffer with a pH between 5.1 and 5.6.
  • an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or a bispecific antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen Formulated in a buffer containing 10 mM citric acid, 100 mM NaCl, 100 mM glycine and 0.01% polysorbate 80, wherein the pH of the formulation was 5.5.
  • Preparations for in vivo administration must be sterile. This is readily accomplished, for example, by filtration using sterile filtration membranes.
  • a method of preventing or treating Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount comprising any one of the compounds described herein that specifically binds Klebsiella pneumoniae Antibodies to O2 antigens, and/or antibodies specifically binding to Klebsiella pneumoniae O1 antigens, and/or bispecific antibodies specifically binding to Klebsiella pneumoniae O2 antigens and O1 antigens, and/or comprising any of the The composition of the bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen, and/or comprising an antibody specifically binding to Klebsiella pneumoniae O2 antigen and specifically binding to Klebsiella pneumoniae O1 antigen Antibody composition.
  • the method of treating a Klebsiella pneumoniae infection further provides a therapeutic or prophylactic effect on a disease and/or condition associated with a Klebsiella pneumoniae infection.
  • a method of preventing Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of a composition comprising any bispecific antibody or pharmaceutical composition described herein.
  • any bispecific antibody or pharmaceutical composition as described above in the preparation of a medicament for treating a disease or disorder.
  • Diseases and/or conditions associated with Klebsiella pneumoniae infection include but are not limited to pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis, diarrhea, soft tissue infection , infection after organ transplantation, surgical infection, wound infection, pulmonary infection, suppurative liver abscess, lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis , ankylosing spondylitis or spondyloarthropathies.
  • the method of preventing or treating Klebsiella pneumoniae infection reduces mortality from Klebsiella pneumoniae infection.
  • a method of preventing or treating Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of a bispecific drug that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen.
  • Antibodies or compositions comprising said bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen and a second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen
  • the antigen-binding domain, wherein the first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: V H , and the V H comprises: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, It comprises the amino acid sequence of SEQ ID NO: 4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 6; and V L comprising: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO: 11, LC - CDR2 comprising the amino acid sequence of SEQ ID NO:14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.
  • the first antigen-binding domain that specifically binds to Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 19 or 28, and V L comprising the amino acid sequence of SEQ ID NO: 19 or 28 ID NO: 24 or 33.
  • the first antigen-binding domain that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH comprising HC in VH having an amino acid sequence of SEQ ID NO: 19 or 28 - CDR1, HC-CDR2 and HC-CDR3; and a VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a VL having the amino acid sequence of SEQ ID NO: 24 or 33.
  • the first antigen-binding domain of the bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising an amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:7; and V L comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:15.
  • the first antigen-binding domain of the bispecific antibody that specifically binds the Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 20 or 29, and V L , It comprises the amino acid sequence of SEQ ID NO:25 or 34.
  • the first antigen-binding domain of the bispecific antibody that specifically binds the O2 antigen of Klebsiella pneumoniae comprises: VH , the VH comprising an amino acid sequence of SEQ ID NO: 20 or 29 HC-CDR1, HC-CDR2, and HC-CDR3 in a VH ; and a VL comprising LC-CDR1, LC-CDR2, and LC-CDR1 in a VL having an amino acid sequence of SEQ ID NO: 25 or 34.
  • VH the VH comprising an amino acid sequence of SEQ ID NO: 20 or 29 HC-CDR1, HC-CDR2, and HC-CDR3 in a VH
  • a VL comprising LC-CDR1, LC-CDR2, and LC-CDR1 in a VL having an amino acid sequence of SEQ ID NO: 25 or 34.
  • the second antigen-binding domain of the bispecific antibody specifically binding to Klebsiella pneumoniae O1 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising an amino acid sequence SEQ ID NO:35, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:37 , and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:39; and V L comprising: LC-CDR1 of Comprising the amino acid sequence of SEQ ID NO:41, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:45.
  • the second antigen-binding domain of the bispecific antibody that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 47 or 51, and V L , It comprises the amino acid sequence of SEQ ID NO:49 or 53.
  • the second antigen-binding domain that specifically binds Klebsiella pneumoniae O1 in the bispecific antibody comprises: V H , and the V H comprises a V having an amino acid sequence of SEQ ID NO: 47 or 51 HC-CDR1, HC-CDR2 and HC-CDR3 in H ; and VL , said VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 in VL having the amino acid sequence SEQ ID NO: 49 or 53 .
  • the second antigen-binding domain of the bispecific antibody specifically binding to Klebsiella pneumoniae O1 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising an amino acid sequence SEQ ID NO:36, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:38 , and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; and V L comprising: LC-CDR1 of Comprising the amino acid sequence of SEQ ID NO:42, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:44, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46.
  • the second antigen-binding domain of the bispecific antibody that specifically binds to the Klebsiella pneumoniae O1 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 48 or 52, and V L , It comprises the amino acid sequence of SEQ ID NO:50 or 54.
  • the second antigen-binding domain that specifically binds to the Klebsiella pneumoniae O1 antigen in the bispecific antibody comprises: V H , and the V H comprises an amino acid sequence of SEQ ID NO: 48 or 52 HC-CDR1, HC-CDR2, and HC-CDR3 in a VH ; and a VL comprising LC-CDR1, LC-CDR2, and LC-CDR1 in a VL having the amino acid sequence of SEQ ID NO: 50 or 54.
  • V H comprises an amino acid sequence of SEQ ID NO: 48 or 52 HC-CDR1, HC-CDR2, and HC-CDR3 in a VH ; and a VL comprising LC-CDR1, LC-CDR2, and LC-CDR1 in a VL having the amino acid sequence of SEQ ID NO: 50 or 54.
  • a method of preventing or treating a Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of a compound that specifically binds to the Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae A bispecific antibody to the O1 antigen or a composition comprising the bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen and specifically binds to the Klebsiella pneumoniae
  • the second antigen-binding domain of O1 antigen wherein said first antigen-binding domain comprises: VH , said VH comprises: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ
  • a method of preventing or treating a Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of a compound that specifically binds to the Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae A bispecific antibody to the O1 antigen or a composition comprising the bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen and specifically binds to the Klebsiella pneumoniae
  • the second antigen-binding domain of O1 antigen wherein said first antigen-binding domain comprises: VH , said VH comprises: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:7; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ
  • a method of preventing or treating a Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of a compound that specifically binds to the Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae A bispecific antibody to the O1 antigen or a composition comprising the bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen and specifically binds to the Klebsiella pneumoniae
  • the second antigen-binding domain of O1 antigen wherein said first antigen-binding domain comprises: VH , said VH comprises: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ
  • a method of preventing or treating a Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of a compound that specifically binds to the Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae A bispecific antibody to the O1 antigen or a composition comprising the bispecific antibody, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to the Klebsiella pneumoniae O2 antigen and specifically binds to the Klebsiella pneumoniae
  • the second antigen-binding domain of O1 antigen wherein said first antigen-binding domain comprises: VH , said VH comprises: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:7; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ
  • a method of preventing and/or treating Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of
  • the composition of the bispecific antibody of the O1 antigen of Klebsiella pneumoniae is more effective than the administration of an equivalent dose of an antibody specifically binding to the O2 antigen of Klebsiella pneumoniae or an equivalent dose of an antibody specifically binding to the O1 antigen of Klebsiella pneumoniae efficient.
  • the method includes administering an effective amount of a bispecific antibody that specifically binds Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae O1 antigen to enhance opsonophagocytosis (OPK) against Klebsiella pneumoniae ) to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100 times, compared with the administration of an equivalent dose of an antibody specifically binding to the Klebsiella pneumoniae O2 antigen or an equivalent dose of an antibody specifically binding to the Klebsiella pneumoniae O1 antigen.
  • OPK opsonophagocytosis
  • the method includes administering an effective amount of a bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae O1 antigen to enhance the neutralizing activity against Klebsiella pneumoniae to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times or more than 100 times, with Compared with the administration of an equivalent dose of an antibody specifically binding to the Klebsiella pneumoniae O2 antigen or an equivalent dose of an antibody specifically binding to the Klebsiella pneumoniae O1 antigen.
  • the method includes administering an effective amount of a bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae O1 antigen to enhance serum bactericidal activity (SBA) against Klebsiella pneumoniae ) to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times or 100 times Above, compared with the administration of an equivalent dose of an antibody specifically binding to the Klebsiella pneumoniae O2 antigen or an equivalent dose of an antibody specifically binding to the Klebsiella pneumoniae O1 antigen.
  • SBA serum bactericidal activity
  • the method includes administering an effective amount of a bispecific antibody that specifically binds to the Klebsiella pneumoniae O2 antigen and the Klebsiella pneumoniae O1 antigen to increase the survival rate of the patient to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times or 100 times more than the specificity of administering an equivalent dose Antibody that specifically binds Klebsiella pneumoniae O2 antigen or an equivalent amount of antibody that specifically binds Klebsiella pneumoniae O1 antigen.
  • Method for preventing or treating diseases using a pharmaceutical composition comprising an antibody specifically binding to Klebsiella pneumoniae O2 antigen and an antibody specifically binding to Klebsiella pneumoniae O1 antigen
  • a method of preventing or treating Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (i) specifically binding to Klebsiella pneumoniae Antibodies or antigen-binding fragments to the O2 antigen of Klebsiella pneumoniae and (ii) antibodies or antigen-binding fragments that specifically bind to the O1 antigen of Klebsiella pneumoniae.
  • a method of preventing or treating a Klebsiella pneumoniae infection in an individual comprising administering to the individual: (i) an antibody or antigen binding agent that specifically binds to the Klebsiella pneumoniae O2 antigen Fragments and (ii) antibodies or antigen-binding fragments that specifically bind to the Klebsiella pneumoniae O1 antigen.
  • the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen is administered concurrently with the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen.
  • the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen is administered sequentially with the antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:5; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:8, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:15.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 17; and V L comprising the amino acid sequence of SEQ ID NO :twenty one.
  • the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising HC-CDR1 in VH having the amino acid sequence of SEQ ID NO:17 , HC-CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO: 21.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:3, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:9, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 18; and V L comprising the amino acid sequence of SEQ ID NO :twenty two.
  • the antibody or antigen - binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1 in VH having the amino acid sequence of SEQ ID NO:18 , HC-CDR2 and HC-CDR3; and a VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a VL having the amino acid sequence of SEQ ID NO: 22.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 19; and V L comprising the amino acid sequence of SEQ ID NO :twenty three.
  • the antibody or antigen- binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1 in VH having the amino acid sequence of SEQ ID NO:19 , HC-CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO: 23.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:4, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:6; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:11, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:16.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 19; and V L comprising the amino acid sequence of SEQ ID NO :twenty four.
  • the antibody or antigen- binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1 in VH having the amino acid sequence of SEQ ID NO:19 , HC-CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO: 24.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen in the method comprises: V H , and the V H comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:1 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:7; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:10, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:15.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 20; and V L comprising the amino acid sequence of SEQ ID NO :25.
  • the antibody or antigen - binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen comprises: VH comprising HC-CDR1 in VH having the amino acid sequence of SEQ ID NO:20 , HC-CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO: 25.
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen in the method comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:35 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:37, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:39; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:41, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:43, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:45.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 47; and V L comprising the amino acid sequence of SEQ ID NO :49.
  • the antibody or antigen - binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen comprises: VH comprising HC-CDR1 in VH having the amino acid sequence of SEQ ID NO:47 , HC-CDR2 and HC-CDR3; and a V L comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a V L having the amino acid sequence of SEQ ID NO:49.
  • the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen in the method comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:36 , HC-CDR2 comprising the amino acid sequence of SEQ ID NO:38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:40; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO:42, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:44, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:46.
  • the antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 48; and V L comprising the amino acid sequence of SEQ ID NO :50.
  • the antibody or antigen- binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen comprises: VH comprising HC-CDR1 in VH having the amino acid sequence of SEQ ID NO:48 , HC-CDR2 and HC-CDR3; and a VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 in a VL having an amino acid sequence of SEQ ID NO:50.
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:11, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:4, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:6; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:11, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 14, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:7; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment specifically binding to Klebsiella pneumoniae O1 antigen
  • the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O2 antigen comprises: VH , said VH comprising: HC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 1, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:7; and V L , which comprises: LC- CDR1 , which comprises the amino acid sequence of SEQ ID NO:10, LC-CDR2, which comprises Amino acid sequence of SEQ ID NO: 12, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and wherein the antibody or antigen-binding fragment specifically binding to the Klebsiella pneumoniae
  • a method of preventing or treating a Klebsiella pneumoniae infection in an individual comprising administering to the individual an effective amount comprising an antibody or antigen-binding fragment that specifically binds Klebsiella pneumoniae O2 antigen and Compositions of antibodies or antigen-binding fragments that specifically bind to the O1 antigen of Klebsiella pneumoniae, the method is more specific than administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to the O2 antigen of Klebsiella pneumoniae or an equivalent dose Antibodies or antigen-binding fragments that specifically bind Klebsiella pneumoniae O1 antigen are more effective.
  • the method comprises administering an effective amount of a combination comprising an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen substance, the method is compared with administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen or an equivalent amount of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen, Enhance opsonophagocytosis (OPK) activity against Klebsiella pneumoniae to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5 times, 10 times, 20 times, 50 times or more than 100 times.
  • OPK Enhance opsonophagocytosis
  • the method comprises administering an effective amount of a combination comprising an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen substance, the method is compared with administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen or an equivalent amount of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen, Enhance serum bactericidal activity (SBA) against Klebsiella pneumoniae to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5 times, 10 times, 20 times, 50 times or more than 100 times.
  • SBA serum bactericidal activity
  • the method comprises administering an effective amount of a combination comprising an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen substance, the method is compared with administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen or an equivalent amount of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen, Enhance neutralizing activity against Klebsiella pneumoniae to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10 times, 20 times, 50 times or more than 100 times.
  • the method comprises administering an effective amount of a combination comprising an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O2 antigen and an antibody or antigen-binding fragment that specifically binds the Klebsiella pneumoniae O1 antigen substance, the method is compared with administering an equivalent dose of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O2 antigen or an equivalent dose of an antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen, Improve the survival rate of patients to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times times or more than 100 times.
  • an article of manufacture comprising a substance capable of preventing or treating a Klebsiella pneumoniae infection in an individual, or for delivering an antibody or antigen-binding fragment (antibodies that specifically bind to the O2 antigen of Klebsiella pneumoniae or antibodies that specifically bind to the O1 antigen of Klebsiella pneumoniae) or bispecific antibodies (such as bispecific antibodies that specifically bind to the O2 and O1 antigens of Klebsiella pneumoniae) Antibody), or a pharmaceutical composition comprising an antibody or an antigen-binding fragment specifically binding to the Klebsiella pneumoniae O2 antigen and an antibody or an antigen-binding fragment specifically binding to the Klebsiella pneumoniae O1 antigen is expressed to the Klebsiella pneumoniae Bacterial O2 antigen or O1 antigen in the cells attached by pathogenic bacteria.
  • the article of manufacture may comprise a container and a label or package insert on or accompanying the container.
  • Suitable containers include, for example, bottles, vials, syringes, and the like.
  • Containers can be made from a variety of materials such as glass or plastic.
  • the container contains a composition effective for treating a disease or condition described herein and has a sterile port (e.g., the container may be an IV bag or a vial with a hypodermic needle pierceable cap) .
  • At least one active substance in the composition is the antibody or antigen-binding fragment or bispecific antibody described in this application.
  • the label or package insert identifies the particular condition that the composition may be used to treat.
  • the label or package insert further comprises instructions for administering the bispecific antibody or pharmaceutical composition to a patient.
  • Articles of manufacture and kits comprising combination therapies described herein are contemplated.
  • Package insert refers to the instructions commonly included in commercial packages of therapeutic products that contain indications, usage, dosage, administration, contraindications and/or warning information pertaining to the use of such therapeutic products.
  • the package insert indicates that the composition is used to treat bacterial infections.
  • the package insert states that the composition is useful for treating Klebsiella pneumoniae infection.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Greene's solution, or dextrose solution.
  • a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Greene's solution, or dextrose solution.
  • kits that can be used for various purposes, such as for the prevention or treatment of Klebsiella pneumoniae infection in individuals, or for the delivery of antibodies that specifically bind to the O2 antigen of Klebsiella pneumoniae, specifically bind to Klebsiella pneumoniae Antibodies against O1 antigen of Klebsiella pneumoniae or bispecific antibodies specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen (such as full-length antibodies specifically binding to Klebsiella pneumoniae O2 antigen, specific A full-length antibody to O1 antigen or a full-length bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen) into cells attached by pathogenic bacteria expressing Klebsiella pneumoniae O2 antigen or O1 antigen, optionally Combined with products.
  • the kit of the present application includes one or more containers comprising an antibody specifically binding to the Klebsiella pneumoniae O2 antigen, an antibody specifically binding to the Klebsiella pneumoniae O1 antigen, or an antibody specifically binding to the Klebsiella pneumoniae O2 antigen Compositions (or single dose forms and/or preparations) and O1 antigen, and in some embodiments, further comprising another medicament (such as the medicament described in this application) and/or with any method described in this application Consistent with the instruction manual.
  • the kit may further include instructions for selecting an individual for treatment.
  • the instructions for use attached to the kit in the present application are usually written instructions on the label or package insert (such as paper sheets included in the kit), machine-readable instructions (such as instructions on a magnetic or optical storage disc) is also acceptable.
  • the kit comprises an antibody that specifically binds Klebsiella pneumoniae O2 antigen, an antibody that specifically binds Klebsiella pneumoniae O1 antigen, or an antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen Bispecific antibodies (such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigens, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigens, or that specifically bind Klebsiella pneumoniae O2 antigens and O1 antigens full-length bispecific antibody) composition.
  • Bispecific antibodies such as full-length antibodies that specifically bind Klebsiella pneumoniae O2 antigens, full-length antibodies that specifically bind Klebsiella pneumoniae O1 antigens, or that specifically bind Klebsiella pneumoniae O2 antigens and O1 antigens full-length bispecific antibody
  • the kit includes: a) comprising any antibody specifically binding to the O2 antigen of Klebsiella pneumoniae, an antibody specifically binding to the O1 antigen of Klebsiella pneumoniae, or a specific binding
  • the effects of antibodies against Klebsiella pneumoniae O1 antigen or bispecific antibodies that specifically bind to Klebsiella pneumoniae O2 antigen and O1 antigen (such as treatment effects, detection effects).
  • the kit includes: a) comprising any antibody specifically binding to the O2 antigen of Klebsiella pneumoniae, an antibody specifically binding to the O1 antigen of Klebsiella pneumoniae, or a specific binding A composition of bispecific antibodies to the Klebsiella pneumoniae O2 antigen and the O1 antigen, and b) administering to the individual an antibody that specifically binds to the Klebsiella pneumoniae O2 antigen, an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen Or an instruction for use of the bispecific antibody composition specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen for treating Klebsiella pneumoniae infection in an individual.
  • the kit includes: a) comprising any antibody specifically binding to Klebsiella pneumoniae O2 antigen, an antibody specifically binding to Klebsiella pneumoniae O1 antigen or an antibody specifically binding to pneumoniae described in this application
  • the composition of the bispecific antibody of Klebsiella O2 antigen and O1 antigen and b) at least one other medicament in an effective amount, which can enhance the antibody specifically binding to Klebsiella pneumoniae O2 antigen, specifically binding pneumonia
  • the effect (such as therapeutic effect, detection effect) of an antibody to the Klebsiella pneumoniae O1 antigen or a bispecific antibody that specifically binds to the Klebsiella pneumoniae O2 antigen and the O1 antigen and c) administering to an individual Antibodies to Bacteria O2 antigens, antibodies specifically binding to Klebsiella pneumoniae O1 antigens or bispecific antibody compositions specifically binding to Klebsiella pneumoniae O2 antigens and O1 antigens and other substances for the treatment of Klebsiella pneumoniae
  • the kit may include one specific composition or two or more compositions, wherein one composition includes an antibody that specifically binds to the Klebsiella pneumoniae O2 antigen, an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, an antibody that specifically binds to the Klebsiella pneumoniae O1 Antibodies to antigens or bispecific antibodies that specifically bind Klebsiella pneumoniae O2 antigen and O1 antigen, another composition includes another agent.
  • the kit comprises one (or a group) encoding an antibody that specifically binds to the Klebsiella pneumoniae O2 antigen, an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, or an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen
  • Bacteria O2 antigen and O1 antigen bispecific antibody for example, the full-length antibody that specifically binds to the Klebsiella pneumoniae O2 antigen, the full-length antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, or the full-length antibody that specifically binds to the Klebsiella pneumoniae O1 antigen
  • the nucleic acid of the full-length bispecific antibody of bacterial O2 antigen and O1 antigen The nucleic acid of the full-length bispecific antibody of bacterial O2 antigen and O1 antigen).
  • the kit comprises: a) one (or a group) encoding an antibody that specifically binds to Klebsiella pneumoniae O2 antigen, an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, or an antibody that specifically binds to pneumoniae A nucleic acid of a bispecific antibody to Klebsiella O2 antigen and O1 antigen, and b) a host cell expressing the nucleic acid (or a set of nucleic acids).
  • the kit comprises: a) one (or a group) encoding an antibody that specifically binds to Klebsiella pneumoniae O2 antigen, an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, or an antibody that specifically binds to pneumoniae
  • the nucleic acid of the bispecific antibody of Klebsiella O2 antigen and O1 antigen and b) instructions for use, suitable for: i) expressing in a host cell an antibody specifically binding to Klebsiella pneumoniae O2 antigen, specifically binding to pneumonia The antibody of Klebsiella pneumoniae O1 antigen or the bispecific antibody that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen, ii) prepares the antibody that specifically binds to Klebsiella pneumoniae O2 antigen, specifically binds to Klebsiella pneumoniae an antibody to the O1 antigen of Klebsiella pneumoniae or a composition of bispecific antibodies specifically binding to the O2 antigen of Kle
  • the kit includes: a) an antibody (or a group) encoding an antibody that specifically binds to the O2 antigen of Klebsiella pneumoniae, an antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae, or an antibody that specifically binds to the pneumoniae O1 antigen
  • the nucleic acid of the bispecific antibody of the Klebsiella O2 antigen and the O1 antigen b) a host cell expressing the nucleic acid (or a set of nucleic acids), and c) instructions for use, suitable for: i) expressing the specific antibody in the host cell
  • kits described herein are packaged in a suitable form.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Kits may optionally provide other components, such as buffers and instructional information.
  • the present application thus also provides articles of manufacture, including vials (eg, sealed vials), bottles, jars, flexible packaging, and the like.
  • compositions comprising an antibody specifically binding to Klebsiella pneumoniae O2 antigen, an antibody specifically binding to Klebsiella pneumoniae O1 antigen or a bispecific antibody composition specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen
  • Instructions usually include information such as dosage, dosing cycle and route of administration.
  • the container can be unit dose, bulk (eg, multi-dose package) or subunit dose.
  • an antibody that specifically binds to the Klebsiella pneumoniae O2 antigen an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, or an antibody that specifically binds to the Klebsiella pneumoniae O2 antigen, as described in the application, comprising a sufficient dose.
  • Antigen and bispecific antibody to O1 antigen (such as a full-length antibody that specifically binds to Klebsiella pneumoniae O2 antigen, a full-length antibody that specifically binds to Klebsiella pneumoniae O1 antigen, or a full-length antibody that specifically binds to Klebsiella pneumoniae O2 antigen
  • Antigen and full-length bispecific antibody to O1 antigen for long-term effective treatment of individuals, such as one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks , 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months or longer.
  • the kit can also comprise multiple unit doses of antibodies specifically binding to the O2 antigen of Klebsiella pneumoniae, antibodies specifically binding to the O1 antigen of Klebsiella pneumoniae, or a double antibody specifically binding to the O2 and O1 antigens of Klebsiella pneumoniae.
  • the specific antibody, pharmaceutical composition and instructions for use are packaged in quantities sufficient for storage and use in pharmacies, eg, hospital pharmacies and compounding pharmacies.
  • KP19173 is used to represent the Klebsiella pneumoniae O1 serotype whose LPS contains two repeating units of D-galactan II and D-galactan I ( Figure 1);
  • KP19213 is used to represent LPS Klebsiella pneumoniae O1 serotype containing D-galactan II and D-galactan III repeating units ( Figure 1);
  • KP19180 represents the pneumonia containing D-galactan I repeating units in LPS Klebsiella O2 serotype ( Figure 2);
  • KP19203 represents the O2 serotype of Klebsiella pneumoniae whose LPS contains D-galactan III repeating units ( Figure 2).
  • the anti-Klebsiella pneumoniae O antigen antibody G3-78 (binding D-galactan III repeating unit) disclosed in the patent application CN107371365A, and/or disclosed in the patent application WO2018/029356Al
  • the anti-Klebsiella pneumoniae O1 antigen antibody MPG-196 (binding D-galactan II repeat unit) was used as a control antibody.
  • Embodiment 1 prepare lipopolysaccharide (LPS), O2 antigen and screen anti-O2 single-chain antibody (scFv)
  • the solution was divided into three layers, which were water layer, phenol layer and insoluble layer from top to bottom.
  • the upper aqueous phase contains LPS, the middle layer is denatured protein, and the lower layer is sediment. Carefully draw the upper aqueous phase into a new tube, and add 6 times the volume of 95% ethanol, and at the same time add sodium acetate (Sinopharm, 10018892) with a final concentration of 0.1%.
  • the mixture was precipitated at -20°C overnight for LPS, and centrifuged at 4000 rpm for 30 min the next day.
  • the precipitate is the crude extract of O2 type LPS of KP19180 strain.
  • KP19180 strain O2 type LPS prepared by the above process, add it to 200mL of 1% acetic acid solution, and let it act in a boiling water bath for 45min. After cooling, ultracentrifuge at 105000g for 2h at 4°C. The precipitate is lipid A and the supernatant is O2 antigen. The supernatant was taken, and the prepared O2 antigen was concentrated by vacuum centrifugation; then dissolved in water for later use.
  • scFvs single-chain antibodies specifically binding to the Klebsiella pneumoniae O2 antigen
  • Single-chain antibody (scFv) specifically binding to Klebsiella pneumoniae O2 antigen was sorted by FACS: the yeast enriched after MACS panning was sorted by flow cytometry fluorescence sorting (FACS). Briefly, after the yeast cells induced in SGCAA medium were precipitated, 1 mL of PBSM was added, centrifuged at 14000 g for 30 sec, and the pellet was washed. Resuspend the yeast cells with 100 ⁇ L of PBSM buffer containing KP19180 strain Bio-O2 antigen, and incubate at room temperature for 1 h.
  • FACS flow cytometry fluorescence sorting
  • An ELISA binding assay was used to identify antibodies that bind to KP19180O2 LPS using O2-type LPS prepared from the KP19180 strain. Briefly, LPS was dissolved in PBS solution (adjusted pH to 7.2, final concentration was 0.2 ⁇ g/mL), coated 96-well plate with 100 ⁇ L/well, and left overnight at 4°C. Wash the 96-well plate 5 times with 200 ⁇ L/well PBST solution before adding the antibody. Add 200 ⁇ L of 10% BSA (Beyotime Biotechnology, ST023-200g) to each well and incubate at 37°C for 1 hour. Wash 5 times with PBST solution.
  • BSA Beyotime Biotechnology, ST023-200g
  • Each antibody sample was first diluted to 1 ⁇ g/mL, followed by a 1:3 serial dilution.
  • the antibody samples after serial dilution were added to 96-well plates, 50 ⁇ L per well, and incubated at 37° C. for 1 h. It was subsequently washed 5 times with PBST solution.
  • Add 100 ⁇ L of primary antibody and secondary antibody mixture (mouse anti-flag (1:2500) and anti-mouse FC-AP (1:2000)) into each well, incubate at 37°C for 1 h, wash 3 times with TBST solution .
  • Add 50 ⁇ L PNPP to each well and incubate at 37°C for 10-20min. The reaction was terminated with 3M NaOH. Read OD450, and generate binding curve by Graphpad Prism, and calculate EC50 value.
  • Example 2 Preparation and characterization of a full-length antibody specifically binding to the Klebsiella pneumoniae O2 antigen
  • the obtained positive scFv antibody was reconstituted into a human IgG1 or IgG4 full-length antibody molecule containing the heavy chain constant region of human IgG1 or IgG4 and the human kappa light chain constant region or human lambda light chain constant region.
  • the extracted plasmids expressing the light chain or heavy chain were co-transfected into 293F cells, cultured at 37° C., 8% CO 2 , and 120 rpm for 5 days, and the culture solution was purified by Protein A affinity chromatography. Briefly, the Protein A chromatography column was first equilibrated with 50 mM PBS buffer (containing 0.15 M NaCl, pH 7.2) at a flow rate of 150 cm/h. The culture supernatant (adjusted to pH 7.2) was passed through the column at a flow rate of 150 cm/h. After the column was further equilibrated, it was eluted with 50 mM sodium citrate buffer (pH 3.5), and the eluate was collected. The obtained full-length antibody was further analyzed for biochemical and biological activity.
  • the full-length antibody molecules prepared above were further subjected to ELISA binding detection. Concrete operation is with embodiment 1. The difference is that after adding the full-length antibody to be detected, the secondary antibody used is goat anti-human IgG-HRP (Beyotime Biotechnology, A0201) 1:10000, and incubated at 37°C for 1 hour. Wash 5 times with PBST solution. Add 100 ⁇ L TMB (southern biotech, 0410-01) to each well, incubate at 37°C for 10-20 minutes, stop the reaction with 2M H 2 SO 4 , read OD450, and generate binding curve by Graphpad Prism to calculate EC50 value.
  • the secondary antibody used is goat anti-human IgG-HRP (Beyotime Biotechnology, A0201) 1:10000, and incubated at 37°C for 1 hour. Wash 5 times with PBST solution. Add 100 ⁇ L TMB (southern biotech, 0410-01) to each well, incubate at 37°C for 10-20 minutes, stop the reaction
  • the commercial reporter cell line HEK-Blue TM hTLR4 (Invivogen, hkb-htlr4) stably expresses human TLR4, MD-2 and CD14 co-receptors, and NF- ⁇ B-induced secreted embryonic alkaline phosphatase (SEAP).
  • Bacterial LPS can trigger Toll-like receptor 4 (TLR-4) signaling in this cell line, leading to the activation of downstream NF- ⁇ B transcription factors, thereby secreting SEAP.
  • TLR-4 Toll-like receptor 4
  • QUANTI-Blue TM solution (Cat#rep-qbs) can be used to detect and quantify SEAP activity.
  • HEK-Blue TM hTLR4 cells were cultured and inoculated into 96-well culture plates with about 2.5 ⁇ 10 4 cells per well, and then 10 ⁇ L of the KP19180LPS prepared in Example 1 ( 10 ⁇ g/mL) and a gradient dilution of the full-length antibody (reconstituted into human IgG1 form) specifically binding to Klebsiella pneumoniae O2 antigen at an initial concentration of 1 mg/mL was pre-mixed, added to a 96-well culture plate, and kept at 37°C , 5% CO 2 incubate for 6-16h.
  • the absorbance value of the well of the antibody of the Klebsiella pneumoniae O2 antigen was calculated, and the inhibition rate of the antibody specifically binding to the Klebsiella pneumoniae O2 antigen at the corresponding concentration was calculated, and the inhibition curve was generated by Graphpad Prism, and the IC50 value was calculated.
  • K1 was selected as the lead antibody, and K1 scFv was used to prepare a scFv phage display library containing mutations in the CDR region.
  • KP19180LPS ELISA binding and LPS neutralization experiments an optimized antibody clone with high affinity and high neutralization activity that specifically binds to the Klebsiella pneumoniae O2 antigen was selected and further constructed into a full-length antibody, which is effective against the lead antibody and a series of The optimized antibodies are further biochemical and biological analyses.
  • Klebsiella pneumoniae are classified into different LPS serotypes according to their unique O antigen structure. O1 and O2 serotype strains are common clinical epidemic strains.
  • the O1 serotype includes two structures, represented by KP19173 and KP19213 respectively, and its specific structure diagram is shown in Figure 1.
  • the O2 serotype also includes two structures, represented by KP19180 and KP19203, and its specific structure diagram is shown in Fig. 2.
  • Antibodies K2, K3 and K5 were selected for binding domain analysis experiments.
  • the ELISA experiment was used to determine the binding ability of the antibody to the LPS of the above four Klebsiella pneumoniae strains with different O antigen structures.
  • the preparation methods of different Klebsiella pneumoniae LPS and the specific operation steps of the ELISA experiment are shown in Example 1.
  • the binding of the above antibodies to the O1-type LPS of the KP19173 strain and the O1-type LPS of the KP19213 strain was further detected.
  • the exemplary antibody specifically binding to the O2 antigen of Klebsiella pneumoniae can specifically bind to the O1-type LPS of the KP19173 strain;
  • MPG196 was the control antibody in this experiment, an exemplary antibody that specifically binds Klebsiella pneumoniae O2 antigen did not bind to KP19213 strain O1 type LPS.
  • the antibody specifically binding to the Klebsiella pneumoniae O2 antigen described in this application specifically binds to the D-half of the Klebsiella pneumoniae O antigen Lactan I domain and does not bind to D-galactan II and D-galactan III domains.
  • Example 4 Antibodies that specifically bind to the Klebsiella pneumoniae O2 antigen bind to the broad spectrum of O2 serotype Klebsiella pneumoniae
  • Example 1 Using the ELISA binding assay described in Example 1 to detect antibodies that specifically bind to the Klebsiella pneumoniae O2 antigen with other Klebsiella pneumoniae O2 serotype strains (KP19002, KP19003, KP19005 and KP19007 strains) isolated from clinical Regarding the combination of LPS, the above-mentioned O2 serotype bacterial strains LPS all express the D-galactan I structure. The preparation methods of LPS from different Klebsiella pneumoniae strains are shown in Example 1.
  • the exemplary antibodies K2, K3 and K5 that specifically bind to the Klebsiella pneumoniae O2 antigen can all be compared with other O2 serotype Klebsiella pneumoniae strains KP19002 (Fig. 4A), KP19003 (Fig. 4B), LPS binding of KP19005 (Fig. 4C) or KP19007 (Fig. 4D). It can be seen that the binding of the antibody specifically binding to the Klebsiella pneumoniae O2 antigen with the Klebsiella pneumoniae O2 serotype strain expressing the D-galactan I structure has a broad spectrum.
  • Example 5 Preventive and protective effects of antibodies specifically binding to Klebsiella pneumoniae O2 antigen in a mouse bacteremia model
  • C57/BL6 mice were purchased from Verton Liver and housed in a specific pathogen-free environment. All animal experiments were performed in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • mice in each group were observed twice a day, and the number of mice died in each group, the time of death and the state of the surviving mice were recorded within 8 days.
  • Graphpad Prism software was used to draw graphs to determine mouse mortality.
  • the exemplary specific binding Klebsiella pneumoniae Antibodies K2 and K5 to bacterial O2 antigen both effectively improved the survival rate and/or prolonged the survival time of mice.
  • the K5 antibody was administered at a dose of 30mpk, the mice did not die until 72h, and the survival rate of the mice remained at about 70% until the end of the experiment at 144h. It can be seen that the antibody specifically binding to the O2 antigen of Klebsiella pneumoniae has a preventive and protective effect on bacteremia induced by Klebsiella pneumoniae.
  • Example 6 Combined therapeutic effect of an antibody specifically binding to Klebsiella pneumoniae O2 antigen and an antibody specifically binding to Klebsiella pneumoniae O1 antigen in a mouse pneumonia model
  • a mouse pneumonia model was constructed, and an antibody specifically binding to Klebsiella pneumoniae O2 antigen and an antibody specifically binding to Klebsiella pneumoniae O1 antigen were used in combination to treat pneumonia infected by Klebsiella pneumoniae in mice.
  • the mice in each group were treated with the negative control antibody HIV-10E8, or the combined application of antibody K5 that specifically binds to Klebsiella pneumoniae O2 antigen and antibody G2 that specifically binds to Klebsiella pneumoniae O1 antigen.
  • K5+G2 1.5mpk+1.5mpk
  • K5+G2 (15mpk+15mpk
  • negative HIV-10E8 application dose is 30mpk.
  • mice were purchased from Victoria Liever and raised in a specific pathogen-free environment. All animal experiments were performed in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC). In this experiment, mice were challenged by the lung delivery route, and a pneumonia model of mouse Klebsiella pneumoniae infection was constructed. Specifically, the mice were first anesthetized and placed on an inclined plate, the mouth was opened, and the throat of the mouse was irradiated with an anesthetized laryngoscope in the left hand, so that the pharynx was clearly visible, and a fixed amount of bacteria was sucked with a micro-liquid intratracheal nebulizer.
  • IACUC Institutional Animal Care and Use Committee
  • mice were put back into the cage, and the antibody that specifically binds to the O2 antigen of Klebsiella pneumoniae and the antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae were injected simultaneously through the tail vein of the mouse, negative control The mice in the group were injected with HIV-10E8.
  • mice were continuously observed until they became awake. Then observe twice a day until the 8th day, record the number and time of death of mice in each group and record the state of surviving mice. Graph the survival data of each group of mice in GraphPad Prism software to determine the mortality of mice
  • Example 7 Preparation of bispecific antibodies with different structures that specifically bind to Klebsiella pneumoniae O2 antigen and O1 antigen
  • V H and V L represent the variable region of the heavy chain and the variable region of the light chain of the antibody respectively;
  • CH represents the constant region of the heavy chain of the antibody, including CH1 , CH2 and CH3 domains ;
  • scFv is an antibody formed by linking the V H and V L of an antibody through a linker;
  • IgG1Fc represents the Fc region of an IgG1 subclass antibody, which includes CH 2 and CH 3 domains;
  • CL represents the light chain constant region.
  • DVD-Ig Dual-variable domain-Ig bispecific antibody, whose structure is that the N-terminals of the light chain and heavy chain of a normal IgG antibody are connected to the VL and VH domains of another antibody, respectively , through the interaction of VH and VL of two antibodies to form an antigen-binding domain, which can simultaneously bind to the corresponding antigen to achieve bispecificity.
  • the bispecific antibody about the DVD-Ig structure is in Wu C, et al. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules. MAbs. 2009Jul-Aug; 1( 4):339-47 also recorded.
  • Table 12-1 shows the compositional design of the DVD-Ig bispecific antibody used in the examples
  • Table 8-1 shows the specific amino acid sequences of its heavy chain and light chain. Entrusted GenScript Biotechnology Co., Ltd. to synthesize bispecific antibody light chain (L chain) and heavy chain (H chain) coding genes, and optimize the coding genes to be suitable for expression in 293F cells.
  • the bispecific antibody samples of DVD-Ig structure after endotoxin removal specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen were subjected to reducing and non-reducing SDS-PAGE electrophoresis detection (results not shown) to ensure its formation correct structure.
  • Table 12-1 Composition design of the heavy chain and light chain of the bispecific antibody that specifically binds Klebsiella pneumoniae O2 antigen and O1 antigen with DVD-Ig structure
  • Bs4Ab structure The bispecific antibody includes a full-length IgG1 structure, and the bispecificity is achieved by inserting another binding unit scFv in its hinge region.
  • the bispecific antibody about the structure of Bs4Ab is in the literature Bezabeh B, et al.Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.MAbs.2017Feb/Mar; 9(2): It is also recorded in 240-256. Its structure diagram is shown in Fig. 7B.
  • Table 12-2 shows the composition design of the bispecific antibody with the Bs4Ab structure used in the examples, and Table 8-2 shows the specific amino acid sequences of its heavy chain and light chain.
  • Entrusted GenScript Biotechnology Co., Ltd. to synthesize bispecific antibody light chain (L chain) and heavy chain (H chain) coding genes, and optimize the coding genes to be suitable for expression in 293F cells.
  • Table 12-2 Composition design of the heavy chain and light chain of the bispecific antibody that specifically binds the O2 antigen and the O1 antigen of Klebsiella pneumoniae with Bs4Ab structure
  • the threonine (T) at position 366 of the CH3 region of an antibody is replaced with tryptophan (W) to form a "knobs" structure
  • the paired threonine at position 366 of the CH3 region of another antibody is Amino acid (T) is replaced by serine (S)
  • 368-position leucine (L) is replaced by alanine (A)
  • 407-position tyrosine (Y) is replaced by valine (V) to form a "hole” ( holes) structure, relying on the reduction of the steric hindrance effect after mutation and the covalent disulfide bond formed in the hinge region to promote the dimerization of the heterologous heavy chain, wherein the numbering is according to the EU index such as Kabat; at the same time, the CrossMab technology is applied to Ensure correct pairing between antibody light and heavy chains.
  • CrossMab technology is based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, either the exchange of the complete Fab domain (CrossMAb Fab), or the exchange of only the variable region in the Fab (CrossMAb V H -V L ) Or exchange of only the constant region (CrossMAb CH 1- CL ).
  • the bispecific antibody about Hetero H, CrossMab structure is also in the document Klein C, et al.The use of CrossMAb technology for the generation of bi-and multispecific antibodies.MAbs.2016Aug-Sep;8(6):1010-20 recorded. In this application, the structure of CrossMab CH 1- CL is specifically described, and the schematic diagram of the structure is shown in Figure 7C.
  • Table 12-3 is the composition design of the Hetero H, CrossMab structure bispecific antibody used in the examples, and the specific amino acid sequences of its heavy chain and light chain are shown in Table 8-3, wherein the Fc comes from human wild Type IgG1 Fc. Entrusted GenScript Biotechnology Co., Ltd. to synthesize bispecific antibody light chain (L chain) and heavy chain (H chain) coding genes, and optimize the coding genes to be suitable for expression in 293F cells.
  • Table 12-3 Composition design of the heavy chain and light chain of the bispecific antibody of Hetero H, CrossMab structure-specific binding to Klebsiella pneumoniae O2 antigen and O1 antigen
  • IgG-(scFv) 2 structural bispecific antibody that is, the scFv fragment of another antibody is connected to the Fc ends of the two heavy chains of an IgG antibody to achieve bispecificity.
  • Bispecific antibodies with IgG-(scFv) 2 structure are also described in Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2): 159-63. The schematic diagram of the structure is shown in Fig. 7D.
  • Table 12-4 shows the composition design of the IgG-(scFv) 2 bispecific antibody used in the examples, and Table 8-4 shows the specific amino acids of its heavy chain and light chain.
  • Entrusted GenScript Biotechnology Co., Ltd. to synthesize bispecific antibody light chain (L chain) and heavy chain (H chain) coding genes respectively, and optimize the coding genes to be suitable for expression in 293F cells.
  • Table 12-4 Composition design of heavy chain and light chain of bispecific antibody with IgG-(scFv) 2 structure specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen
  • Table 12-5 Composition design of the heavy chain and light chain of the bispecific antibody specifically binding to Klebsiella pneumoniae O2 antigen and O1 antigen with scFv-Fab IgG structure
  • Example 8 Determination of EC50 of bispecific antibody binding O1-type LPS and O2-type LPS (D-galactan I)
  • O1-type LPS KP19173 strain O1-type LPS or KP19213 strain O1-type LPS
  • O2-type LPS KP19180O2-type LPS
  • ELISA binding assay For the preparation method of different serotypes of Klebsiella pneumoniae LPS, and the specific operation steps of the ELISA binding experiment, please refer to Example 1.
  • Antibodies G2 and G7 specifically binding to Klebsiella pneumoniae O1 antigen and antibody K5 specifically binding to Klebsiella pneumoniae O2 antigen were used as control antibodies in this experiment.
  • antibody K5 does not bind to KP19213 strain O1 type LPS
  • antibodies G2 and G7 do not bind to KP19180O2 type LPS.
  • 100 ⁇ l LPS (0.2 ⁇ g/mL, derived from KP19173 strain, KP19213 strain or KP19180 strain) was added to each well of a 96-well plate (Corning#9018), and coated overnight at 4°C. The next day, wash with PBST 5 times (200 ⁇ L/well), then add 200 ⁇ l 10% BSA (Beyotime Biotechnology, ST023-200g) to each well, and incubate at 37° C. for 1 h.
  • BSA Beyotime Biotechnology, ST023-200g
  • the exemplary bispecific antibodies of the DVD-Ig structure can all be combined with KP19173 bacterial strain O1 type LPS ( Figure 8A), KP19213 bacterial strain O1 type LPS ( Figure 8B) or KP19180 bacterial strain O2 type LPS ( Figure 8C).
  • exemplary bispecific antibodies of the Bs4Ab structure are capable of binding to KP19173 strain O1-type LPS (Figure 8D), KP19213 strain O1-type LPS ( Figure 8E), or KP19180 strain O2-type LPS ( Figure 8F ).
  • the exemplary bispecific antibody of Hetero H, CrossMab structure can bind to the KP19173 strain O1 type LPS (Fig. 8G), the KP19213 strain O1 type LPS (Fig. 8H) or the KP19180 strain O2 type LPS ( Figure 8I).
  • Exemplary bispecific antibodies of IgG-(scFv) 2 structure were able to interact with KP19173 strain O1 type LPS (Figure 8J), KP19213 strain O1 type LPS ( Figure 8K), or KP19180 strain O2 type LPS binding (Fig. 8L).
  • exemplary bispecific antibodies of the scFv-Fab IgG structure were able to bind to LPS type O1 of KP19173 strain (Figure 8M), LPS type O1 of KP19213 strain ( Figure 8N), or LPS type O2 of KP19180 strain (FIG. 8O).
  • Example 9 Determination of neutralizing activity of bispecific antibody against Klebsiella pneumoniae O1 type LPS and O2 type LPS (D-galactan I)
  • the neutralization activity of bispecific antibodies against O1-type LPS (KP19173 strain O1-type LPS or KP19213 strain O1-type LPS) and O2-type LPS (KP19180O2-type LPS) was detected by LPS neutralization assay. See embodiment 2 for specific operation steps.
  • Antibodies G2 and G7 specifically binding to Klebsiella pneumoniae O1 antigen and antibody K5 specifically binding to Klebsiella pneumoniae O2 antigen were used as control antibodies in this experiment. Among them, antibody K5 has no neutralizing activity against KP19213 strain O1 type LPS, and antibodies G2 and G7 have no neutralizing activity against KP19180 strain O2 type LPS.
  • HEK-Blue TM hTLR4 (approximately 25,000 cells) was added to each well, and then 10 ⁇ L of LPS (10 ⁇ g/mL, derived from KP19173, KP19213 or KP19180 strains were added to each well).
  • LPS 10 ⁇ g/mL, derived from KP19173, KP19213 or KP19180 strains were added to each well.
  • different concentrations of bispecific antibody or control antibody cultured at 37°C for 6-16h, and detected the absorbance value at 620-655nm with a microplate reader.
  • the exemplary bispecific antibody of DVD-Ig structure can inhibit KP19173 strain O1 type LPS (Fig. 9A), KP19213 strain O1 type LPS (Fig. 9B), or KP19180 strain O2 type LPS (Fig. 9C).
  • exemplary bispecific antibodies of the Bs4Ab structure are capable of inhibiting KP19173 strain O1 type LPS ( Figure 9D), KP19213 strain O1 type LPS ( Figure 9E), or KP19180 strain O2 type LPS ( Figure 9F) .
  • the exemplary bispecific antibody of Hetero H, CrossMab structure can inhibit KP19173 bacterial strain O1 type LPS (Fig. 9G), KP19213 bacterial strain O1 type LPS (Fig. 9H), or KP19180 bacterial strain O2 type LPS ( Figure 9I).
  • Exemplary bispecific antibodies of IgG-(scFv) 2 structure were able to inhibit LPS of KP19173 strain O1 (Fig. 9J), KP19213 strain O1 (Fig. 9K), or KP19180 strain O2, as shown in Figure 9J-9L LPS (Fig. 9L).
  • exemplary bispecific antibodies of scFv-Fab IgG structure are able to inhibit KP19173 strain O1 type LPS (Figure 9M), KP19213 strain O1 type LPS ( Figure 9N), or KP19180 strain O2 type LPS ( Figure 9O).
  • Opsonophagocytosis (OPK) activity refers to the death of cells (eg, Klebsiella pneumoniae) due to phagocytosis by immune cells.
  • Opsonophagocytic bactericidal activity was measured using a bioluminescent assay according to the following assay. The experiment was designed to evaluate bispecific antibody-mediated opsonophagocytosis against Klebsiella pneumoniae, exemplified by bispecific antibodies in DVD-Ig format and Bs4Ab format.
  • the plasmid pUC18-mini-Tn7T-Gm-lux expressing complete luciferin was electrotransformed into Klebsiella pneumoniae KP19173 to construct a luminescent Klebsiella pneumoniae strain KP19173-LUX.
  • This method was described in the literature (Choi K H, et al .mini-Tn7insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa [J]. Nature Protocols, 2006, 1(1): 153-161).
  • the OPK activity of the bispecific antibody was evaluated by detecting changes in the relative light units (RLU) of luminescent Klebsiella strains with a microplate reader.
  • RLU relative light units
  • the OPK activity detection method is described in the literature Wang Q, et al. Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit. J Infect Dis. 2016 Jun 1; 213(11): 1800-8. Briefly, the experiment was carried out in a 96-well plate, infant rabbit serum was used as a source of complement, and HL-60 cells were induced to differentiate into macrophages by DMF.
  • Klebsiella pneumoniae KP19173-LUX in the logarithmic growth phase was inoculated into 96 plates, 5 ⁇ 10 5 differentiated macrophages, inactivated baby rabbit serum (1:10 Cedarlane), and different concentrations of The diluted bispecific antibody was mixed evenly, and incubated at 37°C with shaking (250rpm) for 2h. Relative light units (RLU) were then measured using a microplate reader.
  • the RLU value of the well without antibody was used as the reference for the opsonophagocytosis bactericidal activity of 0%, and the RLU value of the well without Klebsiella pneumoniae was used as the reference for the opsonophagocytosis bactericidal activity of 100%.
  • the opsonophagocytosis bactericidal activity (%) of the antibody at different concentrations was calculated from the RLU value of the well, and the IC50 value was calculated by using Graphpad Prism software for drawing.
  • the bispecific antibodies K5-G2-Ig1 and K5-G7-Ig1 in the form of DVD-Ig have strong activity of promoting opsonophagocytosis and bactericidal activity against the KP19173 strain.
  • the bispecific antibodies K5-G2scFv-Ig1 and K5-G7scFv-Ig1 antibodies in the form of Bs4Ab have a strong activity of promoting opsonophagocytosis and bactericidal activity against the KP19173 strain.
  • Table 1 specifically shows the IC50 values of the opsonophagocytosis-promoting bactericidal activity of exemplary bispecific antibodies with different structures against Klebsiella pneumoniae KP19173.
  • the Serum Bactericidal Activity (SBA) assay was designed to evaluate the complement-dependent serum bactericidal activity mediated by bispecific antibodies, with DVD-Ig and Bs4Ab bispecific antibodies as examples.
  • SBA Serum Bactericidal Activity
  • the plasmid pUC18-mini-Tn7T-Gm-lux expressing complete luciferin was electrotransformed into the Klebsiella pneumoniae strain KP19173 to construct the luminescent Klebsiella pneumoniae strain KP19173-LUX.
  • the bispecific antibody-mediated complement-dependent serum bactericidal activity was determined by detecting changes in relative light units (RLU) of luminescent Klebsiella pneumoniae.
  • the detection method of SBA is described in the literature Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils, mBio.2018Mar-Apr;9(2):e00297-18. Briefly, the experiment was carried out in a 96-well plate, and young rabbit serum was used as the source of complement, and the logarithmic growth phase of Klebsiella pneumoniae KP19173-LUX was serially diluted with the bispecific antibody at an initial concentration of 1.3 nM Pre-mixed, inoculated into 96 plate, 37 °C, 5% CO2 incubation for 15min.
  • RLU Relative light units
  • the bispecific antibodies K5-G2-Ig1 and K5-G7-Ig1 in the form of DVD-Ig have antibody-mediated complement-dependent serum bactericidal activity against KP19173 strain.
  • the bispecific antibodies K5-G2scFv-Ig1 and K5-G7scFv-Ig1 antibodies in the form of Bs4Ab have antibody-mediated complement-dependent serum bactericidal activity against the KP19173 strain.
  • Table 14 specifically shows the IC50 values of serum bactericidal activity of bispecific antibodies with different structures against Klebsiella pneumoniae KP19173.
  • Example 12 Therapeutic effect of bispecific antibody on Klebsiella pneumoniae infection (pneumonia)
  • mice C57/BL6 mice were purchased from Verton Liver and housed in a specific pathogen-free environment. All animal experiments were performed in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC). Bispecific antibodies in DVD-Ig format and Bs4Ab format are exemplified. In order to test whether the bispecific antibody has a therapeutic effect in the mouse Klebsiella pneumoniae-infected pneumonia model, this experiment used the lung delivery route to challenge the mice, and established a mouse Klebsiella pneumoniae-infected pneumonia model. The specific preparation method of the mouse pneumonia model is as described in Example 6.
  • the bispecific antibody K5-G2-Ig1 which specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen, through the tail vein of the mouse at a dose of 30 mpk 1 hour later; or
  • the dose of antibody K5 specifically binding to Klebsiella pneumoniae O2 antigen was 30 mpk; or the dose of antibody G2 specifically binding to Klebsiella pneumoniae O1 antigen was 30 mpk; the mice in the model group were injected with antibody Mab601 (30 mpk).
  • Mab601 is an antibody that binds O3 mannose of Klebsiella pneumoniae heterosexually. :617-624. Described. After the antibody injection was completed, the mice were continuously observed until they became awake. Then observe twice a day until the 8th day, record the number and time of death of mice in each group and record the state of surviving mice. The survival data of mice in each group were plotted in GraphPad Prism software to determine the death rate of mice.
  • Example 13 Determination of the antibody specifically binding to the Klebsiella pneumoniae O2 antigen and the Kd value of the bispecific antibody
  • Biacore 3000 (GE) was used to detect the antibodies K4 and K5 (reconstructed adult IgG1 format) that specifically bind to Klebsiella pneumoniae O2 antigen and the bispecific antibody K5- that specifically binds to Klebsiella pneumoniae O2 antigen and O1 antigen Binding affinity of G2-Ig1, G7-K5-CrossMab-Ig1 and K5-G7scFv-Ig1.
  • This method is a conventional method well known to those skilled in the art, and the experimental operation is carried out according to the instruction manual.
  • the diluted biotinylated O antigen of KP19180 strain, O antigen of KP19173 strain or O antigen of KP19213 strain were bound to the SA chip for 20 seconds at a flow rate of 10 ⁇ L/min.
  • the concentration ranges include: 0M, 7.815E-10M, 1.563E-9M, 3.126E-9M, 6.252E-9M, 1.250E-8M, 2.501E-8M, 5.002E-8M .
  • Use the SPR technique to measure the binding and dissociation rate of the antibody and determine the binding affinity.
  • Exemplary antibodies specifically binding to the Klebsiella pneumoniae O2 antigen and specific binding to the Klebsiella pneumoniae O2 antigen are listed in Table 15 and Table 16 respectively. Kon, Koff, and Kd values for binding of exemplary bispecific antibodies to the O1 antigen to different Klebsiella pneumoniae O antigens.

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Abstract

涉及特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段，和特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体以及包含所述抗体或抗原结合片段和/或双特异性抗体的药物组合物，同时也涉及制备所述抗体的方法和用途。

Description

特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体以及组合物
以下提交的序列表的内容通过整体引用并入本申请中：文本名称：特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体以及组合物.xml，记录日期：2022.11.18，大小：161KB)。
技术领域
本申请涉及特异性结合肺炎克雷伯菌(Klebsiella pneumoniae；K.pneumoniae)O2抗原的抗体，特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体，以及包含特异性结合肺炎克雷伯菌O2抗原的抗体和特异性结合肺炎克雷伯菌O1抗原的抗体和/或双特异性抗体的药物组合物，及其制备方法和用途，包括用其预防和治疗克雷伯菌感染的方法。
背景技术
克雷伯(氏)菌属属于革兰氏阴性菌，是一种导致肺炎、脑膜炎、肝脓肿、泌尿系统炎症、伤口感染或败血症等疾病的医院内机会致病菌。克雷伯氏菌感染主要由肺炎克雷伯菌引起，在医学上，其是克雷伯氏菌属中最重要的种类(Podschun,R,and U Ullmann.Clinical microbiology reviews vol.11,4(1998):589-603.)。近年来，肺炎克雷伯氏菌菌株的耐药性越来越强且耐药菌株的比率逐年升高，使得这种细菌感染非常难以治疗。
与克雷伯氏菌毒力相关的因子包括荚膜多糖(capsular polysaccharide，CPS)、脂多糖(lipopolysaccharide，LPS)、黏附因子、铁载体系统及其他毒力因子等。这些毒力因子在细菌粘附到宿主细胞，逃避宿主免疫反应/免疫杀伤等过程中发挥了重要作用。其中脂多糖(LPS)在细菌致病中的作用非常重要，是引起细胞全身炎症反应的重要分子。LPS是所有革兰氏阴性菌细胞膜外叶的主要和必要成分。虽然在不同的细菌之间，LPS的结构有相当大的差异，但其通常由类脂A(lipid A)、核心寡糖(core oligosaccharide)和O抗原(O antigen)组成(Paczosa,Michelle K,and Joan Mecsas.Microbiology and molecular biology reviews:MMBR vol.80,3 629-61.15Jun.2016)。根据独特的O抗原结构，可以划分为不同的克雷伯氏菌LPS血清型，其中最为常见的是O1血清型和O2血清型(Follador,R.et al.The diversity of Klebsiella pneumoniae surface polysaccharides.Microb.Genom.2,e000073(2016))。
现有技术已表明，特异性结合LPS O抗原的抗体在体外促进杀菌的作用是有效的。因此开发新的结合不同LPS O抗原的抗体，或同时结合不同LPS O抗原的双特异性抗体，以及包括结合不同LPS O抗原抗体的药物组合物，对补充抗生素疗法至关重要。
本申请提及的所有出版物、专利、专利申请和已公开的专利申请中披露的内容，以引用方式全部并入本申请中。
申请概述
在一些实施例中，本申请提供了分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其包含：(i)V H，其包含如氨基酸序列SEQ ID NO:17所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:21所示的V L包含的LC-CDR1、LC-CDR2 和LC-CDR3；(ii)V H，其包含如氨基酸序列SEQ ID NO:18所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:22所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；(iii)V H，其包含如氨基酸序列SEQ ID NO:19所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:23所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；(iv)V H，其包含如氨基酸序列SEQ ID NO:19所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；或(v)V H，其包含如氨基酸序列SEQ ID NO:20所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，提供了分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其包含：(i)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；(ii)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；(iii)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；(iv)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；或(v)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，提供了分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其包含：(i)V H，其包含氨基酸序列SEQ ID NO:17或其变体，所述变体与氨基酸序列SEQ ID NO:17具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:21或其变体，所述变体与氨基酸序列SEQ ID NO:21具有至少约80％序列同源性；(ii)V H，其包含氨基酸序列SEQ ID NO:18或其变体，所述变体与氨基酸序列SEQ ID NO:18具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:22或其变体，所述变体与氨基酸序列SEQ ID NO:22具有至少约80％序列同源性；(iii)V H，其包含氨基酸序列SEQ ID NO:19或其变体，所述变体与氨基酸序列SEQ ID NO:19具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:23或其变体，所述变体与氨基酸序列SEQ ID NO:23具有至少约80％序列同源性；(iv)V H，其包含氨基酸序列SEQ ID NO:19或其变体，所述变体与氨基酸序列SEQ ID NO:19具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:24或其变体，所述变体与氨基酸序列SEQ ID NO:24具有至少约80％序列同源性；或(v)V H，其包含氨基酸序列SEQ ID NO:20或其变体，所述变体与氨基酸序列SEQ ID NO:20具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:25或其变体，所述变体与氨基酸序列SEQ ID NO:25具有至少约80％序列同源性。
在一些实施例中，提供了分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其为特异性结合肺炎克雷伯菌LPS侧链内D-半乳聚糖I结构域的抗体或抗原结合片段。在一些实施例中，提供一种分离的特异性结合肺炎克雷伯菌O2抗原的抗体，其与上述任一种分离的抗体竞争性地与具有D-半乳聚糖I结构域的肺炎克雷伯菌特异性结合。在一些实施例中，提供一种分离的特异性结合肺炎克雷伯菌O2抗原的抗体，其与上述任一种分离的特异性结合肺炎克雷伯菌O2抗原的抗体结合相同的表位。
在一些实施例中，如上所述任一种分离的特异性结合肺炎克雷伯菌O2抗原的抗体，其包含Fc区。在一些实施例中，所述分离的特异性结合肺炎克雷伯菌O2抗原的抗体是全长的IgG抗体。在一些实施例中，所述分离的特异性结合肺炎克雷伯菌O2抗原的抗体是全长的IgG1或IgG4抗体。在一些实施例中，所述分离的特异性结合肺炎克雷伯菌O2抗原的抗体是嵌合的、全人的或人源化的抗体。在一些实施例中，所述分离的特异性结合肺炎克雷伯菌O2抗原的抗体是抗原结合片段，所述抗原结合片段选自Fab、Fab’、F(ab)’ 2、Fab’-SH、单链Fv(scFv)、Fv片段、dAb、Fd、纳米抗体(nanobody)、双链抗体(diabody)和线性抗体。
在一些实施例中，提供一种分离的核酸分子，所述核酸分子编码如上所述任一种特异性结合肺炎克雷伯菌O2抗原的抗体。在一些实施例中，提供一种载体，所述载体包含如上所述任一种核酸分子。在一些实施例中，提供一种宿主细胞，所述宿主细胞包含如上所述任一种特异性结合肺炎克雷伯菌O2抗原的抗体、如上所述任一种核酸分子或如上所述任一种载体。在一些实施例中，提供一种制备特异性结合肺炎克雷伯菌O2抗原的抗体的方法，其包含：a)在能有效表达特异性结合肺炎克雷伯菌O2抗原的抗体的条件下培养上述任一种宿主细胞；和b)从宿主细胞中获得所表达的抗体。
在一些实施例中，提供包含如上所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体的药物组合物、试剂盒以及生产制品。
在一些实施例中，提供一种治疗所需个体疾病或病症的方法，包括向所述个体施用有效量的如上所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体或包含其的药物组合物。在一些实施例中，提供如上所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体在制备用于治疗所需个体疾病或病症的药物组合物中的用途。在一些实施例中，提供如上所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体或包含其的药物组合物在制备用于治疗疾病或病症的药物中的用途。在一些实施例中，所述疾病或病症包括与克雷伯菌属感染相关联的病症。在一些实施例中，所述疾病或病症包括肺炎、尿路感染、败血症/菌血症/脓毒症、新生儿败血症/菌血症/脓毒症、腹泻、软组织感染、器官移植后感染、手术感染、伤口感染、肺部感染、化脓性肝脓肿、肺脓肿、蜂窝组织炎、坏死性肌膜炎、肌炎、眼内炎、腹膜炎、脑膜炎、坏死性脑膜炎、强直性脊柱炎或脊柱关节病。
在一些实施例中，本申请提供了特异性结合肺炎克雷伯菌O2抗原和特异性结合肺炎克雷伯菌O1抗原的双特异性抗体，以及包含特异性结合肺炎克雷伯菌O2抗原的抗体和特异性结合肺炎克雷伯菌O1抗原的抗体的药物组合物。还提供其用于预防和治疗肺炎克雷伯菌感染的方法。
一方面，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：(a)重链可变区(V H)，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1；HC-CDR2，其包含氨基酸序列SEQ ID NO:4；和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及轻链可变区(V L)，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11；LC-CDR2，其包含氨基酸序列SEQ ID NO:14；和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；或者(b)重链可变区(V H)，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1；HC-CDR2，其包含氨基酸序列SEQ ID NO:2；和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及轻链可变区(V L)，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10；LC-CDR2，其包含氨基酸序列SEQ ID NO:12；和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述第二抗原结合域包含：(a)重链可变区(V H)，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35；HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及轻链可变区(V L)，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41；LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或者(b)重链可变区(V H)，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36；HC-CDR2，其包含氨基酸序列SEQ ID NO:38；和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及轻链可变区(V L)，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42；LC-CDR2，其包含氨基酸序列SEQ ID NO:44；和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，根据本申请所述的任一双特异性抗体，第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4， 和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；和第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。在一些实施例中，所述第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；和第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。在一些实施例中，所述第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；和第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。在一些实施例中，所述第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；和第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
一方面，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：(a)V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ  ID NO:19或28所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24或33所示的V L包含的LCDR1、LCDR2和LCDR3；或者(b)V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:20或29所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25或34所示的V L包含的LCDR1、LCDR2和LCDR3。在一些实施例中，所述第二抗原结合域包含：(a)V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:47或51所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:49或53所示的V L包含的LCDR1、LCDR2和LCDR3；或者(b)V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:48或52所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:50或54所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；和其第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列。在一些实施例中，所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；和其第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。在一些实施例中，所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；和其第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨 基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列。在一些实施例中，所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；和其第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。
在一些实施例中，所述双特异性抗体的结构选自DVD-Ig、Bs4Ab、Hetero H,CrossMab、IgG-(scFv) 2或scFv-Fab IgG等结构形式。
在一些实施例中，所述双特异性抗体采用DVD-Ig结构。在一些实施例中，所述双特异性抗体包含四条多肽链：其中两条多肽链均包含V H1-L-V H2-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；L是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；另外两条多肽链均包含V L1-L-V L2-C L结构，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；V L2是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；L是连接肽；C L是轻链恒定区；其中，V H1和V L1组成可特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fv)，V H2-C H1和V L2-C L组成可特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)。在另一些实施例中，所述的双特异性抗体，其包含四条多肽链：其中两条多肽链均包含V H1-L-V H2-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；L是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；另外两条多肽链均包含V L1-L-V L2-C L结构，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；V L2是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；L是连接肽；C L是轻链恒定区；其中，V H1和V L1组成可特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fv)，V H2-C H1和V L2-C L组成可特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)。在一些实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:61，或包含与氨基酸序列SEQ ID NO:61具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:62，或包含与氨基酸序列SEQ ID NO:62具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:63，或包含与氨基酸序列SEQ ID NO:63具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:64，或包含与氨基酸序列SEQ ID NO:64具有至少80％序列同源性的变体序列；或(c)氨基酸序列SEQ ID NO:65，或包含与氨基酸序列SEQ ID NO:65具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％序列同源性的变体序列；或(d)氨基酸序列SEQ ID NO:67，或包含与氨基酸序列SEQ ID NO:67具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:68，或包含与氨基酸序列SEQ ID NO:68具有至少80％序列同源性的变体序列。在一些 实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:86，或包含与氨基酸序列SEQ ID NO:86具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:62，或包含与氨基酸序列SEQ ID NO:62具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:87，或包含与氨基酸序列SEQ ID NO:87具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:64，或包含与氨基酸序列SEQ ID NO:64具有至少80％序列同源性的变体序列；或(c)氨基酸序列SEQ ID NO:88，或包含与氨基酸序列SEQ ID NO:88具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％序列同源性的变体序列；或(d)氨基酸序列SEQ ID NO:89，或包含与氨基酸序列SEQ ID NO:89具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:68，或包含与氨基酸序列SEQ ID NO:68具有至少80％序列同源性的变体序列；或(e)氨基酸序列SEQ ID NO:115，或包含与氨基酸序列SEQ ID NO:115具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％序列同源性的变体序列。
在一些实施例中，所述双特异性抗体采用Bs4Ab结构。在一些实施例中，所述双特异性抗体包含四条多肽链：其中两条多肽链从N端到C端包含V H1-C H1-L1-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；L1和L3是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和另外两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)，V H2-L3-V L2组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(scFv)。在一些实施例中，所述双特异性抗体包含四条多肽链：其中两条多肽链从N端到C端包含V H1-C H1-L1-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；L1和L3是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和另外两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)，V H2-L3-V L2组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(scFv)。在一些实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:69，或包含与氨基酸序列SEQ ID NO:69具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:70，或包含与氨基酸序列SEQ ID NO:70具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:71，或包含与氨基酸序列SEQ ID NO:71具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:72，或包含与氨基酸序列SEQ ID NO:72具有至少80％序列同源性的变体序列；或(c)氨基酸序列SEQ ID NO:73，或包含与氨基酸序列SEQ ID NO:73具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列；或(d)氨基酸序列SEQ ID NO:75，或包含与氨基酸序列SEQ ID NO:75具有至少80％序列同源性的变体序列；和/或 氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。在一些实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:90，或包含与氨基酸序列SEQ ID NO:90具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:70，或包含与氨基酸序列SEQ ID NO:70具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:91，或包含与氨基酸序列SEQ ID NO:91具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:72，或包含与氨基酸序列SEQ ID NO:72具有至少80％序列同源性的变体序列；或(c)氨基酸序列SEQ ID NO:92，或包含与氨基酸序列SEQ ID NO:92具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列；或(d)氨基酸序列SEQ ID NO:93，或包含与氨基酸序列SEQ ID NO:93具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
在一些实施例中，所述双特异性抗体采用Hetero H,CrossMab结构。在一些实施例中，所述双特异性抗体包含四条多肽链：其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和其中一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区；和其中一条多肽链从N端到C端包含V H2-C L，其中V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；C L是轻链恒定区；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和其中一条多肽链从N端到C端包含V L2-C H1，其中V L2是特异性结合肺炎克雷伯菌O1抗原的第一个轻链可变区，C H1是重链恒定区C H1结构域。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)，V H2-C L和V L2-C H1组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)。在一些实施例中，所述双特异性抗体包含四条多肽链：其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和其中一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区；和其中一条多肽链从N端到C端包含V H2-C L，其中V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；C L是轻链恒定区；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和其中一条多肽链从N端到C端包含V L2-C H1，其中V L2是特异性结合肺炎克雷伯菌O2抗原的第一个轻链可变区，C H1是重链恒定区C H1结构域。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)，V H2-C L和V L2-C H1组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)。在一些实施例中，所述双特异性抗体包含：(a)；氨基酸序列SEQ ID NO:76，或包含与氨基酸序列SEQ ID NO:76具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:77，或包含与氨基酸序列SEQ ID NO:77具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:78，或包含与氨基酸序列SEQ ID NO:78具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO: 74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:79，或包含与氨基酸序列SEQ ID NO:79具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:80，或包含与氨基酸序列SEQ ID NO:80具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:78，或包含与氨基酸序列SEQ ID NO:78具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。在一些实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:94，或包含与氨基酸序列SEQ ID NO:94具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:77，或包含与氨基酸序列SEQ ID NO:77具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:95，或包含与氨基酸序列SEQ ID NO:95具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:96，或包含与氨基酸序列SEQ ID NO:96具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:80，或包含与氨基酸序列SEQ ID NO:80具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:95，或包含与氨基酸序列SEQ ID NO:95具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
在一些实施例中，所述双特异性抗体采用IgG-(scFv) 2结构。在一些实施例中，所述双特异性抗体包含四条多肽链：其中两条多肽链从N端到C端包含V H1-C H1-C H2-C H3-L-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；L和L3是连接肽；C H1是重链恒定区C H1结构域；C H2是重链恒定区C H2结构域；C H3是重链恒定区C H3结构域；和其中两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)；V H2-L3-V L2组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(scFv)。在一些实施例中，所述双特异性抗体包含四条多肽链：其中两条多肽链从N端到C端包含V H1-C H1-C H2-C H3-L-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；L和L3是连接肽；C H1是重链恒定区C H1结构域；C H2是重链恒定区C H2结构域；C H3是重链恒定区C H3结构域；和其中两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)；V H2-L3-V L2组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(scFv)。在一些实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:97，或包含与氨基酸序列SEQ ID NO:97具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:98，或包含与氨基酸序列SEQ ID NO:98具有至少80％序列同源性的变体序列；和/ 或氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％序列同源性的变体序列。
在一些实施例中，所述双特异性抗体采用scFv-Fab IgG结构。在一些实施例中，所述双特异性抗体包含三条多肽链：其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和其中一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区；和其中一条多肽链从N端到C端包含V H2-L3-V L2，其中V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；L3是连接肽；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)；V H2-L3-V L2组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(scFv)。在一些实施例中，所述双特异性抗体包含三条多肽链：其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和其中一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区；和其中一条多肽链从N端到C端包含V H2-L3-V L2，其中V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；L3是连接肽；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域。其中，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)；V H2-L3-V L2组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(scFv)。在一些实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:81，或包含与氨基酸序列SEQ ID NO:81具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:82，或包含与氨基酸序列SEQ ID NO:82具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:84，或包含与氨基酸序列SEQ ID NO:84具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:82，或包含与氨基酸序列SEQ ID NO:82具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％序列同源性的变体序列。在一些实施例中，所述双特异性抗体包含：(a)氨基酸序列SEQ ID NO:99，或包含与氨基酸序列SEQ ID NO:99具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:100，或包含与氨基酸序列SEQ ID NO:100具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％序列同源性的变体序列；或(b)氨基酸序列SEQ ID NO:101，或包含与氨基酸序列SEQ ID NO:101具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:100，或包含与氨基酸序列SEQ ID NO:100具有至少80％序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％序列同源性的变体序列。
在一些实施例中，所述双特异性抗体包含Fc，其中Fc选自IgGl、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD的Fc。在一些实施例中，所述Fc包含Fc变体。在一些实施例中，所述Fc是糖基化的。在一些实施例中，所述Fc是去糖基化的。在一些实施例中，所述Fc是岩藻糖基化减少的或是无岩藻糖基化。
一方面，本申请提供了一种药物组合物包括：(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段；其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：(a)重链可变区(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；HC-CDR2，其包含氨基酸序列SEQ ID NO:4；和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及轻链可变区(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:11；LC-CDR2，其包含氨基酸序列SEQ ID NO:14；和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；或者(b)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1；HC-CDR2，其包含氨基酸序列SEQ ID NO:2；和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10；LC-CDR2，其包含氨基酸序列SEQ ID NO:12；和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。
一方面，本申请提供了一种在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段；其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：(a)重链可变区(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；HC-CDR2，其包含氨基酸序列SEQ ID NO:4；和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及轻链可变区(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:11；LC-CDR2，其包含氨基酸序列SEQ ID NO:14；和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；或者(b)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1；HC-CDR2，其包含氨基酸序列SEQ ID NO:2；和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10；LC-CDR2，其包含氨基酸序列SEQ ID NO:12；和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。
在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：(a)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35；HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41；LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或者(b)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36；HC-CDR2，其包含氨基酸序列SEQ ID NO:38；和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42；LC-CDR2，其包含氨基酸序列SEQ ID NO:44；和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含 氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
一方面，本申请提供了一种药物组合物包括：(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段；其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：(a)V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:19或28所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24或33所示的V L包含的LCDR1、LCDR2和LCDR3；或者(b)V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:20或29所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25或34所示的V L包含的LCDR1、LCDR2和LCDR3。
一方面，本申请提供了一种在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段；其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：(a)V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:19或28所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24或33所示的V L包含的LCDR1、LCDR2和LCDR3；或者(b)V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:20或29所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25或34所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：(a)V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:47或51所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:49或53所示的V L包含的LCDR1、LCDR2和LCDR3；或者(b)V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少约80％ 序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:48或52所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:50或54所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列。在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列。在一些实施例中，所述药物组合物或方法中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；以及特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段是同时施用的。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段是顺序施用的。
在一些实施例中，提供了在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的本申请所述的任一抗体或抗原结合片段和/或任一药物组合物和/或双特异性抗体。
在一些实施例中，根据本申请所述的任一方法，所述疾病或病症包含由克雷伯(氏)菌感染引起的一种或多种症状。在一些实施例中，所述克雷伯(氏)菌为肺炎克雷伯菌。在一些实施例中，所述的症状包括肺炎、尿路感染、败血症/菌血症/脓毒症、新生儿败血症/菌血症/脓毒症、腹泻、软组织感染、器官移植后感染、手术感染、伤口感染、肺部感染、化脓性肝脓肿、肺脓肿、蜂窝组织炎、坏死性肌膜炎、肌炎、眼内炎、腹膜炎、脑膜炎、坏死性脑膜炎、强直性脊柱炎或脊柱关节病。
在一些实施例中，提供了一种分离的核酸分子，其编码如上所述的任一双特异性抗体。在一些实施例中，提供了一种载体，其包含如上所述的任一核酸分子。在一些实施例中，提供了一种宿主细胞，其包含如上所述的任一双特异性抗体、任一核酸分子或任一载体。在一些实施例中，提供了一种制备特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的方法，包括：a)在有效表达特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的条件下培养如上所述的任一宿主细胞；并且b)在宿主细胞中获得所表达的双特异性抗体。
还提供了包含如上所述的任一种双特异性抗体、核酸分子、载体、或宿主细胞的药物组合物、试剂盒和制品。
附图说明
图1所示为肺炎克雷伯菌O1血清型KP19173菌株LPS和KP19213菌株LPS的结构示意图。
图2所示为肺炎克雷伯菌O2血清型KP19180菌株LPS和KP19203菌株LPS的结构示意图。
图3A所示为特异性结合肺炎克雷伯菌O2抗原的抗体K2、K3或K5与KP19180菌株O2型LPS的ELISA结合结果；图3B所示为特异性结合肺炎克雷伯菌O2抗原的抗体K2、K3或K5与KP19203菌株O2型LPS的ELISA结合结果；图3C所示为特异性结合肺炎克雷伯菌O2抗原的抗体K2、K3或K5与KP19173菌株O1型LPS的ELISA结合结果；图3D所示为特异性结合肺炎克雷伯菌O2抗原的抗体K2、K3或K5与KP19213菌株O1型LPS的ELISA结合结果。
图4A-图4D所示结果为示例性特异性结合肺炎克雷伯菌O2抗原的抗体K2、K3及K5与肺炎克雷伯菌株结合具有广谱性。图4A为特异性结合肺炎克雷伯菌O2抗原的抗体与肺炎克雷伯菌株KP19002O2型LPS的ELISA结合结果；图4B为特异性结合肺炎克雷伯菌O2抗原的抗体与肺炎克雷伯菌株KP19003O2型LPS的ELISA结合结果；图4C为特异性结合肺炎克雷伯菌O2抗原的抗体与肺炎克雷伯菌株KP19005O2型LPS的ELISA结合结果；图4D为特异性结合肺炎克雷伯菌O2抗原的抗体与肺炎克雷伯菌株KP19007O2型LPS的ELISA结合结果。
图5所示结果为在肺炎克雷伯菌KP19180诱导的菌血症模型中，示例性特异性结合肺炎克雷伯菌O2抗原的抗体K2或K5对小鼠菌血症的预防保护效果。
图6所示结果为在肺炎克雷伯菌KP19173诱导的肺炎模型中，联合应用特异性结合肺炎克雷伯菌O2抗原的抗体K5和特异性结合肺炎克雷伯菌O1抗原的抗体G2对小鼠肺炎的治疗效果。
图7A所示为DVD-Ig(Dual-variable domain-Ig)双特异性抗体结构示意图；图7B所示为Bs4Ab双特异性抗体结构示意图；图7C所示为Hetero H,CrossMab双特异性抗体结构示意图；图7D所示为IgG-(scFv) 2双特异性抗体结构示意图；图7E所示为scFv-Fab IgG双特异性抗体结构示意图。
图8A-图8C所示分别为DVD-Ig结构的示例性双特异性抗体与不同血清型肺炎克雷伯菌株LPS的ELISA结合结果。图8A为KP19173菌株O1型LPS的结合结果；图8B为KP19213菌株O1型LPS的结合结果；图8C为KP19180菌株O2型LPS的结合结果。
图8D-图8F所示分别为Bs4Ab结构的示例性双特异性抗体与不同血清型肺炎克雷伯菌株LPS的ELISA结合结果。图8D为KP19173菌株O1型LPS的结合结果；图8E为KP19213菌株O1型LPS的结合结果；图8F为KP19180菌株O2型LPS的结合结果。
图8G-图8I所示分别为Hetero H,CrossMab结构的示例性双特异性抗体与不同血清型肺炎克雷伯菌株LPS的ELISA结合结果。图8G为KP19173菌株O1型LPS的结合结果；图8H为KP19213菌株O1型LPS的结合结果；图8I为KP19180菌株O2型LPS的结合结果。
图8J-图8L所示分别为IgG-(scFv) 2结构的示例性双特异性抗体与不同血清型肺炎克雷伯菌株LPS的ELISA结合结果。图8J为KP19173菌株O1型LPS的结合结果；图8K为KP19213菌株O1型LPS的结合结果；图8L为KP19180菌株O2型LPS的结合结果。
图8M-图8O所示分别为scFv-Fab IgG结构的示例性双特异性抗体与不同血清型肺炎克雷伯菌株LPS的ELISA结合结果。图8M为KP19173菌株O1型LPS的结合结果；图8N为KP19213菌株O1型LPS的结合结果；图8O为KP19180菌株O2型LPS的结合结果。
图9A-图9C所示分别为DVD-Ig结构的示例性双特异性抗体对不同血清型肺炎克雷伯菌株LPS的中和活性结果。图9A为对KP19173菌株O1型LPS的中和活性；图9B为对KP19213菌株O1型LPS的中和活性结果；图9C为对KP19180菌株O2型LPS的中和活性结果。
图9D-图9F所示分别为Bs4Ab结构的示例性双特异性抗体对不同血清型肺炎克雷伯菌株LPS的中和活性结果。图9D为对KP19173菌株O1型LPS的中和活性结果；图9E为对KP19213菌株O1型LPS的中和活性结果；图9F为对KP19180菌株O2型LPS的中和活性结果。
图9G-图9I所示分别为Hetero H,CrossMab结构的示例性双特异性抗体对不同血清型肺炎克雷伯菌株LPS的中和活性结果。图9G为对KP19173菌株O1型LPS的中和活性结果；图9H为对KP19213菌株O1型LPS的中和活性结果；图9I为对KP19180菌株O2型LPS的中和活性结果。
图9J-图9L所示分别为IgG-(scFv) 2结构的示例性双特异性抗体对不同血清型肺炎克雷伯菌株LPS的中和活性结果。图9J为对KP19173菌株O1型LPS的中和活性结果；图9K为对KP19213菌株O1型LPS的中和活性结果；图9L为对KP19180菌株O2型LPS的中和活性结果。
图9M-图9O所示分别为scFv-Fab IgG结构的示例性双特异性抗体对不同血清型肺炎克雷伯菌株LPS的中和活性结果。图9M为对KP19173菌株O1型LPS的中和活性结果；图9N为对KP19213菌株O1型LPS的中和活性结果；图9O为对KP19180菌株O2型LPS的中和活性结果。
图10A所示为DVD-Ig形式的双特异性抗体K5-G2-Ig1针对KP19173菌株的促进调理吞噬杀菌活性结果；图10B所示为DVD-Ig形式的双特异性抗体K5-G7-Ig1针对KP19173菌株的促进调理吞噬杀菌活性结果；图10C所示为Bs4Ab形式的双特异性抗体K5-G2scFv-Ig1针对KP19173菌株的促进调理吞噬杀菌活性结果；图10D所示为Bs4Ab形式的双特异性抗体K5-G7scFv-Ig1针对KP19173菌株的促进调理吞噬杀菌活性结果。
图11A所示为DVD-Ig形式的双特异性抗体K5-G2-Ig1针对KP19173菌株的血清杀菌活性结果；图11B所示为DVD-Ig形式的双特异性抗体K5-G7-Ig1针对KP19173菌株的血清杀菌活性结果；图11C所示为Bs4Ab形式的双特异性抗体K5-G2scFv-Ig1针对KP19173菌株的血清杀菌活性结果；图11D所示为Bs4Ab形式的双特异性抗体K5-G7scFv-Ig1针对KP19173菌株的血清杀菌活性结果。
图12A所示为在小鼠肺炎治疗模型中，双特异性抗体K5-G2-Ig1对肺炎克雷伯菌KP19180感染引起的肺炎的治疗效果；图12B所示为双特异性抗体K5-G2-Ig1对肺炎克雷伯菌KP19173感染小鼠引起的肺炎的治疗效果。
本申请的详细描述
本申请一方面提供特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，一方面提供特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体。一方面提供包含特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的组合物。通过scFv噬菌体库筛选、亲和力成熟以及适当设计的生物化学及生物学实验的组合，鉴定出特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段。同时制备了特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体。当以(i)药物组合物的方式，或(ii)双特异性抗体的方式，或(iii)联合应用的方式治疗疾病时能够覆盖不同血清型肺炎克雷伯菌，达到累积或协同作用，提高抗菌广谱性。
同时还提供编码特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段、或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的核酸，包含特异性结合肺炎克雷伯菌O2抗原的抗体和特异性结合肺炎克雷伯菌O1抗原的抗体的组合物，以及制备和使用特异性结合肺炎克雷伯菌O2抗原的抗体或特异性结合肺炎克雷伯菌O1抗原的抗体或双特异性抗体以及包含其的组合物的方法。
定义
如本申请所述，“O抗原”是指克雷伯(氏)菌LPS的组成部分之一，与类脂A，核心寡糖共同构成LPS。所述“O1抗原”是指O1血清型克雷伯(氏)菌LPS的O抗原部分，所述“O2抗原”是指O2血清型克雷伯(氏)菌LPS的O抗原部分，并且所述O抗原包含D-半乳聚糖-I二糖单元。
如本申请所述，“治疗(treatment)”或“治疗(treating)”是一种获得有益的或期望的结果的方法，包括临床结果。鉴于本申请的目的，所述有益的或期望的临床结果，包括但不限于以下一种或多种： 缓解由疾病引起的一种或多种症状，减轻疾病程度，稳定疾病(例如，预防或延缓疾病恶化)，预防或延缓疾病的扩散(例如，病原菌全身扩散)，预防或延缓疾病复发，延缓或减缓疾病进展，改善疾病状态，缓解疾病(部分或全部)，减少治疗疾病所需的一种或多种其他药物的剂量，延迟疾病进展，改善或提高生活质量，增加体重，和/或延长生存期。同时，“治疗”还包括感染病理结果的减少(例如，宿主细胞裂解或坏死)。本申请的方法考虑了这些治疗的任何一个或多个方面。
术语“预防(prevent)”以及类似的词，如“预防(prevented)”、“预防(preventing)”、“预防(prevention)”或“预防(prophylactic)”等，表示一种预防、抑制或减少疾病或病症(如病原体感染)发生或复发可能性的方法。它还指延缓一种疾病或病症的发生或复发，或延缓一种疾病或病症的症状的发生或复发。正如在此所使用的，“预防(prevention)”和类似的词还包括在疾病或病症发生或复发之前减轻其强度、影响、症状和/或负担。正如在此所使用的，“预防(prevention)”和类似的词还包括减少疾病或病症发生或复发的风险和易感性，例如病原体感染。
抗体或抗原结合片段如本申请所述，术语“抗体”是广义的，包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、单特异性、多特异性抗体(如双特异性抗体)、全长抗体及其抗原结合片段，只要它们表现出所需的抗原结合活性。全长抗体包括两条重链和两条轻链。轻链和重链的可变区负责抗原的结合。两条链中的可变区通常包括3个高变的环，被称为互补决定区(CDRs)，轻链(LC)CDRs包括LC-CDR1、LC-CDR2和LC-CDR3，重链(HC)CDRs包括HC-CDR1、HC-CDR2和HC-CDR3)。本申请所披露的抗体或抗原结合片段的CDR边界可通过Kabat,Chothia或Al-Lazikani惯例来定义或识别(Al-Lazikani 1997；Chothia 1985；Chothia 1987；Chothia 1989；Kabat 1987；Kabat 1991)。重链或轻链的3个CDR区插入到被称为框架区(FRs)的侧翼区段之间，所述框架区比CDR区具有更高的保守性，并形成支撑高变环的支架。重链和轻链的恒定区并不参与抗原结合，但展示出多种效应功能。抗体是基于它们重链恒定区的氨基酸序列进行分类的。抗体的五种主要类别或同种型是IgA、IgD、IgE、IgG和IgM，其特征在于分别具有α、δ、ε、γ和μ型重链。几种主要的抗体类别被分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链n)或IgA2(α2重链)。
如本申请所述，术语“抗原结合片段”包括一种抗体片段，包括，例如，双链抗体(diabody)、Fab、Fab’、F(ab’)2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv) 2、双特异性dsFv(dsFv-dsFv’)、二硫键稳定的双链抗体(ds双链抗体)、单链抗体(scFv)、scFv二聚体(二价双链抗体)，由包含一个或多个CDRs的抗体片段组成的多特异性抗体、单域抗体、纳米抗体、域抗体、二价域抗体或者能够与抗原结合但不包含完整抗体结构的任何其他抗体片段。其中如本申请所述的Fab(fragment antigen-binding)，其是包含抗体的V L结构域、V H结构域、C L结构域、以及C H1结构域的单价片段。抗原结合片段还包括包含上述抗体片段的融合蛋白。抗原结合片段能够与亲本抗体或亲本抗体片段(如亲本scFv)结合相同的抗原。在一些实施例中，抗原结合片段可能包括来自特定人抗体的一个或多个CDRs，该CDRs被移植到来自一个或多个不同人抗体的框架区。
如本申请所述，术语“双特异性抗体”是指在一个抗体分子中对两个不同抗原或表位具有结合特异性。双特异性抗体的生产过程包括完整分子的设计、每个结构域的核苷酸序列的合成和克隆、哺乳动物细胞的表达和最终产品的纯化。示例性的双特异性抗体结构包括本领域所已知的结构，例如，DVD-Ig结构、Bs4Ab结构、Hetero H,CrossMab结构、IgG-(scFv) 2结构或scFv-Fab IgG结构等(例如，参见综述文献Labrijn AF,et al.Nat Rev Drug Discov.2019Aug；18(8):585-608)。
DVD-Ig(Dual-variable domain-Ig)双特异性抗体，其结构是在正常IgG抗体的轻链和重链的N末端分别连接另外一个抗体的V L和V H结构域，通过两个抗体的V H与V L相互作用形成抗原结合域域(Fv)，能够同时与相对应的抗原结合，来实现双特异性。示例性的DVD-Ig结构的双特异性抗体在文献Wu C,et al.Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin(DVD-Ig)molecules.MAbs.2009Jul-Aug；1(4):339-47中也有记载。Bs4Ab结构双特异性抗体是一种双特异性4价抗体，其包括一个全长的IgG1结构，通过在其铰链区插入另一个结合单元scFv，来实现双特异性。关于Bs4Ab结构的双特异性抗体在文献Bezabeh B,et al.Insertion of scFv into the hinge domain of full-length IgG1monoclonal antibody results in tetravalent bispecific molecule with robust properties.MAbs.2017Feb/Mar；9(2):240-256中也有记载。Hetero H,CrossMab结构双特异性抗体，即在Fc区设计了杵臼结构(knobs-in-holes，KIH)，同时引入形成稳定化二硫桥的两个Cys残基突变(S354C在“knob”侧，并且Y349C在“hole”侧)。同时应用CrossMab技术，来确保抗体轻、重链间的正确配对。CrossMab技术是基于双特异性IgG抗体的一个Fab臂内抗体域的交换，可以是完整的Fab域的交换(CrossMAb Fab)，或者是Fab域中仅可变区的交换(CrossMAb V H-V L)或仅恒定区的交换(CrossMAb C H1-C L)。关于Hetero H,CrossMab结构的双特异性抗体在文献Klein C,et al.The use of CrossMAb technology for the generation of bi-and multispecific antibodies.MAbs.2016Aug-Sep；8(6):1010-20中也有记载。IgG-(scFv) 2结构双特异性抗体，即在一个IgG抗体的两条重链的Fc末端连接另一个抗体的scFv片段来实现双特异性。关于IgG-(scFv) 2结构的双特异性抗体在文献Coloma MJ,Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nat Biotechnol.1997Feb；15(2):159-63中也有记载。scFv-Fab IgG结构双特异性抗体，为异二聚体抗体，IgG抗体结构，其中一个Fab臂被替换成scFv结构，其中第一单体包含scFv和IgG Fc，scFv通过连接肽连接在IgG FcC H2域的N末端，第二单体包含Fab和IgG Fc。
如本申请所述，术语“抗原结合域”是指抗原结合分子中与抗原特异性结合的部分。更具体地说，术语“抗原结合域”指的是抗体的一部分，该部分包括一个与部分或全部抗原特异性结合并与之互补的区域。如果是大抗原，抗原结合分子可能只结合抗原的一个特定部分，这个部分被称为抗原表位。例如，抗原结合域可以由一个或多个可变区(也称作可变域)提供。优选地，所述抗原结合域包括抗体轻链可变区(V L)和抗体重链可变区(V H)。一方面，抗原结合域能够结合其抗原并阻断或部分阻断所述抗原的功能。特异性结合肺炎克雷伯菌O2抗原或O1抗原的抗原结合域包括本申请进一步定义的抗体及抗原结合片段。
如本申请所述，术语“表位”是指抗体或抗体部分结合的抗原上特定的原子或氨基酸组。如果两种抗体或抗体部分表现出与某抗原竞争性结合，则它们可能结合抗原上相同表位。
如本申请所述，当第一抗体在等摩尔浓度下抑制第二抗体与肺炎克雷伯菌O2抗原靶标结合至少50％(例如至少55％、60％、65％、70％、75％、80％、85％、90％、95％、98％或99％)时，第一抗体与第二抗体“竞争”结合肺炎克雷伯菌O2抗原靶标，反之亦然。PCT出版物WO 03/48731描述了基于交叉竞争的高通量抗体“表位归类”(binning)方法。
如本申请所述，术语“特异性地结合”、“特异性地识别”或“对…来说是特异性的”是指可测量的和可再现的相互作用，例如靶标与抗体的结合可以确定在异质分子群，包括生物分子中存在该靶标。例如，抗体能够特异性地识别某靶标(可以是表位)是指，与其它靶标结合相比，该抗体与该靶标的结合具有更高的亲和力，亲合力，更容易和/或更持久。在一些实施例中，特异性地识别抗原的抗体与抗原的一个或多个抗原决定簇反应，其结合亲和力是其与其它靶标结合亲和力的至少10倍。
如本申请所述，一种“分离的”抗体是指一种抗体，其(1)与天然存在的蛋白无关，(2)不含相同来源的其他蛋白，(3)由不同种属的细胞所表达，或(4)自然界中不存在。
如本申请所述，术语“分离的核酸”，是指基因组、cDNA或合成来源的核酸或其组合。根据其来源，所述“分离的核酸”(1)与自然界中发现的“分离的核酸”中的全部或部分多核苷酸无关，(2)可与自然状态下不与之相连的多核苷酸可操作性地连接，或(3)在自然界中不作为较长序列的一部分而存在。
如本申请所述，术语“CDR”或“互补决定区”是指重链和轻链多肽的可变区内发现的非连续抗原结合位点。在文献Kabat et al.,J.Biol.Chem.252:6609-6616(1977)；Kabat et al.,U.S.Dept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991)；Chothia et al.,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.et al.,J.Mol.Biol.,273:927-948(1997)；MacCallum et al.,J.Mol.Biol.262:732-745(1996)；Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.et al.,Dev.Comp.Immunol.,27:55-77(2003)；和Honegger and Plückthun,J.Mol.Biol.,309:657-670(2001)中已经描述这些特殊的区域，其中当彼此之间互相比较时，这些定义包括氨基酸残基的重合或子集。然而，采用任何一种定义方式来指示抗体或移植抗体或其变体的CDR，均包括在本申请所定义和使用的术语范围之内。表1中列了由上述引用的各篇参考文献所定义的CDR所包括的氨基酸残基的位置，以示比较。CDR预测的算法和结合界面在本领域是已知的，包括，例如Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008)；Ehrenmann F.et al.,Nucleic Acids Res.,38:D301-D307(2010)；和Adolf-Bryfogle J.et al.,Nucleic Acids Res.,43:D432-D438(2015)中均有描述。本段中所引用的参考文献的内容以其整体引用并入本申请中，以用于本申请和可能包含在本申请中的一个或多个权利要求中。
表1：CDR定义
  Kabat 1 Chothia 2 MacCallum 3 IMGT 4 AHo 5
V H CDR1 31-35 26-32 30-35 27-38 25-40
V H CDR2 50-65 53-55 47-58 56-65 58-77
V H CDR3 95-102 96-101 93-101 105-117 109-137
V L CDR1 24-34 26-32 30-36 27-38 25-40
V L CDR2 50-56 50-52 46-55 56-65 58-77
V L CDR3 89-97 91-96 89-96 105-117 109-137
1氨基酸残基编号参照上述Kabat et al.中的命名方法
2氨基酸残基编号参照上述Chothia et al.中的命名方法
3氨基酸残基编号参照上述MacCallum et al.中的命名方法
4氨基酸残基编号参照上述Lefranc et al.中的命名方法
5氨基酸残基编号参照上述Honegger and Plückthun中的命名方法
术语“嵌合抗体”是指重链和/或轻链的一部分与来自特定种属或属于特定抗体种类或亚类的抗体中的相应序列一致或具有同源性，而这个(些)链的剩余部分与来自另一种属或属于其它抗体种类或亚类的抗体中的相应序列一致或具有同源性的抗体，以及此类抗体的片段，只要其具有本申请中的生物学活性(见U.S.Patent No.4,816,567；and Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。
“Fv”是包含完整抗原识别及结合位点的最小抗体片段。该片段是由一个重链可变区和一个轻链可变区紧密非共价连接形成的二聚体。通过这两个域的折叠衍生出6个高变环(轻链和重链中各3个环)，所述高变环为抗体提供了用于结合抗原的氨基酸残基，并且赋予抗体与抗原结合的特异性。然而，即使单个可变区(或Fv片段的一半，其仅包含对抗原具有特异性的3个CDRs)也具有识别和结合抗原的能力，尽管其亲和力低于完整的结合位点。
“单链Fv”，也可简写成“sFv”或“scFv”，是包含被连接成单一多肽链的V H和V L抗体域的抗体片段。在一些实施例中，scFv多肽进一步包括V H和V L域之间的连接多肽，该连接多肽使得scFv形成抗原结合的理想结构。关于scFv的概述，见Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds.,Springer-Verlag,New York,pp.269-315(1994)。
术语“双链抗体(diabodies)”是指，在V H和V L之间采用短接头(例如5～10个残基)构建scFv片段(见上段内容)制备而成的一种小抗体片段，这样就使得可变区在链间而不是链内进行配对，产生一个双价片段，即具有两个抗原结合位点的片段。双特异性的双链抗体是两个“交叉”scFv片段的异二聚体，其中两个抗体的V H和V L域位于不同的多肽链上。在EP 404,097；WO 93/11161；Hollinger et al.,Proc.Natl.Acad.Sci.USA,90:6444-6448(1993)中全面描述了双链抗体。
非人源(如啮齿类)抗体的“人源化”形式是嵌合抗体，其包括最少的来自非人源抗体的序列。大多数情况下，人源化抗体是人源免疫球蛋白(受体抗体)，其中受体抗体的高变区(HVR)残基被来自非人源种属例如小鼠、大鼠、兔或非人类灵长类动物的且具有理想的抗体特异性，亲和力和性能的高变区残基所取代(供体抗体)。在某些情况下，人源免疫球蛋白框架区(FR)中的残基被相应的非人源残基所取代。另外，人源化抗体可以包括在受体抗体或供体抗体中均不存在的残基。这些修饰能够进一步改善抗体的性能。通常，人源化抗体会包含基本上至少一个，通常两个可变区，其中所有或基本上所有的高变环均与非人免疫球蛋白的高变环相对应，以及所有或基本上所有的框架区均是人免疫球蛋白序列。人源抗体任选地也还包括免疫球蛋白恒定区(Fc)的至少一部分，通常是人免疫球蛋白的恒定区。具体细节可以参考Jones et al.,Nature 321:522-525(1986)；Riechmann et al.,Nature 332:323-329(1988)；和Presta,Curr.Op.Struct.Biol.2:593-596(1992)。
本申请所鉴定的多肽和抗体序列的“氨基酸序列同源性百分比(％)”或“同源性”被定义为：在认为保守性取代属于序列同源性的一部分的情况下进行序列对比，候选序列与待比较多肽序列中相同氨基酸残基所占的百分比。可以通过本领域技术范围内的多种比对方式来确定氨基酸序列同源性百分比，例如，使用如BLAST、BLAST-2、ALIGN、Megalign(DNASTAR)、或MUSCLE软件等可公开获得的计算机软件。本领域技术人员可以确定用于测量比对的合适的参数，包括在所比较序列的全长上实现最大化比对所需的任何算法。然而，为了本申请的目的，氨基酸序列同源性百分比数值是使用序列比对电脑程序MUSCLE(Edgar,R.C.,Nucleic Acids Research 32(5):1792-1797,2004；Edgar,R.C.,BMC Bioinformatics 5(1):113,2004)生成的。
术语“Fc(fragment crystallizable)”或“Fc区”，是指包含完整抗体恒定区的多肽，不包含CH 1结构域，在某些情况下包含部分铰链，无论是单体形式还是多聚体形式。天然Fc的原始免疫球蛋白来源优选地为人类来源，并且可以是任何免疫球蛋白，例如，IgG1、IgG2、IgG3或IgG4。天然Fc由单体多肽组成，单体多肽可以通过共价(即二硫键)和非共价缔合连接成二聚体或多聚体形式。免疫球蛋白的Fc区一般包含重链恒定区的C H2结构域和C H3结构域，且任选包含C H4结构域。
在一些实施例中，Fc二聚体中的两个Fc单体中的每一个包含促进两个单体异源二聚体化的氨基酸取代。在一些实施例中，Fc单体的异源二聚体化可以通过在两个Fc单体中引入不同但相容的取代诸如“knob-into-hole”残基对来促进。“knob-into-hole”技术也公开于美国专利公布第8,216,805号中。在一些实施例中，一个Fc单体包含knob突变T366W，并且另一个Fc单体包含hole突变T366S、L358A和Y407V。在一些实施例中中，引入形成稳定化的二硫桥的两个Cys残基(S354C在“knob”侧，并且Y349C在“hole”侧)。
术语“Fc受体”或“FcR”用于描述结合抗体Fc区的受体。在一些实施例中，本申请所述的FcR是结合IgG抗体(一种γ受体)的FcR，包括FcγRI、FcγRII和FcγRIII亚类的受体，包括这些受体的等位基因变体和可变剪接形式。FcγRII受体包括FcγRIIA(激活受体)和FcγRIIB(抑制受体)，它们具有相似的氨基酸序列，主要在细胞质结构域有所不同。激活受体FcγRIIA的胞质结构域中含有免疫受体酪氨酸活化基序(ITAM)。抑制受体FcγRIIB的胞质结构域中含有免疫受体酪氨酸抑制基 序(ITIM)(见M.in
Figure PCTCN2022133880-appb-000001
Annu.Rev.Immunol.15:203-234(1997))。所述术语还包括同种异型，例如FcγRIIIA同种异型:FcγRIIIA-Phe158、FcγRIIIA-Val158、FcγRIIA-R131和/或FcγRIIA-H131。在Ravetch and Kinet,Annu.Rev.Immunol 9:457-92(1991)和Capel et al.,Immunomethods 4:25-34(1994)；and de Haas et al.,J.Lab.Clin.Med.126:330-41(1995)中对FcRs进行了描述。本申请中术语FcR涵盖其他类型的FcRs，包括将来鉴定的FcRs。术语FcR同时还包括新生儿受体FcRn，其负责向新生儿转移母体IgGs(Guyer et al.,J.Immunol.117:587(1976)and Kim et al.,J.Immunol.24:249(1994))。
术语“FcRn”指新生儿Fc受体(FcRn)。FcRn与主要组织相容性复合体(MHC)在结构上相似，由α链非共价结合到β2微球蛋白上组成。新生儿Fc受体FcRn的多种功能在Ghetie and Ward(2000)Annu.Rev.Immunol.18,739-766.中进行了描述。FcRn在免疫球蛋白IgGs从母体向新生儿的被动转运和调控血清IgG水平中起到重要作用。FcRn作为一种救助受体，可以在细胞内和细胞间以完整的形式结合和运输胞吞化的IgG，并使它们免于经受默认的降解途径。
人IgG重链恒定区的“C H1结构域”通常从118位氨基酸延伸到215位氨基酸(EU编号系统)。
“铰链区”通常被定义为从人IgG1的216位Glu延伸到230位Pro(Burton,Molec.Immunol.22:161-206(1985))。通过将形成重链间二硫键的第一个和最后一个半胱氨酸残基置于与IgG1相同位置后，可以使得其他IgG亚型的铰链区与IgG1序列比对。
人IgG Fc区的“C H2结构域”通常从231位氨基酸延伸到340位氨基酸。C H2结构域的独特之处在于，它不会与另一个区域紧密配对。而是在完整的天然IgG分子的两个C H2结构域之间插入了两条N端连接的支链糖链。据推测，糖类可能作为域与域间配对的替代，有助于保持C H2结构域稳定。Burton,Molec Immunol.22:161-206(1985)。
“C H3”结构域包括在Fc区内从C末端残基延伸到C H2结构域(从341位氨基酸到抗体序列的C末端，通常为IgG的第446或447位氨基酸残基)。
“功能性Fc片段”具有天然Fc区序列所具有的“效应功能”。示例性的“效应功能”包括C1q结合；补体依赖的细胞毒作用(CDC)；Fc受体结合；抗体依赖的细胞介导的细胞毒作用(ADCC)；吞噬作用；细胞表面受体的下调(如B细胞受体；BCR)等。这类效应功能通常需要Fc区与结合结构域(如抗体可变区)结合，并且可以使用本领域公知的多种实验方法进行评估。
具有“改变的”FcR结合亲和力或ADCC活性的IgG Fc变体的抗体，与亲本多肽或包含天然Fc序列的多肽相比，其FcR结合活性和/或ADCC活性增强或减弱。表现出与FcR“结合增强”的Fc变体与亲本多肽或包含天然IgG Fc序列的多肽相比，其与至少一种FcR具有更高的结合亲和力(例如更低的表观Kd或IC50值)。在一些实施例中，与亲本多肽相比，结合能力增强3倍，例如5、10、25、50、60、100、150、200，甚至高达500倍或结合力提高25％到1000％。表现出与FcR“结合降低”的Fc变体，与亲本多肽相比，其与至少一种FcR具有更低的亲和力(例如更高的表观Kd或IC50值)。与亲本多肽相比，其结合能力下降40％或更多。
“抗体依赖的细胞介导的细胞毒作用”或“ADCC”是一种细胞毒性形式，指分泌型的Ig与存在于某些细胞毒性细胞(例如自然杀伤细胞(NK)、中性粒细胞和巨噬细胞)上的Fc受体(FcRs) 结合，使这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后使用细胞毒素杀死靶细胞。抗体“武装”细胞毒性细胞并且是这种杀伤所必需的。介导ADCC的主要细胞类型中，NK细胞只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。在Ravetch and Kinet,Annu.Rev.Immunol 9:457-92(1991)第464页的Table 3中总结了在造血细胞上FcR的表达。评估目标分子的ADCC活性，可以进行体外ADCC实验，在美国专利No.5,500,362或5,821,337中进行了描述。适用于此类实验的效应细胞包括外周血单个核细胞(PBMC)和自然杀伤细胞(NK)。可选地，或者此外，目标分子的ADCC活性也可以在体内进行评估，例如在如Clynes et al.PNAS(USA)95:652-656(1998)中所公开的动物模型中进行了描述。
包含Fc变体的多肽与包含野生型IgG Fc多肽或亲本多肽相比，在人体效应细胞存在下表现出“增强的ADCC活性”或能够更有效的介导ADCC效应，所述包含Fc变体的多肽在实验时与包含野生型IgG Fc多肽(或亲本多肽)数量上基本相同时，无论在体外或体内均能更有效的介导ADCC。通常采用本领域已知的任何体外ADCC实验方法来鉴定此类变体，例如用于鉴定ADCC活性的实验或方法，例如在动物模型中等。在一些实施例中，此类变体与野生型Fc(或亲代多肽)相比，介导ADCC的效率提高5到100倍，例如25到50倍。
“补体依赖的细胞毒作用”或“CDC”是指在补体存在的情况下裂解靶细胞。经典的补体途径的激活是由补体系统第一组分(C1q)与结合同源抗原的抗体(具有适宜结构的亚类)相结合而启动的。为了评估补体激活，可以进行CDC实验，如Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996)中所描述的。在美国专利No.6,194,551B1和WO99/51642中描述了具有改变的Fc区氨基酸序列并增加或降低的C1q结合能力的多肽变体。这些专利出版物的内容通过引用明确地并入本申请中。另见Idusogie et al.J.Immunol.164:4178-4184(2000)。
除非另有说明，一种“编码氨基酸序列的核苷酸序列”包括相互之间互为简并形式且编码相同氨基酸序列的所有核苷酸序列。编码蛋白质或RNA的核苷酸序列也可包括内含子，例如编码蛋白质的核苷酸序列在某些形式中包含内含子。
术语“可操作性地连接”是指调控序列与异源核苷酸序列之间的功能性连接，从而使后者表达。例如，当第一个核苷酸序列与第二个核苷酸序列处于功能性关系时，第一个核苷酸序列与第二个核苷酸序列为可操作性地连接。例如，如果启动子影响编码序列的转录或表达，该启动子与编码序列为可操作性地连接。通常，可操作性连接的DNA序列是连续的，并且在必要时，可以在同一个阅读框中连接两个蛋白质编码区。
“同源”是指两个多肽之间或两个核酸分子之间的序列相似性或序列同源性。如果两个比较序列的同一位置为相同的碱基或氨基酸单体亚基时，例如两个DNA分子的同一位置均为腺嘌呤，则这两个DNA分子在该位置是同源的。两个序列间的同源百分比是指两个序列中共有的匹配或同源位置的数量与位置总数之比再乘以100所得函数。例如，两个序列中如果10个位置中有6个位置是相匹配或同源的，则这两个序列的同源性为60％。举例来说，DNA序列ATTGCC和TATGGC具有50％的同源性。通常来说，在比对两个序列时，以得到最大同源性为目的来进行对比。
本申请所公开的抗体(包括双特异性抗体)或组合物的“有效量”是指足以实现特定目的的量。“有效量”可以凭经验和通过已知的与所述目的相关的方法确定。
术语“治疗有效量”是指本申请所述抗体(包括双特异性抗体)或组合物的量，对“治疗”个体的疾病或病症有效。在肺炎克雷伯菌感染的情况下，本申请公开的抗体或组合物的治疗有效量可以减少感染细胞的数量，抑制(即在一定程度上减缓并最好停止)感染的传播，并/或在一定程度上缓解与感染相关的一种或多种症状。在感染时，本申请公开的抗体或组合物能够抑制肺炎克雷伯菌生长和/或杀死肺炎克雷伯菌，抗体可以是抑制细胞的和/或具有细胞毒性的。在一些实施例中，治疗有效量是指在患者中抑制感染的量。在一些实施例中，治疗有效量是指在患者中完全清除感染的量。
如本申请所用的，“药学上可接受的”或“药理学上相容的”是指无生物学活性或者其它不期望性质的材料，例如该材料能够加入到给予患者的药物组合物中，而不会引起显著的不良生物反应，或者，不与组合物中包含的任何其它组分以有害的方式相互作用。药学上可接受的载体或赋形剂优选满足毒理学或制造检测的所需标准和/或包含在美国食品和药品管理局编制的非活性成分指南中。
本申请中描述的本申请的实施例应理解为包含“由……组成”和/或“基本上由……组成”的实施例。
本申请中提及“约”为一个数值或参数，包含(和描述)针对该值或参数本身的变体。例如，涉及“约X”的描述，包括“X”的描述。
如本申请所用的，提及“不是(not)”一个数值或参数，通常表示并描述“除了(other than)”某一数值或参数之外。例如，该方法不能用于治疗X型感染，意味着该方法通常用于治疗除X型感染之外的其他类型。
除非上下文另有明确说明，本申请和所述权利要求中所采用的单数形式“一”，“一个”和“该”包括复数对象。
特异性结合肺炎克雷伯菌O2抗原的抗体
一方面，本申请提供特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其包括，但不限于，人源化抗体，嵌合抗体，小鼠抗体，人抗体，以及本申请所述的包含重链和/或轻链CDRs的抗体分子。一方面，所述抗体或抗原结合片段是与O2抗原结合的分离的抗体。预期的特异性结合肺炎克雷伯菌O2抗原的抗体或或抗原结合片段包括，特异性结合肺炎克雷伯菌O2抗原的全长抗体(如全长IgG1、IgG2或IgG4)的全部或片段，特异性结合肺炎克雷伯菌O2抗原的单链抗体，多特异性(如双特异性)结合肺炎克雷伯菌O2抗原抗体，特异性结合肺炎克雷伯菌O2抗原的免疫偶联物，以及诸如此类。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段是Fab、Fab’、F(ab)’2、Fab’-SH、单链抗体(scFv)、Fv片段、dAb、Fd、纳米抗体或双链抗体(diabody)。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段是指抗体或抗原结合片段与肺炎克雷伯菌O2抗原结合的亲和力至少是与非靶标结合亲和力的10倍以上(包括例如10、10 2、10 3、10 4、10 5、10 6、或10 7倍)。在一些实施例中，非靶标是指不是肺炎克雷伯菌O2抗原的抗原。
结合亲和力可通过本领域已知的方法来测定，如ELISA，荧光激活细胞分选(FACS)分析或放射免疫沉淀分析(RIA)。Kd值可以通过本领域已知的方法来测定，如表面等离子共振(SPR)技术或生物层干涉(BLI)技术。
在某些方面，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段能够(a)促进、介导或增强肺炎克雷伯菌的调理吞噬杀菌(OPK)，和/或(b)促进、介导或增强补体介导的对肺炎克雷伯菌的血清杀菌活性(SBA)。
尽管本申请广泛地讨论了包含人序列的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段(例如，包含人CDR序列的人重链和轻链可变区)，但同时也考虑了非人抗体。在一些实施例中，非人抗体包括本申请所述的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段的人CDR序列和非人框架区序列，在一些实施例中，非人框架区序列包括任何的用于使用如本申请所述的一种或多种人CDR序列产生重链和/或轻链可变区的序列，包括例如哺乳动物，例如小鼠、大鼠、兔子、猪、牛(例如，牛、公牛、水牛)、鹿、绵羊、山羊、鸡、猫、狗、雪貂、灵长类(例如，狨猴，猕猴)等。在一些实施例中，非人特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包括将一种或多种本申请所述的人CDR序列移植到非人框架区中(例如，鼠或鸡的框架区序列)所产生的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与肺炎克雷伯菌LPS中D-半乳聚糖I结构域结合。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段是肺炎克雷伯菌O2抗原特异性的，并且没有种属交叉反应或不与其它类型的非肺炎克雷伯菌O2抗原发生交叉反应。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与其它类型的非肺炎克雷伯菌O2抗原发生交叉反应。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体，其包含：(i)V H，其包含如氨基酸序列SEQ ID NO:17所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:21所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；或(ii)V H，其包含如氨基酸序列SEQ ID NO:18所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:22所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；或(iii)V H，其包含如氨基酸序列SEQ ID NO:19所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:23所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；或(iv)V H，其包含如氨基酸序列SEQ ID NO:19所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；或(v)V H，其包含如氨基酸序列SEQ ID NO:20所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5，或者所述V H的变体，其HC-CDRs中包含至多约5个氨 基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，其包含氨基酸序列SEQ ID NO:17或其变体，所述变体与氨基酸序列SEQ ID NO:17具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:21或其变体，所述变体与氨基酸序列SEQ ID NO:21具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，其包含氨基酸序列SEQ ID NO:18或其变体，所述变体与氨基酸序列SEQ ID NO:18具有至少约80％(例如至少80％、 85％、90％、95％、96％、97％、98％或99％)序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:22或其变体，所述变体与氨基酸序列SEQ ID NO:22具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，其包含氨基酸序列SEQ ID NO:19或其变体，所述变体与氨基酸序列SEQ ID NO:19具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:23或其变体，所述变体与氨基酸序列SEQ ID NO:23具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，其包含氨基酸序列SEQ ID NO:19或其变体，所述变体与氨基酸序列SEQ ID NO:19具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:24或其变体，所述变体与氨基酸序列SEQ ID NO:24具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：V H，其包含氨基酸序列SEQ ID NO:20或其变体，所述变体与氨基酸序列SEQ ID NO:20具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:25或其变体，所述变体与氨基酸序列SEQ ID NO:25具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，上述氨基酸取代限于本申请表10中所示的“示例性取代”。在一些实施例中，氨基酸取代限于本申请表10中所示的“优选取代”。
在一些实施例中，本申请提供能够与所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体竞争性地结合具有D-半乳聚糖I结构域的肺炎克雷伯菌的抗体。在一些实施例中，本申请提供与所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体竞争性地结合相同表位的抗体。
在一些实施例中，可以利用竞争实验来鉴定与本申请所述的特异性结合肺炎克雷伯菌O2抗原的抗体竞争性结合具有D-半乳聚糖I结构域的肺炎克雷伯菌的单克隆抗体。竞争实验可以通过识别相同的或空间上重叠的表位或者通过一个抗体竞争性抑制另一抗体与抗原结合来确定两个抗体是否结合相同的表位。在某些实施例中，这种竞争性抗体与本申请所述的抗体结合相同的表位。一些示例性的竞争实验包括，但不限于如Harlow and Lane(1988)Antibodies:A Laboratory Manual ch.14(Cold Spring Harbor Laboratory，Cold Spring Harbor,N.Y.)中所提到的常规实验。用于解析抗体结合的表位的详细示例性方法如Morris(1996)"Epitope Mapping Protocols,"in Methods in Molecular Biology vol.66(Humana Press,Totowa,N.J.)中所述。在一些实施例中，如果每种抗体阻断另一种抗体结合的50％或更多，则称其结合相同的表位。在一些实施例中，与本申请所述的特异性结合肺炎克雷伯菌O2抗原的抗体竞争的抗体是嵌合抗体、人源化抗体或全人抗体。
示例性特异性结合肺炎克雷伯菌O2抗原的抗体序列如表2所示，其中根据Kabat定义方式进行CDR编号。本领域技术人员将认识到有多种已知算法(Kabat定义方式)来预测CDR的位置以及界定抗体轻、重链可变区。包含如本申请所述抗体的CDRs、V H和/或V L序列，但基于预测算法而非下表中所示例的抗体也在本申请的范围内。
克雷伯(氏)菌
绝大多数的克雷伯氏菌感染都与住院治疗有关。作为条件致病菌，克雷伯氏菌主要攻击患有严重基础疾病(如糖尿病或慢性肺梗阻)且免疫功能低下的个体。随着时间的推移以及分类学的不断发展，克雷伯氏菌的分类不断修正，出现了三种主要的分类:Cowan、Bascomb和
Figure PCTCN2022133880-appb-000002
其中
Figure PCTCN2022133880-appb-000003
将克雷伯氏菌分类5类，包括肺炎克雷伯菌、产酸克雷伯菌、土生克雷伯菌、植生克雷伯菌和解鸟氨酸克雷伯菌，而肺炎克雷伯菌还包括臭鼻肺炎克雷伯菌和鼻硬结肺炎克雷伯菌亚种(Podschun,R,and U Ullmann.Clinical microbiology reviews vol.11,4(1998):589-603.)。此外，根据其他分类/命名方式出现的肉芽肿克雷伯菌等也在本申请的考虑范围内。在医学上，肺炎克雷伯菌是引起感染最多的种类，也是克雷伯氏菌属中最重要的种类。肺炎克雷伯菌可以引起如败血症、肺炎、泌尿道感染、软骨病等(Podschun,R,and U Ullmann.Clinical microbiology reviews vol.11,4(1998):589-603.)。
脂多糖(LPS)
LPS(Lipopolysaccharide)是革兰氏阴性菌细胞壁外壁的组成成分，其由类脂A、核心寡糖和O抗原组成。O抗原或称为O多糖，是脂多糖的最外层部分，包含不同数量的寡糖重复单元(RU)。独特的O抗原结构定义了克雷伯氏菌属菌株的LPS血清型。根据克雷伯氏菌属O抗原的变异性，目前有9种主要的LPS血清型：O1、O2、O2ac、O3、O4、O5、O7、O8、O12(Hansen,D S et al.Journal of clinical microbiology vol.37,1(1999):56-62.)以及这些血清群中的一些亚型(Kelly,R F,and C Whitfield.Journal of bacteriology vol.178,17(1996):5205-14.)。根据已公布的流行病学数据，O1和O2血清型病原体占所有克雷伯氏菌感染的50-68％(Hansen,D S et al.Journal of clinical microbiology vol.37,1(1999):56-62.；Follador,Rainer et al.Microbial genomics vol.2,8e000073.25Aug.2016；)。O1和O2血清型菌株表达的LPS包含由半乳糖(galactans,gal)均聚物构成的O抗原。O1血清型表达由→3)-β-D-Galf-(1→3)-α-D-Galp-(1→作为重复单元构成的D-半乳聚糖-I(D-gal-I)和抗原性不同的由→3)-α-D-Galp-(1→3)-β-D-Galp-(1→作为重复单元构成的D-半乳聚糖-II(D-gal-II)(Whitfield,C et al.Journal of bacteriology vol.173,4(1991):1420-31.；Kol,O et al.Carbohydrate research vol.236(1992):339-44.)。D-gal-II是O1型LPS特有的结构(Pennini,Meghan E et al.Nature communications vol.8,1 1991.8Dec.2017)。另一方面，O2型LPS仅由D-gal-I组成(Whitfield,C et al.Journal of bacteriology vol.174,15(1992):4913-9.)。对于这两种血清型，D-gal-I的合成是由his连锁的rfb(wb)操纵子编码的(Clarke,B R,and C Whitfield.Journal of bacteriology vol.174,14(1992):4614-21.；Kelly,R F,and C Whitfield.Journal of bacteriology vol.178,17(1996):5205-14.)。此外，从遗传学角度来说，O1血清型菌株携带负责合成D-gal-II的非连锁基因座(wbbYZ)(Hsieh,Pei-Fang et al.Frontiers in microbiology vol.5 608.19Nov.2014)。先前的研究表明，O2血清型的D-gal-I可通过化学计量修饰的或非化学计量添加O- 乙酰基或末端D半乳糖(Kelly等，1995)。最近的研究揭示了在O2血清群中频繁出现末端α-D-Galp残基修饰的gal-I骨架重复单元，即→3)-β-D-Galf-(1→3)-[α-D-Galp-(1→4)]-α-D-Galp-(1→)，称其为D-半乳聚糖-III(gal-III)，且这种修饰的遗传背景已经确定(Szijártó,Valéria et al.International journal of medical microbiology:IJMM vol.306,2(2016):89-98.)。结果表明gal-I到gal-III的转换是由gmlABC编码的，它与编码gal-I的rfb(wb)操纵子相邻。此外，有研究表明，约40％的O1血清型临床分离株携带gmlABC基因(Szijártó,Valéria et al.International journal of medical microbiology:IJMM vol.306,2(2016):89-98.)，这表明在O1血清型内也表达D-gal-III。对萃取的LPS或分离的O抗原进行结构学分析，揭示了可以同时存在D-gal-II和D-gal-III。最终，D-gal-II可以与D-gal-I或D-gal-III均聚物组合，导致O1血清型存在以上两种情况。O2血清型则仅由D-gal-I或D-gal-III均聚物组成(Stojkovic,Katarina et al.Frontiers in microbiology vol.8 684.25Apr.2017)。
特异性结合肺炎克雷伯菌O2抗原的全长抗体
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体是全长的抗体。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的全长抗体是IgA、IgD、IgE、IgG或IgM。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含抗体重链恒定区和抗体轻链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的全长抗体包含IgG恒定区域，例如IgG1、IgG2、IgG3、IgG4或其变体的恒定区域。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含IgG1型重链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含IgG2型重链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含IgG3型重链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含IgG4型重链恒定区。在一些实施例中，所述IgG指的是人IgG。在一些实施例中，所述重链恒定区包含或由氨基酸序列SEQ ID NO:55组成。在一些实施例中，所述重链恒定区包含或由氨基酸序列SEQ ID NO:56组成。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含κ轻链恒定区。在一些实施例中，所述轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含λ轻链恒定区。在一些实施例中，所述轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原抗体或抗原结合片段包含抗体重链可变区和抗体轻链可变区。
在一些实施例中，提供一种包含IgG1恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述IgG1是人IgG1。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成。在一些实施例中，轻链恒定区包含或由氨基酸序列 SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG1恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述IgG1是人IgG1。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG1恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述IgG1是人IgG1。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG1恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述IgG1是人IgG1。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。 在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG1恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述IgG1是人IgG1。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:55组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG4恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述IgG4是人IgG4。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG4恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述IgG4是人IgG4。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸 序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG4恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述IgG4是人IgG4。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG4恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述IgG4是人IgG4。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
在一些实施例中，提供一种包含IgG4恒定区的特异性结合肺炎克雷伯菌O2抗原的全长抗体，其中所述特异性结合肺炎克雷伯菌O2抗原的抗体包含：a)重链可变区，所述重链可变区包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及b)轻链可变区，所述轻链可变区包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述IgG4是人IgG4。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨 基酸序列SEQ ID NO:57组成。在一些实施例中，重链恒定区包含或由氨基酸序列SEQ ID NO:56组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。
特异性结合肺炎克雷伯菌O1抗原的抗体
在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包括，但不限于，人源化抗体，嵌合抗体，小鼠抗体，人抗体，以及本申请所述的包含重链和/或轻链CDRs的抗体分子。一方面，所述抗体或抗原结合片段是与肺炎克雷伯菌O1抗原结合的分离的抗体。预期的特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包括，特异性结合肺炎克雷伯菌O1抗原的全长抗体(如全长IgG1、IgG2或IgG4)的全部或片段，特异性结合肺炎克雷伯菌O1抗原的单链抗体，多特异性(如双特异性)结合肺炎克雷伯菌O1抗原的抗体，特异性结合肺炎克雷伯菌O1抗原的免疫偶联物，以及诸如此类。在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体是Fab、Fab’、F(ab)’2、Fab’-SH、单链抗体(scFv)、Fv片段、dAb、Fd、纳米抗体或双链抗体(diabody)。在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段是指抗体或抗原结合片段与肺炎克雷伯菌O1抗原结合的亲和力至少是与非靶标结合亲和力的10倍以上(包括例如10、10 2、10 3、10 4、10 5、10 6、或10 7倍)。在一些实施例中，非靶标是指不是肺炎克雷伯菌O1抗原的抗原。
结合亲和力可通过本领域已知的方法来测定，如ELISA，荧光激活细胞分选(FACS)分析或放射免疫沉淀分析(RIA)。Kd值可以通过本领域已知的方法来测定，如表面等离子共振(SPR)技术或生物层干涉(BLI)技术。
在某些方面，所述特异性与肺炎克雷伯菌O1抗原结合的抗抗体或抗原结合片段(a)促进、介导或增强肺炎克雷伯菌的调理吞噬杀菌(OPK)活性，和/或(b)促进、介导或增强补体介导的对肺炎克雷伯菌的血清杀菌活性(SBA)。
尽管本申请广泛地讨论了包含人序列的抗体或抗原结合片段(例如，包含人CDR序列的人重链和轻链可变区)，但同时也考虑了非人抗体或抗原结合片段。在一些实施例中，非人抗体或抗原结合片段包括本申请所述的抗体或抗原结合片段的人CDR序列和非人框架区序列，在一些实施例中，非人框架区序列包括任何的用于使用如本申请所述的一种或多种人CDR序列产生重链和/或轻链可变区的序列，包括例如哺乳动物，例如小鼠、大鼠、兔子、猪、牛(例如，牛、公牛、水牛)、鹿、绵羊、山羊、鸡、猫、狗、雪貂、灵长类(例如，狨猴，猕猴)等。在一些实施例中，非人抗体或抗原结合片段包括将一种或多种本申请所述的人CDR序列移植到非人框架区中(例如，鼠或鸡的框架区序列)所产生的抗体或抗原结合片段。
在一些实施例中，本申请所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段，特异性结合肺炎克雷伯菌O1抗原中的一个表位。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段是肺炎克雷伯菌O1抗原特异性的，并且没有种属交叉反应或不与其它类型的非肺炎克雷伯菌O1抗原发生交叉反应。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与其它类型的非肺炎克雷伯菌O1抗原发生交叉反应。
在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含抗体重链恒定区和抗体轻链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含IgG1型重链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含IgG2型重链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含IgG3型重链恒定区。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含IgG4型重链恒定区。在一些实施例中，所述IgG指的是人IgG。在一些实施例中，所述重链恒定区包含或由氨基酸序列SEQ ID NO:55组成。在一些实施例中，所述重链恒定区包含或由氨基酸序列SEQ ID NO:56组成。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含κ轻链恒定区。在一些实施例中，所述轻链恒定区包含或由氨基酸序列SEQ ID NO:57组成。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含λ轻链恒定区。在一些实施例中，所述轻链恒定区包含或由氨基酸序列SEQ ID NO:58组成。在一些实施例中，所述抗O1抗原的抗体或抗原结合片段包含抗体重链可变区和抗体轻链可变区。
在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段可以选自中国专利申请202110980272.2中所述的特异性结合肺炎克雷伯菌O1抗原的抗体。
特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体
一方面，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。
在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35；HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41；LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:17或26，或包含与氨基酸序列SEQ ID NO:17或26具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:21或30，或包含与氨基酸序列SEQ ID NO:21或30具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:17或26所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:21或30所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:18或27，或包含与氨基酸序列SEQ ID NO:18或27具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:22或31，或包含与氨基酸序列SEQ ID NO:22或31具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:18或27所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:22或31所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:23或32，或包含与氨基酸序列SEQ ID NO:23或32具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:19或28所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:23或32所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:19或28所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24或33所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:20或29所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25或34所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:47或51所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:49或53所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少约80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；或者V H，其包含 如氨基酸序列SEQ ID NO:48或52所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:50或54所示的V L包含的LCDR1、LCDR2和LCDR3。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及其中第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包 含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及其中第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；以及其中所述第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；以及其中第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；以及其中第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列。
在一些实施例中，本申请提供了一种双特异性抗体，其包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域，和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包 含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；以及其中第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。
DVD-Ig结构的双特异性抗体
在一些实施例中，根据本申请所述的任一双特异性抗体，其具有双重可变区免疫球蛋白分子(DVD-Ig)结构，该结构是在正常IgG抗体的V L和V H的N末端分别连接另外一个抗体的V L和V H结构域，通过两个抗体的V H与V L相互作用形成抗原结合域，能够同时与相对应的抗原结合，来实现双特异性。在一些实施例中，所述DVD-IgG结构为同源二聚体结构，由两个相同的单体组成，每个单体包括两个抗原结合域，其中一个抗原结合域为Fv，另一个抗原结合域为Fab，上述两个区域通过连接肽(L)串联连接。在一些实施例中，所述DVD-Ig结构还进一步包括Fc。示例性的DVD-Ig结构示在图7A中。
在本申请的一个实施例中，其中一个抗原结合域特异性地与肺炎克雷伯菌O1抗原结合，以及另一个抗原结合域特异性地与肺炎克雷伯菌O2抗原结合。在一些实施例中，所述双特异性抗体可以同时结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原。
在一些实施例中，所述双特异性抗体由两个相同的单体组成，每个单体包括两条多肽链，分别为重链和轻链，共四条多肽链。其中，重链从N端到C端包含V H1-L-V H2-C H1，轻链从N端到C端包含V L1-L-V L2-C L。在一些实施例中，其中所述重链进一步包含Fc，该Fc包含C H2和C H3结构域。在另一些实施例中，所述重链从N端到C端包含V H1-L-V H2-C H1-C H2-C H3，轻链从N端到C端包含V L1-L-V L2-C L。其中V H1和V L1分别是特异性结合一个抗原的重链可变区和轻链可变区；V H2和V L2分别是特异性结合另一个抗原的重链可变区和轻链可变区；L是连接肽；C H1是重链恒定区C H1结构域；C L是轻链恒定区。其中，V H1和V L1组成双特异性抗体的一个抗原结合域(Fv)；V H2-C H1和V L2-C L组成双特异性抗体的另一个抗原结合域(Fab)。
在一些实施例中，所述V H1和V L1分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H1和V L1组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fv)；V H2和V L2分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H2-C H1和V L2-C L组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)。在一些实施例中，所述V H1和V L1分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H1和V L1组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fv)；V H2和V L2分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H2-C H1和V L2-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)。
连接肽(或接头)序列可以是单个氨基酸或多肽序列。在一些实施例中，所述连接肽(或接头)包含或由甘氨酸-丝氨酸接头组成。如本申请所述，术语“甘氨酸-丝氨酸接头”指由甘氨酸和丝氨酸残基组成的肽。示例性的甘氨酸-丝氨酸接头包括通式为(Gly 4Ser)n的氨基酸序列，其中n是正整数(例如，1、2、3、4、5、6、7、8、9或10)。一个优选的甘氨酸-丝氨酸接头为(Gly 4Ser) 2，即GGGGSGGGGS(SEQ ID NO：102)和(Gly 4Ser) 4，即GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：103)。另一个优选的甘氨酸-丝氨酸接头为(Gly 4Ser) 3，即GGGGSGGGGSGGGGS(SEQ ID NO：104)。在其它方面，两个或两个以上的甘氨酸-丝氨酸接头串联在一个连接肽中。在某些方面，所述连接肽包括铰链区的至少一部分(例如，来源于IgGl、IgG2、IgG3或IgG4分子)和一系列的甘氨酸-丝氨酸残基(例如，甘氨酸-丝氨酸接头，例如(Gly 4Ser) n)。在一些实施例中，所述连接肽还可以选择为包含氨基酸序列ASTKGP(SEQ ID NO：105)或氨基酸序列TVAAP(SEQ ID NO：106)。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:61，或包含与氨基酸序列SEQ ID NO:61具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:62，或包含与氨基酸序列SEQ ID NO:62具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:63，或包含与氨基酸序列SEQ ID NO:63具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:64，或包含与氨基酸序列SEQ ID NO:64具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:65，或包含与氨基酸序列SEQ ID NO:65具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:67，或包含与氨基酸序列SEQ ID NO:67具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:68，或包含与氨基酸序列SEQ ID NO:68具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:61，或与氨基酸序列SEQ ID NO:61具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:62，或与氨基酸序列SEQ ID NO:62具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:63，或与氨基酸序列SEQ ID NO:63具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:64，或与氨基酸序列SEQ ID NO:64具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:65，或与氨基酸序列SEQ ID NO:65具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:66，或与氨基酸序列SEQ ID NO:66具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:67，或包含与氨基酸序列SEQ ID NO:67具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:68，或包含与氨基酸序列SEQ ID NO:68具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:86，或包含与氨基酸序列SEQ ID NO:86具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:62，或包含与氨基酸序列SEQ ID NO:62具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:87，或包含与氨基酸序列SEQ ID NO:87具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:64，或包含与氨基酸序列SEQ ID NO:64具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:88，或包含与氨基酸序列SEQ ID NO:88具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:89，或包含与氨基酸序列SEQ ID NO:89具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％ 或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:68，或包含与氨基酸序列SEQ ID NO:68具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:115，或包含与氨基酸序列SEQ ID NO:115具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
Bs4Ab结构的双特异性抗体
在一些实施例中，根据本申请所述的双特异性抗体，其具有Bs4Ab结构，该结构包含两个相同的单体，每个单体均包括两个抗原结合域，其中一个抗原结合域为Fab，另一个抗原结合域为scFv。在一些实施例中，其中所述双特异性抗体进一步包含Fc，该Fc包含C H2和C H3结构域。所述scFv通过第一连接肽(L1)与Fab连接，并通过第二连接肽(L2)与Fc连接。示例性的Bs4Ab结构示在图7B中。
在本申请的一些实施例中，其中一个抗原结合域(Fab或scFv)特异性地与肺炎克雷伯菌O1抗原结合，以及另一个抗原结合域(scFv或Fab)特异性地与肺炎克雷伯菌O2抗原结合。在一些实施例中，所述双特异性抗体可以同时结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原。
在一些实施例中，根据本申请所述的双特异性抗体由两个相同的单体组成，每个单体包含两条多肽链，分别为重链和轻链，共四条多肽链。在一些实施例中，双特异性抗体的重链从N端到C端包含：V H1-C H1-L1-V H2-L3-V L2结构。在另一些实施例中，双特异性抗体的重链从N端到C端包含：V H1-C H1-L1-V L2-L3-V H2结构。在一些实施例中，所述重链还包括Fc，该Fc包含C H2和C H3结构域。在一些实施例中，双特异性抗体的重链从N端到C端包含：V H1-C H1-L1-V H2-L3-V L2-L2-C H2-C H3结构。在另一些实施例中，双特异性抗体的重链从N端到C端包含：V H1-C H1-L1-V L2-L3-V H2-L2-C H2-C H3结构。在一些实施例中，双特异性抗体的轻链从N端到C端包含：V L1-C L结构。其中，V H1和V L1分别是特异性结合其中一个抗原的重链可变区和轻链可变区，V H2和V L2分别是特异性结合另一个抗原的重链可变区和轻链可变区，C H1是重链恒定区C H1结构域，C L是轻链恒定区，L1、L2和L3是连接肽。V H1-C H1和V Ll-C L组成双特异性抗体的其中一个抗原结合域(Fab)，V H2-L3-V L2或V L2-L3-V H2组成双特异性抗体的另一个抗原结合域(scFv)。
在一些实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)；V H2和V L2分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H2-L3-V L2或V L2-L3-V H2组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(scFv)。在另一些实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H1-C H1和V Ll-C L组成特异性结合 肺炎克雷伯菌O2抗原的抗原结合域(Fab)；V H2和V L2分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H2-L3-V L2或V L2-L3-V H2组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(scFv)。在一些实施例中，在一些实施例中，所述双特异性抗体包括C H1，其包含氨基酸序列SEQ ID No:60。在一些实施例中，所述双特异性抗体包括C H2-C H3，其包含氨基酸序列SEQ ID NO:59。
在一些实施例中，根据本申请所述的任一双特异性抗体，特异性结合肺炎克雷伯菌O2抗原或O1抗原的抗原结合域scFv包括基因工程半胱氨酸突变，通过在V H和V L界面引入两个半胱氨酸突变，获得了二硫键稳定型的双特异性抗体。
连接肽(或接头)可用于将双特异性抗体的嵌合重链的结构域和/或结构区连接成一个连续的分子。在一些实施例中，所述双特异性抗体包括至少两个连接肽，L1和L2。在一些实施例中，所述双特异性抗体可能包括额外的接头，例如连接scFv的可变重链和轻链的柔性接头。在一些实施例中，所述双特异性抗体可能包括额外的接头，例如连接scFv的可变重链和轻链的柔性接头以及用于将其他结合单元与双特异性抗体的核心结构连接的其他接头。
连接肽(或接头)的一个典型的、非限制性的例子是包含至少4个残基的多肽链。这类接头的部位可能是柔性的、亲水的，并且它们自己很少或不会形成二级结构(接头部位或柔性接头部位)。在分子组装完成后，至少4个氨基酸的接头可以用于连接彼此靠近的结构域和/或区。也可以使用较长的接头。在一些实施例中，接头可以是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、45、50、100、125、150、175或200个残基。当使用多个接头连接分子的各个部分时，接头可以是相同的或不同的(例如，相同或不同的长度和/或氨基酸序列)。
在某些方面，所述连接肽包含或由甘氨酸-丝氨酸接头组成。如本申请所述，术语“甘氨酸-丝氨酸接头”指由甘氨酸和丝氨酸残基组成的肽。示例性的甘氨酸-丝氨酸接头包括通式为(Gly 4Ser)n的氨基酸序列，其中n是正整数(例如，1、2、3、4、5、6、7、8、9或10)。一个优选的甘氨酸-丝氨酸接头为(Gly 4Ser) 2，即GGGGSGGGGS(SEQ ID NO:102)和(Gly 4Ser) 4，即GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:103)。另一个优选的甘氨酸-丝氨酸接头为(Gly 4Ser) 3，即GGGGSGGGGSGGGGS(SEQ ID NO:104)。在其它方面，两个或两个以上的甘氨酸-丝氨酸接头串联在一个连接肽中。在某些方面，所述连接肽包括铰链区的至少一部分(例如，来源于IgGl、IgG2、IgG3或IgG4分子)和一系列的甘氨酸-丝氨酸残基(例如，甘氨酸-丝氨酸接头，例如(Gly 4Ser) n)。
在一些实施例中，L1和/或L2包括铰链部分和接头部分，例如包括甘氨酸-丝氨酸接头的接头部分。在其它方面，L1和/或L2仅包括铰链部分或仅包括接头部分，例如甘氨酸-丝氨酸接头。在某些方面，L1和L2包括甘氨酸-丝氨酸接头。在某些方面，L1和L2的甘氨酸-丝氨酸接头部分的长度相同，而在其它方面，LI和L2的甘氨酸-丝氨酸接头部分的长度不同。当双特异性抗体包含scFv时，scFv的重链和轻链可以通过柔性接头连接。在一些实施例中，这种柔性接头通常不包括铰链部分， 而是一种甘氨酸-丝氨酸接头或其它柔性接头。可以选择和优化相互连接scFv结构域的柔性接头的长度和氨基酸序列。
在一些实施例中，连接肽(例如L1和/或L2)包含甘氨酸-丝氨酸或全部为甘氨酸的接头以及铰链区域的一部分或经修饰的一部分。在某些方面，双特异性抗体中连接其中一个抗原结合域(如Fab或scFv)和另一个抗原结合域(如scFv或Fab)的连接肽(L1)包含氨基酸序列EPKSDKTGGGGSGGGGS(SEQ ID NO:107)或EPKSCGKTGGGGSGGGGS(SEQ ID NO:108)或EPKSCGGGGSGGGGS(SEQ ID NO:109)。在某些方面，双特异性抗体中连接结抗原结合域scFv与Fc的连接肽(L2)包含氨基酸序列GGGGSGGGGSEPKSDKTHTCPPCP(SEQ ID NO:110)或GGGGSGGGGSCPPCP(SEQ ID NO:111)或GGGGSGGGGSDKTHTCPPCP(SEQ ID NO:112)。
在一些实施例中，所述双特异性抗体除了将一个抗原结合域与另一个抗原结合域连接的连接肽或将其中一个抗原结合域与Fc连接的连接肽(例如L1和L2)，双特异性抗体都可以选择性地包含额外的连接肽。这些额外的连接肽的长度和序列是独立选择的。例如，双特异性抗体还可以包括一个柔性连接肽(L3)，其连接抗原结合域scFv中的可变重链和轻链(V HSCFV和V LSCFV)。这种柔性的连接肽可以包括甘氨酸-丝氨酸接头。通常，这种接头不包括铰链部分。在一些实施例中，连接scFv的可变重链和轻链的柔性连接肽(L3)包含序列GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:103)。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:69，或包含与氨基酸序列SEQ ID NO:69具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:70，或包含与氨基酸序列SEQ ID NO:70具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:71，或包含与氨基酸序列SEQ ID NO:71具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:72，或包含与氨基酸序列SEQ ID NO:72具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:73，或包含与氨基酸序列SEQ ID NO:73具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:75，或包含与氨基酸序列SEQ ID NO:75具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:69，或与氨基酸序列SEQ ID NO:69具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:70，或与氨基酸序列SEQ ID NO:70具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:71，或与氨基酸序列SEQ ID NO:71具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:72，或与氨基酸序列SEQ ID NO:72具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:73，或与氨基酸序列SEQ ID NO:73具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:74，或与氨基酸序列SEQ ID NO:74具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:75，或包含与氨基酸序列SEQ ID NO:75具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:74，或与氨基酸序列SEQ ID NO:74具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，双特异性抗体的嵌合重链从N端到C端包含以下的多肽序列：V H1-C H1-L1-V H2-L3-V L2-L2-C H2-C H3。在一些实施例中，双特异性抗体的轻链从N端到C端包含以下的多肽序列：V L1-C L。在一些实施例中，所述双特异性抗体包含氨基酸序列SEQ ID NO:90或与氨基酸序列SEQ ID NO:90具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或包含氨基酸序列SEQ ID NO:70或与氨基酸序列SEQ ID NO:70具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，双特异性抗体的嵌合重链从N端到C端包含以下的多肽序列：V H1-C H1-L1-V H2-L3-V L2-L2-C H2-C H3。在一些实施例中，双特异性抗体的轻链从N端到C端包含以下的多肽序列：V L1-C L。在一些实施例中，所述双特异性抗体包含氨基酸序列SEQ ID NO:91或与氨基酸序列SEQ ID NO:91具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或包含氨基酸序列SEQ ID NO:72或与氨基酸序列SEQ ID NO:72具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，双特异性抗体的嵌合重链从N端到C端包含以下的多肽序列：V H1-C H1-L1-V H2-L3-V L2-L2-C H2-C H3。在一些实施例中，双特异性抗体的轻链从N端到C端包含以下的多肽序列：V L1-C L。在一些实施例中，所述双特异性抗体包含氨基酸序列SEQ ID NO:92或与氨基酸序列 SEQ ID NO:92具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或包含氨基酸序列SEQ ID NO:74或与氨基酸序列SEQ ID NO:74具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，双特异性抗体的嵌合重链从N端到C端包含以下的多肽序列：V H1-C H1-L1-V H2-L3-V L2-L2-C H2-C H3。在一些实施例中，双特异性抗体的轻链从N端到C端包含以下的多肽序列：V L1-C L。在一些实施例中，所述双特异性抗体包含氨基酸序列SEQ ID NO:93或与氨基酸序列SEQ ID NO:93具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或包含氨基酸序列SEQ ID NO:74或与氨基酸序列SEQ ID NO:74具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
Hetero H,CrossMab结构的双特异性抗体
在一些实施例中，根据本申请所述的任一双特异性抗体，其具有Hetero H,CrossMab结构，其是由异源二聚体构成的二价双特异性抗体，包含两个抗原结合域Fab。在另一些实施例中，其中所述双特异性抗体进一步包含两个Fc，其包含C H2和C H3结构域。在一些实施例中，其中一个Fc中的C H3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基形成“knob”,和另一个Fc中的C H3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基形成“hole”，能够促进异源二聚体的相互结合。其中在Fab中，轻链恒定区(C L)和重链恒定区C H1结构域(C H1)的位置可相互替换；或重链可变区(V H)和轻链可变区(V L)的位置可相互替换；或轻链恒定区(C L)和重链恒定区C H1结构域(C H1)以及重链可变区(V H)和轻链可变区(V L)同时相互替换。示例性的Hetero H,CrossMab结构示在图7C中。
在一些实施例中，所述Fc来源于野生型人IgG1Fc。在另一些实施例中，所述Fc的C H3结构域包含，但不限于如下氨基酸置换：S354C，T366W,Y349C,T366S,L368A,和/或Y407V，其中所述编号依照如Kabat的EU索引。
在本申请的另一些实施例中，所述其中一个抗原结合域特异性地与肺炎克雷伯菌O2抗原结合，以及另一个抗原结合域特异性地与肺炎克雷伯菌O1抗原结合。在一些实施例中，所述双特异性抗体可以同时结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原。
在一些实施例中，本申请所述的双特异性抗体，其具有Hetero H,CrossMab结构，该结构为异源二聚体，由两个不同的单体组成，每个单体包含两条多肽链。其中第一单体包含特异性结合其中一个抗原的抗体的第一重链和第一轻链，以及第二单体包含特异性结合另一个抗原的抗体的第二重链和第二轻链。在一些实施例中，所述双特异性抗体的第一重链从N端到C端包括：V H1-C H1结构；第一轻链从N端到C端包括V L1-C L结构。在一些实施例中，第一重链还包含Fc，该Fc包含C H2和C H3结构域。在一些实施例中，所述双特异性抗体的第一重链从N端到C端包括：V H1-C H1-C H2-C H3结构；第一轻链从N端到C端包括V L1-C L结构。V H1和V L1分别是特异性结合其中一个抗原的重链可变区和轻链可变区，C H1是重链恒定区C H1结构域，C L是轻链恒定区。其中V H1-C H1和V L1- C L组成结合其中一个抗原的抗原结合域(Fab)。在一些实施例中，所述双特异性抗体的第二重链从N端到C端包括：V H2-C L结构；第二轻链从N端到C端包括：V L2-C H1结构。在一些实施例中，第二重链进一步包含Fc，该Fc包含C H2和C H3结构域。在一些实施例中，所述双特异性抗体的第二重链从N端到C端包括：V H2-C L-C H2-C H3结构；第一轻链从N端到C端包括V L2-C H1结构。V H2和V L2分别是特异性结合另一个抗原的重链可变区和轻链可变区，C H1是重链恒定区C H1结构域，C L是轻链恒定区。其中V H2-C L和V L2-C H1组成结合另一个抗原的抗原结合域(Fab)。在一些实施例中，所述双特异性抗体第一单体或第二单体中C L和C H1的位置相互替换。在一些实施例中，V H1和V L1的位置可相互替换。在另一些实施例中，V H2和V L2的位置可相互替换。在一些实施例中，第一重链的C H3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基形成“knob”，和第二重链的C H3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基形成“hole”。在另一些实施例中，第二重链的C H3结构域中氨基酸残基被替换为较大侧链体积的氨基酸残基形成“knob”，和第一重链的C H3结构域中氨基酸残基被替换为较小侧链体积的氨基酸残基形成“hole”。在一些实施例中，所述C H3结构域包含，但不限于如下氨基酸置换：S354C，T366W，Y349C，T366S，L368A和/或Y407V，其中所述编号依照如Kabat的EU索引。
在一些实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区,V H2和V L2分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)，V H2-C L和V L2-C H1组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)。在另一个实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H2和V L2分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)，V H2-C L和V L2-C H1组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:76，或包含与氨基酸序列SEQ ID NO:76具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:77，或包含与氨基酸序列SEQ ID NO:77具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:78，或包含与氨基酸序列SEQ ID NO:78具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:79，或包含与氨基酸序列SEQ ID NO:79具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:80，或包含与氨基酸序列SEQ ID NO:80具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:76，或与氨基酸序列SEQ ID NO:76具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:77，或与氨基酸序列SEQ ID NO:77具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:78，或与氨基酸序列SEQ ID NO:78具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:74，或与氨基酸序列SEQ ID NO:74具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:79，或与氨基酸序列SEQ ID NO:79具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:80，或与氨基酸序列SEQ ID NO:80具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:78，或与氨基酸序列SEQ ID NO:78具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:74，或与氨基酸序列SEQ ID NO:74具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:94，或与氨基酸序列SEQ ID NO:94具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:77，或与氨基酸序列SEQ ID NO:77具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；/或氨基酸序列SEQ ID NO:95，或与氨基酸序列SEQ ID NO:95具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；/或氨基酸序列SEQ ID NO:74，或与氨基酸序列SEQ ID NO:74具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，根据本申请所述的任一双特异性抗体，包含氨基酸序列SEQ ID NO:96，或与氨基酸序列SEQ ID NO:96具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:80，或与氨基酸序列SEQ ID NO:80具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；/或氨 基酸序列SEQ ID NO:95，或与氨基酸序列SEQ ID NO:95具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；/或氨基酸序列SEQ ID NO:74，或与氨基酸序列SEQ ID NO:74具有至少80％例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
IgG-(scFv) 2结构的双特异性抗体
在一些实施例中，本申请所述的双特异性抗体，其具有IgG-(scFv) 2结构，该结构由两个相同的单体组成，每个单体均包含两个抗原结合域，其中一个抗原结合域为Fab，另一个抗原结合域为scFv。在一些实施例中，其中所述双特异性抗体进一步包含Fc，该Fc包含C H2和C H3结构域。所述scFv通过连接肽(L)连接至Fc的羧基末端。示例性的IgG-(scFv) 2结构示在图7D中。
在本申请的一些实施例中，其中一个抗原结合域(Fab或scFv)特异性地与肺炎克雷伯菌O1抗原结合，以及另一个抗原结合域(scFv或Fab)特异性地与肺炎克雷伯菌O2抗原结合。在一些实施例中，所述双特异性抗体可以同时结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原。
在一些实施例中，本申请披露的双特异性抗体包括两个相同的单体，每个单体包含一条重链和一条轻链。在一些实施例中，双特异性抗体的重链从N端到C端包含：V H1-C H1-C H2-C H3结构。在一些实施例中，所述双特异性抗体的轻链从N端到C端包含：V L1-C L。在一些实施例中，所述重链还包括Fc，该Fc包含C H2和C H3结构域。在一些实施例中，双特异性抗体的重链从N端到C端包含：V H1-C H1-C H2-C H3-L-V H2-L3-V L2结构。在另一些实施例中，双特异性抗体的重链从N端到C端包含：V H1-C H1-C H2-C H3-L-V L2-L3-V H2结构。在一些实施例中，双特异性抗体的轻链从N端到C端包含：V L1-C L结构。其中，V H1和V L1分别是特异性结合其中一个抗原的重链可变区和轻链可变区，V H2和V L2分别是特异性结合另一个抗原的重链可变区和轻链可变区，C H1是重链恒定区C H1结构域，C L是轻链恒定区，L和L3是连接肽。V H1-C H1和V L1-C L组成双特异性抗体的其中一个抗原结合域(Fab)，V H2-L3-V L2或V L2-L3-V H2组成双特异性抗体的另一个抗原结合域(scFv)。
在一些实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H1-C H1和V L1-C L组成能够与肺炎克雷伯菌O1抗原特异性结合的抗原结合域(Fab)；V H2和V L2分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H2-L3-V L2或V L2-L3-V H2组成能够与肺炎克雷伯菌O2抗原特异性结合的抗原结合域(scFv)。在一些实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H1-C H1和V L1-C L组成能够与肺炎克雷伯菌O2抗原特异性结合的抗原结合域(Fab)；V H2和V L2分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H2-L3-V L2或V L2-L3-V H2组成能够与肺炎克雷伯菌O1抗原特异性结合的抗原结合域(scFv)。在一些实施例中，在一些实施例中，所述双特异性抗体包括C H1，其包含氨基酸序列SEQ ID No:60。在一些实施例中，所述双特异性抗体包括C H2-C H3，其包含氨基酸序列SEQ ID NO:59。
连接肽(或接头)可用于将双特异性抗体的嵌合重链的结构域和/或区连接成一个连续的分子。在一些实施例中，所述双特异性抗体可能包括额外的接头，例如连接scFv的可变重链和轻链的柔性接头。在一些实施例中，所述双特异性抗体可能包括额外的接头，例如连接scFv的可变重链和轻链的柔性接头以及用于将其他结合单元与双特异性抗体的核心结构连接的其他接头。
接头的一个典型的、非限制性的例子是包含至少4个残基的多肽链。这类接头的部位可能是柔性的、亲水的，并且它们自己很少或不会形成二级结构(接头部位或柔性接头部位)。在分子组装完成后，至少4个氨基酸的接头可以用于连接彼此靠近的结构域和/或区。也可以使用较长的接头。在一些实施例中，接头可以是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、45、50、100、125、150、175或200个残基。当使用多个接头连接分子的各个部分时，接头可以是相同的或不同的(例如，相同或不同的长度和/或氨基酸序列)。
在某些方面，所述连接肽(或接头)包含或由甘氨酸-丝氨酸接头组成。如本申请所述，术语“甘氨酸-丝氨酸接头”指由甘氨酸和丝氨酸残基组成的肽。示例性的甘氨酸-丝氨酸接头包括通式为(Gly 4Ser)n的氨基酸序列，其中n是正整数(例如，1、2、3、4、5、6、7、8、9或10)。一个优选的甘氨酸-丝氨酸接头为(Gly 4Ser) 2，即GGGGSGGGGS(SEQ ID NO:102)和(Gly 4Ser) 4，即GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:103)。另一个优选的甘氨酸-丝氨酸接头为(Gly 4Ser) 3，即GGGGSGGGGSGGGGS(SEQ ID NO:104)。在其它方面，两个或两个以上的甘氨酸-丝氨酸接头串联在一个连接肽中。在某些方面，所述连接肽包括铰链区的至少一部分(例如，来源于IgGl、IgG2、IgG3或IgG4分子)和一系列的甘氨酸-丝氨酸残基(例如，甘氨酸-丝氨酸接头，例如(Gly 4Ser) n)。
在一些实施例中，连接肽包含甘氨酸-丝氨酸或全部为甘氨酸的接头以及铰链区域的一部分或经修饰的一部分。在一些实施例中，双特异性抗体中连接抗原结合域与Fc区羧基末端C H3的连接肽(例如L)包含氨基酸序列GGGGSGGGGTGGGGS(SEQ ID NO:114)。
在一些实施例中，所述双特异性抗体除了将抗原结合域与Fc连接的连接肽(例如L)，双特异性抗体都可以选择性地包含额外的连接肽。这些额外的连接肽的长度和序列是独立选择的。例如，双特异性抗体还可以包括一个柔性连接肽(L3)，其连接scFv的可变重链和轻链(V HSCFV和V LSCFV)。这种柔性的连接肽可以包括甘氨酸-丝氨酸接头。通常，这种接头不包括铰链部分。在一些实施例中，连接scFv的可变重链和轻链的柔性连接肽(L3)包含序列GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:103)。
在一些实施例中，双特异性抗体的嵌合重链从N端到C端包含以下的多肽序列：V H1-C H1-C H2-C H3-L-V H2-L3-V L2。在一些实施例中，双特异性抗体的轻链从N端到C端包含以下的多肽序列：V L1-C L。在一些实施例中，所述双特异性抗体包含氨基酸序列SEQ ID NO:97或与氨基酸序列SEQ ID NO:97具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或包含氨基酸序列SEQ ID NO:83或与氨基酸序列SEQ ID NO:83具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，双特异性抗体的嵌合重链从N端到C端包含以下的多肽序列：V H1-C H1-C H2-C H3-L-V H2-L3-V L2。在一些实施例中，双特异性抗体的轻链从N端到C端包含以下的多肽序列：V L1-C L。在一些实施例中，所述双特异性抗体包含氨基酸序列SEQ ID NO:98或与氨基酸序列SEQ ID NO:98具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或包含氨基酸序列SEQ ID NO:85或与氨基酸序列SEQ ID NO:85具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
scFv-Fab IgG结构的双特异性抗体
在一些实施例中，本申请所述的任一双特异性抗体具有scFv-Fab IgG结构，其为异二聚体形式。所述异二聚体抗体包含第一单体和第二单体，所述第一单体包含能够结合其中一个抗原的抗原结合域(Fab)，所述第二单体包含够结合另一个抗原的抗原结合域(scFv)。在一些实施例中，所述双特异性抗体进一步包含两个Fc，其包含C H2和C H3结构域，两个Fc中进一步包含氨基酸替换，能够促进异源二聚体的相互结合。可变区可变区示例性scFv-Fab IgG结构示在图7E中。
在一些实施例中，所述其中一个抗原结合域(Fab或scFv)特异性地与肺炎克雷伯菌O2抗原结合，以及另一个抗原结合域(scFv或Fab)特异性地与肺炎克雷伯菌O1抗原结合。在一些实施例中，所述双特异性抗体可以同时结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原。
在一些实施例中，本申请所述的任一双特异性抗体具有scFv-Fab IgG结构，其为异二聚体形式。所述异二聚体抗体包含第一单体和第二单体，所述第一单体包含两条多肽链：第一重链和轻链，其中第一重链从N端到C端包含：V H1-C H1结构。轻链包含V L1-C L结构。在一些实施例中，所述第一重链进一步包含Fc，该Fc包含C H2和C H3结构域。在一些实施例中，第一重链从N端到C端包含：V H1-C H1-C H2-C H3结构，轻链包含V L1-C L结构。V H1和V L1分别是特异性结合其中一个抗原的重链可变区和轻链可变区，C H1是重链恒定区C H1结构域，C L是轻链恒定区。V H1-C H1和V L1-C L组成其中一个抗原结合域(Fab)。所述第二单体包含一条多肽链：第二重链。第二重链从N端到C端包含V H2-L3-V L2结构或V L2-L3-V H2结构。在一些实施例中，所述第二重链进一步包含Fc，该Fc包含C H2和C H3结构域。在一些实施例中，第一重链从N端到C端包含：V H2-L3-V L2-C H2-C H3结构或V L2-L3-V H2-C H2-C H3结构。V H2和V L2分别是特异性结合另一个抗原的重链可变区和轻链可变区，L3是连接肽。V H2-L3-V L2或V L2-L3-V H2组成另一个抗原结合域(scFv)。
在一些实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O1抗原的重链可变区和轻链可变区，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(Fab)。V H2和V L2分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H2-L3-V L2或V L2-L3-V H2组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(scFv)。在一些实施例中，V H1和V L1分别是特异性结合肺炎克雷伯菌O2抗原的重链可变区和轻链可变区，V H1-C H1和V L1-C L组成特异性结合肺炎克雷伯菌O2抗原的抗原结合域(Fab)。V H2和V L2分别是特异性结合肺炎克雷伯菌O1抗原的 重链可变区和轻链可变区，V H2-L3-V L2或V L2-L3-V H2组成特异性结合肺炎克雷伯菌O1抗原的抗原结合域(scFv)。
在一些实施例中，所述Fc来源于人野生型IgG1。在另一个实施例中，一个单体中的Fc相对于人野生型IgG1包含，但不限于如下氨基酸置换E357Q、和S364K；另一个单体中的Fc相对于人野生型IgG1包含，但不限于如下氨基酸置换Q295E、L368D、K370S、N384D、Q418E和N421D，其中所述编号依照如Kabat的EU索引。在一些实施例中，示例性连接scFv中V H1与V L1的连接肽(例如L3)包含GKPGSGKPGSGKPGSGKPGS(SEQ ID NO:113)。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:81，或包含与氨基酸序列SEQ ID NO:81具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:82，或包含与氨基酸序列SEQ ID NO:82具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:84，或包含与氨基酸序列SEQ ID NO:84具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:81，或与氨基酸序列SEQ ID NO:81具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:82，或与氨基酸序列SEQ ID NO:82具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:84，或与氨基酸序列SEQ ID NO:84具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:82，或与氨基酸序列SEQ ID NO:82具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:99，或与氨基酸序列SEQ ID NO:99具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:100，或与氨基酸序列SEQ ID NO:100具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
在一些实施例中，如本申请所述任一双特异性抗体，包含氨基酸序列SEQ ID NO:101，或与氨基酸序列SEQ ID NO:101具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:100，或与氨基酸序列SEQ ID NO:100具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列；和/或氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％(例如至少80％、85％、90％、95％、96％、97％、98％或99％)序列同源性的变体序列。
示例性抗体序列如表2-表10所示，其中CDR编号依据Kabat中的EU索引。本领域技术人员将认识到有多种已知算法来预测CDR的位置以及界定抗体轻、重链可变区。包含如本申请所述特异性结合肺炎克雷伯菌O2抗原的抗体，特异性结合肺炎克雷伯菌O1抗原的抗体，和双特异性抗体的CDRs、V H和/或V L序列，但基于预测算法而非下表中所示例的抗体也在本申请的范围内。所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段可以选自中国专利申请202110980272.2中所述的抗O1抗原的抗体，以引用的方式并入本申请中。
表2：示例性特异性结合肺炎克雷伯菌O2抗原的抗体CDR序列
Figure PCTCN2022133880-appb-000004
Figure PCTCN2022133880-appb-000005
表3-1：示例性特异性结合肺炎克雷伯菌O2抗原的抗体V H V L序列
Figure PCTCN2022133880-appb-000006
表3-2：示例性特异性结合肺炎克雷伯菌O2抗原的抗体V H V L半胱氨酸变体序列
Figure PCTCN2022133880-appb-000007
Figure PCTCN2022133880-appb-000008
表4：示例性特异性结合肺炎克雷伯菌O1抗原的抗体CDR序列
Figure PCTCN2022133880-appb-000009
表5-1：示例性特异性结合肺炎克雷伯菌O1抗原的抗体V H V L序列
Figure PCTCN2022133880-appb-000010
Figure PCTCN2022133880-appb-000011
表5-2：示例性特异性结合肺炎克雷伯菌O1抗原的抗体V H V L半胱氨酸变体序列
Figure PCTCN2022133880-appb-000012
表6：示例性抗体恒定区序列
Figure PCTCN2022133880-appb-000013
Figure PCTCN2022133880-appb-000014
表7-1：DVD-Ig结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的部分重链和轻链序列
Figure PCTCN2022133880-appb-000015
Figure PCTCN2022133880-appb-000016
表7-2：Bs4Ab结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的部分重链和轻链序列
Figure PCTCN2022133880-appb-000017
Figure PCTCN2022133880-appb-000018
Figure PCTCN2022133880-appb-000019
表7-3：Hetero H,CrossMab结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的部分重链和轻链序列
Figure PCTCN2022133880-appb-000020
Figure PCTCN2022133880-appb-000021
表7-4：scFv-Fab IgG结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的部分重链和轻链序列
Figure PCTCN2022133880-appb-000022
表8-1：DVD-Ig结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的重链和轻链全长序列
Figure PCTCN2022133880-appb-000023
Figure PCTCN2022133880-appb-000024
Figure PCTCN2022133880-appb-000025
Figure PCTCN2022133880-appb-000026
表8-2：Bs4Ab结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的重链和轻链全长序列
Figure PCTCN2022133880-appb-000027
Figure PCTCN2022133880-appb-000028
表8-3：Hetero H,CrossMab结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的重链和轻链全长序列
Figure PCTCN2022133880-appb-000029
Figure PCTCN2022133880-appb-000030
表8-4：IgG-(scFv) 2结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的重链和轻链全长序列
Figure PCTCN2022133880-appb-000031
Figure PCTCN2022133880-appb-000032
表8-5：scFv-Fab IgG结构特异性结合O2抗原和O1抗原的示例性双特异性抗体的重链和轻链全长序列
Figure PCTCN2022133880-appb-000033
Figure PCTCN2022133880-appb-000034
表9：示例性连接肽(或接头)序列
序列编号 序列
102 GGGGSGGGGS
103 GGGGSGGGGSGGGGSGGGGS
104 GGGGSGGGGSGGGGS
105 ASTKGP
106 TVAAP
107 EPKSDKTGGGGSGGGGS
108 EPKSCGKTGGGGSGGGGS
109 EPKSCGGGGSGGGGS
110 GGGGSGGGGSEPKSDKTHTCPPCP
111 GGGGSGGGGSCPPCP
112 GGGGSGGGGSDKTHTCPPCP
113 GKPGSGKPGSGKPGSGKPGS
114 GGGGSGGGGTGGGGS
特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的抗体的组合
在一方面，本申请提供了一种药物组合物，包括：(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段。
在一些实施例中，提供了一种药物组合物，包括：(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段，其中：特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：(a)重链可变区(V H)，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；或者(b)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；或者(c)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；或者(d)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；或者(e)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，提供了一种药物组合物，包括：(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段，其中：特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：(a)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；或者(b)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
在一些实施例中，所述药物组合物中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:17，以及V L，其包含氨基酸序列SEQ ID NO:21。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:17的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:21的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述药物组合物中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:18，以及V L，其包含氨基酸序列SEQ ID NO:22。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:18的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:22的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述药物组合物中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:19，以及V L，其包含氨基酸序列SEQ ID NO:23。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:19的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:23的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述药物组合物中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:19，以及V L，其包含氨基酸序列SEQ ID NO:24。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:19的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:24的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述药物组合物中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:20，以及V L，其包含氨基酸序列SEQ ID NO:25。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:20的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:25的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述药物组合物中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:47，以及V L，其包含氨基酸序列SEQ ID NO:49。在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:47的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:49的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述药物组合物中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:48，以及V L，其包含氨基酸序列SEQ ID NO:50。在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:48的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:50的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，提供了一种药物组合物包括：特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，提供了一种药物组合物包括：特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列 SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，提供了一种药物组合物包括：特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，提供了一种药物组合物包括：特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
结合亲和力
结合亲和力可以采用Kd、Koff、Kon或Ka来表示。如本申请所用，术语“Koff”是指抗原结合域从抗原结合域/抗原复合物中解离的速率常数，通过动力学选择装置测定。术语“Kon”是指抗体与抗原结合形成抗原结合域/抗原复合物的结合速率常数。本申请所用的解离常数“Kd”是指特定抗体抗原相互作用时的解离常数，是指在抗体分子溶液中，抗原占据所有抗体结合域的一半并且达到平衡时所需的抗原浓度，等于Koff/Kon。Kd的测定假设所有的结合分子均在溶液中。抗原结合域与细胞壁连接的情况，例如在酵母表达系统中，相应的解离速率常数采用EC50来表示，其是Kd的一个良好的近似值。亲和结合常数Ka是解离常数Kd的倒数。
平衡解离常数(Kd)可以作为抗原结合域部分与抗原亲和力的指标。例如，可以通过Scatchard方法使用标记有各种标记物的抗体，和Biacore仪器(由Amersham Biosciences制造)进行简单分析，根据用户手册或附带试剂盒，通过表面等离子体共振来分析生物分子间的相互作用。使用这些方法 得到的Kd值，用单位M来表示。与靶标特异性结合的抗体可能具有，例如≤10 -7M、≤10 -8M、≤10 -9M、≤10 -10M、≤10 -11M、≤10 -12M或≤10 -13M的Kd值。
抗体的结合特异性可以通过本领域已知的方法进行实验测定。这些方法包括，但不限于Western blots、ELISA-、RIA-、ECL-、IRMA-、EIA-、BIAcore测试和肽扫描等。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段特异性结合肺炎克雷伯菌O2抗原靶标，其Kd值为10 -7M至10 -13M(例如10 -7M至10 -13M、10 -8M至10 -13M、10 -9M至10 -13M或10 -10M至10 -12M)。因此，在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与肺炎克雷伯菌O2抗原之间结合的Kd值为10 -7M至10 -13M、1×10 -7M至5×10 -13M、10 -7M至10 -12M、10 -7M至10 -11M、10 -7M至10 -10M、10 -7M至10 -9M、10 -8M至10 -13M、1×10 -8M至5×10 -13M、10 -8M至10 -12M、10 -8M至10 -11M、10 -8M至10 -10M、10 -8M至10 -9M、5×10 -9M至1×10 -13M、5×10 -9M至1×10 -12M、5×10 -9M至1×10 -11M、5×10 -9M至1×10 -10M、10 -9M至10 -13M、10 -9M至10 -12M、10 -9M至10 -11M、10 -9M至10 -10M、5×10 -10M至1×10 -13M、5×10 -10M至1×10 -12M、5×10 -10M至1×10 -11M、10 -10M至10 -13M、1×10 -10M至5×10 -13M、1×10 -10M至1×10 -12M、1×10 -10M至5×10 -12M、1×10 -10M至1×10 -11M、10 -11M至10 -13M、1×10 -11M至5×10 -13M、10 -11M至10 -12M、10 -12M至10 -13M。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与肺炎克雷伯菌O2抗原之间结合的Kd值为10 -7M至10 -13M。
在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与非靶标之间结合的Kd值高于特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与靶标的Kd值，并且本申请中引用的一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与靶标(例如，肺炎克雷伯菌O2抗原)的结合亲和力高于特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与非靶标的结合亲和力。一些实施例中，非靶标是指非肺炎克雷伯菌O2抗原。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与非肺炎克雷伯菌O2抗原靶标结合的Kd值是特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与靶标肺炎克雷伯菌O2抗原之间结合的Kd的至少10倍，例如10-100倍、100-1000倍、10 3-10 4倍、10 4-10 5倍、10 5-10 6倍、10 6-10 7倍、10 7-10 8倍、10 8-10 9倍、10 9-10 10倍、10 10-10 11倍、10 11-10 12倍。
在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段特异性结合肺炎克雷伯菌O1抗原靶标，其Kd值为10 -7M至10 -13M(例如10 -7M至10 -13M、10 -8M至10 -13M、10 -9M至10 -13M或10 -10M至10 -12M)。因此，在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与肺炎克雷伯菌O1抗原之间结合的Kd值为10 -7M至10 -13M、1×10 -7M至5×10 -13M、10 -7M至10 -12M、10 -7M至10 -11M、10 -7M至10 -10M、10 -7M至10 -9M、10 -8M至10 -13M、1×10 -8M至5×10 -13M、10 -8M至10 -12M、10 -8M至10 -11M、10 -8M至10 -10M、10 -8M至10 -9M、5×10 -9M至1×10 -13M、5×10 -9M至1×10 -12M、5×10 -9M至1×10 -11M、5×10 -9M至1×10 -10M、10 -9M至10 -13M、10 -9M至10 -12M、10 -9M至10 -11M、10 -9M至10 -10M、5×10 -10M至1×10 -13M、5×10 -10M至1×10 -12M、5×10 -10M至1×10 -11M、10 -10M至10 -13M、1×10 -10M至5×10 -13M、1×10 -10M 至1×10 -12M、1×10 -10M至5×10 -12M、1×10 -10M至1×10 -11M、10 -11M至10 -13M、1×10 -11M至5×10 -13M、10 -11M至10 -12M、10 -12M至10 -13M。在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与肺炎克雷伯菌O1抗原之间结合的Kd值为10 -7M至10 -13M。
在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与非靶标之间结合的Kd值高于特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与靶标的Kd值，并且本申请中引用的一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与靶标(例如，肺炎克雷伯菌O1抗原)的结合亲和力高于特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与非靶标的结合亲和力。一些实施例中，非靶标是指非肺炎克雷伯菌O1抗原。在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与非肺炎克雷伯菌O1抗原靶标结合的Kd值是特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段与靶标肺炎克雷伯菌O1抗原之间结合的Kd的至少10倍，例如10-100倍、100-1000倍、10 3-10 4倍、10 4-10 5倍、10 5-10 6倍、10 6-10 7倍、10 7-10 8倍、10 8-10 9倍、10 9-10 10倍、10 10-10 11倍、10 11-10 12倍。
核酸
编码特异性结合肺炎克雷伯O2抗原的抗体或抗原结合片段、特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段以及双特异性抗体的核酸分子也被考虑在内。在一些实施例中，提供一种(或一组)编码全长特异性结合肺炎克雷伯菌O2抗原的抗体或特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的核酸，包括本申请所述的任一种特异性结合肺炎克雷伯菌O2抗原的全长抗体、或特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体。在一些实施例中，本申请所述的抗体或抗原结合片段或双特异性抗体的核酸(或一组核酸)还可以包括编码多肽标签的核酸序列(例如蛋白纯化标签，His-标签、HA标签)。
同时本申请还考虑了包含特异性结合肺炎克雷伯菌O2抗原的抗体，特异性结合肺炎克雷伯菌O1抗原的抗体，或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体；或包含编码所述抗体的核酸分子；或包含携带所述核酸分子的载体的分离的宿主细胞。
本申请还包括这些核酸序列的变体。例如，变体包括至少在中等严格杂交条件下与编码本申请的抗体或抗原结合片段或双特异性抗体的核酸序列杂交的核苷酸序列。
本申请同时还提供可将本申请中核酸序列插入到其中的载体。
简言之，将编码抗体或抗原结合片段或双特异性抗体的天然或合成的核酸插入到合适的表达载体中，使得核酸可操作性的连接到5’和3’端调控元件，例如包括启动子(例如淋巴细胞特异性启动子)和3’非翻译区(UTR)，可表达抗体或抗原结合片段或双特异性抗体。所述载体可适用于在真核宿主细胞中复制和整合。典型的克隆与表达载体包含调控目标核酸序列的表达的转录和翻译终止子、起始序列和启动子。
本申请所述的核酸也可以通过使用标准的基因递送方案，用于核酸免疫和基因治疗。核酸递送方法是本领域已知的。例如参见U.S.Pat.Nos.5,399,346、5,580,859、5,589,466，通过引用其全部内容并入本申请。在一些实施例中，本申请还提供基因治疗载体。
可以将核酸克隆到许多类型的载体中。例如，可以将核酸克隆到载体中，所述载体包括，但不限于，质粒、噬菌粒、噬菌体衍生物、动物病毒和柯斯质粒。特别感兴趣的载体包括表达载体、复制载体、探针生成载体和测序载体。
此外，表达载体可以以病毒载体的形式提供给细胞。病毒载体技术是本领域熟知的，并且描述于例如Green and Sambrook(2013,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)，以及其它病毒学或分子生物学手册中。可用作载体的病毒包括，但不限于，逆转录病毒、腺病毒、腺相关病毒、疱疹病毒和慢病毒。通常，合适的载体包括一个在至少一种生物体中起作用的复制起点、启动子序列、方便的限制性内切酶位点以及一个或多个选择标记物(参见例如，WO 01/96584；WO 01/29058；和U.S.Pat.No.6,326,193)。
已经开发了许多基于病毒的系统，用于将基因转移到哺乳动物细胞中。例如，逆转录病毒为基因递送系统提供了便利的平台。可以应用本领域已知的技术，将选择的基因插入载体中并包装在逆转录病毒颗粒中。然后分离重组病毒，在体内或体外递送至受试者的细胞中。许多逆转录病毒系统在本领域中是已知的。在一些实施例中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一些实施例中，使用慢病毒载体。衍生自逆转录病毒的载体，例如慢病毒，是实现长期基因转移的合适工具，因为它们使得转基因长期稳定的整合以及在子代细胞中繁殖。慢病毒载体相对于衍生自肿瘤的逆转录病毒例如小鼠白血病病毒具有额外的优势，因为它们可以转导非分裂细胞，例如肝细胞。同时，其还具有低免疫原性的额外优势。
其它的启动子元件，例如，增强子，调控转录起始频率。通常它们位于起始位点上游30-110bp处，虽然最近发现很多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔通常是灵活的，所以当元件彼此之间位置互换或移动时仍保持启动子的功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔增加到50bp，活性才会开始下降。
合适启动子的一个示例是即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是一个很强的组成型启动子序列，可以驱动任何与其可操作性连接的多核苷酸序列高水平表达。合适启动子的另一个示例是延伸因子1α(EF-1α)启动子。然而，也可以使用其它组成型启动子，包括但不限于，猿猴病毒40(SV40)早期启动子、小鼠乳腺肿瘤病毒(MMTV)、人免病缺陷病毒长末端重复序列(HIV-LTR)启动子、MoMuLV启动子、禽类白血病病毒启动子、Epstein-Barr病毒即刻早期启动子、劳斯氏肉瘤病毒启动子以及人类基因启动子，例如包括但不限于，肌动蛋白启动子、肌球蛋白启动子、血红蛋白启动子和肌酸激酶启动子。此外，不应将本申请局限在仅使用组成型启动子。诱导型启动子也是本申请考虑的部分。诱导型启动子的使用提供了一种分子开关，当需要这种表达时，能启动其与之可操作性连接的多核苷酸序列表达，当不需要时，则关闭表达。诱导型启动子，包含但不局限于，金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
在一些实施例中，所述抗体或抗原结合片段或双特异性抗体的表达是可诱导的。在一些实施例中，编码所述抗体或抗原结合片段或双特异性抗体的核酸序列可操作的连接到诱导型启动子上，包括本申请所述的任一诱导型启动子。
诱导型启动子
诱导型启动子的使用提供了一种分子开关，当需要表达时，可启动与之可操作性连接的多核苷酸序列表达，而在不需要表达时，则关闭表达。真核细胞中适用的示例性诱导型启动子，包括但不限于，激素调节元件(例如，参见Mader,S.and White,J.H.(1993)Proc.Natl.Acad.Sci.USA 90:5603-5607)、合成配体调节元件(参见Spencer,D.M.et al(1993)Science 262:1019-1024)以及电离辐射调控元件(参见Manome,Y.et al.(1993)Biochemistry 32:10607-10613；Datta,R.et al.(1992)Proc.Natl.Acad.Sci.USA 89:1014-10153)。其它适用于体内或体外哺乳动物系统的示例性诱导型启动子参见Gingrich et al.(1998)Annual Rev.Neurosci 21:377-405。在一些实施例中，用于表达抗体或抗原结合片段或双特异性抗体的诱导型启动子系统为Tet系统。在一些实施例中，表达抗体或抗原结合片段或双特异性抗体的诱导型启动子系统为大肠杆菌lac抑制系统。
本申请所采用的一个示例性诱导型启动子系统为Tet系统。该系统是基于Gossen等(1993)描述的Tet系统。在一个示例性实施例中，目标多核苷酸由包含一个或多个Tet操纵子(TetO)位点的启动子控制。在非激活状态，Tet阻遏物(TetR)与TetO位点结合并抑制启动子的转录。在激活状态，例如，在存在诱导剂如四环素(Tc)、无水四环素、多西环素(Dox)或其活性类似物的情况下，诱导剂会使TetR从TetO上释放，从而导致转录发生。多西环素是四环素抗生素家族中的一员，其化学名为1-二甲氨基-2,4a,5,7-五羟基-11-甲基-4,6-二氧基-1,4a,11,11a,12,12a-六氢四烯-3-甲酰胺。
在一个实施例中，TetR经密码子优化适用于在哺乳动物细胞中表达，例如小鼠或人类细胞。由于遗传密码的简并性，大多数氨基酸由不止一个密码子编码，从而使得给定核酸的序列具有大量的变体，而其编码的氨基酸序列没有任何改变。然而，许多生物体在密码子使用方面存在差异，也称为“密码子偏好”(即，给定氨基酸使用特定密码子的偏好)。密码子偏好通常与特定密码子的优势tRNA种类的存在有关，反过来又提高了mRNA翻译的效率。因此可以通过密码子优化来定制源自特定物种的编码序列(例如，原核生物)，以提高其在不同物种(例如，真核生物)中的表达。
Tet系统的其它具体变体，包括以下的“Tet-Off”和“Tet-On”系统。在Tet-off系统中，转录在Tc或Dox存在下是失活的。在该系统中，由TetR与单纯疱疹病毒VP16强转录激活结构域融合组成的四环素调控的转录激活蛋白(tTA)，在四环素反应启动子元件(TRE)转录控制下调控靶核酸的表达。TRE元件由TetO序列串联与启动子(通常是来源于人巨细胞病毒即刻早期启动子的最小启动子序列)融合组成。在不存在Tc或Dox的情况下，tTA结合TRE并激活靶基因的转录。在存在Tc或Dox的情况下，tTA不能结合TRE，靶基因不能表达。
相反，在Tet-On系统中，转录在Tc或Dox存在下是激活的。Tet-On系统是基于反向四环素调控的转录激活因子rtTA。与tTA一样，rtTA是由TetR阻遏物与VP16反式激活域组成的融合蛋白。然而，TetR的DNA结合区中4个氨基酸的变化改变了rtTA的结合特性，使其在存在Dox的情况下 只能识别靶转基因TRE上的tetO序列。所以在Tet-On系统中，只有在存在Dox的情况下，rtTA才能激活TRE调控的靶基因的转录。
另一种诱导型启动子系统是大肠杆菌的lac阻遏物系统(参见Brown et al.,Cell 49:603-612(1987))。Lac阻遏物系统通过调控与包含lac操纵子(lacO)的启动子可操作性连接的目标多核苷酸的转录发挥功能。Lac阻遏物(lacR)与LacO结合，进而阻止目标多核苷酸的转录。通过合适的诱导剂来诱导目标多核苷酸的表达，例如，异丙基-β-D硫代半乳糖吡喃苷(IPTG)。
为了评估多肽或其部分的表达，待导入细胞的表达载体还可包含选择标记基因或报告基因或二者都有，以便于从病毒载体转染或感染的细胞群体中识别和选择表达细胞。在其他方面，选择标记可以携带在单独的DNA片段上并在共转染实验中使用。选择标记基因或报告基因都可侧接于合适的调控序列，使其在宿主细胞中能够表达。有用的选择标记包括，例如，抗生素耐药基因，如neo以及类似基因。
报告基因可用于鉴定潜在的转染细胞和评价调控序列的功能。通常，报告基因是不存在于受体生物体或组织中或不由受体生物体或组织表达的基因，其编码一种多肽，其表达表现为一些易于检测的特性，例如酶活性。当DNA导入受体细胞后，在合适的时间检测报告基因的表达。合适的报告基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌碱性磷酸酶或绿色荧光蛋白的基因(e.g.,Ui-Tel et al.,2000FEBS Letters 479:79-82)。合适的表达系统是公知的，可以通过已知的技术制备或通过商业途径获得。通常，把具有能够显示报告基因最高表达水平的最小5'侧翼区的构建体认定为启动子。此类启动子区可以与报告基因连接，并用于评估某些物质在调节启动子驱动的转录中能力。
在一些实施例中，提供编码本申请所述的任一种抗体或抗原结合片段或双特异性抗体的核酸。在一些实施例中，所述核酸包括编码抗体或抗原结合片段或双特异性抗体重链和轻链的一个或多个核酸序列。在一些实施例中，所述一个或多个核酸序列中的每一个包含在单独的载体中。在一些实施例中，至少有一些核酸序列包含在同一载体中。在一些实施例中，所有核酸序列包含在同一载体中。载体可以选自，例如，哺乳动物表达载体和病毒载体(如源自逆转录病毒、腺病毒、腺相关病毒、疱疹病毒和慢病毒的载体)。
将基因导入细胞并表达的方法在本领域是已知的。在涉及表达载体的上下文中，通过本领域的任何方法载体可以很容易地导入宿主细胞中，如哺乳动物细胞、细菌、酵母或昆虫细胞。例如表达载体可以通过物理、化学或生物方法导入宿主细胞。
将多核苷酸导入宿主细胞的物理方法包括磷酸钙沉淀、脂质体转染、基因枪法、显微注射、电穿孔法以及诸如此类。制备包含载体和/或外源核酸的细胞的方法在本领域是熟知的。参见例如Green and Sambrook(2013,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。在一些实施例中，通过磷酸钙转染法将多核苷酸导入宿主细胞。
将目标多核苷酸导入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已成为将基因插入哺乳动物细胞，例如人类细胞中的最广泛使用的方法。其它病毒 载体可以源自慢病毒、痘病毒、单纯疱疹病毒1型、腺病毒和腺相关病毒等。参见如U.S.Pat.Nos.5,350,674和5,585,362。
将多核苷酸导入宿主细胞的化学方法包括胶体分散系统，例如高分子复合物、纳米胶囊、微球、磁珠和以脂质为基础的系统，其包括水包油乳剂、胶团、混合胶团和脂质体。一种在体内和体外被用作递送载体的示例性胶体系统是脂质体(例如，人工膜囊)。
在使用非病毒递送系统的情况下，示例性的递送载体是脂质体。考虑使用脂质制剂将核酸导入宿主细胞(体外、离体或体内)。在另一方面，所述核酸可以与脂质结合。与脂质结合的核酸可被包裹进脂质体的水性内部，散布在脂质体的脂质双层内，通过与脂质体和寡核苷酸结合的连接分子连接在脂质体，包埋在脂质体中，与脂质体形成复合物，分散在含有脂质的溶液中，与脂质混合，与脂质结合，悬浮在脂质中，包含在胶束中或与胶束混合，或以其它方式与脂质结合。脂质、脂质/DNA或脂质/表达载体相关的组合物在溶液中不限于任何特定结构。例如，它们可能以双分子层结构、以胶束或以“塌陷”结构存在。它们也可以简单的分散在溶液中，可能形成大小或形状不均匀的聚集体。脂质是脂肪物质，可以是天然存在的或是合成的脂质。例如，脂质包括天然存在于细胞质中的脂肪滴，以及含有长链脂肪烃及其衍生物的一类化合物，例如脂肪酸、醇、胺、氨基醇和醛。
无论采用何种方法将外源核酸导入宿主细胞中或以其他方式将细胞暴露于本申请的抑制剂中，为了确认重组DNA序列存在于宿主细胞中，可以进行多种实验。这类实验包括例如本领域技术人员熟知的“分子生物学”实验。例如Southern和Northern blotting，RT-PCR和PCR；“生物化学”实验，例如检测某一特定多肽是否存在或不存在，例如通过免疫学方法(ELISAs和Western blots)或者通过本申请所述的实验来进行鉴定均落入本申请范围内。
抗体或抗原结合片段和双特异性抗体的制备
在一些实施例中，所述抗体或抗原结合片段(例如，特异性结合肺炎克雷伯菌O2抗原或O1抗原的抗体或抗原结合片段)是单克隆抗体。在一些实施例中，所述抗体或抗原结合片段或双特异性抗体是源于单克隆抗体。在一些实施例中，所述抗体或抗原结合片段或双特异性抗体包括来自单克隆抗体的V H和V L，或者其变体。在一些实施例中，所述，所述抗体或抗原结合片段或双特异性抗体进一步包括来自单克隆抗体的C H1和C L区域，或者其变体。单克隆抗体可以应用例如本领域已知的方法制备，包括杂交瘤细胞法、酵母展示、噬菌体展示方法或应用重组DNA法。此外，示例性的酵母展示和噬菌体展示法在本申请及以下的实施例中进行了描述。双特异性抗体可以应用例如本领域已知的方法制备，包括化学偶联、杂交瘤法和基因工程法等。
在杂交瘤细胞法中，通常用免疫剂免疫仓鼠、小鼠或其他适合的宿主动物，以引发产生或能够产生与免疫剂特异性结合的抗体的淋巴细胞。或者，可以在体外免疫淋巴细胞。免疫剂可包括目标蛋白的多肽或融合蛋白。通常，如果需要人源细胞，采用外周血淋巴细胞(PBLs)，而如果需要非人哺乳动物来源细胞，则会使用脾细胞或淋巴结细胞。使用适当的融合剂将淋巴细胞与永生细胞系进行融合，例如聚乙二醇，以形成杂交瘤细胞。永生细胞系通常是转化的哺乳动物细胞，尤其是啮齿类、牛科和人源的骨髓瘤细胞。通常使用大鼠或小鼠骨髓瘤细胞系。杂交瘤细胞可以在合适的培 养基中进行培养，所述培养基优选含有一种或多种抑制未融合永生细胞生长或存活的物质。例如，如果亲本细胞缺乏次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HGPRT或HPRT)，则杂交瘤细胞培养基通常包括次黄嘌呤、氨蝶呤和胸苷(HAT培养基)，该培养基能阻止HGPRT缺陷细胞生长。
在一些实施例中，永生化细胞系有效融合，通过所选择的抗体生产细胞保证抗体高水平稳定表达，并且对某些培养基敏感，例如HAT培养基。在一些实施例中，永生细胞系是小鼠骨髓瘤细胞系，可以从例如，加利福尼亚圣地亚哥的索尔克细胞保藏中心和弗吉尼亚马纳萨斯的美国典型培养物保藏中心获得。同时还描述了人骨髓瘤和鼠-人杂交骨髓瘤细胞系用于制备人源单克隆抗体。
然后可以测定培养杂交瘤细胞的培养基中是否存在针对多肽的单克隆抗体。由杂交瘤细胞产生的单克隆抗体的结合特异性可以通过免疫沉淀法或体外结合实验确定，如放射性免疫测定法(RIA)或酶联免疫吸附法(ELISA)。此类技术或分析方法在本领域是已知的。单克隆抗体的结合亲和力可以通过例如Munson and Pollard,Anal.Biochem.,107:220(1980)中所述的斯卡查德(Scatchard)分析确定。
在鉴定出所需的杂交瘤细胞后，可以通过有限稀释法对目标克隆进行亚克隆，并通过标准方法进行培养。基于此目的适合的培养基包括，例如改良Eagle培养基(DMEM)和RPMI-1640培养基。或者，杂交瘤细胞可以在哺乳动物体内以腹水的形式生长。
亚克隆分泌的单克隆抗体可以通过常规免疫球蛋白纯化方法从培养基或腹水中分离或纯化，例如蛋白A-琼脂糖凝胶、羟基磷灰石色谱层析、凝胶电泳、透析或亲和层析。
在一些实施例中，根据本申请所述的任一抗体或抗原结合片段或双特异性抗体，所述抗体或抗原结合片段或双特异性抗体包含选自抗体文库(例如展示scFv或Fab片段的噬菌体文库)的克隆的序列。所述克隆可以通过筛选具有所需活性的抗体片段组合文库的方法进行鉴定。例如，本领域已知多种方法用于产生噬菌体展示文库以及筛选这些文库来获得所需结合特性的抗体。这些方法在例如Hoogenboom et al.,Methods in Molecular Biology 178:1-37(O'Brien et al.,ed.,Human Press,Totowa,N.J.,2001)中进行了综述，并且在例如McCafferty et al.,Nature 348:552-554；Clackson et al.,Nature 352:624-628(1991)；Marks et al.,J.Mol.Biol.222:581-597(1992)；Marks and Bradbury,Methods in Molecular Biology 248:161-175(Lo,ed.,Human Press,Totowa,N.J.,2003)；Sidhu et al.,J.Mol.Biol.338(2):299-310(2004)；Lee et al.,J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004)；and Lee et al.,J.Immunol.Methods 284(1-2):119-132(2004)中进行了进一步描述。
在某些噬菌体展示方法中，通过聚合酶链式反应(PCR)分别克隆V H和V L基因的所有组成成分，并在噬菌体文库中随机重组，然后筛选能够结合抗原的噬菌体，如Winter et al.,Ann.Rev.Immunol.,12:433-455(1994)中所述。噬菌体通常以scFv片段或以Fab片段形式展示抗体片段。免疫来源的文库噬菌体提供针对免疫原的高亲和力抗体而不需要构建杂交瘤细胞。或者，可以克隆天然库(例如来自人)，来提供针对多种非自身抗原和自身抗原的单一抗体来源，而不需任何免疫，如Griffiths et al.,EMBO J,12:725-734(1993)中所述。最后，天然文库也可以通过克隆来自干细胞的非重排V-gene片段，并使用包含随机序列的PCR引物编码CDR3高变区并且在体外完成重排的方法进行制备，如 Hoogenboom and Winter,J.Mol.Biol.,227:381-388(1992)中所述。描述人抗体噬菌体文库的专利出版物包括，例如U.S.Pat.No.5,750,373、和US Patent Publication Nos.2005/0079574、2005/0119455、2005/0266000、2007/0117126、2007/0160598、2007/0237764、2007/0292936和2009/0002360。
通过噬菌体展示筛选文库中能够特异性结合靶标(例如，肺炎克雷伯菌O2抗原或O1抗原)的抗原结合部分的方法来制备所述的抗体或抗原结合片段或双特异性抗体。该文库可以是人scFv噬菌体展示文库，具有至少1×10 9(例如至少1×10 9、2.5×10 9、5×10 9、7.5×10 9、1×10 10、2.5×10 10、5×10 10、7.5×10 10或1×10 11)种多样性的独特的人抗体片段。在一些实施例中，所述文库是人天然文库，通过从健康受试者的PMBCs和脾脏中提取的DNA构建，包含所有人重链和轻链亚家族。在一些实施例中，所述文库是人天然文库，通过从各种疾病患者体内分离的PMBCs中提取的DNA构建，例如自身免疫病的患者、癌症患者和感染性疾病的患者。在一些实施例中，所述文库是半合成的人文库，其中重链CDR3完全是随机的，所有氨基酸(除了半胱氨酸)以相同的概率存在于任何给定的位置。(参见，例如，Hoet,R.M.et al.,Nat.Biotechnol.23(3):344-348,2005)。在一些实施例中，半合成的人文库的重链CDR3长度在5到24个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24个)氨基酸之间。在一些实施例中，所述文库是全合成的噬菌体展示文库。在一些实施例中，所述文库是非人噬菌体展示文库。
对靶标抗原(例如，肺炎克雷伯菌O2抗原或O1抗原)具有高亲和力的噬菌体克隆可以通过噬菌体与靶标抗原的迭代结合进行筛选，所述靶标抗原与固相支持物结合(例如用于溶液淘选的珠子或用于细胞淘选的哺乳动物细胞)，接下来去除未结合的噬菌体，并洗脱特异性结合噬菌体。随后，洗脱结合的噬菌体克隆并用于感染合适的宿主细胞，例如E.coli XL1-Blue，进行表达和纯化。可以通过多轮淘选(例如，2、3、4、5、6或更多轮)，例如溶液淘选、细胞淘选或两者结合以富集特异性结合靶标抗原的噬菌体克隆。富集的噬菌体克隆与靶标抗原的特异性结合可以通过本领域已知的任何方法进行检测，包括例如ELISA和FACS。
单克隆抗体也可以通过重组DNA方法进行制备，例如U.S.Patent No.4，816,567中所述。编码本申请中所述单克隆抗体的DNA可以通过常规方法(例如通过能特异性结合编码鼠源抗体轻链和重链基因的寡聚核苷酸探针)轻易的分离和测序。如上所述的杂交瘤细胞或本申请的抗原特异性噬菌体克隆可以作为这种DNA的来源。分离后，可将DNA置于表达载体中，然后该载体转染入宿主细胞，例如猿猴COS细胞、中华仓鼠卵巢癌(CHO)细胞或不产生免疫球蛋白的骨髓瘤细胞中，获得在重组宿主细胞中合成的单克隆抗体。所述DNA也可以被修饰，例如用人类重链和轻链恒定结构和/或框架区域的编码序列代替同源的非人类序列(美国专利号4,816,567；Morrison et al.，同上)，或者将非免疫球蛋白多肽的全部或部分编码序列共价连接到免疫球蛋白编码序列上。这种非免疫球蛋白多肽可以取代本申请中抗体的恒定区，或可以取代本申请中抗体可变区中的一个抗原结合位点，形成嵌合的二价抗体。在一些实施例中，针对不同表位或抗原的额外的可变区可以被包括进来以产生嵌合的双特异性抗体。
所述抗体可以是单价抗体。制备单价抗体的方法是本领域已知的。例如，一种涉及免疫球蛋白轻链和修饰重链的重组表达方法。通常在Fc区的任意位置截短重链，以阻止重链相互交联。或者，相关的半胱氨酸残基被其它氨基酸残基取代或被缺失以防止交联。
体外方法也适用于制备单价抗体。消化抗体产生抗体片段，特别是Fab片段，可以使用任何本领域已知的方法完成。
化学偶联法化学偶联是最早应用于制备双特异性抗体的技术。1985年，Brennan首次利用两个单克隆抗体G1片段的化学结合来制备双特异性抗体(Brennan M,et al.Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments[J].Science,1985,229(4708):81-83)。化学偶联法主要有两种方式，一是将两种单抗抗体或其衍生物直接偶联形成双特异性抗体，二是先通过各种理化方式先将两种单抗解离成游离的轻链和重链，再将这些轻链和重链重新组合。化学偶联法的优点是快速，操作简便，回收率高，但也容易破坏抗体的抗原结合域，影响抗体活性，且容易形成多聚体。
利用杂交瘤细胞系来制备双特异性抗体，是指通过细胞融合技术，将两种不同的杂交瘤细胞系进行融合，再对可产生具有特定治疗性抗体的细胞进行鉴定和分离(Kohler，G，et al.Continuous cultures of fused cells secreting antibody of predefined specificity[J].J Immunol.,2005,174(5):2453-2455)。因为两种杂交瘤细胞可以产生两种不同的轻-重链，而这些轻-重链可以随机组合，故用此方法制备的双特异性抗体具有较大的随机性，制备效率低。
基因工程技术目前也被用于制备多种双特异性抗体(Roland E K.Antibody-cytokine fusion proteins[J].Arch Biochem Biophys.,2012,526(2):194-205)。利用基因工程来编辑重组抗体，可以通过多种方式限制两对轻－重链结合的选择性来解决随机组合的问题。KiH(Knob into hole)和CrossMab是目前被应用于改善轻-重链配对问题的两种常用技术。KiH技术即在C H3结构域引入不对称的突变结构(“knob”突变指在C H3结构域中，用一个大的氨基酸残基替换一个较小的残基，而“hole”突变指使用小的氨基酸残基来替换较大的残基)。经过改造的双特异性抗体的Fc区由于空间位阻的影响更倾向于发生异二聚化而不是同二聚化(Ridgway J B，et al.“Knobs-into-holes”engineering of antibody C H3 domains for heavy chain heterodimerization[J].Protein Eng.1996，9(7):617-621)。而在糖基化的C H3结构域中引入一个Y349C突变，可以使糖基化重链间形成二硫键，增强KiH的稳定性(Kuglstatter A,et al.Structural differences between glycosylated,disulfide-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products[J].Protein Eng Des Sel.,2017,30(9):649-656)。
除了空间位阻效应外，氨基酸残基的电荷效应也被用于提高双特异性抗体两条重链之间的异源二聚。通过结构模拟和分子设计改造，使一个链中产生“正电”，配对链中产生“负电”，通过同种电荷相斥、异种电荷相吸的模式促进重链异二聚体形成。两条链分别突变K409D和D399K，K409D/K392D和D399K/E356K，或者E356K/E357K/D399K和K370E/K409D/K439E等模式，均可在一定程度上提高异二聚体形成(IGAWA T,et al.Methods for producing polypeptides by regulating  polypeptide；association:US,20100015133A1[P].2006)。将KiH空间位阻效应和电荷效应相结合，也是进下提高异源二聚的策略之一。
CrossMab技术是Roche公司基于KiH技术基础上开发的一种新的抗体配对技术，通过将双特异性抗体的一条Fab的轻链与重链进行结构域交换，而另一条不交换。交换后的轻链中会含有一部分同源重链的片段，使其不能与未经交换的重链配对，从而确保了轻链与重链之间的正确组合(Schaefer W,et al.Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies[J].Proc Natl Acad Sci USA，2011,108(27):11187-11192)。包括“CrossMab Fab”，“CrossMab V H-V L”或“CrossMab C H1-C L”等形式。
具有所需结合特异性(抗体-抗原结合位点)的抗体可变区可以与免疫球蛋白恒定区融合。优选与免疫球蛋白重链恒定区进行融合，其包括至少部分铰链，C H2和C H3结构域。在一些实施例中，包含轻链结合必要位点的重链恒定区C H1结构域至少出现在一种融合体中。编码免疫球蛋白重链融合体的DNA，如果需要，还可以包括编码免疫球蛋白轻链的DNA，被插入进独立的表达载体中，并共转染至合适的宿主生物中。在一些实施例中，针对不同抗原表位或不同抗原的抗体可变区可与免疫球蛋白恒定区序列融合以产生嵌合的双特异性抗体。
全人和人源化抗体
所述抗体或抗原结合片段或双特异性抗体可以是人源化抗体或全人抗体。非人(如小鼠)抗体部分的人源化形式是嵌合的免疫球蛋白、免疫球蛋白链或其片段(例如Fv、Fab、Fab’、F(ab’) 2、scFv或抗体的其他抗原结合子序列)，其通常包括最少的源于非人免疫球蛋白的序列。人源化抗体包括人免疫球蛋白、免疫球蛋白链或其片段(受体抗体)，其中受体CDR的残基被具有所需特异性、亲和力和性能的非人源(供体抗体)CDR残基取代，例如小鼠、大鼠或兔子的CDR。在一些实施例中，人免疫球蛋白Fv框架区残基被相应的非人源残基取代。人源化抗体还可以包含既不属于受体抗体也不在引入的CDR或框架区序列中的氨基酸残基。通常，人源化抗体包含至少一个，通常两个可变区，其中全部或基本上全部CDR区对应于非人免疫球蛋白的CDR区，全部或基本上全部框架区是人免疫球蛋白共有序列。
通常，人源化抗体含有一个或多个从非人源引入的氨基酸残基。那些非人源氨基酸残基通常被称为“移入”残基，通常来自“移入”可变区。根据一些实施例，人源化基本上可以按照Winter和其同事的如下方法进行(Jones et al.,Nature,321:522-525(1986)；Riechmann et al.,Nature,332:323-327(1988)；Verhoeyen et al.,Science,239:1534-1536(1988))，通过用啮齿动物CDRs或CDR序列取代人源抗体的相应序列。因此，这种“人源化”抗体部分(U.S.Patent No.4,816,567)，其基本上少于完整的人源抗体，其可变区已被来自非人源的相应序列所取代。在实际中，人源化抗体部分是典型的人源抗体部分，其中一些CDR残基和可能的一些框架区残基被来自啮齿类抗体中类似位点的残基所取代。
产生人抗体部分是人源化的一种替代方式。例如，目前可以制备在免疫后能够产生完整的全人抗体文库而不产生内源性免疫球蛋白的转基因动物(例如，小鼠)。例如，已有报道，嵌合和种系突 变小鼠中抗体重链连接区(JH)基因的纯合子缺失，完全抑制了内源性抗体的产生。将人种系免疫球蛋白基因阵列转移到这种种系突变小鼠体内，可在抗原刺激下产生全人抗体，参见，例如akobovits et al.,PNAS USA,90:2551(1993)；Jakobovits et al.,Nature,362:255-258(1993)；Bruggemann et al.,Year in Immunol.,7:33(1993)；U.S.Patent Nos.5,545,806,5,569,825,5,591,669；5,545,807；和WO 97/17852。或者，可以通过将人类免疫球蛋白基因座引入转基因动物中(例如内源性免疫球蛋白基因已经被部分或全部沉默的小鼠)来制备全人抗体。抗原刺激后，可以发现全人抗体的产生在各个方面都与其在人类中的产生非常相似，包括基因重排、组装和抗体文库。这种方法在例如U.S.Patent Nos.5,545,807；5,545,806；5,569,825；5,625,126；5,633,425；and 5,661,016,and Marks et al.,Bio/Technology,10:779-783(1992)；Lonberg et al.,Nature,368:856-859(1994)；Morrison,Nature,368:812-813(1994)；Fishwild et al.,Nature Biotechnology,14:845-851(1996)；Neuberger,Nature Biotechnology,14:826(1996)；Lonberg and Huszar,Intern.Rev.Immunol.,13:65-93(1995)中进行了描述。
人抗体或人抗体部分也以通过体外活化B细胞(见U.S.Patents 5,567,610和5,229,275)或通过使用本领域已知的各种技术来产生，包括噬菌体展示文库。Hoogenboom and Winter,J.Mol.Biol.,227:381(1991)；Marks et al.,J.Mol.Biol.,222:581(1991).Cole et al.和Boerner et al.等人的技术也可以用于制备全人单克隆抗体。见Cole et al.,Monoclonal Antibodies and Cancer Therapy,Alan R.Liss,p.77(1985)and Boerner et al.,J.Immunol.,147(1):86-95(1991)。
抗体或抗原结合片段的变体
在一些实施例中，本申请提供的抗体或抗原结合片段(例如，特异性结合肺炎克雷伯菌O2抗原或O1抗原的抗体，或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体)的变体的氨基酸序列也在考虑中。例如，可能需要改善抗体或抗原结合片段的结合亲和力和/或其它生物学活性。抗原结合实体变体的氨基酸序列可以通过在编码抗原结合实体的核苷酸序列中引入适当的修饰或通过肽合成来制备。此类修饰包括例如，抗原结合实体氨基酸序列中残基的缺失和/或插入和/或取代。可以通过氨基酸残基缺失、插入和取代的任一组合来完成最终的构建，使其具有所需的特征。例如，抗原结合性。
在一些实施例中，提供具有一个或多个氨基酸取代的抗体或抗原结合片段的变体。取代突变的目标位点包括高变区(HVRs)和框架区(FRs)。可以在目标抗体中引入氨基酸取代，筛选所需活性的产物，例如，改善的生物活性，保持/改善抗原结合能力，降低的免疫原性，减少的病原菌结合或改善对病原菌的调理吞噬杀伤(OPK)，例如，肺炎克雷伯菌。在一些实施例中，本申请所述的氨基酸取代仅限于本申请表10中的“示例性取代”。在一些实施例中，所述氨基酸取代仅限于本申请表10中的“优选取代”。
保守取代如下表10所示。
Figure PCTCN2022133880-appb-000035
Figure PCTCN2022133880-appb-000036
根据侧链性质将氨基酸分为不同类别：
a.疏水氨基酸：去甲亮氨酸Norleucine、蛋氨酸Met、丙氨酸Ala、缬氨酸Val、亮氨酸Leu、异亮氨酸Ile；
b.中性亲水性氨基酸：半胱氨酸Cys、丝氨酸Ser、苏氨酸Thr、天冬酰胺Asn、谷氨酰胺Gln；
c.酸性氨基酸：天冬氨酸Asp、谷氨酸Glu；
d.碱性氨基酸：组氨酸His、赖氨酸Lys、精氨酸Arg；
e.影响链方向的氨基酸：甘氨酸Gly、脯氨酸Pro；
f.芳香族氨基酸：色氨酸Trp、酪氨酸Tyr、苯丙氨酸Phe。
非保守氨基酸的取代包含将以上一种类别取代为另一种类别。
一种示例性的取代变体是亲和力成熟的抗体，可采用例如以噬菌体展示为基础的亲和力成熟技术而方便地产生。简言之，将一个或多个CDR残基进行突变，变体抗体部分展示在噬菌体上，并筛选具有特定生物活性(例如，基于RBC细胞裂解抑制实验或结合亲和力)的变体。可以在HVRs区进行改变(例如，取代)来获得改善的基于RBC裂解抑制实验或抗体亲和力。可以在HVR的“热点区”产生改变，即在体细胞成熟过程中发生高频突变的密码子编码的残基(参见，例如Chowdhury,Methods Mol.Biol.207:179-196(2008))，和/或在特异的决定性残基(SDRs)，检测所得变体V H和V L的结合亲和力。从二级文库中构建和重新选择亲和力成熟的方法已经在一些文献中进行描述，例如，Hoogenboom et al.in Methods in Molecular Biology 178:1-37(O'Brien et al.,ed.,Human Press,Totowa,NJ,(2001))。
在一些亲和力成熟的实施例中，通过多种方法中的任一种(例如易错PCR，链改组或寡核苷酸定向突变)，将多样性引入选择的用于亲和力成熟的可变基因中。然后创建二级文库。对该文库进行筛选，鉴定出具有所需亲和力的抗体变体。另一种引入多样性的方法包括HVR介导的方式，其中几个HVR残基(例如，一次4-6个残基)被随机化。涉及抗原结合的HVR残基被特异性地识别，例如，采用丙氨酸扫描诱变或建模。通常CDR-H3和CDR-L3区域尤其是重点靶标。
在一些实施例中，取代、插入或缺失可能发生在一个或多个HVRs内，只要这种改变基本上不降低抗体结合抗原的能力。例如，可以在HVRs中产生基本上不降低结合亲和力的保守性改变(例如，本申请中提供的保守性取代)。这些改变可能发生在HVR“热点区”或SDRs区域之外。在一些实施例中上文提供的变体V H和V L序列，每一个HVR或者是未发生改变，或者包含不超过1个、2个或3个氨基酸取代。
一种通过突变的方法，用于鉴定出抗体中能够结合靶标的氨基酸或区域的方法称为“丙氨酸扫描突变”，如Cunningham and Wells(1989)Science,244:1081-1085中所述。在该方法中，一个或一组目标残基(例如，带电残基如精氨酸、天冬氨酸、组氨酸、赖氨酸和谷氨酸)被中性或带负电荷氨基酸(例如，丙氨酸或谷氨酸)取代，以此来确定抗体与抗原相互作用是否受到影响。可以在氨基酸的位置进一步引入取代，来证明该位置对初始取代具有功能敏感性。或者/另外，通过抗原-抗体复合物的晶体结构来鉴定抗体和抗原之间的接触位点。这些接触位点残基和邻近残基可作为取代候选物而被靶向或消除。筛选变体，确定它们是否具有所需要的性质。
氨基酸序列的插入，包括在氨基端和/或羧基末端的融合，长度范围从1个残基到包含100个或更多个残基的多肽，还包括在序列内插入1个或多个氨基酸残基。末端插入的例子包括N末端具有甲硫氨酰残基的抗原结合部分。抗原结合部分的其它插入变体，包括在抗原结合部分N-末端或C-末端融合一个酶(例如，ADEPT)或增加抗原结合部分血清半衰期的多肽。
Fc变体
在一些实施例中，将一个或多个氨基酸修饰引入本申请所述的抗体或抗原结合片段(例如，特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体、特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体，或包含所述抗体或抗原结合片段或双特异性抗体的融合蛋白)的Fc区，从而产生Fc变体。在一些实施例中，Fc变体具有增强的ADCC效能，通常与结合Fc的受体(FcRs)有关。在一些实施例中，Fc变体具有降低的ADCC效能。有很多关于Fc序列的改变或突变影响其效能的例子，例如，WO 00/42072和Shields et al.J Biol.Chem.9(2):6591-6604(2001)描述了与FcRs的结合增强或减弱的抗体变体。这些出版物的内容通过引用并入本申请。
抗体依赖的细胞介导的细胞毒作用(ADCC)是治疗性抗体针对肿瘤细胞的作用机制。ADCC是细胞介导的免疫防御，当靶细胞膜表面的抗原被特异性抗原结合部分(例如，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体)结合，免疫系统的效应细胞主动裂解靶细胞(例如，感染的细胞)。通 常ADCC效应涉及由抗体激活的NK细胞。NK细胞表达Fc受体CD16。该受体识别并结合与靶细胞表面相结合的抗体分子的Fc部分。NK细胞表面最常见的Fc受体为CD16或FcγRIII。Fc受体与抗体Fc区的结合导致NK细胞的活化，细胞裂解颗粒的释放，及随后靶细胞的凋亡。
在一些实施例中，本申请还提供特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体变体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体的变体)，其包含具有一种或多种效应功能的Fc区，这使得它成为一个理想的申请候选抗体，在特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的体内半衰期是重要的，但某些效应功能(如CDC和ADCC)是不必要或有害的。通过进行体外和/或体内的细胞毒性试验，来确认CDC和/或ADCC活性的降低/消除。例如，可以进行Fc受体(FcR)结合实验来确认抗体缺乏FcγR结合(因此可能缺乏ADCC活性)，但是保留FcRn结合能力。介导ADCC的主要细胞中，NK细胞仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。Ravetch and Kinet Annu.Rev.Immunol.9:457-492(1991)第464页的表3中总结了FcR在造血细胞上的表达。在体外评估目标分子的ADCC活性的非限制性实例在U.S.Pat.No.5,500,362中进行了描述(参见例如Hellstrom,I.et al.Proc.Nat'l Acad.Sci.USA 83:7059-7063(1986))and Hellstrom,I et al.,Proc.Nat'l Acad.Sci.USA 82:1499-1502(1985)；U.S.Pat.No.5,821,337(see Bruggemann,M.et al.,J.Exp.Med.166:1351-1361(1987))。或者，可以采用非放射性检测方法(参见，例如ACTI TM流式细胞术非放射性细胞毒性检测(CellTechnology,Inc.Mountain View,Calif.)和CYTOTOX 96 TM非放射性细胞毒性检测(Promega,Madison,Wis.))。此类检测实验采用的效应细胞包括外周血单核细胞(PBMC)和自然杀伤细胞(NK)。或者，另外地，目标分子的ADCC活性在体内进行检测，例如，在动物模型中，如Clynes et al.Proc.Nat'l Acad.Sci.USA 95:652-656(1998)中所述。同时还可以进行C1q结合试验来确认抗体不能与C1q结合，从而缺乏CDC活性。参见，例如WO2006/029879和WO 2005/100402中C1q和C3c结合ELISA。为了评估补体激活情况，可进行CDC检测(参见，例如Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996)；Cragg,M.S.et al.,Blood 101:1045-1052(2003)；和Cragg,M.S.and M.J.Glennie,Blood 103:2738-2743(2004))。使用本领域已知的方法来测定FcRn结合和体内清除/半衰期(参见，例如，Petkova,S.B.et al.,Int'l.Immunol.18(12):1759-1769(2006))。
具有降低的效应功能的抗体，包括在Fc区残基238、265、269、270、297、327和329位进行一个或多个残基的取代(U.S.Pat.No.6,737,056)。这些Fc变体包括在265、269、270、297和327位进行两个或多个残基的取代的Fc变体，包括被称为“DANA”的Fc变体，其在265和297位残基取代为丙氨酸(U.S.Pat.No.7,332,581)。
这类与FcRs结合能力提高或降低的抗体变体已有描述(参见例如U.S.Pat.No.6,737,056；WO2004/056312,和Shields et al.,J.Biol.Chem.9(2):6591-6604(2001))。
在一些实施例中，Fc区的改变导致调理作用的改变(即增强或减弱)，参见Moore et al.,MAbs.2(2):181–189(2010)中所述。
在一些实施例中，提供一种特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)变体，其包含具有一个或多个氨基酸取代的Fc变体，能够延长半衰期和/或增强与Fc受体(FcRn)的结合。具有延长半衰期和改善FcRn结合的抗体在US2005/0014934A1(Hinton等)中有所描述。这些抗体Fc区包含一个或多个氨基酸取代，增强了Fc区与FcRn的结合。这些Fc变体在Fc区包含238、256、265、272、286、303、305、307、311、312、317、340、356、360、362、376、378、380、382、413、424或434位的残基中的一个或多个取代，例如Fc区434位残基的取代(U.S.Pat.No.7,371,826)。
同时参见Duncan&Winter,Nature 322:738-40(1988)；U.S.Pat.No.5,648,260；U.S.Pat.No.5,624,821和WO 94/29351中提供其它Fc变体的例子。
同时考虑了包括本申请所述的任一种Fc变体或其组合的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)。
糖基化变体
在一些实施例中，对本申请所提供的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)进行改变，以增加或降低特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体糖基化的程度。通过改变特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体或其多肽部分的氨基酸序列以此来增加或去除一个或多个糖基化位点，可以方便地实现添加或删除特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体上的糖基化位点。
其中特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体包含Fc区，可以改变与其连接的糖。由哺乳动物细胞产生的天然抗体通常包含分支的双触角寡糖，该寡糖通常通过N-连接与Fc区C H2结构域Asn297连接，参见例如Wright et al.，TIBTECH 15:26-32(1997)。所述寡糖可包含多种糖类，例如甘露糖、N-乙酰氨基葡萄糖苷(GlcNAc)、半乳糖和唾液酸，以及与双触角寡糖结构“茎”部的GlcNAc相连接的海藻糖。在一些实施例中，可对本申请的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性 结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体进行寡糖修饰，从而产生具有某些改进特性的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体变体。
与Fc区的C H2结构域连接的N-聚糖是异质的。CHO细胞中产生的抗体或Fc融合蛋白通过岩藻糖基转移酶活性被岩藻糖基化，参见Shoji-Hosaka et al.,J.Biochem.2006,140:777-83。通常，可以在人血清中检测出一小部分天然存在的非岩藻糖基化IgGs。Fc区的N-糖基化对于其与FcγR结合很重要；而非岩藻糖基化的N-聚糖增强了Fc与FcγRIIIa的结合能力。与FcγRIIIa结合能力增强使得ADCC效应增强，这在需要细胞毒性的某些抗体治疗应用中是有利的。
在一些实施例中，当不需要Fc介导的细胞毒作用时，增强的效应功能可能是有害的。在一些实施例中，Fc片段或C H2结构域是非糖基化的。在一些实施例中，通过对C H2结构域中的N-糖基化位点进行突变以阻止其糖基化。
在一些实施例中，提供特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)变体，其包含Fc区，其中连接于Fc区的糖类结构具有减少的岩藻糖或缺乏岩藻糖，这可能会增强ADCC功能。具体地，本申请提供特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体变体，其相对于野生型CHO细胞产生的相同的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体具有减少的岩藻糖。也就是说，它们的特征在于，与天然CHO细胞(例如，产生天然糖基化形式的CHO细胞，含有天然FUT8基因的CHO细胞)产生的抗体相比，具有更少量的岩藻糖。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的N-连接聚糖具有少于50％、40％、30％、20％、10％或5％的岩藻糖。例如，该变特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的岩藻糖含量可能是1％-80％、1％-65％、5％-65％或20％-40％。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的N-连接聚糖不包含岩藻糖，即，其中特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体完全不含岩藻糖，或没有岩藻糖或是去岩藻糖基化。岩藻糖的含量是通过计算连接到Asn297上的糖链内岩藻糖平均含量相对于通过MALDI-TOF质谱测量的所有连接在Asn297上的糖结构(如复合、杂交或甘露糖结构)的总量来确定的，如WO 2008/077546所述。Asn297是指位于Fc区297位的天冬酰胺残基(EU Fc区残基编号体系)。然而，由于抗体的微小序列变化，Asn297也可位于297位的上游或下游±3个氨基酸，即在294和300位之间。这些 岩藻糖基化变体可能具有增强的ADCC功能。参见例如US Patent Publication Nos.US 2003/0157108(Presta,L.),US 2004/0093621(Kyowa Hakko Kogyo Co.,Ltd)。与“去岩藻糖基化”或“岩藻糖缺乏”的抗体变体相关的出版物的实例，包括US 2003/0157108；WO 2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/0093621；US 2004/0132140；US 2004/0110704；US 2004/0110282；US 2004/0109865；WO 2003/085119；WO 2003/084570；WO 2005/035586；WO 2005/035778；WO2005/053742；WO2002/031140；Okazaki et al.J.Mol.Biol.336:1239-1249(2004)；Yamane-Ohnuki et al.Biotech.Bioeng.87:614(2004)。能够产生去岩藻糖基化抗体的细胞系包括缺乏蛋白岩藻糖基化功能的Lec13CHO细胞(Ripka et al.Arch.Biochem.Biophys.249:533-545(1986)；US Pat Appl No US 2003/0157108A1,Presta,L；和WO 2004/056312A1,Adams et al.,尤其是实施例11)，和基因敲除细胞系，例如α-1,6-岩藻糖基转移酶基因，FUT8基因敲除的CHO细胞(参见Yamane-Ohnuki et al.Biotech.Bioeng.87:614(2004)；Kanda,Y.et al.,Biotechnol.Bioeng.,94(4):680-688(2006)；和WO2003/085107)。
特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)变体进一步提供二等分寡糖，例如，其中连接于特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体Fc区的双触角寡糖被GlcNAc等分。这种特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)变体可能具有减少的岩藻糖基化和/或增强的ADCC功能。这类抗体变体的实例在WO 2003/011878(Jean-Mairet et al.)；U.S.Pat.No.6,602,684(Umana et al.)；US 2005/0123546(Umana et al.),和Ferrara et al.,Biotechnology and Bioengineering,93(5):851-861(2006)中有所描述。还提供特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)变体，其在与Fc区连接的寡糖中具有至少一个半乳糖残基。这类特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体变体可能具有增强的CDC功能。这类变体在例如WO 1997/30087(Patel et al.)；WO 1998/58964(Raju,S.)；和WO 1999/22764(Raju,S.)中有所描述。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯 菌O2抗原和O1抗原的全长双特异性抗体)变体包含能与FcγRIII相结合的Fc区。在一些实施例中，包含Fc区的所述特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)变体在人效应细胞(例如T细胞)存在下具有ADCC活性，或者与具有人野生型IgG1Fc区的其他相同特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)相比，在人效应细胞存在下，具有增强的ADCC活性。
半胱氨酸工程变体
在一些实施例中，需要制备半胱氨酸工程化的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)，在该抗体中一个或多个氨基酸残基被半胱氨酸残基取代。在一些实施例中，取代残基出现在特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的可及位点。通过用半胱氨酸取代那些残基，具有活性的巯基基团位于特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的可及位点，可以用于将该特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体与其它部分偶联，例如药物部分或接头-药物部分，来制备如本申请中进一步描述的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体免疫偶联物。半胱氨酸工程化的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如，特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)可以按照例如U.S.Pat.No.7,521,541所述进行制备。
衍生物
在一些实施例中，本申请所提供的特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)可进一步修饰以包含本领域已知并且容易获得的其它非蛋白部分。适用于衍生化特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的部分包括但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、右旋糖酐、聚乙烯醇、聚乙烯吡咯烷酮、聚 -1,3-二氧戊烷、聚-1,3,6-三氧杂环已烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、右旋糖酐或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、环氧丙烷/环氧乙烷共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇及其混合物。聚乙二醇丙醛由于其在水中的稳定性，在制造中具有优势。聚合物可以具有任意分子量，可以是支链或非支链的。连接在特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体上的聚合物数量可以变化，并且如果连接多于一个多聚物，它们可以是相同的或不同的分子。通常，用于衍生化的聚合物的数量和/或类型可基于以下考虑因素来确定，包括但不限于，需要改进特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的特性或功能，特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体衍生物是否用于特定条件下的治疗等。
药物组合物
本申请还提供包含任一种特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)、编码抗体的核酸、包含编码抗体的核酸的载体或者包含本申请所述的核酸或载体的宿主细胞的组合物(例如药物组合物，在这里也称为制剂)。在一些实施例中，提供一种药物组合物，包含本申请所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体和药学上可接受的载体。
可通过混合具有所需纯度的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体与任选的药学上可接受的载体、赋形剂或稳定剂(Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980))获得合适的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体制剂，制备成冻干制剂或液体制剂形式。可接受的载体、赋形剂或稳定剂在所用剂量和浓度下对接受者无毒，包括缓冲剂如：磷酸盐、柠檬酸和其它有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；六甲基氯化铵；苯扎氯铵；苄索氯铵；苯酚；丁醇或苄醇；对羟基苯甲酸烷基酯，如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇和间甲酚)；低分子量(少于10个残基)多肽；蛋白质，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其它碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂如EDTA；糖类，如蔗糖、甘露醇、海藻糖或山梨糖醇；成盐反离子如钠；金属复合物(如锌-蛋白复合物)；和/或非离子表面活性剂如TWEEN TM，PLURONICS TM或聚乙二醇(PEG)；示例性制剂如WO98/56418中所述，并通过引用明确并入本申请。适合皮下给药的冻干制剂在WO97/04801中有所描述。这类冻干制剂可通过合适的稀释剂重构成高蛋白浓度的制剂，并且重构的制剂可以通过皮下给药的方式给予本申请中待治疗个体。阳离子脂质体或脂质体可以用于将本申请中的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎 克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体递送至细胞。
本申请所述的制剂除包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)之外，还可以包含一种或多种治疗特定病症所必要的其它活性物质，优选具有活性互补且彼此无不良反应的物质。例如，除了特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体之外，可能需要进一步包含其它具有治疗活性的物质，例如，抗生素类药物。这些分子以对预期目的有效的量组合存在。其它活性物质的有效量取决于制剂中的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的含量，疾病或病症或治疗方式等，以及如上所述的其它因素。这些药物通常以与本申请描述的相同剂量和给药途径使用，或者以目前应用剂量的1％至99％使用。
所述特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如，特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)也可以包埋在例如通过凝聚技术和界面聚合制备的微胶囊中，例如分别在胶体药物递送系统(例如，脂质体、白蛋白微球、微乳液、纳米颗粒和纳米胶囊)中或粗乳液中的羟甲基纤维素或明胶-微胶囊和聚(甲基丙烯酸甲酯)微胶囊。可以制备缓释制剂。
可以制备特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如，特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)的缓释制剂。缓释制剂的适合的实例包括含有抗体(或其片段)的固体疏水聚合物半透性基质，这些基质是成型制品的形式，例如，薄膜或微胶囊。缓释基质的实例包括聚酯、水凝胶(例如，聚(2-羟乙基甲基丙烯酸酯)或聚(乙烯醇))、聚乳酸(U.S.Pat.No.3,773,919)，L-谷氨酸和L-谷氨酸乙酯共聚物，不可降解的乙烯-醋酸乙烯酯，可降解的乳酸-乙醇酸共聚物如LUPRON DEPOT TM(由乳酸-乙醇酸共聚物和醋酸亮丙瑞林组成的可注射微球)以及聚-D(-)-3-羟基丁酸。虽然诸如乙烯-醋酸乙烯酯和乳酸-乙醇酸之类的聚合物可以使分子的释放超过100天，某些水凝胶可以在更短的时间内释放蛋白质。当包封的抗体在体内长时间停留时，它们会因暴露于37℃的潮湿环境中发生变性或聚集，可能导致生物活性的丧失或免疫原性的改变。可以根据相应的机制，设计合理的策略来稳定特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体。例如，如果发现聚集机制是通过硫代二硫化物交换形成分子间S-S键，则可以通过修饰巯基残基、在酸性溶液中冻干、控制含水量、使用适当的添加剂、以及开发特定的聚合物基质组合物来实现稳定化。
在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)配制在含有柠檬酸盐、氯化钠、乙酸盐、琥珀酸盐、甘氨酸、聚山梨酯80(吐温80)或上述任何组合的缓冲液中。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体配制在包含约100mM至约150mM甘氨酸的缓冲液中。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体配制在包含约50mM至约100mM NaCl的缓冲液中。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体配制在包含约10mM至约50mM醋酸盐的缓冲液中。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体配制在包含约10mM至约50mM琥珀酸的缓冲液中。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体配制在包含约0.005％至约0.02％聚山梨醇酯80的缓冲液中。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体配制在pH值介于5.1和5.6之间的缓冲液中。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体配制在包含10mM柠檬酸、100mM NaCl、100mM甘氨酸和0.01％聚山梨醇酯80的缓冲液中，其中制剂的pH为5.5。
用于体内给药的制剂必须是无菌的。这可以通过例如应用无菌过滤膜过滤而容易地实现。
预防或治疗肺炎克雷伯菌感染的方法
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的包含本申请所述的任一特异性结合肺炎克雷伯菌O2抗原的抗体，和/或特异性结合肺炎克雷伯菌O1抗原的抗体，和/或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体，和/或包含任一所述特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物，和/或包含特异性结合肺炎克雷伯菌O2抗原的抗体和特异性结合肺炎克雷伯菌O1抗原的抗体的组合物。在一些实施例中，治疗肺炎克雷伯菌感染的方法进一步提供了与肺炎克雷伯菌感染相关的疾病和/或病症治疗性或预防性的效果。在某些方面，提供了一种预防个体中肺炎克雷伯菌感染的方法，包括向个体施用有效量的组合物，该组合物包含本申请所述的任一双特异性抗体或药物组合物。在一些实施例中，提供了如上所述任一双特异性抗体或者药物组合物在制备用于治疗疾病或病症的药物中的用途。
与肺炎克雷伯菌感染相关的疾病和/或病症包括但不限于肺炎、尿路感染、败血症/菌血症/脓毒症、新生儿败血症/菌血症/脓毒症、腹泻、软组织感染、器官移植后感染、手术感染、伤口感染、肺部感染、化脓性肝脓肿、肺脓肿、蜂窝组织炎、坏死性肌膜炎、肌炎、眼内炎、腹膜炎、脑膜炎、坏死性脑膜炎、强直性脊柱炎或脊柱关节病。在一些实施例中，预防或治疗肺炎克雷伯菌感染的方法降低了肺炎克雷伯菌感染导致的死亡率。
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体或包含所述双特异性抗体的组合物，其中双特异性抗体包含特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，以及V L，其包含氨基酸序列SEQ ID NO:24或33。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:19或28的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:24或33的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中的双特异性抗体特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述双特异性抗体中特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:20或29，以及V L，其包含氨基酸序列SEQ ID NO:25或34。在一些实施例中，所述双特异性抗体中特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:20或29的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:25或34的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中的双特异性抗体特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。在一些实施例中，所述双特异性抗体中特异性结 合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，以及V L，其包含氨基酸序列SEQ ID NO:49或53。在一些实施例中，所述双特异性抗体中特异性结合肺炎克雷伯菌O1的第二抗原结合域包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:47或51的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:49或53的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中的双特异性抗体特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。在一些实施例中，所述双特异性抗体中特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，以及V L，其包含氨基酸序列SEQ ID NO:50或54。在一些实施例中，所述双特异性抗体中特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:48或52的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:50或54的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体或包含所述双特异性抗体的组合物，其中双特异性抗体包含特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中所述第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中所述第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体或包含所述双特异性抗体的组合物，其中双特异性抗体包含特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中所述第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包 含氨基酸序列SEQ ID NO:15；以及其中所述第二抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体或包含所述双特异性抗体的组合物，其中双特异性抗体包含特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中所述第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中所述第二抗原结合域包含：V H，所述V H包含HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包括：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体或包含所述双特异性抗体的组合物，其中双特异性抗体包含特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中所述第一抗原结合域包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及其中所述第二抗原结合域包含：V H，所述V H包含HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包括：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，提供了一种预防和/或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的包含特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体的组合物，比施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或等价剂量的特异性结合肺炎克雷伯菌O1抗原的抗体更有效。在一些实施例中，所述方法包括施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体增强针对肺炎克雷伯菌的调理吞噬杀菌 (OPK)活性至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上，与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或等价剂量的特异性结合肺炎克雷伯菌O1抗原的抗体相比。在一些实施例中，所述方法包括施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体增强对肺炎克雷伯菌的中和活性至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上，与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或等价剂量的特异性结合肺炎克雷伯菌O1抗原的抗体相比。在一些实施例中，所述方法包括施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体增强针对肺炎克雷伯菌的血清杀菌活性(SBA)至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上，与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或等价剂量的特异性结合肺炎克雷伯菌O1抗原的抗体相比。在一些实施例中，所述方法包括施用有效量的特异性结合肺炎克雷伯菌O2抗原和肺炎克雷伯菌O1抗原的双特异性抗体提高病人的生存率至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上，与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或等价量的特异性结合肺炎克雷伯菌O1抗原的抗体相比。
应用包含特异性结合肺炎克雷伯菌O2抗原的抗体和特异性结合肺炎克雷伯菌O1抗原的抗体的药物组合物预防或治疗疾病的方法
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的药物组合物，其包括：(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段。
在一些实施例中，提供了一种预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用：(i)特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和(ii)特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段同时施用。在一些实施例中，特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段与特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段顺序施用。
在一些实施例中，所述方法中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:17；以及V L，其包含氨基酸序列SEQ ID NO:21。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H， 所述V H包含具有氨基酸序列SEQ ID NO:17的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:21的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:18；以及V L，其包含氨基酸序列SEQ ID NO:22。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:18的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:22的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:19；以及V L，其包含氨基酸序列SEQ ID NO:23。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:19的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:23的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:19；以及V L，其包含氨基酸序列SEQ ID NO:24。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:19的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:24的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体 或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:20；以及V L，其包含氨基酸序列SEQ ID NO:25。在一些实施例中，所述特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:20的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:25的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:47；以及V L，其包含氨基酸序列SEQ ID NO:49。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:47的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:49的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述方法中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，其包含氨基酸序列SEQ ID NO:48；以及V L，其包含氨基酸序列SEQ ID NO:50。在一些实施例中，所述特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含具有氨基酸序列SEQ ID NO:48的V H中的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，所述V L包含具有氨基酸序列SEQ ID NO:50的V L中的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，提供了一种包括特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的药物组合物，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，提供了一种包括特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的药物组合物，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，提供了一种包括特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的药物组合物，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，提供了一种包括特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的药物组合物，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，提供了一种包括特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的药物组合物，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID  NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45。
在一些实施例中，提供了一种包括特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的药物组合物，其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
在一些实施例中，提供了预防或治疗个体中肺炎克雷伯菌感染的方法，所述方法包括向个体施用有效量的包含特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的组合物，该方法比施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或等价剂量的特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段更有效。在一些实施例中，所述方法包括施用有效量的包含特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的组合物，该方法与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或等价量的特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段相比，增强针对肺炎克雷伯菌的调理吞噬杀菌(OPK)活性至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。在一些实施例中，所述方法包括施用有效量的包含特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的组合物，该方法与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或等价量的特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段相比，增强针对肺炎克雷伯菌的血清杀菌活性(SBA)至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。在一些实施例中，所述方法包括施用有效量的包含特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的组合物，该方法与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或等价量的特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段相比，增强针对肺炎克雷伯菌的中和活 性至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。在一些实施例中，所述方法包括施用有效量的包含特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的组合物，该方法与施用等价剂量的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或等价剂量的特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段相比，提高病人的生存率至约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、2倍、5倍、10倍、20倍、50倍或100倍以上。
制品及试剂盒
在本申请的一些实施例中，提供一种制品，所述制品包含一种物质，所述物质能够用于预防或治疗个体中的肺炎克雷伯菌感染，或者用于递送抗体或抗原结合片段(特异性结合肺炎克雷伯菌O2抗原的抗体或特异性结合肺炎克雷伯菌O1抗原的抗体)或双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体)，或者包含特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段的药物组合物到被表达肺炎克雷伯菌O2抗原或O1抗原的病原菌附着的细胞中。所述制品可以包括一种容器以及在容器上或随该容器附带的标签或包装说明书。合适的容器包括，例如瓶子、小瓶、注射器等。容器可以由多种材料制成，例如玻璃或塑料。通常，该容器内装有能够有效治疗本申请所述疾病或病症的组合物，并且具有一个无菌端口(例如该容器可以是一个静脉输液袋或是一个具有皮下注射针头可刺穿盖子的小瓶)。组合物中的至少一种活性物质即为本申请所述的抗体或抗原结合片段或双特异性抗体。标签或包装说明书标示了该组合物可以用于治疗的特定病症。标签或包装说明书进一步包含给患者施用双特异性抗体或药物组合物的说明书。包括本申请所述的包含联合治疗的制品和试剂盒均在考虑范围之内。
包装说明书是指通常包含在治疗产品的商业包装内的说明书，其包含关于与这些治疗产品使用有关的适应症、用法、剂量、施用、禁忌症和/或警告信息。在一些实施例中，包装说明书标明该组合物可以用于治疗细菌感染。在一些实施例中，包装说明书标明该组合物可用于治疗肺炎克雷伯菌感染。
此外，所述制品还可以包括第二容器，其包含药学上可接受的缓冲液，例如抑菌性注射用水(BWFI)、磷酸盐缓冲液、格林氏溶液或葡萄糖溶液。还可以包括从商业和用户角度而言所需的其他材料，包括其他缓冲液、稀释液、过滤器、针头和注射器。
同时还提供可用于各种目的的试剂盒，例如用于预防或治疗个体中肺炎克雷伯菌感染，或者用于递送特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)到被表达肺炎克雷伯菌O2抗原或O1抗原的病原菌附着的细胞中，任选与制品组合。本申请的试剂盒包括一个或多个容器，其包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的组合物 (或单剂量形式和/或制品)，并且在一些实施例中，进一步包含另一种药剂(例如本申请所述的药剂)和/或与本申请所述任一方法相一致的使用说明书。该试剂盒可进一步包括选择适合治疗个体的描述。本申请中试剂盒中所附带的使用说明书通常是标签或包装说明书上的书面说明(例如包含在试剂盒内的纸页)，机器可读的说明(例如，磁性或光学储存光盘上的说明)也是可以接受的。
例如，在一些实施例中，试剂盒包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)的组合物。在一些实施例中，试剂盒包括：a)包含本申请所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物，和b)至少一种有效量的其它药剂，其能够增强特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的效果(如治疗效果、检测效果)。在一些实施例中，试剂盒包括：a)包含本申请所述的任一种特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物，和b)向个体施用特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体组合物用于治疗个体中肺炎克雷伯菌感染的使用说明书。在一些实施例中，试剂盒包括：a)包含本申请所述的任一种特异性结合肺炎克雷伯菌O2抗原抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物，和b)至少一种有效量的其它药剂，其能够增强特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的效果(如治疗效果、检测效果)和c)向个体施用特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体组合物和其它物质用于治疗个体中肺炎克雷伯菌感染的使用说明书。所述特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体和其他物质可以存在于独立的容器或同一个容器中。例如，该试剂盒可以包括一种特定组合物或两种或更多种组合物，其中一种组合物包括特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体，另一种组合物包括另一种药剂。
在一些实施例中，试剂盒包含一种(或一组)编码特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)的核酸。在一些实施例中，试剂盒包含：a)一种(或一组)编码特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克 雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的核酸，和b)一种表达核酸(或一组核酸)的宿主细胞。在一些实施例中，试剂盒包含：a)一种(或一组)编码特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的核酸，和b)使用说明书，适用于：i)在宿主细胞中表达特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体，ii)制备包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物，和iii)向个体施用包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物来预防或治疗个体中肺炎克雷伯菌感染。在一些实施例中，试剂盒包括：a)一种(或一组)编码特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的核酸，b)一种表达核酸(或一组核酸)的宿主细胞，和c)使用说明书，适用于：i)在宿主细胞中表达特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体，ii)制备包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物，和iii)向个体施用包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的组合物来预防或治疗个体中肺炎克雷伯菌感染。
本申请所述的试剂盒以合适的形式进行包装。合适的包装包括，但不限于，小瓶、瓶子、广口瓶、软包装(例如密封的聚酯薄膜或塑料袋)等。试剂盒可以任选地提供其它的组分，例如缓冲液和说明信息。本申请因此还提供制品，其包括小瓶(例如密封的小瓶)、瓶子、广口瓶、软包装等等。
关于包含特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体组合物的使用说明书，通常包括一些信息，诸如，剂量，给药周期和给药途径。容器可以是单位剂量的，大包装的(如，多剂量包装)或亚单位剂量的。例如，提供一种包含足够剂量的如本申请所述的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体(例如特异性结合肺炎克雷伯菌O2抗原的全长抗体、特异性结合肺炎克雷伯菌O1抗原的全长抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的全长双特异性抗体)的试剂盒以对个体进行长期有效的治疗，例如一周、8天、9天、10天、11天、12天、13天、2周、3周、4周、6周，8周，3个月、4个月、5个月、7个月、8个月、9个月或更长时间。试剂盒还可包含多单位剂量的特异性结合肺炎克雷伯菌O2抗原的抗体、特异性结合肺炎克雷伯菌O1抗原的抗体或特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体、药物组合物和使用说明书，并且以足够在药房中储存和使用的量进行包装，例如，医院药房和复合药房。
本领域的技术人员将认识到在本申请的范围和宗旨内可能的若干实施例。现在将通过参考以下非限制性实施例来更详细地描述本申请。以下实施例进一步阐明本申请，但不应解释为以任何方式进行限制其范围。
具体实施方式
在下述公开的实施例中，以KP19173代表LPS包含D-半乳聚糖Ⅱ和D-半乳聚糖Ⅰ两类重复单元的肺炎克雷伯菌O1血清型(图1)；以KP19213代表LPS包含D-半乳聚糖Ⅱ和D-半乳聚糖Ⅲ两类重复单元的肺炎克雷伯菌O1血清型(图1)；以KP19180代表LPS包含D-半乳聚糖Ⅰ重复单元的肺炎克雷伯菌O2血清型(图2)；以KP19203代表LPS包含D-半乳聚糖Ⅲ重复单元的肺炎克雷伯菌O2血清型(图2)。在本发明的实施例中，以专利申请CN107371365A中披露的抗肺炎克雷伯菌O抗原抗体G3-78(结合D-半乳聚糖Ⅲ重复单元)、和/或专利申请WO2018/029356Al中披露的抗肺炎克雷伯菌O1抗原抗体MPG-196(结合D-半乳聚糖II重复单元)作为对照抗体。
实施例1：制备脂多糖(LPS)、O2抗原并筛选抗O2的单链抗体(scFv)
LPS粗提取
挑取肺炎克雷伯O2型菌株KP19180单克隆接种到2×YT培养基中进行扩大培养。4000rpm、室温离心收集细菌，弃去上清。细菌沉淀加入无菌水重悬后，放入-80℃冰箱反复冻融3次、超声破裂细胞壁。采用热酚水法提取LPS：将等体积的90％(w/v)苯酚溶液加入到细菌悬液中，于68℃水浴中振荡混匀30min后冷却至室温，4000rpm、离心30min。离心后溶液分为3层，从上至下依次为水层、酚层和不溶层。上层水相含LPS，中层为变性蛋白，下层为沉淀。小心吸取上层水相到新管中，并加入6倍体积的95％乙醇，同时加入终浓度为0.1％的乙酸钠(国药，10018892)。将混合物-20℃过夜沉淀LPS，次日4000rpm、离心30min。沉淀即为KP19180菌株O2型LPS粗提物。
LPS纯化
将上述KP19180菌株O2型LPS粗提物应用100mM Tris-HCI(pH 8)进行复溶，加入终浓度为100μg/mL的DNaseI(Solarbio,D8071-25mg)和50μg/mL RNaseA(Solarbio,R8020-25mg)，37℃消化过夜。将消化后的LPS溶液沸水煮沸10min，冷却至室温后加入终浓度为100μg/mL的Proteinase K(Roche,14059723)，65℃作用2h。加入水饱和酚后混匀，4000rpm、离心30min，取上清液置于透析袋中4℃下应用蒸馏水透析过夜。将透析后溶液置于冷冻干燥机进行冷冻干燥，称取干重，-20℃保存备用。对LPS纯品进行如SDS-PAGE、DNA残留、RNA残留和蛋白浓度等质量检测，并对其进行LPS内毒素活性实验，以确定其内毒素活性。
制备KP19180 O2抗原
取1g由上述过程中制备的KP19180菌株O2型LPS，加入200mL 1％的乙酸溶液中，于沸水浴中作用45min。冷却后，4℃、105000g超速离心2h。沉淀为类脂A，上清为O2抗原。取上清，采用抽真空离心的方法浓缩所制备的O2抗原；随后用水溶解备用。
制备生物素化标记的O2抗原
取适量的O2抗原溶液加入3KDa超滤管中，4000rpm离心20-30min。向超滤管中加入0.2M的硼酸缓冲液(pH＝9.0)，4000rpm离心，此步骤整体重复3次。采用0.2M的硼酸缓冲液(pH＝9.0)溶解O2抗原，利用硫酸-苯酚比色法检测O2抗原溶液中多糖浓度，根据溶液体积计算出多糖总量(张青,张天民.苯酚-硫酸比色法测定多糖含量[C].中国药学会全国生化新药研究与临床应用学术会议论文集.青岛:中国药学会生化与生物技术药物专业委员会,2004:176-178.)。按照Amine-PEG11-Biotin(Thermo，26136)与O2抗原质量比为1.35:1，称取适量的Amine-PEG11-Biotin加入O2抗原溶液中，37℃孵育4d，分别在1h、24h、48h、72h，向每毫升反应液中加入180μL ALD Coupling Buffer(Sterogene，9704)。采用3KDa超滤管进行换液，将生物素化标记的O2抗原的溶剂更换为PBS，所得溶液即为生物素化标记的O2抗原(命名为Bio-O2抗原)。
筛选特异性结合肺炎克雷伯菌O2抗原的单链抗体(scFv)
构建scFv抗体酵母展示文库：从2000份人血液样本中提取RNA，经逆转录获得cDNA。采用V H和V L特异性引物扩增V H和V L片段，经过胶回收纯化后，连接V H和V L，构建scFv。将其克隆到酵母展示质粒PYD1中，随后将该质粒电转至酵母菌中，获得scFv抗体酵母展示文库。
筛选特异性结合肺炎克雷伯菌O2抗原的单链抗体(scFv)：经几轮淘选后，从酵母展示文库中分离结合肺炎克雷伯菌KP19180O2抗原的scFvs。简言之，采用MACS磁珠分选，对结合肺炎克雷伯菌O2抗原的酵母细胞进行富集。将酵母细胞在2500g下离心5min，获得的细胞沉淀按照OD600＝1的起始浓度，用1L SGCAA培养基进行重悬，并于20℃、250rpm培养条件下诱导表达40-48h。将细胞培养液离心，并用PBSM溶液清洗后，用5-10倍体积的含有1μM Bio-O2抗原的PBSM溶液重悬细胞沉淀，4℃孵育1h。离心后应用PBSM洗涤，未结合的抗原被PBSM溶液洗去。加入磁珠后，充分混匀，随后置于4℃悬转仪上孵育30min。2500g离心5min，弃去上清，用5-10倍体积的PBSM溶液重悬沉淀。每次取7mL细胞悬液上样到柱子中，直到所有的细胞悬液流穿柱子。洗脱并收集结合到柱子上的细胞，用于培养以及后续FACS分选。
采用FACS分选特异性结合肺炎克雷伯菌O2抗原的单链抗体(scFv)：对经MACS淘选后富集的酵母应用流式细胞荧光分选技术(FACS)进行分选。简言之，对SGCAA培养基中诱导的酵母细胞进行沉淀后加入1mL PBSM，14000g离心30sec，洗涤沉淀。用100μL含有KP19180菌株Bio-O2抗原的PBSM缓冲液重悬酵母细胞，室温孵育1h。洗涤后，用Streptavidin-PE(BD Biosciences，554061)和抗V5标签抗体[iFluor 488](GenScript，A01803-100)对细胞进行染色，筛选出前1％的双阳性染色细胞，并将其分选到培养基中，进行细胞扩增。应用由KP19180菌株获得的Bio-O2抗原重复进行筛选2-3个循环。通过进一步的FACS分析，对单个克隆进行检测。在筛选过程结束后，获得一组阳性scFv抗体，对其进行ELISA结合实验检测。
ELISA结合实验：
应用由KP19180菌株制备的O2型LPS进行ELISA结合试验，该试验用于鉴定结合KP19180O2型LPS的抗体。简言之，将LPS溶解在PBS溶液中(调节pH至7.2，终浓度为0.2μg/mL)， 按照100μL/孔包被96孔板，4℃过夜。在加入抗体之前，用200μL/孔PBST溶液洗涤96孔板5次。每孔加入200μL 10％BSA(Beyotime Biotechnology，ST023-200g)，37℃孵育1小时。用PBST溶液洗涤5次。首先将每个抗体样品稀释至1μg/mL，随后以1：3的比例进行梯度稀释。将梯度稀释后的抗体样品分别加入96孔板中，每孔50μL，37℃孵育1h。随后用PBST溶液洗涤5次。将100μL一抗和二抗混合物(小鼠抗-flag(1:2500)和抗-小鼠FC-AP(1:2000))加入每个孔中，37℃孵育1h，用TBST溶液洗涤3次。每孔加入50μL PNPP，37℃孵育10-20min。用3M NaOH终止反应。读取OD450，并通过Graphpad Prism生成结合曲线，计算EC50值。
实施例2：制备和表征特异性结合肺炎克雷伯菌O2抗原的全长抗体
制备全长的特异性结合O2抗原的抗体
将获得的阳性scFv抗体重构成含有人IgG1或IgG4的重链恒定区和人kappa轻链恒定区或人lambda轻链恒定区的人IgG1或IgG4全长抗体分子。从酵母表达载体中扩增V L和V H，分别构建入真核表达载体pTT5-L(包含kappa恒定区或包含lambda恒定区)和pTT5-H1(包含IgG1重链恒定区)或pTT5-H4(包含IgG4重链恒定区)中。将分别提取的表达轻链或重链的质粒，共转染293F细胞，37℃、8％CO 2、120rpm培养5d，用Protein A亲和层析柱纯化培养液。简言之，首先采用50mM PBS缓冲液(包含0.15M NaCl，pH7.2)以150cm/h的流速平衡Protein A层析柱。培养液上清(调节pH至7.2)以150cm/h流速流穿柱子。进一步平衡该柱后，采用50mM柠檬酸钠缓冲液(pH3.5)洗脱，收集洗脱液。将获得的全长抗体进行进一步的生化和生物活性分析。
ELISA结合试验
将上述制备的全长抗体分子进一步进行ELISA结合检测。具体操作同实施例1。不同之处在于，加入待检测全长抗体后，所采用二抗为山羊抗人IgG-HRP(Beyotime Biotechnology，A0201)1:10000，37℃孵育1小时。用PBST溶液洗涤5次。每孔加入100μL TMB(southern biotech，0410-01)，37℃孵育10-20分钟，用2M H 2SO 4终止反应，读取OD450，并通过Graphpad Prism生成结合曲线，计算EC50值。
LPS中和试验
商业报告细胞系HEK-Blue TM hTLR4(Invivogen，hkb-htlr4)稳定表达人TLR4、MD-2和CD14共受体，以及NF-κB诱导的分泌型胚胎碱性磷酸酶(SEAP)。细菌LPS可以触发该细胞系Toll-样受体4(TLR-4)信号传导，导致下游NF-κB转录因子的激活，从而分泌SEAP。QUANTI-Blue TM溶液(Cat#rep-qbs)可以用于检测和量化SEAP活性。简言之，根据制造商的操作说明书，培养HEK-Blue TM hTLR4细胞，并接种到96孔培养板中，每孔接种约2.5×10 4个细胞，之后将10μL实施例1中制备的KP19180LPS(10μg/mL)和起始浓度为1mg/mL的全长特异性结合肺炎克雷伯菌O2抗原的抗体(重构成人IgG1形式)的梯度稀释液预先混合，加入96孔培养板中，37℃、5％CO 2孵育6-16h。然后，将40μL细胞上清与160μL预热的QUANTI-Blue TM(Invivogen)溶液混合，孵育60-90min，读取620-655nm处的吸光度(OD)值。将本实验中仅应用LPS刺激而不加入抗体的孔的吸光度值作为 抑制率为0％的参照，无LPS刺激的孔的吸光度值作为抑制率为100％的参照，根据加入不同浓度特异性结合肺炎克雷伯菌O2抗原的抗体的孔的吸光度值，计算相应浓度下特异性结合肺炎克雷伯菌O2抗原的抗体的抑制率，经Graphpad Prism生成抑制曲线，计算IC50值。
亲和力成熟
根据全长抗体分子ELISA结合实验以及LPS中和实验结果，选取K1为先导抗体，用K1scFv制备包含CDR区突变的scFv噬菌体展示文库。通过KP19180LPS ELISA结合及LPS中和实验，选取具有较高亲和力和较高中和活性的优化后的特异性结合肺炎克雷伯菌O2抗原的抗体克隆，进一步构建成全长抗体，对先导抗体以及一系列优化后的抗体进行进一步的生物化学和生物学分析。
ELISA结合实验及KP19180LPS中和实验具体操作步骤如上文所述，先导抗体及优化后抗体的结合能力和中和能力实验结果见表11。
如表11所示，经过亲和力成熟实验后，一系列优化后的抗体均能够与KP19180O2型LPS结合并保持对KP19180O2型LPS的中和活性，且与K1相比，其结合能力和中和活性均有所提高。
表11
Figure PCTCN2022133880-appb-000037
实施例3：特异性结合肺炎克雷伯菌O2抗原的抗体结合域分析
肺炎克雷伯菌根据其独特的O抗原结构被分为不同的LPS血清型。O1和O2血清型菌株是目前常见的临床流行菌株。
同时O1血清型又包括2种结构，分别以KP19173和KP19213为代表，其具体结构示意图见图1，同样O2血清型也包括2种结构，分别以KP19180和KP19203为代表，其具体结构示意图见图2。
选取抗体K2、K3及K5进行结合域分析实验。应用ELISA实验测定抗体与上述4种具有不同O抗原结构的肺炎克雷伯菌株LPS的结合能力，不同肺炎克雷伯菌株LPS的制备方法和ELISA实验的具体操作步骤见实施例1。
通过ELISA结合实验确认示例特异性结合肺炎克雷伯菌O2抗原的抗体K2、K3及K5均可以与KP19180菌株O2型LPS特异性结合，如图3A所示；进一步测定示例性特异性结合肺炎克雷伯菌O2抗原的抗体与KP19203菌株O2型LPS的结合情况，G3-78为该实验中的对照抗体。如图3B所示，示例性特异性结合肺炎克雷伯菌O2抗原的抗体不与KP19203菌株O2型LPS结合。
进一步检测上述抗体与KP19173菌株O1型LPS以及KP19213菌株O1型LPS的结合情况，如图3C所示，示例性特异性结合肺炎克雷伯菌O2抗原的抗体能够特异性结合KP19173菌株O1型 LPS；如图3D所示，MPG196为该实验中的对照抗体，示例性特异性结合肺炎克雷伯菌O2抗原的抗体不与KP19213菌株O1型LPS结合。
根据上述不同血清型菌株的结构组成，以及ELISA结合实验结果，推断本申请所述的特异性结合肺炎克雷伯菌O2抗原的抗体特异性地结合肺炎克雷伯菌O抗原中的D-半乳聚糖I结构域，并且不与D-半乳聚糖Ⅱ和D-半乳聚糖Ⅲ结构域结合。
实施例4：特异性结合肺炎克雷伯菌O2抗原的抗体结合O2血清型肺炎克雷伯菌的广谱性
应用实施例1中所述的ELISA结合试验，检测特异性结合肺炎克雷伯菌O2抗原的抗体与来自临床分离的其他肺炎克雷伯菌O2血清型菌株(KP19002、KP19003、KP19005和KP19007菌株)LPS的结合情况，上述O2血清型菌株LPS均表达D-半乳聚糖I结构。不同肺炎克雷伯菌株LPS的制备方法如实施例1所示。
如图4A-图4D所示，示例性特异性结合肺炎克雷伯菌O2抗原的抗体K2、K3及K5均可以与来自其他O2血清型肺炎克雷伯菌株KP19002(图4A)、KP19003(图4B)、KP19005(图4C)或KP19007(图4D)的LPS结合。由此可见，特异性结合肺炎克雷伯菌O2抗原的抗体与表达D-半乳聚糖I结构的肺炎克雷伯菌O2血清型菌株的结合具有广谱性。
实施例5：特异性结合肺炎克雷伯菌O2抗原的抗体在小鼠菌血症模型中的预防保护作用
C57/BL6小鼠购买自维通利华并饲养在无特殊病原体的环境中。所有动物实验都是依照研究机构动物护理和使用委员会(IACUC)方案和指导进行的。
参照文献描述(参见Cross-specificity of protective human antibodies against Klebsiella pneumoniae LPS O-antigen，Nat Immunol.2018Jun；19(6):617-624.)制备小鼠肺炎克雷伯菌菌血症模型。
具体而言，取对数生长期的KP19180菌株悬液，4000rpm、10min,4℃离心后小心弃去培养液上清，加入灭菌PBS，涡旋振荡重悬，离心后弃去上清，重复2次。加入灭菌PBS重悬，调整菌悬液浓度为1×10 8CFU/100μL，备用。首先对小鼠通过腹腔注射100μl D-(+)-半乳糖胺盐酸盐(GalN，200mg/mL in pH＝7.4PBS,sigma,G1639-5G)，再通过静脉注射300μL KP19180菌悬液，并以此剂量作为后续实验中的造模剂量。
为了确定特异性结合肺炎克雷伯菌O2抗原的抗体是否具有预防保护性作用，在小鼠造模前24h，通过腹腔注射不同浓度的特异性结合肺炎克雷伯菌O2抗原的抗体K2或K5(重构成人IgG1形式)，抗体注射剂量分别为15mpk或30mpk，阴性对照为抗HIV抗体(Broad and potent neutralization of HIV-1by a gp41-specific human antibody Nature 491(7424),406-412(2012))，本文中称作HIV-10E8，注射剂量为30mpk。抗体注射24h后，按上述方法进行小鼠肺炎克雷伯菌血症模型造模。每天观察各组小鼠两次，记录8天内各组小鼠死亡数量、死亡时间及存活小鼠的状态。利用Graphpad Prism软件绘图，确定小鼠死亡率。
如图5所示，在KP19180菌株诱导的肺炎克雷伯菌血症模型中，与阴性对照抗体HIV-10E8相比，无论采用低剂量15mpk或高剂量30mpk，示例性特异性结合肺炎克雷伯菌O2抗原的抗体K2和K5均有效提高了小鼠的生存率和/或延长生存时间。当给予K5抗体，剂量为30mpk时，直到72h 小鼠才出现死亡，并且直到144h实验结束时，小鼠的存活率一直维持在70％左右。由此可见，特异性结合肺炎克雷伯菌O2抗原的抗体对肺炎克雷伯菌诱导的菌血症具有预防保护作用。
实施例6：特异性结合肺炎克雷伯菌O2抗原的抗体与特异性结合肺炎克雷伯菌O1抗原的抗体在小鼠肺炎模型中的联合治疗作用
构建小鼠肺炎模型，联合应用特异性结合肺炎克雷伯菌O2抗原的抗体和特异性结合肺炎克雷伯菌O1抗原的抗体对小鼠肺炎克雷伯菌感染的肺炎进行治疗。应用阴性对照抗体HIV-10E8、或联合应用特异性结合肺炎克雷伯菌O2抗原的抗体K5和特异性结合肺炎克雷伯菌O1抗原的抗体G2对各组小鼠进行处理，抗体组应用剂量如下：K5+G2(1.5mpk+1.5mpk)，以及K5+G2(15mpk+15mpk)，阴性HIV-10E8应用剂量为30mpk。
小鼠肺炎模型的制备：简言之，C57/BL6小鼠购买自维通利华并饲养在无特殊病原体的环境中。所有动物实验都是依照研究机构动物护理和使用委员会(IACUC)方案和指导进行的。本实验采用肺递送途径攻毒小鼠，构建了小鼠肺炎克雷伯菌感染的肺炎模型。具体而言，首先将小鼠麻醉后置于倾斜的平板上，打开口腔，同时左手持麻醉咽喉镜照射小鼠喉部，使会咽清晰可见，用微量液体气管内雾化器吸取固定量菌液(KP19173，OD600＝0.32)，然后将喷射头温和地插入气管，快速推注注射器使菌液喷射至气管内(Using Klebsiella pneumoniae to Model Acute Lung Inflammation in Mice，Methods Mol Biol.2019；1960:169-180.)。菌液递送剂量为25μL/小鼠。菌液递送完成后，将小鼠放回笼内，1h后通过小鼠尾静脉同时注射特异性结合肺炎克雷伯菌O2抗原的抗体和特异性结合肺炎克雷伯菌O1抗原的抗体，阴性对照组中的小鼠注射HIV-10E8。抗体注射完成后，持续观察至小鼠清醒。而后每天观察2次，直到第8天，记录各组小鼠死亡数量和时间并记录存活小鼠的状态。在GraphPad Prism软件中对各组小鼠的存活数据绘图，确定小鼠死亡率
如图6所示，当联合应用特异性结合肺炎克雷伯菌O2抗原的抗体与特异性结合肺炎克雷伯菌O1抗原抗体时，对小鼠肺炎克雷伯菌感染造成的肺炎具有治疗作用，即使在抗体使用剂量为1.5mpk+1.5mpk时，对小鼠肺炎感染后的144h生存率仍然维持在70％左右，与阴性对照相比具有统计学意义(p<0.05)。
实施例7：不同结构的特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体的制备
下述序列设计中:V H和V L分别代表抗体的重链可变区和轻链可变区；C H代表抗体重链恒定区，包括C H1、C H2和C H3结构域；scFv是由抗体的V H和V L通过连接肽(Linker)连接而成的抗体；IgG1Fc代表IgG1亚类抗体的Fc区域，其包含C H2和C H3结构域；C L代表轻链恒定区。
7.1 DVD-Ig结构双特异性抗体的构建：
1)序列设计：DVD-Ig(Dual-variable domain-Ig)双特异性抗体，其结构是在正常IgG抗体的轻链和重链的N末端分别连接另外一个抗体的V L和V H结构域，通过两个抗体的V H与V L相互作用形成抗原结合域，能够同时与相对应的抗原结合，来实现双特异性。关于DVD-Ig结构的双特异性抗体在文献Wu C,et al.Molecular construction and optimization of anti-human IL-1alpha/beta dual variable  domain immunoglobulin(DVD-Ig)molecules.MAbs.2009Jul-Aug；1(4):339-47中也有记载。其结构示意图如图7A所示。表12-1为实施例中所使用的DVD-Ig结构的双特异性抗体的组成设计，表8-1中示出了其重链和轻链的具体氨基酸序列。委托金斯瑞生物科技有限公司分别合成双特异性抗体轻链(L链)以及重链(H链)编码基因，并对编码基因进行优化，以适于在293F细胞中表达。
2)双特异性抗体的表达纯化：通过限制性内切酶进行酶切后，将编码双特异性抗体的轻、重链基因分别亚克隆到pTTα1载体中。提取表达抗体轻、重链的重组质粒，共转染293F细胞。37℃，5％CO 2，120rpm培养7天，用protein A亲和层析柱纯化培养液。简言之，首先采用6倍柱体积的包含0.15M NaCl的50mM PBS缓冲液(pH7.2)以150cm/h的流速平衡蛋白A柱。调节培养液上清的pH至7.2，以150cm/h流速上样。上样结束后，继续平衡该柱，最后采用50mM柠檬酸-柠檬酸纳缓冲液(pH3.5)洗脱，收集洗脱液。用30kDa超滤管浓缩，更换Buffer为PBS溶液。应用去内毒素试剂盒(SA031K，常州天地人和生物科技有限公司)去除内毒素后，分别测量蛋白的浓度和内毒素的含量。将去除内毒素后的DVD-Ig结构特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体样品分别进行还原和非还原SDS-PAGE电泳检测(结果未显示)，以确保其形成正确的结构。
表12-1：DVD-Ig结构特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体重链和轻链的组成设计
Figure PCTCN2022133880-appb-000038
7.2 Bs4Ab结构双特异性抗体的构建：
1)序列设计：Bs4Ab结构双特异性抗体包括一个全长的IgG1结构，通过在其铰链区插入另一个结合单元scFv来实现双特异性。关于Bs4Ab结构的双特异性抗体在文献Bezabeh B,et al.Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.MAbs.2017Feb/Mar；9(2):240-256中也有记载。其结构示意图如图7B所示。表12-2为实施例中所使用的Bs4Ab结构的双特异性抗体的组成设计，表8-2中示出了其重链和轻链的具体氨基酸序列。委托金斯瑞生物科技有限公司分别合成双特异性抗体轻链(L链)以及重链(H链)编码基因，并对编码基因进行优化，以适于在293F细胞中表达。
2)双特异性抗体的表达纯化：具体的操作步骤如同7.1中所述。
表12-2：Bs4Ab结构特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体重链和轻链的组成设计
Figure PCTCN2022133880-appb-000039
7.3 Hetero H,CrossMab结构双特异性抗体的构建：
1)序列设计：Hetero H,CrossMab结构双特异性抗体，即在Fc区设计了杵臼结构(knobs-in-holes，KIH)，同时引入形成稳定化二硫桥的两个Cys残基突变(S354C在“knob”侧，并且Y349C在“hole”侧)。通过KIH方法，即将一个抗体C H3区366位苏氨酸(T)替换为色氨酸(W)形成“杵”(knobs)结构，并将配对的另一个抗体C H3区366位苏氨酸(T)替换为丝氨酸(S)，368位亮氨酸(L)替换为丙氨酸(A)，407位酪氨酸(Y)替换为缬氨酸(V)形成“臼”(holes)结构，依靠突变后空间位阻效应的下降以及铰链区形成的共价二硫键，促进异源重链二聚化，其中所述编号依照如Kabat的EU索引；同时应用CrossMab技术，来确保抗体轻、重链间的正确配对。CrossMab技术是基于双特异性IgG抗体的一个Fab臂内抗体域的交换，可以是完整的Fab域的交换(CrossMAb Fab)，或者是Fab中仅可变区的交换(CrossMAb V H-V L)或仅恒定区的交换(CrossMAb C H1-C L)。关于Hetero H,CrossMab结构的双特异性抗体在文献Klein C,et al.The use of CrossMAb technology for the generation of bi-and multispecific antibodies.MAbs.2016Aug-Sep；8(6):1010-20中也有记载。本申请以CrossMab C H1-C L结构进行具体说明，该结构示意图如图7C所示。表12-3为实施例中所使用的Hetero H,CrossMab结构的双特异性抗体的组成设计，表8-3中示出了其重链和轻链的具体氨基酸序列，其中Fc来自于人野生型IgG1Fc。委托金斯瑞生物科技有限公司分别合成双特异性抗体轻链(L链)以及重链(H链)编码基因，并对编码基因进行优化，以适于在293F细胞中表达。
2)双特异性抗体的表达纯化：具体的操作步骤如同7.1中所述。
表12-3：Hetero H,CrossMab结构特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体重链和轻链的组成设计
Figure PCTCN2022133880-appb-000040
7.4 IgG-(scFv) 2结构双特异性抗体的构建：
1)序列设计：IgG-(scFv) 2结构双特异性抗体，即在一个IgG抗体的两条重链的Fc末端连接另一个抗体的scFv片段来实现双特异性。关于IgG-(scFv) 2结构的双特异性抗体在文献Coloma MJ,Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nat Biotechnol.1997Feb；15(2):159-63中也有记载。该结构示意图如图7D所示。表12-4为实施例中所使用的IgG-(scFv) 2结构的双特异性抗体的组成设计，表8-4中示出了其重链和轻链的具体氨基酸。委托金斯瑞生物科技有限公司分别合成双特异性抗体轻链(L链)以及重链(H链)编码基因，并对编码基因进行优化，以适于在293F细胞表达。
2)双特异性抗体的表达纯化：具体的操作步骤如同7.1中所述。
表12-4：IgG-(scFv) 2结构特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体重链和轻链的组成设计
Figure PCTCN2022133880-appb-000041
7.5 scFv-Fab IgG结构双特异性抗体的构建：
1)序列设计：scFv-Fab IgG结构双特异性抗体，为异二聚体抗体，IgG抗体结构，其中一个Fab臂被替换成scFv结构，其中第一单体包含scFv和Fc，scFv通过连接肽连接在Fc C H2结构域的N末端，第二单体包含Fab和Fc。该结构示意图如图7E所示。表12-5为实施例中所使用的scFv-Fab IgG结构的双特异性抗体的组成设计，表8-5中示出了其重链和轻链的具体氨基酸。委托金斯瑞生物科技有限公司分别合成双特异性抗体轻链(L链)以及重链(H链)编码基因，并对编码基因进行优化，以适于在293F细胞中表达。
2)双特异性抗体的表达纯化：具体的操作步骤如同7.1中所述。
表12-5：scFv-Fab IgG结构特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体重链和轻链的组成设计
Figure PCTCN2022133880-appb-000042
实施例8：双特异性抗体结合O1型LPS和O2型LPS(D-galactanⅠ)的EC50测定
应用ELISA结合实验分别检测双特异性抗体与O1型LPS(KP19173菌株O1型LPS或KP19213菌株O1型LPS)或O2型LPS(KP19180O2型LPS)的结合情况。不同血清型肺炎克雷伯菌LPS制备方法，及ELISA结合实验具体操作步骤参见实施例1。特异性结合肺炎克雷伯菌O1抗原的抗体G2和G7以及特异性结合肺炎克雷伯菌O2抗原的抗体K5作为本实验的对照抗体。其中抗体K5不与KP19213菌株O1型LPS结合，抗体G2和G7不与KP19180O2型LPS结合。简言之，在96孔板(Corning#9018)中每孔加入100μl LPS(0.2μg/mL,分别来源于KP19173菌株、KP19213菌株或者KP19180菌株)，4℃包被过夜。第二天用PBST清洗5次(200μL/孔)，随后每孔加入200μl10％BSA(Beyotime Biotechnology，ST023-200g)，37℃孵育1h。PBST清洗5次后(200μL/孔)，每孔加入梯度稀释的双特异性抗体或对照抗体，37℃孵育1h。PBST清洗5次后，每孔加入100μL稀释后的辣根过氧化物酶标记山羊抗人IgG(Beyotime Biotechnology，A0201)，37℃孵育1h。PBST清洗5次后(200μL/孔)，加入TMB显色底物(Southern Biotech，0410-01)反应15min后，用2M H 2SO 4终止反应。酶标仪(Spark 10M,Tecan)检测450nm处吸光度值(OD450)。并通过GraphPad Prism生成结合曲线，计算抗体结合EC50值。
如图8A-图8C所示，DVD-Ig结构的示例性双特异性抗体均能够与KP19173菌株O1型LPS(图8A)、KP19213菌株O1型LPS(图8B)或KP19180菌株O2型LPS结合(图8C)。
如图8D-图8F所示，Bs4Ab结构的示例性双特异性抗体能够与KP19173菌株O1型LPS(图8D)、KP19213菌株O1型LPS(图8E)、或KP19180菌株O2型LPS结合(图8F)。
如图8G-图8I所示，Hetero H,CrossMab结构的示例性双特异性抗体能够与KP19173菌株O1型LPS(图8G)、KP19213菌株O1型LPS(图8H)或KP19180菌株O2型LPS结合(图8I)。
如图8J-图8L所示，IgG-(scFv) 2结构的示例性双特异性抗体能够与KP19173菌株O1型LPS(图8J)、KP19213菌株O1型LPS(图8K)、或KP19180菌株O2型LPS结合(图8L)。
如图8M-图8O所示，scFv-Fab IgG结构的示例性双特异性抗体能够与KP19173菌株O1型LPS(图8M)、KP19213菌株O1型LPS(图8N)、或KP19180菌株O2型LPS结合(图8O)。
实施例9：双特异性抗体对肺炎克雷伯菌O1型LPS和O2型LPS(D-galactanⅠ)的中和活性测定
应用LPS中和实验分别检测双特异性抗体对O1型LPS(KP19173菌株O1型LPS或KP19213菌株O1型LPS)和O2型LPS(KP19180O2型LPS)的中和活性。具体操作步骤参见实施例2。特异性结合肺炎克雷伯菌O1抗原的抗体G2和G7以及特异性结合肺炎克雷伯菌O2抗原的抗体K5作为本实验的对照抗体。其中抗体K5对KP19213菌株O1型LPS不具有中和活性，抗体G2和G7对KP19180菌株O2型LPS不具有中和活性。简言之，首先在96孔板中，每孔加入180μL HEK-Blue TM hTLR4(约25,000个细胞)，然后每孔再加入10μL LPS(10μg/mL,分别来源于KP19173菌株、KP19213菌株或者KP19180菌株)和不同浓度的双特异性抗体或对照抗体，37℃培养6-16h，应用酶标仪检测620-655nm的吸光度值。将本实验中无抗体加入的孔的吸光度值作为抑制率为0％ 的参照，将无LPS加入的孔的吸光度值作为抑制率为100％的参照，计算不同浓度下双特异性抗体的抑制率，利用Graphpad Prism软件绘图，计算IC50值。
如图9A-图9C所示，DVD-Ig结构的示例性双特异性抗体能够抑制KP19173菌株O1型LPS(图9A)、KP19213菌株O1型LPS(图9B)、或KP19180菌株O2型LPS(图9C)。
如图9D-图9F所示，Bs4Ab结构的示例性双特异性抗体能够抑制KP19173菌株O1型LPS(图9D)、KP19213菌株O1型LPS(图9E)、或KP19180菌株O2型LPS(图9F)。
如图9G-图9I所示，Hetero H,CrossMab结构的示例性双特异性抗体能够抑制KP19173菌株O1型LPS(图9G)、KP19213菌株O1型LPS(图9H)、或KP19180菌株O2型LPS(图9I)。
如图9J-图9L所示，IgG-(scFv) 2结构的示例性双特异性抗体能够抑制KP19173菌株O1型LPS(图9J)、KP19213菌株O1型LPS(图9K)、或KP19180菌株O2型LPS(图9L)。
如图9M-图9O所示，scFv-Fab IgG结构的示例性双特异性抗体能够抑制KP19173菌株O1型LPS(图9M)、KP19213菌株O1型LPS(图9N)、或KP19180菌株O2型LPS(图9O)。
实施例10：双特异性抗体介导的调理吞噬杀菌(OPK)活性
调理吞噬杀菌实验(OPK)活性是指由于免疫细胞的吞噬而发生的细胞(例如，肺炎克雷伯菌)的死亡。调理吞噬杀菌活性根据如下实验采用生物发光测定进行测量。该实验设计用于评价双特异性抗体介导的针对肺炎克雷伯菌的调理吞噬杀菌作用，以DVD-Ig形式和Bs4Ab形式的双特异性抗体作为示例。首先将表达完整荧光素的质粒pUC18-mini-Tn7T-Gm-lux电转化入肺炎克雷伯菌KP19173中，构建发光的肺炎克雷伯菌株KP19173-LUX，该方法在文献(Choi K H,et al.mini-Tn7insertion in bacteria with single attTn7 sites:example Pseudomonas aeruginosa[J].Nature Protocols,2006,1(1):153-161)中有所描述。通过酶标仪检测发光克雷伯菌株的相对光单位(RLU)的变化来评估双特异性抗体的OPK活性。OPK活性检测方法在文献Wang Q,et al.Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit.J Infect Dis.2016Jun 1；213(11):1800-8中有所描述。简言之，该实验在96孔板中进行，幼兔血清作为补体来源，HL-60细胞经DMF诱导分化为巨噬细胞。将对数生长期的发光肺炎克雷伯菌KP19173-LUX接种到96板中、每孔加入5×10 5分化的巨噬细胞、灭活的幼兔血清(1:10Cedarlane)、以及不同浓度倍比稀释的双特异性抗体，混合均匀，于37℃，振荡(250rpm)孵育2h。然后利用酶标仪测量相对光单位(RLU)。以无抗体加入的孔的RLU值作为调理吞噬杀菌活性为0％的参照，以无肺炎克雷伯菌加入的孔的RLU值作为调理吞噬杀菌活性为100％的参照，根据加入双特异性抗体的孔的RLU值计算不同浓度下抗体的调理吞噬杀菌活性(％)，利用Graphpad Prism软件绘图，计算IC50值。
如图10A-10B所示，DVD-Ig形式的双特异性抗体K5-G2-Ig1、K5-G7-Ig1对KP19173菌株具有较强的促进调理吞噬杀菌的活性。
如图10C-10D所示，Bs4Ab形式的双特异性抗体K5-G2scFv-Ig1、K5-G7scFv-Ig1抗体对KP19173菌株具有较强的促进调理吞噬杀菌的活性。
表1中具体展示了不同结构的示例性双特异性抗体对肺炎克雷伯菌KP19173的促进调理吞噬杀菌活性IC50值。
表13
Figure PCTCN2022133880-appb-000043
实施例11：双特异性抗体介导的血清杀菌活性
血清杀菌活性(Serum Bactericidal Activity,SBA)实验设计用于评价双特异性抗体介导的补体依赖性血清杀菌活性，以DVD-Ig形式和Bs4Ab形式的双特异性抗体作为示例。首先将表达完整荧光素的质粒pUC18-mini-Tn7T-Gm-lux电转入肺炎克雷伯菌株KP19173菌株中，构建发光肺炎克雷伯菌株KP19173-LUX。通过检测发光肺炎克雷伯菌的相对光单位(RLU)的变化，来确定双特异性抗体介导的补体依赖性血清杀菌活性。SBA的检测方法在文献Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils，mBio.2018Mar-Apr；9(2):e00297-18中有所描述。简言之，该实验在96孔板中进行，幼兔血清作为补体来源，将对数生长期的发光肺炎克雷伯菌KP19173-LUX与起始浓度为1.3nM的双特异性抗体梯度稀释液预先混合，接种到96板中，37℃、5％CO2孵育15min。每孔加入25μL稀释的幼兔血清(1:10Cedarlane)和35μL PBS缓冲液后，于37℃、振荡(250rpm)、孵育3h。通过酶标仪测量相对光单位(RLU)。以无抗体加入的孔的RLU值作为血清杀菌滴度为0％的参照，以无肺炎克雷伯菌加入的孔的RLU值作为血清杀菌滴度为100％的参照，根据加入双特异性抗体的孔的RLU值来计算不同浓度下抗体的血清杀菌滴度，利用GraphPad Prism软件绘图，确定各抗体的血清杀菌活性IC50值。
如图11A-11B所示，DVD-Ig形式的双特异性抗体K5-G2-Ig1、K5-G7-Ig1体针对KP19173菌株具有抗体介导的补体依赖的血清杀菌活性。
如图11C-11D所示，和Bs4Ab形式的双特异性抗体K5-G2scFv-Ig1、K5-G7scFv-Ig1抗体针对KP19173菌株具有抗体介导的补体依赖的血清杀菌活性。
表14中具体展示了不同结构双特异性抗体对肺炎克雷伯菌KP19173的血清杀菌活性IC50值。
表14
Figure PCTCN2022133880-appb-000044
实施例12：双特异性抗体对肺炎克雷伯菌感染(肺炎)的治疗作用
C57/BL6小鼠购买自维通利华并饲养在无特殊病原体的环境中。所有动物实验都是依照研究机构动物护理和使用委员会(IACUC)方案和指导进行的。以DVD-Ig形式和Bs4Ab形式的双特异性抗 体作为示例。为了检测双特异性抗体在小鼠肺炎克雷伯菌感染肺炎模型中是否有治疗作用，本实验采用肺递送途径攻毒小鼠，构建了小鼠肺炎克雷伯菌感染的肺炎模型。小鼠肺炎模型的具体制备方法如实施例6所述。其中菌液递送剂量为：25μL KP19173(OD600＝0.32)/小鼠或25μL KP19180(OD600＝0.5)菌液/小鼠。菌液递送完成后，将小鼠放回笼内，1h后通过小鼠尾静脉注射特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体K5-G2-Ig1，剂量为30mpk；或特异性结合肺炎克雷伯菌O2抗原的抗体K5，剂量为30mpk；或特异性结合肺炎克雷伯菌O1抗原的抗体G2，剂量为30mpk；模型组中的小鼠注射抗体Mab601(30mpk)。Mab601是一种持异性结合肺炎克雷伯菌O3甘露糖的抗体，在文献Rollenske T,et al.Cross-specificity of protective human antibodies against Klebsiella pneumoniae LPS O-antigen.Nat Immunol.2018Jun；19(6):617-624.中有所描述。抗体注射完成后，持续观察至小鼠清醒。而后每天观察2次，直到第8天，记录各组小鼠死亡数量和时间并记录存活小鼠的状态。在GraphPad Prism软件中对各组小鼠的存活数据绘图，确定小鼠死亡率。
结果如图12A所示，在肺炎克雷伯菌KP19180感染肺炎模型中，与模型组相比，双特异性抗体K5-G2-Ig1显著提高了小鼠的存活率，延长小鼠生存时间。
结果如图12B所示，在肺炎克雷伯菌KP19173感染肺炎模型中，双特异性抗体K5-G2-Ig1对小鼠提供了100％的生存保护率。
以上结果表明，本发明的双特异性抗体对各个不同的肺炎克雷伯菌株引起的肺炎均具有治疗效果。
实施例13：特异性结合肺炎克雷伯菌O2抗原的抗体以及双特异性抗体亲和力Kd值的测定
采用Biacore 3000(GE)检测特异性结合肺炎克雷伯菌O2抗原的抗体K4和K5(重构建成人IgG1形式)以及特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体K5-G2-Ig1、G7-K5-CrossMab-Ig1和K5-G7scFv-Ig1的结合亲和力。该方法为本领域技术人员熟知的常规方法，根据使用说明书进行实验操作。简言之，将稀释后的生物素化的KP19180菌株的O抗原、KP19173菌株的O抗原或KP19213菌株的O抗原分别与SA芯片结合20s，流速10μL/min。检测不同浓度下侯选抗体的亲和力，浓度范围包括：0M、7.815E-10M、1.563E-9M、3.126E-9M、6.252E-9M、1.250E-8M、2.501E-8M、5.002E-8M。用SPR技术测量抗体的结合和解离速率并确定结合亲和力，表15和表16中分别列出了特异性结合肺炎克雷伯菌O2抗原的示例性抗体以及特异性结合肺炎克雷伯菌O2抗原和O1抗原的示例性双特异抗体与不同肺炎克雷伯菌株O抗原结合的Kon、Koff、以及Kd值。
表15
Figure PCTCN2022133880-appb-000045
Figure PCTCN2022133880-appb-000046
表16
Figure PCTCN2022133880-appb-000047

Claims (76)

  1. 一种分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其包含：
    (i)V H，其包含如氨基酸序列SEQ ID NO:17所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:21所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；
    (ii)V H，其包含如氨基酸序列SEQ ID NO:18所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:22所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；
    (iii)V H，其包含如氨基酸序列SEQ ID NO:19所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:23所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；
    (iv)V H，其包含如氨基酸序列SEQ ID NO:19所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；或
    (v)V H，其包含如氨基酸序列SEQ ID NO:20所示的V H包含的HC-CDR1、HC-CDR2和HC-CDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
  2. 根据权利要求1中所述的分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其包含：
    (i)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:5，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:8，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；
    (ii)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:3，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:9，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；
    (iii)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:13，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；
    (iv)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代；或
    (v)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7，或者所述V H的变体，其HC-CDRs中包含至多约5个氨基酸的取代；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15，或者所述V L的变体，其LC-CDRs中包含至多约5个氨基酸的取代。
  3. 根据权利要求1或2中所述的分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其包含：
    (i)V H，其包含氨基酸序列SEQ ID NO:17或其变体，所述变体与氨基酸序列SEQ ID NO:17具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:21或其变体，所述变体与氨基酸序列SEQ ID NO:21具有至少约80％序列同源性；
    (ii)V H，其包含氨基酸序列SEQ ID NO:18或其变体，所述变体与氨基酸序列SEQ ID NO:18具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:22或其变体，所述变体与氨基酸序列SEQ ID NO:22具有至少约80％序列同源性；
    (iii)V H，其包含氨基酸序列SEQ ID NO:19或其变体，所述变体与氨基酸序列SEQ ID NO:19具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:23或其变体，所述变体与氨基酸序列SEQ ID NO:23具有至少约80％序列同源性；
    (iv)V H，其包含氨基酸序列SEQ ID NO:19或其变体，所述变体与氨基酸序列SEQ ID NO:19具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:24或其变体，所述变体与氨基酸序列SEQ ID NO:24具有至少约80％序列同源性；或
    (v)V H，其包含氨基酸序列SEQ ID NO:20或其变体，所述变体与氨基酸序列SEQ ID NO:20具有至少约80％序列同源性；以及V L，其包含氨基酸序列SEQ ID NO:25或其变体，所述变体与氨基酸序列SEQ ID NO:25具有至少约80％序列同源性。
  4. 一种分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其与权利要求1-3中任一项所述的分离的抗O2抗原的抗体或抗原结合片段竞争性地与具有D-半乳聚糖I结构域的肺炎克雷伯菌特异性结合，或与权利要求1-3中任一项所述的分离的抗O2抗原的抗体或抗原结合片段特异性地结合相同的表位。
  5. 根据权利要求1-4中任一项分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其中所述抗体或抗原结合片段包含Fc。
  6. 根据权利要求5中的分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其中所述抗体或抗原结合片段是全长的IgA、IgD、IgE、IgG或IgM抗体。
  7. 根据权利要求6中的分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其中所述抗体或抗原结合片段是全长的IgG1、IgG2、IgG3或IgG4抗体。
  8. 根据权利要求1-7中任一项的分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其中所述抗体或抗原结合片段是嵌合的、人的或人源化的抗体。
  9. 根据权利要求1-8中任一项的分离的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段，其中所述抗原结合片段选自Fab、Fab’、F(ab)’ 2、Fab’-SH、单链Fv(scFv)、Fv片段、dAb、Fd、纳米抗体(nanobody)、双链抗体(diabody)和线性抗体。
  10. 一种双特异性抗体，包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中第一抗原结合域包含权利要求1-9中任一项所述的特异性结合肺炎克雷伯菌O2抗原的抗体。
  11. 根据权利要求10中的双特异性抗体，其中所述第一抗原结合域包含：
    (a)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:4；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:11；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:14；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:16；或者
    (b)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:2；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:10；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:12；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:15。
  12. 根据权利要求10-11中任一项的双特异性抗体，其中所述第二抗原结合域包含：
    (a)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:35；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:41；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或者
    (b)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:36；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:38；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:42；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:44；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
  13. 根据权利要求10-12中任一项所述的双特异性抗体，其中第一抗原结合域包含：
    (a)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；
    以及其中第二抗原结合域包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或
    (b)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；
    以及其中第二抗原结合域包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46；或
    (c)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；
    以及其中第二抗原结合域包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或
    (d)V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述 V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；
    以及其中第二抗原结合域包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
  14. 根据权利要求10-13中任一项的双特异性抗体，包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中所述第一抗原结合域包含：
    (a)V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:19或28所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24或33所示的V L包含的LCDR1、LCDR2和LCDR3；或
    (b)V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:20或29所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25或34所示的V L包含的LCDR1、LCDR2和LCDR3。
  15. 根据权利要求10-14中任一项的双特异性抗体，其中所述第二抗原结合域包含：
    (a)V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:47或51所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:49或53所示的V L包含的LCDR1、LCDR2和LCDR3；或
    (b)V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:48或52所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:50或54所示的V L包含的LCDR1、LCDR2和LCDR3。
  16. 根据权利要求10-15中任一项所述的双特异性抗体，其中：
    (a)所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；以及其中
    所述第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列；或
    (b)所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；以及其中
    所述第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列；或
    (c)所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；以及其中
    所述第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸 序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列；或
    (d)所述第一抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；以及其中
    所述第二抗原结合域包含：V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。
  17. 一种双特异性抗体，包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中所述第二抗原结合域包含：
    (a)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:35；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:41；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或者
    (b)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:36；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:38；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:42；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:44；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
  18. 根据权利要求17中的双特异性抗体，包括特异性结合肺炎克雷伯菌O2抗原的第一抗原结合域和特异性结合肺炎克雷伯菌O1抗原的第二抗原结合域，其中所述第二抗原结合域包含：
    (a)V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:47或51所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:49或53所示的V L包含的LCDR1、LCDR2和LCDR3；或
    (b)V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:48或52所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:50或54所示的V L包含的LCDR1、LCDR2和LCDR3。
  19. 根据权利要求10-18中任一项所述的双特异性抗体，其结构选自DVD-Ig、Bs4Ab、Hetero H,CrossMab、IgG-(scFv) 2、或scFv-Fab IgG等结构。
  20. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中两条多肽链从N端到C端包含V H1-L-V H2-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；L是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和
    另外两条多肽链从N端到C端包含V L1-L-V L2-C L结构，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；V L2是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；L是连接肽；C L是轻链恒定区。
  21. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中两条多肽链从N端到C端包含V H1-L-V H2-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；L是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和
    另外两条多肽链从N端到C端包含V L1-L-V L2-C L结构，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；V L2是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；L是连接肽；C L是轻链恒定区。
  22. 根据权利要求20-21中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:61，或包含与氨基酸序列SEQ ID NO:61具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:62，或包含与氨基酸序列SEQ ID NO:62具有至少80％序列同源性的变体序列。
  23. 根据权利要求20-21中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:63，或包含与氨基酸序列SEQ ID NO:63具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:64，或包含与氨基酸序列SEQ ID NO:64具有至少80％序列同源性的变体序列。
  24. 根据权利要求20-21中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:65，或包含与氨基酸序列SEQ ID NO:65具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％序列同源性的变体序列。
  25. 根据权利要求20-21中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:67，或包含与氨基酸序列SEQ ID NO:67具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:68，或包含与氨基酸序列SEQ ID NO:68具有至少80％序列同源性的变体序列。
  26. 根据权利要求20-21中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:86，或包含与氨基酸序列SEQ ID NO:86具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:62，或包含与氨基酸序列SEQ ID NO:62具有至少80％序列同源性的变体序列。
  27. 根据权利要求20-21中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:87，或包含与氨基酸序列SEQ ID NO:87具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:64，或包含与氨基酸序列SEQ ID NO:64具有至少80％序列同源性的变体序列。
  28. 根据权利要求20-21中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:88，或包含与氨基酸序列SEQ ID NO:88具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％序列同源性的变体序列。
  29. 根据权利要求20-21中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:89，或包含与氨基酸序列SEQ ID NO:89具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:68，或包含与氨基酸序列SEQ ID NO:68具有至少80％序列同源性的变体序列。
  30. 根据权利要求20-21中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:115，或包含与氨基酸序列SEQ ID NO:115具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:66，或包含与氨基酸序列SEQ ID NO:66具有至少80％序列同源性的变体序列。
  31. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中两条多肽链从N端到C端包含V H1-C H1-L1-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；L1和L3是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和
    另外两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区。
  32. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中两条多肽链从N端到C端包含V H1-C H1-L1-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区； L1和L3是连接肽；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和
    另外两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区。
  33. 根据权利要求31-32中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:69，或包含与氨基酸序列SEQ ID NO:69具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:70，或包含与氨基酸序列SEQ ID NO:70具有至少80％序列同源性的变体序列。
  34. 根据权利要求31-32中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:71，或包含与氨基酸序列SEQ ID NO:71具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:72，或包含与氨基酸序列SEQ ID NO:72具有至少80％序列同源性的变体序列。
  35. 根据权利要求31-32中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:73，或包含与氨基酸序列SEQ ID NO:73具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  36. 根据权利要求31-32中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:75，或包含与氨基酸序列SEQ ID NO:75具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  37. 根据权利要求31-32中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:90，或包含与氨基酸序列SEQ ID NO:90具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:70，或包含与氨基酸序列SEQ ID NO:70具有至少80％序列同源性的变体序列。
  38. 根据权利要求31-32中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:91，或包含与氨基酸序列SEQ ID NO:91具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:72，或包含与氨基酸序列SEQ ID NO:72具有至少80％序列同源性的变体序列。
  39. 根据权利要求31-32中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:92，或包含与氨基酸序列SEQ ID NO:92具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  40. 根据权利要求31-32中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:93，或包含与氨基酸序列SEQ ID NO:93具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  41. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；
    一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区；
    一条多肽链从N端到C端包含V H2-C L，其中V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；C L是轻链恒定区；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；以及
    一条多肽链从N端到C端包含V L2-C H1，其中V L2是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C H1是重链恒定区C H1结构域。
  42. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；
    一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区；
    一条多肽链从N端到C端包含V H2-C L，其中V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；C L是轻链恒定区；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；以及
    一条多肽链从N端到C端包含V L2-C H1，其中V L2是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C H1是重链恒定区C H1结构域。
  43. 根据权利要求41-42中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:76，或包含与氨基酸序列SEQ ID NO:76具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:77，或包含与氨基酸序列SEQ ID NO:77具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:78，或包含与氨基酸序列SEQ ID NO:78具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  44. 根据权利要求41-42中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:79，或包含与氨基酸序列SEQ ID NO:79具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:80，或包含与氨基酸序列SEQ ID NO:80具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:78，或包含与氨基酸序列SEQ ID NO:78具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  45. 根据权利要求41-42中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:94，或包含与氨基酸序列SEQ ID NO:94具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:77，或包含与氨基酸序列SEQ ID NO:77具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:95，或包含与氨基酸序列SEQ ID NO:95具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  46. 根据权利要求41-42中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:96，或包含与氨基酸序列SEQ ID NO:96具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:80，或包含与氨基酸序列SEQ ID NO:80具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:95，或包含与氨基酸序列SEQ ID NO:95具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:74，或包含与氨基酸序列SEQ ID NO:74具有至少80％序列同源性的变体序列。
  47. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中两条多肽链从N端到C端包含V H1-C H1-C H2-C H3-L-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；L和L3是连接肽；C H1是重链恒定区C H1结构域；C H2是重链恒定区C H2结构域；C H3是重链恒定区C H3结构域；和
    另外两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区。
  48. 根据权利要求10-19中任一项所述的双特异性抗体，其包含四条多肽链：
    其中两条多肽链从N端到C端包含V H1-C H1-C H2-C H3-L-V H2-L3-V L2结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；L和L3是连接肽；C H1是重链恒定区C H1结构域；C H2是重链恒定区C H2结构域；C H3是重链恒定区C H3结构域；和
    另外两条多肽链从N端到C端包含V L1-C L结构，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区。
  49. 根据权利要求47-48中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:97，或包含与氨基酸序列SEQ ID NO:97具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％序列同源性的变体序列。
  50. 根据权利要求47-48中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:98，或包含与氨基酸序列SEQ ID NO:98具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％序列同源性的变体序列。
  51. 根据权利要求10-19中任一项所述的双特异性抗体，其包含三条多肽链：
    其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O2抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；
    一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O2抗原的轻链可变区，C L是轻链恒定区；以及
    一条多肽链从N端到C端包含V H2-L3-V L2，其中V H2是特异性结合肺炎克雷伯菌O1抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O1抗原的轻链可变区；L3是连接肽；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域。
  52. 根据权利要求10-19中任一项所述的双特异性抗体，其包含三条多肽链：
    其中一条多肽链从N端到C端包含V H1-C H1结构，其中V H1是特异性结合肺炎克雷伯菌O1抗原的重链可变区；C H1是重链恒定区C H1结构域；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域；和
    其中一条多肽链从N端到C端包含V L1-C L，其中V L1是特异性结合肺炎克雷伯菌O1抗原的轻链可变区，C L是轻链恒定区；和
    其中一条多肽链从N端到C端包含V H2-L3-V L2，其中V H2是特异性结合肺炎克雷伯菌O2抗原的重链可变区；V L2是特异性结合肺炎克雷伯菌O2抗原的轻链可变区；L3是连接肽；其中所述多肽链进一步包含Fc，该Fc包含C H2和C H3结构域。
  53. 根据权利要求51-52中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:81，或包含与氨基酸序列SEQ ID NO:81具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:82，或包含与氨基酸序列SEQ ID NO:82具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％序列同源性的变体序列。
  54. 根据权利要求51-52中任一项所述的双特异性抗体，其包含：
    氨基酸序列SEQ ID NO:84，或包含与氨基酸序列SEQ ID NO:84具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:82，或包含与氨基酸序列SEQ ID NO:82具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％序列同源性的变体序列。
  55. 根据权利要求51-52中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:99，或包含与氨基酸序列SEQ ID NO:99具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:100，或包含与氨基酸序列SEQ ID NO:100具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:83，或包含与氨基酸序列SEQ ID NO:83具有至少80％序列同源性的变体序列。
  56. 根据权利要求51-52中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    氨基酸序列SEQ ID NO:101，或包含与氨基酸序列SEQ ID NO:101具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:100，或包含与氨基酸序列SEQ ID NO:100具有至少80％序列同源性的变体序列；和/或
    氨基酸序列SEQ ID NO:85，或包含与氨基酸序列SEQ ID NO:85具有至少80％序列同源性的变体序列。
  57. 一种药物组合物，包括：特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段；其中特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    (a)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:4；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:11；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:14；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:16；或者
    (b)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:2；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:10；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:12；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:15。
  58. 一种在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段，其中特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    (a)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:4；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:11；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:14；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:16；或者
    (b)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:1；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:2；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:10；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:12；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:15。
  59. 根据权利要求57中所述的药物组合物或权利要求58中所述的方法，其中特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    (a)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:35；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:41；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或者
    (b)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:36；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:38；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:42；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:44；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
  60. 根据权利要求57、59中任一项所述的药物组合物或权利要求58-59中任一项所述的方法，
    (a)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和 LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或
    (b)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:2，和HC-CDR3，其包含氨基酸序列SEQ ID NO:7；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:10，LC-CDR2，其包含氨基酸序列SEQ ID NO:12，和LC-CDR3，其包含氨基酸序列SEQ ID NO:15；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和LC-CDR3，其包含氨基酸序列SEQ ID NO:46；或
    (c)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:35，HC-CDR2，其包含氨基酸序列SEQ ID NO:37，和HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:41，LC-CDR2，其包含氨基酸序列SEQ ID NO:43，和LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或
    (d)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:1，HC-CDR2，其包含氨基酸序列SEQ ID NO:4，和HC-CDR3，其包含氨基酸序列SEQ ID NO:6；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:11，LC-CDR2，其包含氨基酸序列SEQ ID NO:14，和LC-CDR3，其包含氨基酸序列SEQ ID NO:16；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:36，HC-CDR2，其包含氨基酸序列SEQ ID NO:38，和HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:42，LC-CDR2，其包含氨基酸序列SEQ ID NO:44，和 LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
  61. 根据权利要求57，59-60中任一项所述的药物组合物或权利要求58-60中任一项所述的方法，其中特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    (a)V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:19或28所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:24或33所示的V L包含的LCDR1、LCDR2和LCDR3；或
    (b)V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:20或29所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:25或34所示的V L包含的LCDR1、LCDR2和LCDR3。
  62. 根据权利要求57，59-61中任一项所述的药物组合物或权利要求58-61中任一项所述的方法，其中特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    (a)V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:47或51所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:49或53所示的V L包含的LCDR1、LCDR2和LCDR3；或
    (b)V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:48或52所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:50或54所示的V L包含的LCDR1、LCDR2和LCDR3。
  63. 根据权利要求57，59-62中任一项所述的药物组合物或权利要求58-62中任一项所述的方法，
    (a)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列；或
    (b)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:20或29，或包含与氨基酸序列SEQ ID NO:20或29具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:25或34，或包含与氨基酸序列SEQ ID NO:25或34具有至少80％序列同源性的变体序列；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列；
    (c)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少80％序列同源性的变体序列；或
    (d)其中特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:19或28，或包含与氨基酸序列SEQ ID NO:19或28具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:24或33，或包含与氨基酸序列SEQ ID NO:24或33具有至少80％序列同源性的变体序列；
    以及其中特异性结合肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52 具有至少80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少80％序列同源性的变体序列。
  64. 一种药物组合物，包括：特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段；其中特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含:
    (a)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:35；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:41；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或者
    (b)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:36；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:38；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:42；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:44；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
  65. 一种在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段，其中特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    (c)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:35；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:37；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:39；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:41；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:43；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:45；或者
    (d)重链可变区(V H)，所述V H包含：
    重链互补决定区(HC-CDR)1，其包含氨基酸序列SEQ ID NO:36；
    HC-CDR2，其包含氨基酸序列SEQ ID NO:38；和
    HC-CDR3，其包含氨基酸序列SEQ ID NO:40；以及
    轻链可变区(V L)，所述V L包含：
    轻链互补决定区(LC-CDR)1，其包含氨基酸序列SEQ ID NO:42；
    LC-CDR2，其包含氨基酸序列SEQ ID NO:44；和
    LC-CDR3，其包含氨基酸序列SEQ ID NO:46。
  66. 根据权利要求64中所述的药物组合物或权利要求65中所述的方法，其中特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段包含：
    (a)V H，其包含氨基酸序列SEQ ID NO:47或51，或包含与氨基酸序列SEQ ID NO:47或51具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:49或53，或包含与氨基酸序列SEQ ID NO:49或53具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:47或51所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:49或53所示的V L包含的LCDR1、LCDR2和LCDR3；或
    (b)V H，其包含氨基酸序列SEQ ID NO:48或52，或包含与氨基酸序列SEQ ID NO:48或52具有至少约80％序列同源性的变体序列；以及V L，其包含氨基酸序列SEQ ID NO:50或54，或包含与氨基酸序列SEQ ID NO:50或54具有至少约80％序列同源性的变体序列；或者V H，其包含如氨基酸序列SEQ ID NO:48或52所示的V H包含的HCDR1、HCDR2和HCDR3；以及V L，其包含如氨基酸序列SEQ ID NO:50或54所示的V L包含的LCDR1、LCDR2和LCDR。
  67. 根据权利要求58-63和65-66中任一项所述的方法，其中特异性识别肺炎克雷伯菌O2抗原的抗体或抗原结合片段和特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段向个体同时施用或向个体顺序施用。
  68. 一种编码权利要求1-9中任一项所述的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或权利要求10-56中任一项所述的双特异性抗体的分离的核酸分子。
  69. 一种包含权利要求68中所述的核酸分子的载体。
  70. 一种分离的宿主细胞，其包含权利要求1-9中任一项所述的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或权利要求10-56中任一项所述的双特异性抗体、权利要求68中所述的核酸分子或权利要求69中所述的载体。
  71. 一种制备权利要求1-9中任一项所述的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或权利要求10-56中任一项所述的双特异性抗体的方法，其包含：
    a)在能有效表达抗体的条件下培养权利要求70中所述的宿主细胞；和
    b)从宿主细胞中获得表达的抗体。
  72. 一种药物组合物，包含权利要求1-9中任一项所述的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段或权利要求10-56中任一项所述的特异性结合肺炎克雷伯菌O2抗原和O1抗原的双特异性抗体、权利要求68中所述的核酸分子、权利要求69中所述的载体或权利要求70中所述的分离的宿主细胞，以及药学上可接受的载体或辅料。
  73. 一种在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的权利要求57，59-64，66，或72中所述的药物组合物、权利要求10-56中所述的特异性结合肺炎克雷伯O1抗原和O2抗原的双特异性抗体和权利要求1-9中任一项所述的特异性结合肺炎克雷伯菌O2抗原的抗体或抗原结合片段。
  74. 根据权利要求73或权利要求58-63，65-67中所述的方法，其中所述的疾病或病症包含由克雷伯(氏)菌属感染引起的一种或多种症状。
  75. 根据权利要求74中所述的方法，其中所述的克雷伯(氏)菌属为肺炎克雷伯菌。
  76. 根据权利要求75中所述的方法，其中所述的疾病或病症包括肺炎、尿路感染、败血症/菌血症/脓毒症、新生儿败血症/菌血症/脓毒症、腹泻、软组织感染、器官移植后感染、手术感染、伤口感染、肺部感染、化脓性肝脓肿、肺脓肿、蜂窝组织炎、坏死性肌膜炎、肌炎、眼内炎、腹膜炎、脑膜炎、坏死性脑膜炎、强直性脊柱炎或脊柱关节病。
PCT/CN2022/133880 2021-12-06 2022-11-24 特异性结合肺炎克雷伯菌o2抗原和o1抗原的双特异性抗体以及组合物 WO2023103788A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109689090A (zh) * 2016-08-05 2019-04-26 免疫医疗有限责任公司 抗o2抗体及其用途
CN109843917A (zh) * 2016-10-19 2019-06-04 免疫医疗有限责任公司 抗o1抗体及其用途
CN111448204A (zh) * 2017-11-30 2020-07-24 瓦希隆股份公司 抗肺炎克雷伯菌的疫苗

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109689090A (zh) * 2016-08-05 2019-04-26 免疫医疗有限责任公司 抗o2抗体及其用途
CN109843917A (zh) * 2016-10-19 2019-06-04 免疫医疗有限责任公司 抗o1抗体及其用途
CN111448204A (zh) * 2017-11-30 2020-07-24 瓦希隆股份公司 抗肺炎克雷伯菌的疫苗

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ARATO VANESSA, RASO MARIA MICHELINA, GASPERINI GIANMARCO, BERLANDA SCORZA FRANCESCO, MICOLI FRANCESCA: "Prophylaxis and Treatment against Klebsiella pneumoniae: Current Insights on This Emerging Anti-Microbial Resistant Global Threat", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 22, no. 8, 14 April 2021 (2021-04-14), pages 4042, XP093072190, DOI: 10.3390/ijms22084042 *
SARA E HESTER;JIHYE PARK;LAURA L GOODFIELD;HEATHER A FEAGA;ANDREW PRESTON;ERIC T HARVILL: "Horizontally acquired divergent O-antigen contributes to escape from cross-immunity in the classical bordetellae", BMC EVOLUTIONARY BIOLOGY, BIOMED CENTRAL LTD., LONDON, GB, vol. 13, no. 1, 25 September 2013 (2013-09-25), GB , pages 209, XP021158423, ISSN: 1471-2148, DOI: 10.1186/1471-2148-13-209 *
SUN YUE-PENG, LIANG SUN: "Selective protective effect of hydrogen water on free radical injury of athletes after high-intensity exercise", BIOMEDICAL RESEARCH, SCIENTIFIC PUBL. OF INDIA,, INDIA, vol. 28, no. 10, 14 March 2017 (2017-03-14), India , pages 4558 - 4561, XP093072184, ISSN: 0970-938X *

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